TGF-βRII binding proteins

Abstract

To provide a new pharmaceutical agent for the treatment of human disease, in particular for the treatment of cancer.SOLUTION: The present invention relates to an antibody or antibody fragment thereof that specifically binds to the extracellular domain of human TGF-βRII. The present invention further relates to a vector comprising a polynucleotide encoding the antibody or antibody fragment of the invention, an isolated cell producing the antibody or antibody fragment of the invention, and a pharmaceutical composition comprising the antibody or antibody fragment of the invention. The antibody or antibody fragment of the invention can be used to treat cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of medicine. More specifically, the present invention relates to proteins that bind to transforming growth factor-beta receptor II (TGF-βRII) and their use in human therapy, particularly in the treatment of cancer. [Background technology]

[0002] Transforming growth factor beta (TGF-β) is a signaling molecule part of the TGF-β superfamily. There are three TGF-β ligands (TGF-β1, 2, and 3) that regulate various cellular functions. TGF-β signaling has a tumor-suppressive role in normal cells but can have a tumor-promoting role in malignant cells. It is involved in processes such as proliferation, migration, differentiation, apoptosis, angiogenesis, and epithelial-mesenchymal transition (Bierie and Moses, 2006; Siegel and Massague, 2003). These signals are mediated by binding to TGF-β receptor type 2 (TGF-βRII), which leads to dimerization with TGF-βRI and its phosphorylation. This heterotetrameric complex, consisting of two TGF-βRIIs and two TGF-βRIs, then recruits and phosphorylates SMAD2 and SMAD3, and then recruits SMAD4 to bind to co-SMAD molecules to form a SMAD / co-SMAD complex, which translocates to the nucleus, where it regulates the transcription of TGF-β target genes ( Hata and Chen, 2016 ; Vander Ark, Cao et al., 2018 ).

[0003] High levels of TGF-β expression have been shown to be associated with poor prognosis in cancer. TGF-β can induce epithelial-mesenchymal transition, which promotes cancer cell migration and metastasis. Gene expression profiles have shown that TGF-β signaling is a significant pathway in colorectal cancer liver metastasis (Jung, Staudacher et al., 2017). Furthermore, stromal-epithelial interactions have been reported to be involved in cancer progression. Cancer-associated fibroblasts (CAFs), an abundant stromal cell component found in solid tumors, contribute to immune evasion and promote tumor initiation and growth, in addition to building and remodeling the extracellular matrix that can serve as a tumor scaffold (Quail and Joyce, 2013). Tumor-derived TGF-β1 induces the transdifferentiation of fibroblasts into "activated" CAFs by binding to TGFβRII, which can promote a pro-tumorigenic stromal environment.

[0004] Existing approaches targeting the TGF-β signaling pathway to inhibit aberrant TGF-β signaling characteristics of CAFs and tumors have so far had suboptimal efficacy and / or developmental challenges. One anti-TGF-βRII monoclonal antibody, LY3022859, disclosed in WO 2010 / 053814, has been reported to block the extracellular domain of TGF-βRII. However, a Phase I dose-escalation trial found that it caused cytokine release syndrome and was deemed unsafe for patients with advanced solid tumors. Another agent, disclosed in WO 2012 / 093125, is an anti-TGF-βRII single variable domain antibody with a shorter half-life, and the exact mechanism of action or binding domain of this molecule has not been established. To date, no anti-TGF-βRII antibody has successfully entered clinical trials due to the complexity and versatility of TGF-β tumor regulation, which makes drug development difficult (Hao, Baker et al., 2019). Herein, we describe novel heavy chain variable region, heavy chain, Fab, and full-length IgG monospecific antibodies that can bind to TGF-βRII. These antibodies, or antibodies containing these heavy chains or heavy chain variable regions, target novel locations on TGF-βRII and block the interaction between the receptor and its ligand, an improvement over existing antibodies. Summary of the Invention

[0005] It is an object of the present invention to provide new pharmaceutical agents for the treatment of human diseases, particularly cancer. This object is met by the provision of antibodies or antibody fragments thereof, and specific binding domains, that specifically bind to the extracellular domain of human TGF-βRII, as described and claimed herein.

[0006] In a first aspect, the present invention relates to an antibody or antibody fragment thereof that specifically binds to the extracellular domain of human TGF-βRII, wherein the antibody or antibody fragment binds to an epitope in the extracellular domain of human TGF-βRII, and phenylalanine (F) at position 25 of isoform A of human TGF-βRII or position 50 of isoform B of human TGF-βRII is an essential residue for binding.

[0007] Preferably, such an antibody or antibody fragment binds to TGF-βRII and blocks or inhibits the binding of human TGF-β to human TGF-βRII, thereby inhibiting signal transmission to cells and blocking or inhibiting heterodimerization of TGF-βRII.

[0008] In a second aspect, the present invention relates to a vector comprising a polynucleotide encoding either or both of the heavy and light chains of an antibody or antibody fragment described herein.

[0009] In a third aspect, the present invention relates to a cell that produces an antibody or antibody fragment described herein.

[0010] In a fourth aspect, the invention relates to a pharmaceutical composition comprising an antibody or antibody fragment described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0011] In a fifth aspect, the present invention relates to a pharmaceutical for preventing, inhibiting the progression or recurrence of symptoms of, and / or treating cancer, comprising an antibody or antibody fragment thereof as described herein as an active ingredient.

[0012] In a sixth aspect, the present invention relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or antibody fragment described herein, or a pharmaceutical composition described herein.

[0013] In a seventh aspect, the present invention relates to a method for blocking the binding of human TGF-β to human TGF-βRII in a cell, the method comprising providing to a cell an antibody or antibody fragment described herein and allowing the antibody or antibody fragment to bind to human TGF-βRII in the cell, thereby blocking the binding of human TGF-β to human TGF-βRII in the cell.

[0014] In an eighth aspect, the present invention relates to a method for inhibiting signal transduction to a cell induced by binding of human TGF-β to human TGF-βRII in the cell, the method comprising providing to the cell an antibody or antibody fragment described herein, and allowing the antibody or antibody fragment to bind to human TGF-βRII in the cell, thereby inhibiting signal transduction to the cell.

[0015] In a ninth aspect, the present invention relates to a method for preventing or inhibiting metastasis, comprising administering to a subject an effective amount of an antibody or antibody fragment described herein, or a pharmaceutical composition described herein. DETAILED DESCRIPTION OF THE INVENTION

[0016] Generally, the present invention relates to a protein, specifically an antibody or antibody fragment thereof, that specifically binds to the extracellular domain of human TGF-βRII. Preferably, the antibody is isolated. Preferably, the antibody is a monoclonal antibody. More preferably, the antibody is an isolated monoclonal antibody.

[0017] An "isolated monoclonal antibody" refers to an antibody produced by a clonal cell. Examples of isolated antibodies include, but are not limited to, affinity-purified antibodies, antibodies produced in vitro in a hybridoma or other cell line, and human antibodies derived from transgenic non-human animals.

[0018] The term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains: two heavy (H) and two light (L) chains. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain variable region comprises three heavy chain complementarity-determining regions (HCDRs) and four framework regions (FRs) and is preferably human or humanized. The heavy chain constant region consists of three domains, CH1, CH2, and CH3, and can be derived from any organism, preferably human. The CH1 and CH2 domains of the heavy chain constant region are connected via a flexible hinge region. Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The light chain variable region can be of two types: kappa (K) or lambda (λ), and, like the VH, comprises three light chain complementarity-determining regions (LCDRs) and four framework regions. The light chain variable region is preferably human or humanized. The light chain constant region consists of one domain, CL, and can be derived from any organism, preferably human. The two heavy chains are linked to each other by a disulfide bond between the two hinge regions, and each heavy chain is paired with a light chain by a disulfide bond between the CH1 and CL regions. In conventional antibodies, the two heavy chains and the two light chains are identical, providing the antibody with two identical antigen-binding sites.

[0019] Antibody binding has different qualities, including specificity and affinity. Specificity determines which antigen or epitope is specifically bound by the antibody binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope.

[0020] A "human" antibody refers to an antibody in which all antibody domains are derived from human germline immunoglobulin sequences. Human antibodies used in the present invention can be produced by methods using mice transgenic to produce human antibodies, such as Humab mice, KM mice, Xeno mice, Tc mice, or MeMo® mice (WO 2009 / 157771). Human antibodies can also be prepared using SCID mice in which human immune cells have been reconstituted to generate a human antibody response upon immunization.

[0021] A "humanized" antibody region refers to an antibody prepared by grafting complementarity-determining region (CDR) sequences of an antibody derived from the germline of a non-human animal, such as a mouse or chicken, onto human framework sequences of a human antibody. Humanized antibodies can also be produced by linking nucleic acids encoding the CDR regions of an antibody isolated from an antibody-producing hybridoma to nucleic acids encoding the framework regions of a human-derived antibody using well-known methods.

[0022] An antibody can bind to an antigen via its heavy and / or light chain variable region, specifically via its specific CDR. The CDR of the heavy and / or light chain variable region of an antibody binds to an "epitope" (also called an "antigenic determinant") of the antigen. An epitope can be formed from non-contiguous amino acids juxtaposed by tertiary folding of a protein, so-called linear epitopes and conformational epitopes, respectively. Epitopes formed from adjacent linear amino acids typically retain their conformation upon exposure to denaturing solvents, while epitopes formed by tertiary folding typically lose their conformation upon treatment with denaturing solvents. An epitope can typically contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0023] An "antigen" is typically a molecule that can induce an immune response in a host organism, thereby producing antibodies specific to the antigen. At the molecular level, an antigen is characterized by its ability to be bound by the antigen-binding site of an antibody. As mentioned above, the antigen-binding site of an antibody is formed by its heavy and / or light chain variable regions, specifically by its CDRs. An antigen contains at least one epitope, but often contains more.

[0024] Also, a mixture of antigens can be considered an "antigen," and one skilled in the art will understand that a tumor cell lysate or virus particle may also be referred to as an "antigen," while many antigenic determinants are present in such a tumor cell lysate or virus particle preparation.

[0025] In the present invention, the antigen is human TGF-βRII. Human TGF-βRII is a transmembrane protein that exists in different isoforms. The amino acid sequence of human TGF-βRII isoform A is provided as SEQ ID NO: 101, and the amino acid sequence of the extracellular domain of human TGF-βRII isoform A is provided as SEQ ID NO: 102. Human TGF-βRII isoform B is a splice variant that encodes a longer isoform due to an insertion in the extracellular domain. The amino acid sequence of human TGF-βRII isoform B is provided as SEQ ID NO: 103, and the amino acid sequence of the extracellular domain of human TGF-βRII isoform B is as set forth in SEQ ID NO: 104.

[0026] TGF-βRII is a member of the serine / threonine protein kinase family and the TGFβ receptor subfamily. It is known by various synonyms, including TGFBR2, AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC, TAAD2, TGFR-2, TGFβ-RII, transforming growth factor beta receptor 2, and TBR-ii and TBRII. TGF-βRII binds to TGF-β by forming a heterodimeric complex with another receptor protein. This receptor / ligand complex phosphorylates the protein, which then enters the nucleus and regulates the transcription of a subset of genes related to cell proliferation.

[0027] An antibody binds to an antigen with a particular binding affinity. "Binding affinity" is defined by the formula: k off / k on The dissociation constant (K D ) and refers to the strength of the antibody-antigen interaction. Depending on the desired biological activity, antibodies may have high k onSpeed ​​and / or low k off The selection can be based on speed.

[0028] "Antibody fragments" include, but are not limited to, functional fragments of heavy and / or light chains. Such functional fragments contain at least one CDR derived from or synthesized based on a CDR from an antibody heavy or light chain and are capable of specifically recognizing an antigen. Antibody fragments can be, for example, but are not limited to, Fab, F(ab')2, scFv, minibody, or sdAb. "Antibody fragment" also refers to a proteinaceous portion comprising a functional portion of an antibody. In this case, it is at least the heavy chain variable region or one or more of the HCDRs described herein. The antibody fragment can be any binding agent, including but not limited to, a single chain Fv, single chain or tandem diabody (TandAb®), VHH, Anticalins®, Nanobodies®, BiTE®, Fab, ankyrin repeat protein or DARPIN®, Avimer®, DART, TCR-like antibody, Adnectin®, Affilin®, Trans-Body®, Affibody®, TrimerX®, MicroProtein, Fynomer®, Centyrin®, or KALBITOR®.

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

[0030] "F(ab')2" typically refers to a binding domain comprising two Fab domains joined together by a hinge region.

[0031] The term "single-chain variable fragment" (scFv) typically refers to a binding domain comprising a VH domain and a VL domain connected via a linker, for example, a peptide linker of about 10 to about 25 amino acids in length.

[0032] By "minibody" is meant a binding domain that typically comprises two scFv and a CH3 domain.

[0033] The term "single domain antibody" (sdAb) typically refers to a binding domain containing only the VH or VL domain of an antibody, usually fused to or otherwise linked to an Fc region. Like a whole antibody, this antibody can selectively bind to a specific antigen. Single domain antibody fragments can be engineered from heavy chain antibodies found in camelids, and these are sometimes called VHH fragments (Nanobodies®). Some fish also have heavy chain-only antibodies (IgNAR, "immunoglobulin novel antigen receptor"), from which single domain antibody fragments called VNAR fragments can be derived. An alternative approach is to split the dimeric variable domain from common immunoglobulin G (IgG) of human or mouse origin into monomers. While most research on single domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to be capable of binding to target epitopes. Therefore, nanobodies are also encompassed by the present invention.

[0034] "Antibody fragment" also refers to at least one CDR, which is a portion of a protein construct that exhibits binding specificity to an antigen. Preferably, at least one CDR is an HCDR3. In one embodiment, the protein construct comprises HCDR1, HCDR2, and HCDR3. In another embodiment, the protein construct comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The protein construct may further comprise a portion of an Fc region, including CH2 and / or CH3. The protein construct may also comprise a CH1 region. The CH1, CH2, and / or CH3 regions can be engineered to obtain any desired properties with respect to antigen binding and / or effector function.

[0035] The present invention also encompasses variants of the antibodies or antibody fragments of the present invention. A "variant" of an antibody or antibody fragment of the present invention includes at least a functional portion of an antibody or antibody fragment described herein, or a derivative or analog thereof. Such a functional portion, derivative, or analog comprises at least the binding domain of an antibody or antibody fragment described herein, including a heavy chain variable region and / or a light chain variable region comprising the CDRs disclosed herein. A variant may contain one to five amino acid substitutions within one or more of the CDRs. For example, amino acid residues may be replaced with conservative amino acid residues. A variant may also contain one or more amino acid substitutions within one or more framework regions. Preferably, a variant comprises a VH region having at least 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99% sequence identity to the VH amino acid sequence of an antibody or antibody fragment of the present invention. Preferably, the variant comprises a VL region having at least 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99% sequence identity to the VL amino acid sequence of an antibody or antibody fragment of the invention.

[0036] "Percent identity (%)" herein with respect to a nucleic acid or amino acid sequence is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after aligning the sequences for optimal comparison purposes. To optimize the alignment between the two sequences, gaps can be introduced into either of the two sequences being compared. Such alignment can be performed over the entire length of the sequences being compared. Alternatively, alignment can be performed over a shorter length, for example, about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Sequence identity is the percentage of perfect matches between the two sequences over the reported aligned region.

[0037] The comparison of sequences and the determination of the sequence identity percentage between two sequences can be achieved by using mathematical algorithms.Those skilled in the art will be aware of the fact that there are several different computer programs available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp.1-44 Addison Wesley).

[0038] Specifically, the percent sequence identity according to the present invention described herein between two nucleic acid sequences can be determined using the AlignX application of the Vector NTI Program Advance 11.5.2 software using the default settings, which employ a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc. Acid Res. 22:4673-4680), a swgapdnamt scoring matrix, a gap opening penalty of 15, and a gap extension penalty of 6.66. Amino acid sequences can be aligned using the AlignX application of the Vector NTI Program Advance 11.5.2 software using the default settings, which employ a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, 1994), a blosum62mt2 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.1.

[0039] The variants maintain the binding specificity, e.g., antigen specificity, of the antibodies or antibody fragments of the invention. However, the binding affinity of the variants may differ from the binding affinity of the original antibodies or antibody fragments of the invention. The variants may, for example, have a lower or higher K than the antibodies or antibody fragments described herein. on Velocity and / or K off It may have a speed.

[0040] The functional derivative of the antibody or antibody fragment of the present invention can be an antibody mimic, a polypeptide, an aptamer, or a combination thereof.These proteins or aptamers typically bind to one target.It should be understood that any combination of these antibodies, antibody mimics, polypeptides, and aptamers can be linked together by methods known in the art.For example, in some embodiments, the antibody or antibody fragment of the present invention is part of a conjugate or fusion protein.

[0041] Antibody mimetics are polypeptides that can specifically bind to antigens similar to antibodies, but are not structurally related to antibodies. Antibody mimetics are typically artificial peptides or proteins with a molar mass of approximately 3-20 kDa. Non-limiting examples of antibody mimetics include affibody molecules (typically based on the Z domain of protein A), affins (typically based on gamma-B crystals or ubiquitin), affimers (typically based on cystatins), affitins (typically based on Sac7d from Sulfolobus acidocaldarius), alphabodies (typically based on triple-helical coiled-coils), anticalins (typically based on lipocalins), avimers (typically based on the A domains of various membrane receptors), DARPins (typically based on the ankyrin repeat motif), finomers (typically based on the SH3 domain of Fyn7), Kunitz domain peptides (typically based on the Kunitz domains of various protease inhibitors), and monobodies (typically based on the type III domain of fibronectin).

[0042] Monobodies are synthetic binding proteins that use fibronectin type III domain (FN3) as a molecular scaffold.Monobodies are an alternative to antibodies for creating target-binding proteins.Monobodies and other antibody mimics are typically generated from combinatorial libraries, where a portion of the scaffold is diversified using molecular display and directed evolution techniques, such as phage display, mRNA display, and yeast surface display.

[0043] Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules. Aptamers are usually created by selecting them from large pools of random sequences, but natural aptamers also exist in riboswitches. As macromolecules, aptamers can be used for both basic research and clinical purposes.

[0044] The antibody of the present invention is preferably an IgG antibody, preferably an IgG1 or IgG4 antibody. Most preferably, the antibody is an IgG1 antibody. Such full-length IgG antibodies are preferred due to their favorable half-life and the desire to remain close to fully self (human) molecules for immunogenicity reasons. IgG1 is preferred due to its long circulating half-life in humans.

[0045] The term "full-length IgG" or "full-length antibody" according to the present invention is defined to include essentially a complete IgG, but not necessarily possessing all of the functions of an intact IgG. For the avoidance of doubt, a full-length IgG comprises two heavy chains and two light chains. Each of these chains contains a constant (C) region and a variable (V) region, which can be divided into domains designated CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens via the variable region domains contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system via the constant domains, primarily via the Fc portion. Full-length antibodies according to the present invention encompass IgG molecules in which mutations that provide desired characteristics may be present.

[0046] Full-length IgGs may contain mutations in the constant region that modulate effector functions, including antibody-dependent cellular cytotoxicity (ADCC) or cell-dependent cytotoxicity (CDC), both increasing and attenuating such functions, increase homodimerization or heterodimerization to generate monospecific or multispecific antibodies from host cells containing nucleic acids encoding different heavy chains, and facilitate the separation of antibodies or antibody fragments produced by such host cells.

[0047] For example, leucine at position 235 according to the EU numbering system can be substituted with glycine, and / or glycine at position 236 according to the EU numbering system can be substituted with arginine. Such modifications ensure that binding to Fc receptors and / or effector functions is eliminated or reduced. Other mutations in the CH2 and Fc regions are also encompassed by the present invention.

[0048] A full-length IgG should not have a deletion of a substantial portion of any region. However, IgG molecules in which one or more amino acid residues have been deleted without essentially altering the binding properties of the resulting IgG molecule are encompassed by the term "full-length IgG." For example, such IgG molecules can have a deletion of 1 to 10 amino acid residues, preferably within the non-CDR regions, where the deleted amino acids are not essential for the binding specificity of the IgG.

[0049] For example, the lysine at position 447 according to the EU numbering system can be deleted. Such deletion reduces antibody heterogeneity. Furthermore, to suppress swapping within IgG4 antibody molecules, the serine at position 228 according to the EU numbering system in the hinge region can be substituted with proline.

[0050] Suitable light chains for use in the antibodies or antibody fragments of the present invention include light chains produced in response to immunization with an antigen, referred to as cognate light chains, or synthetically produced light chains based thereon. Suitable light chains include common light chains (cLCs), such as those that can be identified by screening for the most commonly used light chains in existing antibody libraries (wet libraries or in silico), which do not substantially interfere with the affinity and / or selectivity of the epitope-binding domains of the heavy chains, but are also suitable for pairing with an array of heavy chains. Common light chains are preferably encoded by a germline sequence of V and J gene segments that have been rearranged but have not undergone or have undergone minimal somatic hypermutation. For example, suitable light chains include those derived from transgenic non-human animals, such as MeMo®, which incorporate a common light chain into their genome and have diversity in their heavy chains, and can be used to purify a large panel of common light chain antibodies capable of specifically binding to an antigen of interest upon exposure to that antigen.

[0051] Thus, the term "common light chain" refers to a light chain that can associate with two or more different heavy chains and exhibits antigen-binding ability (see, e.g., WO2009 / 157771, WO2019 / 190327, and WO2014 / 051433). A preferred light chain V gene for such a common light chain is IGKV1-39. Preferred light chain J genes are jk1 and jk5. The combined sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5, with alternative names being IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (designation according to the IMGT database at www.imgt.org). Preferred examples of common light chains include the light chain encoded by the human kappa light chain IgVκ1-39*01 / IGJκ1*01 (IMGT database designation) germline gene (hereinafter abbreviated as "IGVK1-39 / JK1 common light chain") and IgVκ1-39*01 / IGJκ5. The various MeMo® transgenic animals described above contain the IGVK1-39 / JK1 common light chain.

[0052] A common light chain according to the present invention refers to a light chain that may be identical or may have some amino acid sequence differences, but the binding specificity of the antibody or antibody fragment of the present invention is not affected. Those skilled in the art will recognize that "common" also refers to functional equivalents of light chains that do not have identical amino acid sequences. Variants of the light chain exist in which changes exist compared to the parent common light chain that do not substantially affect the formation of a functional binding region. Such variants can therefore bind to different heavy chains and form functional antigen-binding domains. For example, within the definition of a common light chain used herein, it is possible to prepare or find variable light chains that are not identical but are still functionally equivalent by introducing and testing, for example, conservative amino acid changes, amino acid changes in regions that do not contribute, or only partially contribute, to binding specificity when paired with a cognate chain. Such variants can therefore bind to different cognate chains and form functional antigen-binding domains. Thus, the term "common light chain" as used herein refers to a light chain that may be identical or may have some amino acid sequence differences, but that retains the binding specificity of the resulting antibody after pairing with a heavy chain. The combination of a particular common light chain and such a functionally equivalent variant is encompassed by the term "common light chain." For detailed descriptions of the use of common light chains, see WO2004 / 009618, WO2019 / 190327, and WO2009 / 157771. Preferably, a common light chain that is a germline-like light chain, more preferably a germline light chain, preferably a rearranged germline human kappa light chain, most preferably a rearranged germline human kappa light chain IgVκ1-39 / Jκ or IGVκ3-20 / Jκ, is used in the present invention.

[0053] Antibodies or antibody fragments can be produced, for example, from hybridomas using hybridoma technology. Alternatively, antibodies or antibody fragments can be produced by secretion from mammalian cells co-expressing the heavy and / or light chains, or fragments thereof. Preferably, antibodies or antibody fragments are produced by immunizing non-human animals. These and other methods are known in the art and can be suitably used to produce the antibodies or antibody fragments of the present invention.

[0054] Antibodies can be produced from various animal species. The MeMo® transgenic animals described above, which contain the IGVK1-39 / JK1 common light chain, are suitable for use. Generally, transgenic mice are immunized with human target DNA and / or protein, followed by booster immunizations, thereby eliciting an immune response that includes the production of antigen-specific antibodies. Serum titers from immunized mice can be determined by ELISA and FACS analysis. The spleens and draining lymphoid material of the immunized mice can then be collected and single-cell suspensions generated. RNA encoding the heavy chain and / or variable regions of such antibodies can be isolated and cDNA synthesized. VH and / or VL family-specific PCR can then be performed to generate a phage display library. Human target protein-binding Fabs can then be selected using, for example, a Kingfisher selection robot. Nucleic acids encoding the heavy and / or light chain variable regions of the target protein-binding Fabs can be used to produce antibodies in host cells.

[0055] Although antibodies are generated by immunization of transgenic organisms, there are cases in which such antibodies are not cross-reactive with humans and transgenic animals, reducing the efficiency of assays and testing of such antibodies. Therefore, it may be beneficial to obtain binding domains or antibodies, including chimeric or humanized binding domains or antibodies, containing variable regions and / or complementary determining regions (CDRs), as well as nucleic acids encoding such variable regions and / or CDRs that are based on, derived from, or obtained from nucleic acids of species evolutionarily distant from humans. One potential source of such variable regions may be domestic birds, such as chickens, ducks, or ostriches. The antibody repertoire generated by immunization of birds, such as chickens, ducks, or ostriches, may identify unique epitopes compared to antibodies generated by immunization of mice or other species evolutionarily closer to humans (e.g., rodents and cynomolgus monkeys).

[0056] Primary DNA or protein immunizations are performed, followed by additional booster immunizations. IgY antibodies can be isolated from the yolk of harvested eggs or from the serum of immunized birds. Spleens and / or bone marrow are removed from birds that show a significant humoral response to the target protein. RNA can then be isolated and cDNA synthesized. The resulting cDNA sample is then used as a template to amplify and digest the VH and / or VL genes using two primers. The PCR product is then cloned into a phagemid vector for display of Fab fragments on phage, essentially as described by de Haard et al. (J. Biol. Chem. (1999), Vol. 274(26), pp. 18218-18230). Nucleic acids encoding the VH and / or VL regions are ligated into the vector, and the resulting ligation vector is transformed into cells to obtain a library. Panning selection using KingFisher Flex can then be performed to select phages that bind to the target protein. Screening can be performed by FACS using cells expressing human or mouse target proteins. The VH and / or VL genes of all clones that specifically bind to the human and / or mouse target proteins are sequenced. Nucleic acids encoding the heavy and / or light chain variable regions of the binding domains that bind to the target proteins can be used to produce antibodies in host cells.

[0057] Antibody fragments can be produced by methods known in the art.

[0058] scFv antibodies can be produced by isolating mRNA from hybridomas or mammalian cells and then reverse transcribing it into cDNA for PCR amplification. This produces a large library containing a wide range of VH and VL antibody genes (Marks and Hoogenboom, Journal of Molecular Biology (1991), Vol. 222, pp. 581-597). During the construction of ScFvs, the domains can be ordered as VH-linker-VL or VL-linker-VH (Hu, O'Dwyer and Wall, Journal of Biotechnology (2005), Vol. 120, pp. 38-45). An example of a linker is the classical (G4S)3 linker (Huston et al., Proc Natl Acad Sci USA (1988), Vol. 85, pp. 5879-5883). In the case of a minibody, the DNA fragment encoding the hinge-CH3 domain can be based on the sequence of, for example, human IgG1 (Kim et al., PLOS One (2014), Vol. 9 (12), e113442). The scFv and hinge-CH3 regions can be assembled by either ligating the cohesive ends generated by XhoI and SALI, or by using PCR to bring the XhoI restriction site of the scFv product together with the hinge to the NH2 terminus of CH3 (Hu et al., Cancer Research (1996), Vol. 56, pp. 3055-3061). After PCR, the antibody V domains can be recombined by in vitro recombination of plasmids or phagemids (Lilley et al., Journal of Immunological Methods (1994), Vol. 171, pp. 211-226; Hogrefe et al., Gene (1993), Vol. 128, pp. 119-126).This phage display technology relies on fusing antibody fragments to the phage minor coat protein pIII or its C-terminal domain (Smith, Science (1985), Vol. 228, pp. 1315-1317; Hoogenboom, Nature Biotechnology (2005), Vol. 23, pp. 1105-1116). In addition to displaying scFv fragments, phage display is now widely used to display Fab fragments as well (Wieland et al., Veterinary Immunology and Immunopathology (2006), Vol. 110, pp. 129-140).

[0059] Fab fragments can be generated from monoclonal antibodies by enzymatic digestion using papain or pepsin. Papain digestion of antibodies produces three distinct fragments: two antigen-binding fragments called Fab fragments or regions, each with a single antigen-binding site, and an Fc region resulting from cleavage below the CH1 domain (Porter, Biochem (1959), Vol. 73, pp. 119-126). Pepsin digestion of antibodies produces two fragments: an F(ab')2 fragment containing two antigen-binding regions connected via disulfide bridges in the hinge region, and an Fc fragment (Valedkarimi et al., Human Antibodies (2018), Vol. 26, pp. 171-176). These Fab and F(ab')2 fragments can be purified by a combination of the following techniques: ion exchange, protein A or G affinity, antigen affinity, or gel filtration chromatography (Mage and Lamoyi, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker Inc, New York, 1987, pp. 79-97). F(ab')n or (modified F(ab')n) can be produced in connection with the invention described herein and can be obtained using any suitable enzymatic cleavage and / or digestion technique. In certain embodiments, antibody fragments can be obtained by cleavage with IdeS protease, an IgG-degrading enzyme from Streptococcus pyogenes, which cleaves human IgG1 at a specific site below the hinge to leave intact F(ab')n antibody fragments, where n is the number of antibody domains present on the IgG, and where one heavy chain of the F(ab')n is paired with the other heavy chain at their respective C-termini, and this pairing comprises two or more disulfide bridges. Methods for generating bispecific and multispecific antibodies where n is 2 or greater have been previously described. See PCT / NL2019 / 050199, PCT / NL2013 / 050294, PCT / NL2013 / 050293, which are incorporated herein by reference.

[0060] Alternatively, antibody fragments lacking the Fc region can be obtained by using a cysteine ​​protease from Porphyoromonas gingivalis, which digests human IgG1 at specific sites above the hinge (KSCDK / THTCPPC) to generate intact Fab and Fc fragments. Antibody fragments can be formed by this technique by expressing a heavy chain (e.g., CH1, CH2, and / or CH3) comprising variable and constant domains connected to additional variable domains via linkers as described herein, or paired with a light chain connected to additional variable domains via linkers as described herein, and a proteolytic enzyme, such as that from Porphyoromonas gingivalis, cleaves the constant domain of the heavy chain, leaving intact cleaved antibody-binding fragments.

[0061] The generation of sdAbs has been achieved by phage display and by using repertoires of naive or synthetic VH or VL dAbs based on the incorporation of solubilizing residues from camelid sdAbs into human VH (Tanha et al., J. Biol. Chem (2001), Vol. 276, pp. 24774-24780. Davies and Riechmann, Biotechnology (1995), Vol. 13, pp. 475-479). Human sdAbs have also been achieved without engineering by a reversible evolution and affinity-based selection method that yielded several VHs from a synthetic human VH phage display library (Jespers at al, Nat. Biotechnol (2004), Vol. 337, pp. 893-903). Using another phage selection method, exclusively non-aggregating human VH domains can be obtained from naive human VH display libraries (To et al., J Biol Chem (2005), Vol. 280, pp. 41395-41403). This same technique can be applied to the resulting VL (Kim et al., Landes Bioscience (2014), Vol. 6:1, pp. 219-235).

[0062] The antibody or antibody fragment of the present invention can be used to treat cancer by administering an effective amount of the antibody or antibody fragment to a subject in need of cancer treatment.

[0063] As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal (e.g., a patient, such as a human patient with cancer), such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc.

[0064] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject or administering to a subject an active agent or combination of active agents for the purpose of curing or ameliorating a disease or its symptoms, including reversing, alleviating, ameliorating, suppressing, or delaying symptoms, complications, conditions, or biochemical manifestations associated with a disease, as well as preventing the onset, progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease.

[0065] As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that results in a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder, e.g., cancer. A beneficial effect can take the form of an improvement over a baseline, including an improvement over measurements or observations made before initiating treatment according to the method. For example, a beneficial effect can take the form of delaying, stabilizing, stopping, or reversing the progression of cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or markers of cancer. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, delay or stop tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0066] The term "therapeutic amount" or "effective amount" refers to an amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic result. The result can be reduction, amelioration, remission, lessening, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. 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.

[0067] A therapeutic amount of a drug or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, slow, slow to some extent, and stop cancer cell invasion into peripheral organs, (iv) inhibit tumor metastasis, (v) inhibit tumor growth, (vi) prevent or slow tumor onset and / or recurrence, and / or (vii) relieve to some extent one or more of the symptoms associated with cancer.

[0068] The therapeutic amount may vary depending on factors such as the medical condition, age, sex, and weight of the individual being treated, and the ability of the agent or agent combination to elicit a desired response in the individual.

[0069] A therapeutic amount can be administered in one or more administrations.

[0070] A therapeutic amount also includes an amount that balances any toxic or detrimental effects of an agent or combination of agents with the therapeutically beneficial effects.

[0071] More specifically, the present invention provides an antibody or antibody fragment thereof that specifically binds to the extracellular domain of human TGF-βRII, wherein the antibody or antibody fragment binds to an epitope in the extracellular domain of human TGF-βRII and the phenylalanine (F) at position 25 is an essential residue for binding.

[0072] In the research leading to the present invention, an antibody that binds to a specific epitope in the extracellular domain of human TGF-βRII was identified. This epitope includes at least the amino acid residue at position 25 in the extracellular domain of isoform A of human TGF-βRII. The amino acid sequence of the extracellular domain of isoform A of human TGF-βRII is set forth as SEQ ID NO: 102. The amino acid at position 25 in this sequence is phenylalanine (F) (bold and underlined). The database accession number for the gene encoding the human TGF-βRII protein and isoform A is GenBank NM_001024847.2 (protein sequence reference number is NP_001020018.1 (SEQ ID NO: 102)). The splice variant encoding a longer isoform resulting from an insertion in the extracellular domain is isoform B. The gene encoding isoform B is GenBank NM_003242.6 (protein sequence reference is NP_003233.4 (SEQ ID NO: 103), with the insertion underlined). The amino acid at position 25 of the extracellular domain of isoform A corresponds to the amino acid at position 50 of the extracellular domain of isoform B. The amino acid sequence of the extracellular domain of human TGF-βRII isoform B is set forth as SEQ ID NO: 104. The amino acid at position 50 in this sequence, phenylalanine (F), is bolded and underlined. These accession numbers are provided primarily to provide further methods of identifying TGF-βRII proteins as targets; the actual sequence of the TGF-βRII protein bound by the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers.

[0073] Wherever this description refers to amino acid position 25 of human TGF-βRII, it refers to amino acid position 25 of the extracellular domain of isoform A of human TGF-βRII, as well as the corresponding position in another isoform of human TGF-βRII, specifically position 50 of isoform B. The same applies to other amino acid positions identified herein.

[0074] The amino acid residue at position 25 of the extracellular domain of human TGF-βRII was found to be an essential residue for binding of the antibody of the present invention, as determined by alanine scanning. In alanine scanning, amino acids at each position of an antigen, in this case TGF-βRII, are substituted with alanine one by one. If this weakens or significantly reduces the binding of the antibody to the unmodified antigen, the substituted amino acid contributes to binding and is considered to be an essential residue. Thus, an antibody or antibody fragment specifically binds to an epitope containing such a residue. In this context, an amino acid residue is considered to be an essential residue if its binding activity or reactivity is reduced by more than 50% compared to the unmodified amino acid sequence. Therefore, "significantly reduced" means that the binding activity or reactivity is reduced by at least 50% compared to the unmodified amino acid sequence of the extracellular domain of human TGF-βRII.

[0075] Thus, the antibody or antibody fragment of the present invention has less than 50%, preferably less than 40%, 30%, 20%, 10%, 5%, or 2% of the binding activity or reactivity of the wild-type amino acid sequence of the extracellular domain of human TGFβRII when the phenylalanine (F) at position 25 of the amino acid sequence of the extracellular domain of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) is replaced with alanine (A).

[0076] Thus, the antibody or antibody fragment of the present invention specifically binds to an epitope of human TGF-βRII that contains a phenylalanine (F) at position 25 of the amino acid sequence of the extracellular domain of human TGF-βRII. Preferably, the binding of the antibody or antibody fragment to this epitope is determined by alanine scanning, wherein an antibody or antibody fragment that binds to this epitope has less than 50%, preferably less than 40%, 30%, 20%, 10%, 5%, or 2% of the binding activity or reactivity when an alanine (A) is present at position 25 of the amino acid sequence of the extracellular domain of human TGF-βRII compared to when a phenylalanine (F) is present at position 25.

[0077] Another way to define an antibody or antibody fragment of the present invention is that it binds to an epitope in the extracellular domain of human TGFβRII where substitution of the phenylalanine (F) residue at position 25 of isoform A of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) with alanine (A) reduces binding of the antibody or antibody fragment by at least 50%, preferably at least 60%, 70%, 80%, 90%, 95%, or 98%.

[0078] The antibody or antibody fragment of the present invention may be defined as an antibody or antibody fragment that binds to an epitope in the extracellular domain of human TGF-βRII, which epitope includes the phenylalanine (F) residue at position 25 of isoform A of human TGF-βRII (whose wild-type sequence is set forth in SEQ ID NO: 102). In addition to the essential amino acid residue at position 25, an aspartic acid (D) at position 119 of the epitope in the extracellular domain of isoform A of human TGF-βRII has been identified as a further essential residue for binding. The amino acid at position 119 of isoform A of human TGF-βRII corresponds to the amino acid at position 144 in the extracellular domain of isoform B (SEQ ID NO: 104). The antibody or antibody fragment of the present invention preferably has a binding reactivity of less than 10%, more preferably less than 5%, 4%, 3%, or 2%, compared to the wild-type amino acid sequence of the extracellular domain of human TGF-βRII when aspartic acid (D) at position 119 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A).

[0079] In certain embodiments, the antibody or antibody fragment preferably has a sequence similar to that of the wild-type amino acid sequence of the extracellular domain of human TGFβRII: - less than 5%, preferably less than 3%, of the binding reactivity when phenylalanine (F) at position 25 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A); When aspartic acid (D) at position 119 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A), the binding reactivity is less than 5%, preferably less than 3%.

[0080] Thus, the present invention encompasses antibodies or antibody fragments that specifically bind to an epitope of human TGF-βRII that comprises a phenylalanine (F) at position 25 and an aspartic acid (D) at position 119 of the amino acid sequence of the extracellular domain of human TGF-βRII. Preferably, the binding of the antibody or antibody fragment to this epitope is determined by alanine scanning, wherein an antibody or antibody fragment that binds to this epitope has less than 50%, preferably less than 5% or 3%, of the binding activity or reactivity when an alanine is present at position 25 of the amino acid sequence of the extracellular domain of human TGF-βRII, and less than 10%, preferably less than 5% or 3%, of the binding activity or reactivity when an alanine (A) is present at position 119 of the amino acid sequence of the extracellular domain of human TGF-βRII, compared to when a phenylalanine (F) and an aspartic acid (D) are present at each of those positions, and each change is tested alone and not as a combination of the two changes compared to the wild-type extracellular domain.

[0081] Another way to define an antibody or antibody fragment of the invention is to define it as: - substitution of the phenylalanine (F) residue at position 25 of isoform A of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) with alanine (A) reduces the binding of the antibody or antibody fragment by at least 50%, preferably at least 95% or 97%; and - binds to an epitope in the extracellular domain of human TGFβRII where substitution of the aspartic acid (D) residue at position 119 of isoform A of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) with alanine (A) reduces binding of the antibody or antibody fragment by at least 90%, preferably at least 95% or 97%.

[0082] The antibody or antibody fragment of the present invention may also be defined as an antibody or antibody fragment that binds to an epitope within the extracellular domain of human TGF-βRII, which epitope includes the phenylalanine (F) and aspartic acid (D) residues at positions 25 and 119, respectively, of isoform A of human TGF-βRII (whose wild-type sequence is set forth in SEQ ID NO: 102).

[0083] In another embodiment, the antibody or antibody fragment binds to an epitope in the extracellular domain of human TGF-βRII, and the threonine (T) at position 52 of the epitope in the extracellular domain of isoform A of human TGF-βRII is an additional essential residue for binding. The amino acid at position 52 of isoform A of human TGF-βRII corresponds to amino acid at position 77 of isoform B. In this embodiment, the antibody or antibody fragment preferably has a similar sequence as compared to the wild-type amino acid sequence of the extracellular domain of human TGF-βRII: - less than 5%, preferably less than 2%, of the binding reactivity when phenylalanine (F) at position 25 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A); - a binding reactivity of less than 60%, preferably less than 40% or 30%, more preferably less than 20% when the threonine (T) at position 52 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A); and When aspartic acid (D) at position 119 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A), the binding reactivity is less than 3%.

[0084] Therefore, the present invention encompasses antibodies or antibody fragments that specifically bind to an epitope of human TGF-βRII that contains phenylalanine (F) at position 25, threonine (T) at position 52, and aspartic acid (D) at position 119 of the amino acid sequence of the extracellular domain of human TGF-βRII. Preferably, the binding of an antibody or antibody fragment to this epitope is determined by alanine scanning, wherein an antibody or antibody fragment that binds to this epitope has less than 50%, preferably less than 5% or 2% of the binding activity or reactivity when alanine (A) is present at position 25 of the amino acid sequence of the extracellular domain of human TGF-βRII, less than 60%, preferably less than 40%, 30%, or 20% of the binding activity or reactivity when alanine (A) is present at position 52 of the amino acid sequence of the extracellular domain of human TGF-βRII, compared to when phenylalanine (F), threonine (T), and aspartic acid (D) are present at each of those positions, and less than 10%, preferably less than 3% of the binding activity or reactivity when alanine (A) is present at position 119 of the amino acid sequence of the extracellular domain of human TGF-βRII, wherein each change is tested alone and not as a combination of the two changes compared to the wild-type extracellular domain.

[0085] Another way to define an antibody or antibody fragment of the invention is to define it as: - substitution of the phenylalanine (F) residue at position 25 of isoform A of human TGFβRII (whose wild-type sequence is set forth in SEQ ID NO: 102) with alanine (A) reduces the binding of the antibody or antibody fragment by at least 50%, preferably at least 95% or 98%, - substitution of the threonine (T) residue at position 52 of isoform A of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) with alanine (A) reduces the binding of the antibody or antibody fragment by at least 60%, preferably at least 70% or 80%, and - binding to an epitope in the extracellular domain of human TGFβRII where substitution of the aspartic acid (D) residue at position 119 of isoform A of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) with alanine (A) reduces binding of the antibody or antibody fragment by at least 90%, preferably at least 97%.

[0086] The antibody or antibody fragment of the present invention may also be defined as an antibody or antibody fragment that binds to an epitope within the extracellular domain of human TGF-βRII, which epitope includes the phenylalanine (F), threonine (T), and aspartic acid (D) residues at positions 25, 52, and 119, respectively, of isoform A of human TGF-βRII (whose wild-type sequence is set forth in SEQ ID NO: 102).

[0087] In another embodiment, the antibody or antibody fragment binds to an epitope in the extracellular domain of human TGF-βRII, and the isoleucine (I) at position 54 and the glutamic acid (E) at position 120 of the epitope in the extracellular domain of isoform A of human TGF-βRII are additional essential residues for binding. The amino acids at positions 54 and 120 of isoform A of human TGF-βRII correspond to the amino acids at positions 79 and 145 of isoform B, respectively. In this embodiment, the antibody or antibody fragment preferably has a sequence similar to that of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII: - less than 10% of the binding reactivity when phenylalanine (F) at position 25 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A); - less than 20% of the binding reactivity when isoleucine (I) at position 54 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A); - a binding reactivity of less than 3%, preferably less than 2%, more preferably less than 1% when aspartic acid (D) at position 119 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A); and When glutamic acid (E) at position 120 of the wild-type amino acid sequence of the extracellular domain of human TGF-βRII is substituted with alanine (A), the binding reactivity is less than 80%, preferably less than 70%, 60%, 50%, or 40%, more preferably less than 30%, and most preferably less than 10% or 5%.

[0088] Thus, the present invention encompasses antibodies or antibody fragments that specifically bind to an epitope of human TGF-βRII that contains phenylalanine (F) at position 25, isoleucine (I) at position 54, aspartic acid (D) at position 119, and glutamic acid (E) at position 120 of the amino acid sequence of the extracellular domain of human TGF-βRII. Preferably, the binding of the antibody or antibody fragment to this epitope is determined by alanine scanning, wherein an antibody or antibody fragment that binds to this epitope has less than 50%, preferably less than 10%, of the binding activity or reactivity when alanine (A) is present at position 25 of the amino acid sequence of the extracellular domain of human TGF-βRII compared to when phenylalanine (F), threonine (T), aspartic acid (D), and glutamic acid (E) are present at each of these positions, and when alanine (A) is present at position 54 of the amino acid sequence of the extracellular domain of human TGF-βRII. if an alanine (A) is present at position 119 of the amino acid sequence of the extracellular domain of human TGF-βRII, it has less than 20% of the binding activity or reactivity; if an alanine (A) is present at position 119 of the amino acid sequence of the extracellular domain of human TGF-βRII, it has less than 10%, preferably less than 3%, 2%, or 1% of the binding activity or reactivity; and if an alanine (A) is present at position 120 of the amino acid sequence of the extracellular domain of human TGF-βRII, it has less than 80%, preferably less than 70%, 60%, 50%, 40%, 30%, 10%, or 5% of the binding activity or reactivity, where each change is tested alone and not as a combination of the two changes compared to the wild-type extracellular domain.

[0089] Another way to define an antibody or antibody fragment of the invention is to define it as: - substitution of the phenylalanine (F) residue at position 25 of isoform A of human TGFβRII (whose wild-type sequence is set forth in SEQ ID NO: 102) with alanine (A) reduces the binding of the antibody or antibody fragment by at least 50%, preferably at least 90%, - substitution of the isoleucine (I) residue at position 54 of isoform A of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) with alanine (A) reduces the binding of the antibody or antibody fragment by at least 80%; - substitution of the aspartic acid (D) residue at position 119 of isoform A of human TGFβRII (whose wild-type sequence is set forth in SEQ ID NO: 102) with alanine (A) reduces binding of the antibody or antibody fragment by at least 90%, preferably at least 97%, 98%, or 99%; and - binds to an epitope in the extracellular domain of human TGFβRII where substitution of the glutamic acid (E) residue at position 120 of isoform A of human TGFβRII (the wild-type sequence of which is set forth in SEQ ID NO: 102) with alanine (A) reduces binding of the antibody or antibody fragment by at least 30%, preferably at least 40%, 50%, 60%, 70%, 90%, or 95%.

[0090] The antibody or antibody fragment of the present invention may also be defined as an antibody or antibody fragment that binds to an epitope within the extracellular domain of human TGF-βRII, which epitope includes phenylalanine (F), isoleucine (I), aspartic acid (D), and glutamic acid (E) residues at positions 25, 54, 119, and 120, respectively, of isoform A of human TGF-βRII (whose wild-type sequence is set forth in SEQ ID NO: 102).

[0091] The present inventors have identified several antibodies comprising heavy chain variable regions that specifically bind to an epitope of extracellular TGF-βRII in which phenylalanine (F) at position 25 is essential for binding. One example of such an antibody comprises a heavy chain variable region (VH) having the following: (a) VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and (c) a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; In this case, 1 to 5 amino acid residues may be changed to their conservative amino acids in any one or more of the CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3. This antibody is encompassed by the present invention.

[0092] Another example of such an antibody is a heavy chain variable region (VH) having: (a) VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; (b) a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and (c) a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; In this case, 1 to 5 amino acid residues may be changed to their conservative amino acids in any one or more of the CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3. This antibody is also encompassed by the present invention.

[0093] Another example of such an antibody is a heavy chain variable region (VH) having: (a) VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7; (b) a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; and (c) a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; In this case, 1 to 5 amino acid residues may be changed to their conservative amino acids in any one or more of the CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3. This antibody is also encompassed by the present invention.

[0094] Thus, the heavy chain variable region of an antibody or antibody fragment of the present invention may comprise a CDR1 selected from the group consisting of the sequences set forth in SEQ ID NOs: 1, 4, and 7, a CDR2 selected from the group consisting of the sequences set forth in SEQ ID NOs: 2, 5, and 8, and / or a CDR3 selected from the group consisting of the sequences set forth in SEQ ID NOs: 3, 6, and 9, wherein 1 to 5 amino acid residues may be changed to their conservative amino acids within any one or more of the CDRs selected from CDR1, CDR2, and CDR3.

[0095] The framework region of the heavy chain variable region can be selected from any suitable framework region. Examples of suitable framework regions are those encoded by human V genes IGHV3-15 and IGHV3-23. These germline V genes can contain one or more somatic mutations.

[0096] In one embodiment of the invention, the antibody or antibody fragment thereof comprises a VH having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS (SEQ ID NO: 10), or a VH amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity thereto.

[0097] In another embodiment, the antibody or antibody fragment thereof comprises a VH having the amino acid sequence: QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS (SEQ ID NO: 11), or a VH amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity thereto.

[0098] In another embodiment, the antibody or antibody fragment thereof comprises a VH having the amino acid sequence: QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAASGKNYFDPWGQGTLVTVSS (SEQ ID NO: 12), or a VH amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity thereto.

[0099] The heavy chain variable region can have 0 to 10, preferably 0 to 5, amino acid mutations relative to the amino acid sequence shown. In a preferred embodiment, the heavy chain variable region contains 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, and more preferably 0 amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof at positions other than the CDRs. A combination of insertions, additions, deletions, or substitutions is permitted if no more than 10 positions, preferably no more than 5 positions, of the aligned sequences differ. A gap in one aligned sequence corresponds to the same number of amino acids skipped in the other sequence. Amino acid substitutions, if present, are preferably conservative amino acid substitutions. Substitutions within the heavy chain variable region may be in addition to the substitutions within one or more CDRs described above. Conservative amino acids are known in the art based on the similarity of their respective functional groups, which are based on characteristics such as charge, hydrophobicity, hydrophilicity, acidity, basicity, and size.

[0100] The amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof, preferably do not occur at the binding interface of the heavy and light chains.

[0101] When an amino acid is changed at the interface of heavy / light chain interaction, the corresponding amino acid in the other chain is preferably changed to accommodate the change. The insertion or addition of an amino acid preferably does not involve the insertion or addition of a proline.

[0102] The present invention encompasses antibodies or antibody fragments comprising variants of the antibodies or antibody fragments described herein. Specifically, the present invention encompasses antibodies or antibody fragments comprising a VH having any one of SEQ ID NOS: 22-91 and 93, i.e., any one of the VH sequences shown in Figure 4. Antibodies or antibody fragments comprising at least HCDR3 or all CDRs of any one of SEQ ID NOS: 22-91 and 93 are also encompassed. Preferred antibodies or antibody fragments comprise a heavy chain variable region having or derived from the amino acid sequence set forth in SEQ ID NOS: 40, 43, 46, 47, 48, or 54, which are variants of antibodies comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOS: 11. Other preferred antibodies or antibody fragments comprise a heavy chain variable region having or derived from the amino acid sequence set forth in SEQ ID NOS: 67, 70, 75, 76, 77, 78, 83, 84, or 88, which are variants of antibodies comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOS: 12. Other preferred antibodies or antibody fragments comprise a heavy chain variable region having or derived from the amino acid sequence set forth in SEQ ID NO: 24 or 26, which are variants of antibodies comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. Most preferred variants are those comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40, 43, 46, 47, 48, 54, 67, 70, 75, 76, 77, 78, 83, 84, or 88.

[0103] The heavy chain variable region of such variants can have 0 to 10 amino acid mutations relative to the amino acid sequence shown, preferably 0 to 5. In a preferred embodiment, the heavy chain variable region contains 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, and more preferably 0 amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof at positions other than the CDRs relative to the amino acid sequence shown.

[0104] The antibody or antibody fragment, or binding domain of the invention may comprise any suitable light chain, such as, for example, a cognate light chain or a consensus light chain as defined herein.

[0105] In one embodiment, the light chain variable region comprises the amino acid sequence of the IgVκ1-39*01 gene segment shown in Figure 1B, with 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof. IgVκ1-39 is an abbreviation for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, or IGKV1-39. The external identifiers for this gene are HGNC:5740, Entrez Gene:28930, and Ensembl:ENSG00000242371. A preferred amino acid sequence of IgVκ1-39 is presented in Figure 1A. This figure lists the sequence of the V region. The V region can be combined with one of five J regions. Figures 1B and 1C depict two preferred sequences of IgVκ1-39 combined with a J region. The aligned sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5, with alternative names IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (designation according to the IMGT database at www.imgt.org).

[0106] Preferably, IgVκ1-39*01 comprising the light chain variable region is a germline sequence. More preferably, IGJκ1*01 or IGJκ5*01 comprising the light chain variable region is a germline sequence. More preferably, IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0107] Mature B cells that produce antibodies with light chains often produce light chains that have undergone one or more mutations relative to the germline sequence, which refers to the normal sequence in non-lymphoid cells of an organism. The process involved in these mutations is called somatic hypermutation. The resulting light chain is called an affinity-matured light chain. When such a light chain is derived from the germline IgVκ1-39*01 sequence, it is an IgVκ1-39*01-derived light chain. As used herein, the phrase "IgVκ1-39*01" includes IgVκ1-39*01-derived light chains. Mutations introduced into nucleic acids encoding light chains by somatic hypermutation can also be artificially introduced in the laboratory. Other mutations in the light chain, not necessarily in terms of type but not amount, can also be introduced in the laboratory without affecting the properties of the light chain. A light chain is at least an IgVκ1-39*01 light chain if it comprises the sequence shown in FIG. 1A, 1B, or 1C with 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof. Preferably, the IgVκ1-39*01 light chain comprises the sequence shown in FIG. 1A, 1B, or 1C with 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof, more preferably the sequence shown in FIG. 1A, 1B, or 1C with 0 to 5, preferably 0 to 4, 0 to 3, 0 to 2, or 0 to 1 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof, even more preferably the sequence shown in FIG. 1A, 1B, or 1C with 0 to 3, preferably 0 to 3, 0 to 4, 0 to 3, 0 to 2, or 0 to 1 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof, even more preferably the sequence shown in FIG. 1A, 1B, or 1C with 0 to 3, 1A, 1B, or 1C having 0 to 2 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof, more preferably having 0 to 1 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof, and most preferably having 0 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0108] In one embodiment, the antibody or antibody fragment thereof, or the binding domain has a light chain variable region (VL) comprising: (a) VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19; (b) a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and (c) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21; Here, 1 to 5 amino acid residues may be substituted with their conservative amino acids in any one or more of the CDRs selected from VL-CDR1, VL-CDR2, and VL-CDR3.

[0109] The light chain variable region of an antibody or antibody fragment, or binding domain of the present invention preferably comprises the CDR1, CDR2, and CDR3 regions comprising the amino acid sequences CDR1-QSISSY (SEQ ID NO: 13), CDR2-AAS (SEQ ID NO: 14), and CDR3-QQSYSTPPT (SEQ ID NO: 15), i.e., the CDRs of IGKV1-39 (according to IMGT), allowing for 0 to 5 amino acid substitutions relative to the amino acid sequence shown. According to Kabat numbering, the amino acid sequences are CDR1-RASQSISSYLN (SEQ ID NO: 19), CDR2-AASSLQS (SEQ ID NO: 20), and CDR3-QQSYSTPPT (SEQ ID NO: 21). Any amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof are preferably absent in the CDR3 region of the light chain variable region, and preferably not in the CDR1 or CDR2 regions of the light chain variable region. Amino acid substitutions are preferably conservative amino acid substitutions.

[0110] Specifically, the light chain variable region of an antibody or antibody fragment, or binding domain of the present invention preferably comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK (SEQ ID NO: 16), or a VL amino acid sequence having at least 80% identity thereto. This therefore allows for 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof. Amino acid substitutions are preferably conservative amino acid substitutions.

[0111] The antibody of the present invention can be of any isotype: IgA, IgM, IgG, IgD, or IgE. Preferably, the antibody is IgG, specifically IgG1 or IgG4. Most preferably, the antibody is IgG1.

[0112] The constant region of the antibody of the present invention is preferably a human constant region, but may be that of any animal. It may be a mouse or rat constant region. Depending on the host organism to be immunized or the screening method used to select recombinant antibodies generated in response to the antigen, mouse or rat constant regions may be advantageously used.

[0113] The antibody or antibody fragment thereof may comprise a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17. The antibody or antibody fragment thereof may comprise a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18. The constant region may comprise one or more, preferably ten or fewer, preferably five or fewer amino acid differences from the constant region of a conventional human antibody.

[0114] Some antibodies have modified CH2 / lower hinge regions, for example, to reduce Fc receptor interaction or to reduce ADCC, C1q binding, or other effector activity. The antibodies of the present invention may be IgG antibodies with variant CH2 and / or lower hinge domains such that the antibody has reduced interaction with Fc-gamma receptors. Such mutant CH2 and / or lower hinge domains preferably contain amino acid mutations at positions 235 and / or 236 (EU numbering), preferably residues at positions 235G and / or 236R.

[0115] The isolated monoclonal antibody can comprise two heavy chain constant regions of an IgG1 antibody, wherein the lysine at position 447 according to the EU numbering system is deleted.

[0116] Preferred antibodies or antibody fragments of the invention comprise a combination of heavy chain variable region CDRs and light chain variable region CDRs listed in Table 1. [Table 1]

[0117] A preferred antibody or antibody fragment of the present invention comprises a heavy chain having a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 22 to 91, and 93, specifically a preferred VH described herein, and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16. This antibody or antibody fragment may comprise any of the constant domains described herein.

[0118] In one embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0119] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO:16.

[0120] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0121] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 43, and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0122] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 75, and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0123] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 70 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0124] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 84 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0125] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 88 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0126] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 40, and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0127] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 83, and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0128] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 78 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0129] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0130] In another embodiment, the antibody or antibody fragment thereof described herein comprises a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 76 and a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0131] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:10 and two light chain variable regions of SEQ ID NO:16.

[0132] In another embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:11 and two light chain variable regions of SEQ ID NO:16.

[0133] In another embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:12 and two light chain variable regions of SEQ ID NO:16.

[0134] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:43 and two light chain variable regions of SEQ ID NO:16.

[0135] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:75 and two light chain variable regions of SEQ ID NO:16.

[0136] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:70 and two light chain variable regions of SEQ ID NO:16.

[0137] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:84 and two light chain variable regions of SEQ ID NO:16.

[0138] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:88 and two light chain variable regions of SEQ ID NO:16.

[0139] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:40 and two light chain variable regions of SEQ ID NO:16.

[0140] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:83 and two light chain variable regions of SEQ ID NO:16.

[0141] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:78 and two light chain variable regions of SEQ ID NO:16.

[0142] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:47 and two light chain variable regions of SEQ ID NO:16.

[0143] In one embodiment, an antibody or antibody fragment thereof described herein comprises two heavy chain variable regions of SEQ ID NO:76 and two light chain variable regions of SEQ ID NO:16.

[0144] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0145] In another embodiment, the antibody of the invention comprises: (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0146] In another embodiment, the antibody of the invention comprises: (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0147] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 43 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0148] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 75 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0149] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 70 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0150] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 84 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0151] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 88 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0152] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 40 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0153] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 83 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0154] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 78 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0155] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0156] In one embodiment, the antibody of the invention (A) a heavy chain having a VH comprising the amino acid sequence set forth in SEQ ID NO: 76 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17; and (B) A light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0157] The present invention provides a binding domain that specifically binds to human TGF-βRII, A heavy chain variable region (VH) chosen from: (A) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 12; (B) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 26; (C) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 30; (D) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 40; (E) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 61; (F) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 65; (G) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 70; (H) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 76; (I) a VH having VH-CDR1, VH-CDR2, and VH-CDR3, the VH having the amino acid sequence set forth in SEQ ID NO: 85; and (J) A VH having VH-CDR1, VH-CDR2, and VH-CDR3, wherein the VH has the amino acid sequence set forth in SEQ ID NO: 86; Further provided herein is a binding domain in which 1 to 5 amino acid residues may be substituted with their conserved amino acids in any one or more of the CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3.

[0158] These VH-CDR1, VH-CDR2, and VH-CDR3 sequences are shown in bold and underlined in the sequence listing provided herein.

[0159] In one embodiment, a binding domain of the invention comprises a VH amino acid sequence selected from SEQ ID NOs: 12, 26, 30, 40, 61, 65, 70, 76, 85, and 86, or a VH amino acid sequence having at least 80% identity thereto.

[0160] The binding domains of the invention have been shown to be useful in monovalent and multivalent forms, and offer a variety of applications in monovalent or bivalent molecules, or as one or more binding domains incorporated into multispecific molecules. The binding domains in monovalent form have superior TGF-βRII blocking ability compared to monovalent control antibodies, making them suitable for the above applications.

[0161] In certain embodiments, the present invention provides antibodies, preferably multispecific antibodies, comprising a binding domain of the invention in a monovalent form, which have equivalent receptor-blocking activity at equimolar concentrations compared to a bivalent, monospecific control antibody, preferably measured in the same assay. In certain embodiments, the control antibody comprises two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 97 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 135. In certain embodiments, the assay is a TGFβ-reporter assay, preferably the TGFβ-reporter assay described in Example 5. In certain embodiments, equivalent receptor-blocking activity comprises a 5- to 2-fold, preferably 3-fold, deviation from the receptor-blocking activity of the control antibody.

[0162] The antibodies and binding domains described and claimed herein, such as those described in the above embodiments, are preferably isolated antibodies or binding domains. Preferably, they are monoclonal antibodies. More preferably, they are isolated monoclonal antibodies.

[0163] The antibodies or antibody fragments, or binding domains of the invention interfere with the binding of the ligands TGF-β1, TGF-β2, and / or TGF-β3 to TGF-βRII. The term "interferes with binding" means that the antibody or antibody fragment is directed against an epitope on TGF-βRII and competes with TGF-β1, TGF-β2, and / or TGF-β3 for binding to TGF-βRII. The antibodies or antibody fragments, or binding domains may weaken ligand binding, displace ligand binding if it is already bound to TGF-βRII, or at least partially prevent the ligand from binding to TGF-βRII, e.g., by steric hindrance, and / or interfere with or prevent TGF-βRI-TGF-βRII complex formation. The antibody or antibody fragment, or the binding domain may attenuate the formation of a complex between TGF-βRI and TGF-βRII, displace such a complex if it has already formed, or at least partially prevent TGF-βRI from complexing with TGF-βRII, for example, by steric hindrance. The term "interfere with binding" means that the antibody or antibody fragment, or binding domain of the invention blocks the binding of the ligands TGF-β1, TGF-β2, and / or TGF-β3 to TGF-βRII.

[0164] The present invention further provides expression vectors comprising polynucleotides encoding either or both of the heavy and light chains of the antibodies or antibody fragments, or binding domains described herein. Examples of vectors include plasmids, phagemids, cosmids, viruses, and phage nucleic acids, or other nucleic acid molecules, that can replicate in prokaryotic or eukaryotic host cells, such as mammalian cells. The vector may be an expression vector in which the polynucleotide encoding either or both of the heavy and light chains of the antibody or antibody fragment of the present invention is operably linked to an expression control element. Typical expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the polynucleotide.

[0165] Various methods for producing antibodies exist in the art. Antibodies are typically produced by cells expressing nucleic acid encoding the antibody. Thus, the present invention also provides isolated cells or cells in tissue culture that produce and / or contain the antibody or antibody fragment of the present invention. Typically, these are in vitro cells, isolated cells, or recombinant cells. Such cells contain nucleic acid encoding the antibody or antibody fragment of the present invention. The cells are preferably animal cells, more preferably mammalian cells, more preferably primate cells, and most preferably human cells. For purposes of the present invention, a suitable cell is any cell that can contain, preferably produce, an antibody according to the present invention, and / or contain nucleic acid according to the present invention. Preferably, the cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, an NSO cell, or a PER.C6 cell. It is particularly preferred that the cell is a CHO cell.

[0166] Further provided is a cell culture or cell line comprising a cell according to the invention. Cell lines developed for industrial scale production of proteins and antibodies are further referred to herein as industrial cell lines.

[0167] The present invention further provides a method for producing an antibody or antibody fragment, or binding domain of the present invention, comprising culturing a cell of the present invention and harvesting the antibody or antibody fragment, or binding domain from the culture. The cells may be cultured in serum-free medium. Preferably, the cells are suitable for suspension growth. The antibody or antibody fragment, or binding domain may be purified from the culture medium. Preferably, the antibody or antibody fragment, or binding domain is affinity purified.

[0168] The present invention further provides pharmaceutical compositions comprising an antibody or antibody fragment, or binding domain described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0169] When the antibody or antibody fragment of the present invention is formulated for use as an injection or infusion solution for intravenous drip, the injection or infusion solution may be any form of aqueous solution, suspension, or emulsion, or may be formulated as a solid drug together with a pharmaceutically acceptable carrier so that the drug is dissolved, suspended, or emulsified in the solvent at the time of use. Examples of solvents used for injection or infusion solutions for intravenous drip include distilled water for injection, saline, glucose solution, and isotonic solutions (e.g., in which sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc. are soluble).

[0170] Examples of pharmaceutically acceptable carriers include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, disinfectants, pH adjusters, and antioxidants. Stabilizing agents include various amino acids, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, and dibutylhydroxytoluene. Solubilizing agents include alcohols (e.g., ethanol), polyols (e.g., propylene glycol and polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 20®, Polysorbate 80®, and HCO-50). Suspending agents include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Emulsifying agents include gum arabic, sodium alginate, and tragacanth. Sedatives include benzyl alcohol, chlorobutanol, and sorbitol. Buffers include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, and epsilon aminocaproic acid buffer. Preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Disinfectants include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. pH adjusters include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.Antioxidants that can be used include (1) aqueous antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium bisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, and α-tocopherol; or (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoric acid.

[0171] Infusion solutions for infusion or infusion can be produced by sterilizing or aseptically operating, for example, sterilizing by filtration through a filter, in the final process, and then filling into a sterile container. Infusion solutions for infusion or infusion can be used by dissolving vacuum-dried or freeze-dried sterile powder (which may contain a pharmaceutically acceptable carrier powder) in a suitable solvent at the time of use.

[0172] The present invention further provides a method of treating cancer in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or antibody fragment, binding domain, or pharmaceutical composition described herein. Accordingly, the present invention provides an antibody or antibody fragment, or binding domain described herein for use in treating cancer in a subject. The present invention further provides a pharmaceutical product for preventing cancer, suppressing the progression or recurrence of cancer symptoms, and / or treating cancer, comprising as an active ingredient an antibody or antibody fragment thereof, or binding domain described herein.

[0173] Cancer patients usually have abnormal cells that are removed from the body.The term "abnormal cell" as used herein includes tumor cells, more specifically tumor cells present on any type of cancerous tissue associated with cancer type that correlates with higher than normal TGF-βRII expression.Abnormal cell herein also refers to cells that have increased signal transduction as a result of increased TGF-β expression and / or TGF-β release, and also to cells that form a suppressive environment that reduces the effectiveness of tumor immunity due to abnormal TGF-β signal transduction and / or expression, or higher than normal TGF-β, TGF-βRI, and / or TGF-βRII expression, and / or higher than normal latent TGF-β release.

[0174] The antibodies or antibody fragments, or binding domains of the invention may be effective in treating several cancer types, including, for example, cancer types correlated with higher than normal TGF-β signaling, particularly higher than normal TGF-βRII expression, including, but not limited to, breast cancer, colon cancer, colorectal cancer, gastric cancer, glioblastoma, cervical cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, myelodysplastic syndrome, pancreatic cancer, prostate cancer, and renal cancer.

[0175] The antibodies or antibody fragments, or binding domains of the invention block the binding of cellular human TGF-β to human TGF-βRII, thereby inhibiting TGF-βRII signaling, resulting in reduced proliferation of harmful or abnormal cells and / or enhanced tumor immunity and other beneficial effects.

[0176] Thus, the present invention also provides a method for blocking the binding of human TGF-β to human TGF-βRII in a cell, the method comprising providing a cell with an antibody or antibody fragment, or binding domain described herein, and allowing the antibody or antibody fragment to bind to human TGF-βRII in the cell, thereby blocking the binding of human TGF-β to human TGF-βRII in the cell. This may be an in vitro method.

[0177] Depending on the desired activity, the antibodies of the present invention may have regulated effector functions. Antibody-dependent cellular cytotoxicity (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is a cell-mediated immune defense mechanism whereby effector cells of the immune system actively lyse target cells whose membrane surface antigens are bound by specific antibodies. ADCC effector function is typically mediated by Fc receptors (FcRs). These receptors are the primary immunoregulatory receptors connecting antibody-mediated (humoral) immune responses to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM), and FcδR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high-affinity receptor (KD in the nanomolar range), while FcγRII and FcγRIII are low- to intermediate-affinity receptors (KD in the micromolar range). In antibody-dependent cellular cytotoxicity (ADCC), FcvRs on the surface of effector cells (natural killer cells, macrophages, monocytes, and eosinophils) bind to the Fc region of IgG that binds to target cells. Upon binding, signaling pathways are triggered that result in the secretion of various substances, such as lytic enzymes, perforin, granzymes, and tumor necrosis factor, which mediate target cell destruction. The level of ADCC effector function varies among human IgG subtypes. Briefly, depending on the allotype and the particular FcvR, ADCC effector function is high for human IgG1 and IgG3 and low for IgG2 and IgG4. The knowledge that binding sites for FcvRs exist on antibodies has led to engineered antibodies that lack ADCC effector function.

[0178] Another type of effector function, independent of effector cells, is called complement-dependent cytotoxicity (CDC). This is the effector function of IgG and IgM antibodies. It is another mechanism by which therapeutic antibodies or antibody fragments can achieve antitumor effects. CDC is initiated when C1q, the initiating component of the classical complement pathway, binds to the Fc portion of a target-bound antibody. This is the first step in the complex complement activation cascade, which can ultimately result in the lysis of antibody-labeled cells.

[0179] In the antibodies of the present invention, ADCC activity can be enhanced by slightly modifying the constant region through different techniques, one of which is the removal of fucose. The removal of fucose has resulted in increased antitumor activity in several in vivo models (Junttila et al., Cancer Research (2010), Vol. 70 (22), pp. 4481-4489). Afucosylation technology may also be applied, which prevents fucosylation of N-linked carbohydrate structures within the Fc region.

[0180] In the antibody of the present invention, effector function can be reduced or eliminated. For example, leucine at position 235 according to the EU numbering system can be substituted with glycine, and / or glycine at position 236 according to the EU numbering system can be substituted with arginine. Such modifications ensure that binding to Fc receptors and / or effector function is eliminated or reduced. Other substitutions, deletions, or insertions known in the art for the same purpose are also encompassed by the present invention.

[0181] The present invention further provides a method for inhibiting signal transduction to a cell induced by binding of human TGF-β to human TGF-βRII in the cell, the method comprising providing the cell with an antibody or antibody fragment, or binding domain described herein, and allowing the antibody or antibody fragment to bind to human TGF-βRII in the cell, thereby inhibiting signal transduction to the cell. This may be an in vitro method.

[0182] The present invention also provides a method for preventing or inhibiting metastasis, comprising administering to a subject an effective amount of an antibody or antibody fragment, binding domain, or pharmaceutical composition described herein.

[0183] The antibody or antibody fragment, or binding domain of the present invention can be used for cancer treatment as a monotherapy, but can also be combined with other anti-cancer drugs.Other anti-cancer drugs include, but are not limited to, therapeutic antibodies that target the same or different tumor antigens or modulate immune system elements, drugs used in chemotherapy (e.g., cyclophosphamide), and drugs used in local administration applications, including hormone therapy or oncolytic viruses.Treatment with the antibody or antibody fragment, or binding domain of the present invention can also be combined with other anti-cancer treatments, such as surgery or radiation therapy.The combination of treatments can be simultaneous, separate, or sequential.

[0184] Throughout this specification and the appended claims, the words "comprise," "include," and "having," as well as variations such as "comprises," "comprising," "includes," and "including," are intended to be interpreted inclusively, that is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the content permits.

[0185] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., one or at least one) of the grammatical object of the article. By way of example, "an element" can mean one element or more than one element.

[0186] "Plurality" means two or more.

[0187] It should be noted that, unless otherwise specified, amino acid positions assigned to CDRs and frameworks in the variable regions of antibodies or antibody fragments are designated herein by Kabat numbering (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Amino acids in the constant regions are designated by the EU numbering system based on Kabat amino acid positions (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

[0188] The accession number is provided primarily to provide a method of further target identification, and the actual sequence of the bound protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The antigen-binding site binds to an antigen and its various variants, such as those expressed by some antigen-positive immune cells or tumor cells.

[0189] When a gene, protein is referred to herein, it preferably refers to the human form of that gene or protein. When a gene or protein is referred to herein, it refers to the native gene or protein as well as variants of that gene or protein that can be detected in tumors, cancers, etc., preferably human tumors, cancers, etc.

[0190] HGNC stands for HUGO Gene Nomenclature Committee. The number after the abbreviation is an accession number that allows information about the gene and the protein encoded by the gene to be retrieved from the HGNC database. Entrez Gene provides an accession number or gene ID that allows information about the gene or the protein encoded by the gene to be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensemble provides an accession number that allows information about the gene or the protein encoded by the gene to be obtained from the Ensemble database. Ensembl is a collaborative project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system that generates and maintains automated annotations for selected eukaryotic genomes. [Brief explanation of the drawings]

[0191] [Figure 1] The amino acid sequences of the IGKV1-39 light chain variable V region (Figure 1A), the IGKV1-39 / jk1 light chain variable region (Figure 1B), and the IGKV1-39 / jk5 light chain variable region (Figure 1C) are presented. [Figure 2] Figure 2 shows FACS data of antibody binding to endogenously expressed TGF-βRII on CCD18Co cells. Figure 2A shows the MFI PE of antibodies comprising heavy chain variable regions having SEQ ID NO:92 (negative control), SEQ ID NO:93, and SEQ ID NO:94. Figure 2B shows the MFI PE of antibodies comprising heavy chain variable regions having SEQ ID NO:92 (negative control), SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:95, and SEQ ID NO:96. [Figure 3] ELISA data for the ligand blocking activity of antibodies comprising heavy chain variable regions having SEQ ID NO: 92 (negative control), SEQ ID NO: 93, and SEQ ID NO: 94 (Figure 3A), and antibodies comprising heavy chain variable regions having SEQ ID NO: 92 (negative control), SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 95, and SEQ ID NO: 96 (Figure 3B) are presented. [Figure 4]Amino acid sequence alignments generated using the AlignX application of Vector NTI Program Advance 11.5.2 software for heavy chains comprising heavy chain variable regions having SEQ ID NO: 10 (center alignment), SEQ ID NO: 11 (bottom alignment), and SEQ ID NO: 12 (top alignment) are presented along with their respective affinity matured variants. Identical amino acids are shown in black letters against a white background, slightly similar amino acids are shown in white letters against a gray background, conservative changes are shown in black letters against a gray background, and dissimilar amino acids are shown in white letters against a dark gray background. [Figure 5] Figure 1 shows the binding affinity of the parent antibody (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12) and affinity matured variants (SEQ ID NO: 10 variant, SEQ ID NO: 11 variant, SEQ ID NO: 12 variant). ka is the on rate in 1 / msec and kd is the off rate in 1 / sec. [Figure 6] Data from luciferase reporter assays of affinity matured variants are shown. The X-axis shows antibody concentration (μg / mL). The Y-axis shows fold induction of Smad complexes as a measure of TGF-β signaling inhibition. Graphs A-K each compare the activity of affinity matured variants to a control antibody (SEQ ID NO: 97). Basal TGF-β is included as a control for TGF-β stimulation of cells. [Figure 7] Data from alanine scanning are shown. Figure 7A provides an overview of how essential residues are identified and mapped. Figure 7B shows the mean fluorescence, as a percentage, of SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 93 compared to wild type. Figure 7C is a table showing the binding reactivity, as a percentage, of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 93, and SEQ ID NO: 98 (control) compared to wild type. Residues essential for binding are shown in a rectangle. Figure 7D shows the mapping of essential residues on TGFβRII. [Figure 8]

[0033] Figure 1 shows results from screening various bispecific antibodies comprising a TGF-βRII-binding domain of the present invention in a TGF-β reporter assay. The X-axis shows antibody concentration (μg / mL). The Y-axis shows fold induction of Smad signaling. Graphs A-E each compare the activity of a bispecific antibody comprising a single TGF-βRII-binding domain to a positive control antibody known to block ligand binding to TGF-βRII(+), as described in Example 1. Basal TGF-β is included as a control for TGF-β stimulation of cells. A) Δ ​​is a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 76; □ is a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 61; and ● is a TGF-βRII-binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 26. B) ○ is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 70, * is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 70, □ is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 61, ● is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 86, and Δ is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 65. C) * is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 65, ○ is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40, ● is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12, □ is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 76, and Δ is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 26. D) ● is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12, and □ is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 85.E) ● is a TGF-βRII binding domain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:86.

[0192] The following examples are illustrative of the present invention and are not intended to limit the invention in any way. [Example]

[0193] Example 1 - Antibody production Transgenic mice (MeMo® mice) containing the common IGKV1-39 light chain were immunized with human TGF-βRII (isoform A), thereby generating an immune response that included the production of human anti-TGF-βRII-specific antibodies. Lymphoid material from the immunized mice was collected, nucleic acid extracted from it, and used to synthesize cDNA encoding the heavy chain variable region of such antibodies. The cDNA was used to generate a phage display library, from which human TGF-βRII-binding Fabs were selected using a Kingfisher selection robot.

[0194] Two rounds of affinity-driven selection with different concentrations of biotinylated recombinant protein were performed using a Kingfisher robot. Human TGF-βRII-Fc was biotinylated using the EZ-Link™ Sulfo-NHS-Biotin Kit (ThermoFisher, catalog no. 21217) according to the manufacturer's protocol, aliquoted into equal amounts, and stored at -20°C until further use. Two subsequent rounds of in-solution selection were performed using the Kingfisher robot with biotinylated human TGF-βRII-Fc. Three different concentrations of protein were used in the first selection round. Selection without antigen was included as a negative control. 50 μg / mL of total human IgG (Sigma, catalog no. 1456) was added to the solution during all phage library incubations to minimize selection of Fc binders. After washing, bound phage were eluted with trypsin. Phage output was determined according to the spot method, in which TG1 cells were infected, plated on LB-Amp / Glu agar plates, and single colonies were picked for screening. A second selection round was performed using the output from the first round. In the second selection round, TGF-βRII-Fc-biotin was used at decreasing protein amounts, starting from the concentration used for each first selection round output.

[0195] Colonies were picked into 96-well plates, and periplasmic extracts containing soluble Fab were prepared. The resulting Fab-containing fractions were used to identify TGF-βRII-specific clones using FACS.

[0196] HEK293T cells transiently transfected with human TGF-βRII were used for FACS screening. The final Fab concentration was 0.5–5 μg / mL. Fab binding to TGF-βRII was detected with a goat anti-kappa light chain antibody (Ab0646, 5 μg / mL) followed by a rabbit anti-goat PE antibody (Ab0330, 1 / 100 dilution).

[0197] Two rounds of immunization were performed to obtain a large panel of Fabs for further characterization. In the second round of immunization, a different vector was used to increase expression levels, and mice were co-immunized with human TGF-βRI. This may have contributed to the higher immune responses observed in mice during the second round of immunization.

[0198] Based on information obtained from US2010 / 0119516, a positive control antibody was produced. This positive control antibody contains two heavy chain variable regions and two light chain variable regions having the amino acid sequences of mAb TGF1 described therein (SEQ ID NO: 97 and SEQ ID NO: 135, respectively). Antibodies containing these heavy chain variable regions and light chain variable regions have been reported to block ligand binding to TGF-βRII.

[0199] A negative control antibody against RSV was generated that contained two heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO: 136 and two light chain variable regions set forth in SEQ ID NO: 16.

[0200] Example 2 - Antibody Characterization The VH fragment of the TGF-βRII binding Fab identified in Example 1 was recloned into an IgG expression format, and IgG was expressed and purified from 293T Freestyle cells.

[0201] antigen binding The antibodies were screened for binding to endogenously expressed human TGF-βRII on CCD18Co cells using FACS. The results are shown in Figure 2. All antibodies tested showed similar binding to CCD18Co cells.

[0202] The Fabs obtained from the first immunization round, which contain a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11, include those obtained from the second immunization round, which contain a heavy chain variable region having the same HCDR3 sequence as the Fabs obtained from the second immunization round, for example, the amino acid sequence set forth in SEQ ID NO: 93. This indicates that VDJ gene segment recombination in mice is nearly identical in response to this antigen.

[0203] binding affinity The binding affinities of antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 93, and SEQ ID NO: 12 were determined by SPR. For this purpose, the antibodies were reformatted as bivalent IgG monospecific against TGFβRII.

[0204] Anti-TGF-βRII IgG antibody was captured onto a CM5 sensor chip surface using immobilized anti-CH1 antibody (Ab0669, BD catalog no. BD555784), followed by the addition of human recombinant TGF-βRII (R&D systems catalog no. 241-R2 / CF, region Ile24-Aps195). Measurements were performed at 25°C. Coupling was performed at pH 4.5. Results are shown in Table 2. [Table 2]

[0205] Ligand blockade The ligand blocking activity of the IgG samples was determined using an ELISA assay.

[0206] ELISA plates were coated with 0.4 μg / mL TGF-β1. Human TGF-βRII-Fc (R&D, Cat. No. 341-BR) was added to a final concentration of 0.01 μg / mL. Antibodies were incubated over a 3-fold concentration range, starting at a final concentration of 10 μg / mL. An antibody that specifically binds to tetanus toxoid containing a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 92 was included as a control. Antibodies binding to human TGF-βRII-Fc were detected with a biotin-conjugated anti-human Fc antibody (1:10,000) and streptavidin-HRP (1:2000).

[0207] As shown in Figure 3, all tested antibodies exhibit good ligand-blocking activity compared to the control antibody. The IC50 values ​​of selected antibodies are presented in Table 3. [Table 3]

[0208] Example 3 - Affinity Maturation A Fab phage display library of variant heavy chains containing variable heavy chain regions having the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 that specifically bind to TGF-βRII and block ligand interaction with TGF-βRII and heterodimerization between TGF-βRII and TGF-βRI was generated to evaluate whether antibodies with higher affinity could be produced. This library was designed to generate heavy chain variable regions with increased affinity and typically contain three to four mutations, i.e., one in CDR1, one in CDR2, and one or two in CDR3, with a certain number of mutations containing more than four mutations overall. A summary of these variants is provided as a sequence alignment in Figure 4.

[0209] Variants with higher affinity were selected using two different selection methods, both using biotinylated human TGF-βRII-Fc as the antigen, and both using the Kingfisher selection robot. Several selection rounds were performed. The first selection round involved affinity-based selection as described in Example 1, using different concentrations of biotinylated TGF-βRII-Fc. Only the output from the selection with the lowest antigen concentration showing clear enrichment was then used further in two separate selection methods. The first selection method involved two additional affinity-based selection rounds, but this time using a reduced concentration of antigen compared to the optimal antigen concentration used in the previous selection round. The second selection method also involved two additional affinity-based selection rounds using reduced concentrations of antigen, and also involved an off-rate-based selection that included a wash step in the presence of an excess of non-biotinylated antigen.

[0210] 50 μg / mL total human IgG (Sigma, Cat. No. I4506) was added to the solution during all phage library incubations to minimize selection of Fc binders. After washing, bound phages were eluted with trypsin. Phage output was determined according to the spot method, in which TG1 cells were infected with serial dilutions of the phage output, plated on LB-Amp / Glu agar plates, and colonies were counted the next day.

[0211] The phage output was rescued by bacterial infection, generating sufficient phage for subsequent selection, and plates with bacterial colonies were also generated. Colonies were picked into 96-well plates for antibody production. The resulting antibodies were used to prepare periplasmic extracts containing soluble Fab.

[0212] The affinity-matured variants were reformatted as bivalent IgG monospecific against TGF-βRII. SPR analysis was performed to determine the affinity of the variants for TGF-βRII. The results are shown in Table 4 and Figure 5. [Table 4-1] [Table 4-2] [Table 4-3]

[0213] Variant reporter assays The affinity matured variants were reformatted into a bivalent IgG format as described in Example 2. The resulting antibodies were used in a TGF-βRII luciferase-based reporter assay to test the ability of the antibodies to inhibit ligand-induced activation of the receptor.

[0214] Reporter CAGA in 293F Freestyle cells 12Luciferase was transiently transfected. The following day, the transfected cells were plated and stimulated with 1 ng / mL hTGF-β1 in the presence or absence of a six-point half-log serial dilution of anti-TGF-βRII antibody. The anti-TGF-βRII antibody was added simultaneously. The starting and maximum antibody concentrations were 10 μg / mL, and the minimum concentration was 0.03 μg / mL. The mixture of 293F Freestyle cells, hTGF-β1, and anti-TGF-βRII antibody was incubated for 3 hours in a 37°C, 5% CO2 incubator. Each plate A negative competitor control containing a heavy chain variable region having SEQ ID NO: 97 and one well titration without TGF-β were performed. As a readout, Steady-Glo Luciferase Assay System detection reagent was added and luciferase was measured on an EnVision after a 5-minute incubation. The folding response was calculated as follows:

number

[0215] The results are shown in Figure 6. The data indicate that some of the variants exhibit improved ligand-blocking activity compared to their parent antibodies. The IC50 values ​​of selected antibodies are presented in Table 5. [Table 5]

[0216] Example 4 - Epitope Mapping To identify the residues of TGF-βRII that are part of the epitope bound by the antibodies generated herein, shotgun mutagenesis experiments were performed using standard techniques (Davidson and Doranz, 2014). As a control in the shotgun mutagenesis approach, an antibody containing a heavy chain variable region having SEQ ID NO: 98 was used. This antibody was unable to block ligand binding to the receptor and was able to bind to the receptor in the presence of all functional TGF-βRII antibodies. The results are shown in Figure 7.

[0217] Example 5 - TGF-βRII Blocking Activity of Antibodies Containing a TGF-βRII Binding Domain RSVxTGF-βRII and antigen AxTGF-βRII antibodies were screened in a TGFβ reporter assay.

[0218] Bispecific antibodies were produced comprising a first binding domain that binds to TGF-βRII, comprising the VH region of a series of antibodies described herein, and a second binding domain that binds to RSV, comprising the VH region having the amino acid sequence set forth in SEQ ID NO: 136, or a second binding domain that binds to an antigen (antigen A) expressed on the same cells as TGF-βRII. Antigen A is an arbitrarily selected antigen that does not react with TGFβ or affects the signal transduction cascade tested in the TGFβ reporter assay. Positive and negative control antibodies described in Example 1 were also included.

[0219] The TGFβ reporter assay detects the CAGA gene, which contains 12 copies of the CAGA box, a SMAD3- and SMAD4-binding sequence. 12 A luciferase vector is used [Dennler et al, 1998]. Binding of TGF-β to TGF-βRII results in the phosphorylation of TGF-βRI. Phosphorylation of TGF-βRI initiates a signaling cascade that leads to the phosphorylation and activation of SMAD2 and SMAD3, which then form a complex with SMAD4. The SMAD complex then translocates to the nucleus and binds to SMAD-binding elements (SBEs) in the nucleus, resulting in the transcription and expression of TGF-β / SMAD-responsive genes. CAGA 12 A luciferase reporter can be used to monitor activation by TGFβ and to screen for TGF-βRII blocking antibodies after transfection of mammalian cells with the reporter.

[0220] Cryopreserved 293FF cells stably transfected to express antigen A were transiently transfected with the TGFβ reporter. IgG was tested at final concentrations ranging from 10 μg / mL to 100 pg / mL. IgG was added in a final volume of 25 μL (4x concentrated). 25 μL of hTGFβ1 (4x concentrated) was then added at a final concentration of 1 ng / mL. 50 μL of the transfected cell suspension (5 x 10 4 Cells) were added. 100 μL of Steady-Glo™ substrate was added to each well and incubated for 5 minutes. Luminescence was measured on an EnVision and results were analyzed using Graphpad Prism.

[0221] The results are shown in Figure 8. Bispecific antibodies comprising a monovalent binding domain for TGF-βRII and a control binding domain for RSV, as well as bispecific antibodies comprising monovalent binding domains for TGF-βRII and antigen A, block the interaction between TGF-β and TGF-βRII. Bispecific antibodies targeting TGF-βRII and RSV comprising a TGF-βRII binding domain comprising a VH region having the amino acid sequences set forth in SEQ ID NOs: 76 and 70 are nearly as potent as bivalent monospecific positive control antibodies. Bispecific antibodies targeting TGF-βRII and antigen A comprising a TGF-βRII binding domain comprising a VH region having the amino acid sequences set forth in SEQ ID NOs: 70, 61, 86, 65, 12, and 76 are more potent than bivalent monospecific positive control antibodies. Thus, the anti-TGF-βRII binding domains of the present disclosure exhibit comparable, comparable, and superior TGF-βRII blocking potency in monovalent form, offering a variety of uses as one or more valencenes incorporated into monovalent, bivalent, or multispecific molecules.

[0222] Heavy chain variable region sequencing The nucleic acids encoding the VH regions of a series of antibodies identified as binding to human TGF-βRII and blocking its interaction with its ligand were sequenced, and the sequence information is provided below. array SEQ ID NO: 1: HCDR1 according to Kabat IYAMT SEQ ID NO: 2: HCDR2 according to Kabat VISGSGGTTYYADSVKG SEQ ID NO: 3: HCDR3 according to Kabat RGQYRDIVGATDY SEQ ID NO: 4: HCDR1 according to Kabat NAWMS SEQ ID NO: 5: HCDR2 according to Kabat RIKTTISGGATDFAAPVKG SEQ ID NO: 6: HCDR3 according to Kabat DLRDY SEQ ID NO: 7: HCDR1 according to Kabat RYAMS SEQ ID NO: 8: HCDR2 according to Kabat AISASGDRTHNTDSVKG SEQ ID NO: 9: HCDR3 according to Kabat GIAASGKNYFDP SEQ ID NO: 10: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS SEQ ID NO: 11: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS SEQ ID NO: 12: Heavy chain variable region [ka] QSISSY SEQ ID NO: 14: LCDR2 by IMGT AAS SEQ ID NO: 15: LCDR3 by IMGT QQSYSTPPT SEQ ID NO: 16: Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK SEQ ID NO: 17: Heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 18: Light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 19: LCDR1 according to Kabat RASQSISSYLN SEQ ID NO: 20: LCDR2 according to Kabat AASSLQS SEQ ID NO: 21: LCDR3 according to Kabat QQSYSTPPT SEQ ID NO: 22: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDINAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS SEQ ID NO: 23: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDIQAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS SEQ ID NO: 24: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYRMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGQYREIVGATDYWGQGTLVTVSS SEQ ID NO: 25: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFYFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRSQYRDKVGATDYWGQGTLVTVSS SEQ ID NO: 26: Heavy chain variable region [ka] EVQLVESGGGLVQPGGSLRLSCAASGFAFDIYAMTWVRQAPGKGLEWVSVISGSGGTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS SEQ ID NO: 28: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTVYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIAGGTDYWGQGTLVTVSS SEQ ID NO: 29: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFDFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRSQYRDKVGATDYWGQGTLVTVSS SEQ ID NO: 30: Heavy chain variable region [ka] EVQLVESGGGLVQPGGSLRLSCAASGFTFDINAMTWVRQAPGKGLEWVSVISGSGGTTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRYIAGATDYWGQGTLVTVSS SEQ ID NO: 32: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDITAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIAGATDYWGQGTLVTVSS SEQ ID NO: 33: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFSFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRAQYRDKVGATDYWGQGTLVTVSS SEQ ID NO: 34: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRYVVGATDYWGQGTLVTVSS SEQ ID NO: 35: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFYFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRHIAGATDYWGQGTLVTVSS SEQ ID NO: 36: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFRFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS SEQ ID NO: 37: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFDINAMTWVRQAPGKGLEWVSVISGSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGQYREIQGANDYWGQGTLVTVSS SEQ ID NO: 38: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSRAWMSWVRQAPGKGLEWVGRIKTTISGGATQFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRNYWGQGTLVTVSS SEQ ID NO: 39: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSRAWMSWVRQAPGKGLEWVGRIKTTVSGGATAFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRAYWGQGTLVTVSS SEQ ID NO: 40: Heavy chain variable region [ka] QVQLVESGGGLVEPGGSLRLSCAASGFTFSRAWMSWVRQAPGKGLEWVGRIKTTYSGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRKYWGQGTLVTVSS SEQ ID NO: 42: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSRAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRAYWGQGTLVTVSS SEQ ID NO: 43: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFKFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATQFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS SEQ ID NO: 44: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTYSGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS SEQ ID NO: 45: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTYSGGATEFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRKYWGQGTLVTVSS SEQ ID NO: 46: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRAYWGQGTLVTVSS SEQ ID NO: 47: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSRAWMSWVRQAPGKGLEWVGRIKTTISGAATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS SEQ ID NO: 48: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFANAWMSWVRQAPGKGLEWVGRIKTTYSGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRKYWGQGTLVTVSS SEQ ID NO: 49: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFQFSNAWMSWVRQAPGKGLEWVGRIKTTYSGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS SEQ ID NO: 50: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAHMSWVRQAPGKGLEWVGRIKTTYSGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRQYWGQGTLVTVSS SEQ ID NO: 51: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFANAWMSWVRQAPGKGLEWVGRIKTTYSGGATEFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRTYWGQGTLVTVSS SEQ ID NO: 52: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTYSGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRSYWGQGTLVTVSS SEQ ID NO: 53: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFQFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATEFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS SEQ ID NO: 54: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFVFSNAWMSWVRQAPGKGLEWVGRIKTTFSGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRKYWGQGTLVTVSS SEQ ID NO: 55: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFHFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRAYWGQGTLVTVSS SEQ ID NO: 56: Heavy chain variable region QVQLVESGGGLVEPGGSLRLSCAASGFKFSNAWMSWVRQAPGKGLEWVGRIKTTISGGKTEFAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTLDLRRYWGQGTLVTVSS SEQ ID NO: 57: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFAFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAKRGKNYFDPWGQGTLVTVSS SEQ ID NO: 58: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFQFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAKSGKNYFDPWGQGTLVTVSS SEQ ID NO: 59: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAKSGKNYFDPWGQGTLVTVSS SEQ ID NO: 60: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFKFRRYAMSWVRQAPGKGLEWVSSISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGLAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 61: Heavy chain variable region [ka] QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGRNYFDPWGQGTLVTVSS SEQ ID NO: 63: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFAFRRYAMSWVRQAPGKGLEWVSSISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 64: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIFASGKHYFDPWGQGTLVTVSS SEQ ID NO: 65: Heavy chain variable region [ka] QVQLVESGGGLVQPGGSLRLSCAVSGFQFRRYAMSWVRQAPGKGLEWVSDISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIARSGKNYFDPWGQGTLVTVSS SEQ ID NO: 67: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTLNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAARGKNYFDPWGQGTLVTVSS SEQ ID NO: 68: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFAFRRYAMSWVRQAPGKGLEWVSAISAFGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGKNFFDPWGQGTLVTVSS SEQ ID NO: 69: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGKNFFDPWGQGTLVTVSS SEQ ID NO: 70: Heavy chain variable region [ka] QVQLVESGGGLVQPGGSLRLSCAVSGFQFRRYAMSWVRQAPGKGLEWVSAISAHGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 72: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTIRRYAMSWVRQAPGKGLEWVSYISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTANSGKNYFDPWGQGTLVTVSS SEQ ID NO: 73: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAARGKNYFDPWGQGTLVTVSS SEQ ID NO: 74: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFERYAMSWVRQAPGKGLEWVSAISASGDRTQNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGRNYFDPWGQGTLVTVSS SEQ ID NO: 75: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFEFRRYAMSWVRQAPGKGLEWVSAISAGGDRTANTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAARGKNYFDPWGQGTLVTVSS SEQ ID NO: 76: Heavy chain variable region [ka] QVQLVESGGGLVQPGGSLRLSCAVSGFSFRRYAMSWVRQAPGKGLEWVSAISASGDRTLNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAARGKNYFDPWGQGTLVTVSS SEQ ID NO: 78: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFEFRRYAMSWVRQAPGKGLEWVSAISASGDRTDNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIARSGKNFFDPWGQGTLVTVSS SEQ ID NO: 79: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFNFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVTGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGLAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 80: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFAFRRYAMSWVRQAPGKGLEWVSAISAHGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 81: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGLAASGKNFFDPWGQGTLVTVSS SEQ ID NO: 82: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFAFRRYAMSWVRQAPGKGLEWVSSISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGLASSGKNYFDPWGQGTLVTVSS SEQ ID NO: 83: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFQFRRYAMSWVRQAPGKGLEWVSSISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGLAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 84: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFQFRRYAMSWVRQAPGKGLEWVSAISASGDRYHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 85: Heavy chain variable region [ka] [ka] QVQLVESGGGLVQPGGSLRLSCAVSGFTFKRYAMSWVRQAPGKGLEWVSAISASGDRSHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGLAARGKNYFDPWGQGTLVTVSS SEQ ID NO: 88: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFNFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGTAARGKNYFDPWGQGTLVTVSS SEQ ID NO: 89: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAASGKNFFDPWGQGTLVTVSS SEQ ID NO: 90: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAARGKNYFDPWGQGTLVTVSS SEQ ID NO: 91: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAVSGFRFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYFCAKGIAARGKNFFDPWGQGTLVTVSS SEQ ID NO: 92: Heavy chain variable region EVQLVETGAEVKKPGASVKVSCKASDYIFTKYDINWVRQAPGQGLEWMGWMSANTGNTGYAQKFQGRVTMTRDTSINTAYMELSSLTSGDTAVYFCARSSLFKTETAPYYHFALDVWGQGTTVTVSS SEQ ID NO: 93: Heavy chain variable region EVQLVESGGDLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRVKTTVSGGTTDYAAAVKGRFTISRDDSKNTLYLQMNSLKTEDTAIYYCTIDLRDYWGQGTLVTVSS SEQ ID NO: 94: Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVSSINTSGGNTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAKGIAATGKNYFDPWGQGTLVTVSS SEQ ID NO: 95: Heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVSSINTSGGNTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGIAAAGKNWFGPWGQGTLVTVSS SEQ ID NO: 96: Heavy chain variable region QVQLVESGGGLVQPGGSLSLSCAASGFTFSRYAMSWVRQAPGKGLEWVSSINTSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGIAASGKNYFDPWGQGTLVTVSS SEQ ID NO: 97: Heavy chain variable region EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKEGWSFDSSGYRSWFDSWGQGTLVTVSS SEQ ID NO: 98: Heavy chain variable region QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSFRGGYTAFDVWGQGTLVTVSS SEQ ID NO: 99: IGKV1-39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP SEQ ID NO: 100: IGKV1-39 / jk5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK SEQ ID NO: 101: Isoform A of human TGF-βRII [ka] [ka] [ka] [ka] NAWMS SEQ ID NO: 106: HCDR2 according to Kabat RIKTTISGGATQFAAPVKG SEQ ID NO: 107: HCDR3 according to Kabat DLRDY SEQ ID NO: 108: HCDR1 according to Kabat RYAMS SEQ ID NO: 109: HCDR2 according to Kabat AISAGGDRTANTDSVKG SEQ ID NO: 110: HCDR3 according to Kabat GTAARGKNYFDP SEQ ID NO: 111: HCDR1 according to Kabat RYAMS SEQ ID NO: 112: HCDR2 according to Kabat AISASGDRTKNTDSVKG SEQ ID NO: 113: HCDR3 according to Kabat GTAAAGKNYFDP SEQ ID NO: 114: HCDR1 according to Kabat RYAMS SEQ ID NO: 115: HCDR2 according to Kabat AISASGDRYHNTDSVKG SEQ ID NO: 116: HCDR3 according to Kabat GTAASGKNYFDP SEQ ID NO: 117: HCDR1 according to Kabat RYAMS SEQ ID NO: 118: HCDR2 according to Kabat AISASGDRTHNTDSVKG SEQ ID NO: 119: HCDR3 according to Kabat GTAARGKNYFDP SEQ ID NO: 120: HCDR1 according to Kabat NYWMS SEQ ID NO: 121: HCDR2 according to Kabat RIKTTYSGGATDFAAPVKG SEQ ID NO: 122: HCDR3 according to Kabat DLRDY SEQ ID NO: 123: HCDR1 according to Kabat RYAMS SEQ ID NO: 124: HCDR2 according to Kabat SISASGDRTHNTDSVKG SEQ ID NO: 125: HCDR3 according to Kabat GLAASGKNYFDP SEQ ID NO: 126: HCDR1 according to Kabat RYAMS SEQ ID NO: 127: HCDR2 according to Kabat AISASGDRTDNTDSVKG SEQ ID NO: 128: HCDR3 according to Kabat GIARSGKNFFDP SEQ ID NO: 129: HCDR1 according to Kabat RAWMS SEQ ID NO: 130: HCDR2 according to Kabat RIKTTISGAATDFAAPVKG SEQ ID NO: 131: HCDR3 according to Kabat DLRDY SEQ ID NO: 132: HCDR1 according to Kabat RYAMS SEQ ID NO: 133: HCDR2 according to Kabat AISASGDRTLNTDSVKG SEQ ID NO: 134: HCDR3 according to Kabat GTAARGKNYFDP SEQ ID NO: 135: Light chain variable region EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK SEQ ID NO: 136: Heavy chain variable region EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKEGWSFDSSGYRSWFDSWGQGTLVTVSS

Claims

1. An antibody or antibody fragment thereof that specifically binds to the extracellular domain of human TGF-βRII, wherein the antibody or antibody fragment binds to an epitope in the extracellular domain of human TGF-βRII, and phenylalanine (F) at position 25 of isoform A of human TGF-βRII or position 50 of isoform B of human TGF-βRII is an essential residue for binding.

2. 2. The antibody or antibody fragment of claim 1, wherein the aspartic acid (D) at position 119 of said epitope in the extracellular domain of isoform A of human TGF-βRII or at position 144 of isoform B of human TGF-βRII is an additional essential residue for binding.

3. The antibody or antibody fragment of claim 1 or 2, wherein the threonine (T) at position 52 of said epitope in the extracellular domain of isoform A of human TGF-βRII or at position 77 of isoform B of human TGF-βRII is an additional essential residue for binding.

4. 3. The antibody or antibody fragment of claim 1 or 2, wherein the isoleucine (I) at position 54 and the glutamic acid (E) at position 120 of the epitope in the extracellular domain of isoform A of human TGF-βRII or the isoleucine (I) at position 79 and the glutamic acid (E) at position 145 of isoform B of human TGF-βRII are further essential residues for binding.

5. the antibody or antibody fragment comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 of a VH having an amino acid sequence set forth in any one of SEQ ID NOs: 25, 29-31, 33-35, 37, 38, 41, 44-47, 49, 50, 54, 55, 57-59, 62, 64, 66, 68, 69, 71, 73, 75, 78, 80, 82, 84-86, 88-91, and 94-96; The antibody or antibody fragment of any one of claims 1 to 4, wherein 1 to 5 amino acid residues may be substituted with their conservative amino acids in any one or more of the CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3.

6. The antibody or antibody fragment thereof according to any one of claims 1 to 4, wherein the antibody comprises a VH amino acid sequence selected from any one of SEQ ID NOs: 25, 29-31, 33-38, 41, 44-47, 49, 50, 54, 55, 57-60, 62, 64, 66, 68, 69, 71, 73, 75, 77, 78, 80, 82, 84-86, 88-91, and 94-96, or a VH amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein any amino acid mutation, insertion, deletion, substitution, addition, or combination thereof is located at a position other than the CDRs with respect to the sequence of the SEQ ID NO.

7. An antibody or antibody fragment thereof comprising VH-CDR1, VH-CDR2, and VH-CDR3 having an amino acid sequence set forth in any one of SEQ ID NOs: 94 to 96, wherein 1 to 5 amino acid residues may be substituted with conservative amino acids thereof in any one or more of the CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3.

8. The antibody or antibody fragment thereof of claim 7, wherein the antibody comprises a VH amino acid sequence selected from any one of SEQ ID NOs: 94-96, or a VH amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

9. The antibody or antibody fragment, A light chain variable region (VL), (a) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19; (b) a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20; and (c) further comprising a VL having a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21; The antibody or antibody fragment thereof according to any one of claims 1 to 8, wherein 1 to 5 amino acid residues may be substituted with their conservative amino acids in any one or more of the CDRs selected from VL-CDR1, VL-CDR2, and VL-CDR3.

10. The antibody has the VL amino acid sequence: The antibody or antibody fragment thereof according to any one of claims 1 to 9, comprising a VL amino acid sequence having at least 80% identity thereto: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSTPPTFGQGTKVEIK (SEQ ID NO: 16).

11. The antibody or antibody fragment thereof according to any one of claims 1 to 10, wherein the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 25, 29-31, 33-38, 41, 44-47, 49, 50, 54, 55, 57-60, 62, 64, 66, 68, 69, 71, 73, 75, 77, 78, 80, 82, 84-86, 88-91, and 94-96, and a light chain variable region of SEQ ID NO:

16.

12. The antibody or antibody fragment thereof according to any one of claims 1 to 11, wherein the antibody comprises two heavy chain variable regions of any one of SEQ ID NOs: 25, 29-31, 33-38, 41, 44-47, 49, 50, 54, 55, 57-60, 62, 64, 66, 68, 69, 71, 73, 75, 77, 78, 80, 82, 84-86, 88-91, and 94-96, and two light chain variable regions of SEQ ID NO:

16.

13. The antibody according to any one of claims 1 to 12, wherein the antibody is an IgG antibody.

14. The antibody of any one of claims 1 to 13, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

15. The antibody according to any one of claims 1 to 14, wherein the antibody is an IgG1 antibody.

16. The antibody or antibody fragment thereof according to any one of claims 1 to 15, wherein the antibody or antibody fragment further comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

17.

17. The antibody or antibody fragment thereof according to any one of claims 1 to 16, wherein the antibody or antibody fragment further comprises a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

18.

18. The antibody of any one of claims 1 to 17, wherein binding of the antibody to Fc receptors is eliminated or reduced.

19. An antibody that specifically binds to human TGF-βRII, (A) a heavy chain having a VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 94 to 96, and a heavy chain constant region comprising an amino acid sequence set forth in SEQ ID NO: 17; and (B) An antibody comprising a light chain having a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

18.

20. The antibody or fragment thereof according to any one of claims 1 to 19, wherein the antibody is a monoclonal antibody.

21. A binding domain that specifically binds to human TGF-βRII, A heavy chain variable region (VH) selected from: (A) a VH having VH-CDR1, VH-CDR2, and VH-CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO:94; (B) a VH having VH-CDR1, VH-CDR2, and VH-CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO:95; and (C) a VH having VH-CDR1, VH-CDR2, and VH-CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 96; Any one of the following: wherein 1 to 5 amino acid residues may be replaced with their conservative amino acids in any one or more of said CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3.

22. the binding domain comprises a VH amino acid sequence selected from SEQ ID NOs: 94, 95, and 96; or A VH amino acid sequence having at least 80% identity thereto The binding domain of claim 21 , comprising:

23. the antibody or antibody fragment, or the binding domain, A light chain variable region (VL), (a) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19; (b) a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20; and (c) further comprising a VL having a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21; 23. The binding domain of claim 21 or 22, wherein 1 to 5 amino acid residues may be substituted with their conservative amino acids in any one or more of the CDRs selected from VL-CDR1, VL-CDR2, and VL-CDR3.

24. The antibody or binding domain has the VL amino acid sequence:

24. The binding domain of any one of claims 21 to 23, comprising a VL amino acid sequence having at least 80% identity thereto: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSTPPTFGQGTKVEIK (SEQ ID NO: 16).

25. A vector comprising a polynucleotide encoding either or both of the heavy and light chains of the antibody or antibody fragment of any one of claims 1 to 20, or the binding domain of any one of claims 21 to 24.

26. A cell producing an antibody or antibody fragment according to any one of claims 1 to 20, or a binding domain according to any one of claims 21 to 24.

27. 27. The cell of claim 26, wherein the cell is a recombinant cell transformed with the vector of claim 25.

28. A pharmaceutical composition comprising an antibody or antibody fragment according to any one of claims 1 to 20, or a binding domain according to any one of claims 21 to 24, and a pharma- ceutically acceptable carrier, diluent or excipient.

29. A pharmaceutical for use in preventing cancer, suppressing the progression or recurrence of cancer symptoms, and / or treating cancer, comprising as an active ingredient an antibody or antibody fragment thereof according to any one of claims 1 to 20, or a binding domain according to any one of claims 21 to 24.

30. 30. The method of claim 29, wherein the cancer is a cancer type that correlates with higher than normal TGF-β signaling, in particular higher than normal TGF-βRII expression.

31. 31. The pharmaceutical product of claim 29 or 30, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, colorectal cancer, gastric cancer, glioblastoma, cervical cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, myelodysplastic syndrome, pancreatic cancer, prostate cancer, and renal cancer.

32. A method for blocking binding of human TGF-β to human TGF-βRII of a cell, comprising providing to the cell an antibody or antibody fragment of any one of claims 1 to 20, or a binding domain of any one of claims 21 to 24, and allowing the antibody or antibody fragment, or the binding domain to bind to the human TGF-βRII of the cell, thereby blocking binding of human TGF-β to human TGF-βRII of the cell.

33. A method for inhibiting signal transduction to a cell induced by binding of human TGF-β to human TGF-βRII, the method comprising providing to the cell an antibody or antibody fragment of any one of claims 1 to 20, or a binding domain of any one of claims 21 to 24, and allowing the antibody or antibody fragment, or the binding domain to bind to the human TGF-βRII of the cell, thereby inhibiting the signal transduction to the cell.

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