Tgfβrii extracellular domain variants and uses thereof

Abstract

Provided herein are polypeptides comprising a variant human TGFβRII extracellular domain and fusions thereof with antibody molecules. Also provided are polynucleotides encoding these polypeptides and fusion molecules, vectors and host cells, and methods of making and using these polypeptides and fusion molecules.

Description

[0001] Cross-referencing This application claims priority to U.S. Provisional Application 63 / 587,852, filed October 4, 2023, which is incorporated herein by reference in its entirety.

[0002] Reference sequence list The contents of the sequence list (name: 213071_Seqlisting_ST26.XML; size: 35,046 bytes; and creation date: October 2, 2024), which was submitted electronically in the form of an .XML file with this application, are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to polypeptides comprising the extracellular region of a variant human TGFβRII and fusions thereof with antigen-binding molecules. Also provided are polynucleotides encoding these polypeptides and fusion molecules, vectors and host cells, and methods for preparing and using these polypeptides and fusion molecules. Background Technology

[0004] TGFβ is a pluripotent cytokine that is a potent inducer of reactive phenotypes in fibroblasts, and all three TGFβ isoforms (1-3) play context-dependent roles in regulating cell proliferation, differentiation, apoptosis, angiogenesis, fibrosis, and immune activation. TGFβ is synthesized as a homodimeric proprotein composed of both a C-terminal mature growth factor dimer and an N-terminal prodomain. Members of the TGFβ superfamily transfect signal through transmembrane receptors containing serine / threonine kinase domains, known as type I (TGFβRI) and type II. These receptors significantly influence the regulation of TGFβ signaling and its associated responses. TGFβRII is constitutively active, while TGFβRI acts as a downstream element in the signaling pathway, playing a crucial role in determining the specificity of intracellular signals induced by cytokines from the TGFβ superfamily. Binding of ligands to TGFβR complexes triggers recruitment of signal transduction mechanisms to promote downstream functional activity. TGFβRII exhibits high affinity binding to the active forms of TGFβ1 and TGFβ3. TGFβ has been reported to be important in tumor-stromal crosstalk and a key factor in inducing fibroblast transdifferentiation into cancer-associated fibroblasts (CAF). Furthermore, both CAF and TGFβ are associated with cancer resistance to chemotherapy, targeted therapy, and immunotherapy. Therefore, therapies targeting the TGFβ pathway in CAF are needed.

[0005] During the production of biotherapeutic agents, recombinant proteins often undergo undesirable product fragmentation, commonly referred to as "shearing." Protein shearing occurs through enzymatic or chemical disruption of peptide bonds. Peptide bonds are generally resistant to non-enzymatic hydrolysis; however, cleavage of specific side chain residues (such as aspartic acid, glycine, serine, threonine, cysteine, or asparagine) has been described, particularly when solvent-exposed residues are under acidic or alkaline conditions and at high temperatures. However, the shearing observed during biotherapeutic agent production is primarily due to the action of intracellular or extracellular host cell proteases (such as metalloproteinases, serine proteases, and cathepsin D). This shearing can produce heterogeneous products, leading to negative impacts on yield, downstream processing requirements, immunogenicity risks, pharmacokinetics, and ultimately, higher costs for commercially available biotherapies. Fusion proteins (such as the antibody-TGFβRII fusion described herein) may be more difficult to produce than classic IgG molecules, exhibiting reduced yields compared to conventional IgG and increased susceptibility to shearing during production.

[0006] Therefore, there is a need to develop improved forms of human TGFβRII that exhibit lower levels of protease-mediated cleavage upon fusion with antibodies. Summary of the Invention

[0007] This disclosure provides polypeptides comprising the extracellular region of a variant human TGFβRII and fusions thereof with antigen-binding molecules. It also provides polynucleotides encoding these polypeptides and fusion molecules, vectors, and host cells, as well as methods for preparing and using these polypeptides and fusion molecules. Compared to the corresponding wild-type (WT) human TGFβRII, the extracellular region of the variant human TGFβRII disclosed herein exhibits substantially less proteolytic cleavage during protein production, which in turn results in higher final product yield, simpler downstream processing, more homogeneous products, and lower commercial costs compared to WT human TGFβRII.

[0008] Therefore, in one aspect, this disclosure provides a polypeptide comprising the extracellular region of a variant human TGFβRII, wherein the amino acid sequence of the extracellular region of the variant human TGFβRII is: (a) The amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1 is missing; (b) The amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1 is missing; (c) Contains threonine and proline at the positions corresponding to amino acids 5 and 9 of SEQ ID NO: 1, respectively; (d) Contains isoleucine and alanine at the positions corresponding to amino acids 33 and 35 of SEQ ID NO: 1, respectively; (e) Contains isoleucine, alanine and alanine at the positions corresponding to amino acids 33, 35 and 36 of SEQ ID NO: 1, respectively; (f) Contains threonine, proline, isoleucine and alanine at the positions corresponding to amino acids 5, 9, 33 and 35 of SEQ ID NO: 1, respectively; (g) Contains threonine, proline, isoleucine, alanine and alanine at the positions corresponding to amino acids 5, 9, 33, 35 and 36 of SEQ ID NO: 1, respectively; (h) contains isoleucine and alanine at the positions corresponding to amino acids 33 and 35 of SEQ ID NO:1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-15 of SEQ ID NO:1; (i) Contains isoleucine and alanine at the positions corresponding to amino acids 33 and 35 of SEQ ID NO:1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-22 of SEQ ID NO:1; (j) Contains isoleucine, alanine, and alanine at positions corresponding to amino acids 33, 35, and 36 of SEQ ID NO: 1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-15 of SEQ ID NO: 1; or (k) contains isoleucine, alanine, and alanine at the positions corresponding to amino acids 33, 35, and 36 of SEQ ID NO: 1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-22 of SEQ ID NO: 1.

[0009] In one embodiment, the extracellular region of the variant human TGFβRII is at least 75% identical to SEQ ID NO: 1 in the full length of the human TGFβRII extracellular region variant (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0010] In one embodiment, the amino acid sequence of the extracellular region of the variant human TGFβRII consists of the amino acid sequences shown in any of SEQ ID NO: 2-12.

[0011] In one embodiment, the polypeptide includes an antigen-binding region. In one embodiment, the antigen-binding region includes scFv, VH, VL, VHH, an antibody heavy chain, or an antibody light chain.

[0012] In one implementation, the polypeptide contains an antibody constant region.

[0013] In one implementation, the extracellular region of the variant human TGFβRII is linked to the antigen-binding region or the antibody constant region via a linker sequence.

[0014] In one embodiment, the polypeptide comprises, from the N-terminus to the C-terminus: an antibody heavy chain or its Fc fragment; and the extracellular region of the variant human TGFβRII.

[0015] In one embodiment, the polypeptide comprises, from the N-terminus to the C-terminus: an antibody heavy chain or its Fc fragment; a linker sequence; and the extracellular region of the variant human TGFβRII.

[0016] In one embodiment, the adapter sequence comprises the amino acid sequence shown in SEQ ID NO: 13. In another embodiment, the adapter sequence comprises the amino acid sequence shown in SEQ ID NO: 14.

[0017] In one embodiment, the polypeptide comprises the amino acid sequence shown in any of SEQ ID NO: 15-25.

[0018] In one aspect, this disclosure provides an antibody molecule comprising two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from its N-terminus to its C-terminus: a variable region; a constant region; a linker sequence; and a variant human TGFβRII extracellular region consisting of an amino acid sequence shown in any of SEQ ID NO: 2-12. In one embodiment, the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 13. In one embodiment, the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 14. In one embodiment, the antibody molecule comprises the amino acid sequence shown in any of SEQ ID NO: 15-25. In one embodiment, the antibody molecule is a full-length heterotetrameric antibody molecule comprising two heavy chain polypeptides and two light chain polypeptides. In one embodiment, the antibody molecule is a full-length heavy chain antibody molecule comprising two heavy chain polypeptides and not comprising light chain polypeptides. In one embodiment, the constant region is a human heavy chain constant region. In one embodiment, the human heavy chain constant region is an isotype of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM.

[0019] In one aspect, this disclosure provides a polynucleotide encoding a polypeptide or antibody molecule disclosed herein.

[0020] In one aspect, this disclosure provides a vector comprising the polynucleotides disclosed herein.

[0021] In one aspect, this disclosure provides an engineered cell comprising the polynucleotides or vectors disclosed herein.

[0022] In one aspect, this disclosure provides a method for generating a polypeptide, the method comprising culturing the engineered cells disclosed herein under conditions that cause the expression of polynucleotides and the generation of polypeptides.

[0023] In one aspect, this disclosure provides a method for inhibiting the activity of human TGFβ, the method comprising contacting human TGFβ with a polypeptide or antibody molecule disclosed herein.

[0024] In one aspect, this disclosure provides a method for inhibiting the activity of human TGFβ in a subject, wherein the subject is administered a polypeptide or antibody molecule disclosed herein. Attached Figure Description

[0025] Figure 1A-Figure 1B This is a graph showing the effect of the anti-FAP-TGFβRII bispecific variant on SMAD-driven luciferase reporter gene activity, which is related to TGFβ1 ( Figure 1A ) or TGFβ-3 ( Figure 1B ) is activated during signal transduction. Reporter gene activity is expressed as relative luminescent units (RLU) (y-axis) varying with the concentration of the anti-FAP-TGFβRII bispecific variant (x-axis).

[0026] Figure 2 This is a graph showing the effect of the anti-FAP-TGFβRII bispecific variant on TGFβ1-induced vimentin expression levels in A549-vimentin-RFP reporter cells. Vimentin % (y-axis) is shown as a function of the anti-FAP-TGFβRII bispecific variant concentration (x-axis).

[0027] Figure 3 This is a graph illustrating the binding kinetics of the anti-FAP-TGFβRII bispecific variant AF1-TGFβRII (V03) with human FAP and human TGFβ1. Arrows point to different binding events consistent with the simultaneous binding of the antibody to human FAP and human TGFβ1, with human FAP or TGFβ1 injected first. Detailed Implementation

[0028] This disclosure provides peptides comprising an improved variant of the extracellular region of human TGFβRII and fusions thereof with antibody molecules. It also provides polynucleotides encoding these peptides and fusion molecules, vectors, and host cells, as well as methods for preparing and using these peptides and fusion molecules. Compared to the corresponding wild-type (WT) human TGFβRII, the disclosed variant of human TGFβRII exhibits substantially less proteolytic cleavage during protein production, which in turn results in higher final product yield, simpler downstream processing, more homogeneous products, and lower commercial costs compared to WT human TGFβRII.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. It should be understood that the foregoing general description and the following detailed description are merely illustrative and exemplary and are not intended to limit any of the claimed subject matter. In this application, the singular is used to include the plural unless otherwise specified. It must be noted that, as used in this specification and the appended claims, the singular forms “a” and “the” include the plural referent unless the context clearly specifies otherwise. In this application, the use of “or” means “and / or” unless otherwise specified. Furthermore, the use of the terms “comprising” and other forms such as “containing” and “including” is not restrictive. Section headings used herein are for organizational purposes only and should not be construed as limiting the described subject matter.

[0030] As used herein, the terms “about” and “approximately” when used to modify a numerical value or range indicate that a deviation of 5% to 10% (e.g., up to 5% to 10% above) and 5% to 10% (e.g., down to 5% to 10%) below the value or range is still within the intended meaning of the value or range.

[0031] As used herein, the term “FAP” refers to fibroblast activation protein α. As used herein, the term “human FAP” refers to the FAP protein encoded by the wild-type human FAP gene (e.g., RefSeq accession number NG_027991.1). Exemplary immature wild-type human FAP amino acid sequences are provided in RefSeq accessions NP_004451.2 and NP_001278736.1.

[0032] As used in this article, the terms "transforming growth factor β" and "TGFβ" refer to the substances produced by the metabolism of β-transforming growth factor in humans. TGFB1 , TGFB2 and TGFB3 Any TGFβ family protein encoded by a gene. As used herein, the term "human TGFβ1" refers to a protein derived from human TGFβ. TGFB1 Genes (e.g., wild-type humans) TGFB1 The TGFβ1 protein encoded by the human TGFβ1 gene. An exemplary wild-type human TGFβ1 protein is provided in RefSeq accession number NP_000651.3. As used herein, the term "human TGFβ2" refers to the protein encoded by the human TGFβ1 gene. TGFB2 Genes (e.g., wild-type humans) TGFB2 The TGFβ2 protein encoded by the human TGFβ2 gene. Exemplary wild-type human TGFβ2 proteins are provided in RefSeq accessions NP_001129071.1 and NP_003229.1. As used herein, the term "human TGFβ3" refers to the protein encoded by the human TGFβ2 gene. TGFB3 Genes (e.g., wild-type humans) TGFB3 The TGFβ3 protein encoded by the gene. Exemplary wild-type human TGFβ3 proteins are provided in RefSeq accessions NP_003230.1, NP_001316868.1 and NP_001316867.1.

[0033] As used in this article, the terms "transforming growth factor β receptor" and "TGFβ receptor" refer to receptors formed in humans by... TGFBR1 , TGFBR2 and TGFBR3 Any TGFβ receptor family protein encoded by a gene. As used herein, the terms "human TGFβR1" and "human TGFβRI" refer to proteins from the human TGFβ receptor family. TGFBR1 Genes (e.g., wild-type humans) TGFBR1 The TGFβR1 protein is encoded by the gene. An exemplary wild-type human TGFβR1 protein is available from GenBank. ™ Accession numbers NP_004603.1, NP_001124388.1, and NP_001293139.1 are provided. As used herein, the terms "human TGFβR2" and "human TGFβRII" refer to substances produced by humans. TGFB2 Genes (e.g., wild-type humans) TGFB2 The TGFβRII protein is encoded by the gene. An exemplary wild-type human TGFβR2 protein is available from GenBank. ™ Accessions NP_001129071.1 and NP_003229.1 are provided. As used herein, the terms "human TGFβR3" and "human TGFβRIII" refer to substances produced by humans. TGFB3 Genes (e.g., wild-type humans) TGFB3 The TGFβRIII protein encoded by the gene. An exemplary wild-type human TGFβ3 protein is available from GenBank. ™ Login numbers NP_003230.1, NP_001316868.1 and NP_001316867.1 are provided.

[0034] As used herein, the term "variant human TGFβRII extracellular region" refers to a polypeptide containing one or more insertions, deletions, or substitutions relative to the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide is capable of binding to human TGFβ1. In some embodiments, the variant human TGFβRII extracellular region is at least 75% identical to SEQ ID NO: 1 in its full length (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical).

[0035] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDR, VH region, and / or VL region. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camel-derived antibodies, affinity molecules, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG...). 2a or IgG 2b The antibody is an immunoglobulin molecule. In some embodiments, the antibody described herein is an IgG antibody, or its class (e.g., human IgG1 or IgG4) or subclass. In one specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody. In some embodiments, the antibody as described herein comprises a full-length antibody or its antigen-binding fragment linked to a ligand-binding moiety specifically binding to TGFβ (i.e., the extracellular region of human TGFβRII). In some embodiments, the antibody is a chimeric antigen receptor comprising the VH and / or VL disclosed herein linked to a transmembrane domain and one or more intracellular domains capable of signaling T cell activation.

[0036] A "multispecific antibody" is an antibody that specifically binds to two or more different antigens or two or more different regions of the same antigen (e.g., a bispecific antibody). Multispecific antibodies include bispecific antibodies containing two different antigen-binding sites (excluding the Fc region). Multispecific antibodies can include, for example, recombinant antibodies, human antibodies, humanized antibodies, surface-remodeled antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, heteroconjugated antibodies, linked single-chain antibodies or linked single-chain Fv (scFv), camel-derived antibodies, affinity molecules, linked Fab fragments, F(ab')2 fragments, chemically linked Fvs, and disulfide-linked Fvs (sdFvs). Multispecific antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some embodiments, the multispecific antibodies described herein are IgG antibodies, or their class (e.g., human IgG1, IgG2, or IgG4) or subclass.

[0037] As used herein, an "antigen-binding region" refers to a portion of the polypeptide disclosed herein containing amino acid residues that confer specificity against an antigen. Examples of antigen-binding regions include antibody complementarity-determining regions (CDRs), heavy chain variable regions, light chain variable regions, heavy chains, light chains, and any fragments thereof. Antigen-binding regions can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans.

[0038] As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable region of both heavy-chain and light-chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entirety, wherein the definition includes overlap or subsets of amino acid residues when compared to one another. In some embodiments, the term “CDR” is as described by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Antibody Engineering The term "CDR" is defined as follows: Martin A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," edited by Kontermann and Düibel, Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some embodiments, the term "CDR" is as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In some embodiments, the heavy chain CDR and light chain CDR of the antibody are defined using different conventions. In some embodiments, the heavy chain CDR and / or light chain CDR are defined by performing structural analysis of the antibody and identifying residues in the variable region predicted to contact the epitope region of the target molecule. CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0039] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, usually a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain. These amino acids vary considerably in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within a variable domain are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a human frame region (FR). In a particular embodiment, the variable region is a primate (e.g., a non-human primate) variable region. In some implementations, the variable region includes the rodent or mouse CDR and the primate (e.g., non-human primate) frame region (FR).

[0040] As used herein, the terms “VH” and “VL” refer to the variable regions of the antibody heavy chain and light chain, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0041] As used herein, the terms “constant region” and “constant domain” are interchangeable and are common in the art. A constant region is an antibody portion, such as the carboxyl-terminal portion of the light chain and / or heavy chain, that does not directly participate in the binding of the antibody to the antigen but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fcγ receptors).

[0042] As used herein, the term “heavy chain” when referring to antibody use can refer to any different type of amino acid sequence based on a constant domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and muon (µ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0043] As used herein, the term "light chain," when referring to antibody use, can refer to any different type of amino acid sequence based on a constant domain, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0044] As used herein, the terms “specific binding,” “specific recognition,” “immunospecific binding,” and “immunospecific recognition” are similar terms in the context of antibodies and refer to molecules that bind to antigens (e.g., epitopes or immune complexes) as understood by those skilled in the art. For example, molecules that specifically bind to antigens may bind to antigens that typically have properties such as those obtained by, for example, immunoassays, BIAcores, etc. ® Other peptides or polypeptides with lower affinity determined by a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID.) or other assays known in the art. In one specific embodiment, the affinity is lower than that when the molecule nonspecifically binds to another antigen. A K is at least 2 logarithms (e.g., multiples of 10), 2.5 logarithms, 3 logarithms, 4 logarithms, or greater. A A molecule that specifically binds to an antigen.

[0045] As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can typically be represented by the dissociation constant (Kd). Affinity can be measured using methods commonly known in the art, including those described herein.

[0046] As used herein, the term “EU numbering system” refers to the EU numbering convention for antibody constant regions, as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0047] As used herein, the term "linked with" refers to a covalent or non-covalent bond between two molecules or portions. Those skilled in the art will understand that when a first molecule or portion is linked to a second molecule or portion, the link need not be direct, but can occur via an insert molecule or portion. For example, when the heavy chain variable region of a full-length antibody is linked to a ligand-binding portion, the ligand-binding portion can (e.g., via peptide bonds) bind to the constant region of the full-length antibody (e.g., the heavy chain constant region), rather than directly to the heavy chain variable region.

[0048] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of one or more polypeptides (i.e., conformational, nonlinear, discontinuous, or non-continuous epitopes). In some embodiments, the epitope to which the antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange coupled mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays (e.g., using CLIPS (peptide-to-scaffold chemical linkage) to confine peptides to map discontinuous or conformational epitopes) and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is incorporated herein by reference in its entirety). Antibody-antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), each of which is incorporated herein by reference in its entirety. Mutagenesis mapping studies can be performed using any method known to those skilled in the art.See, for example, Champe M et al., (1995) JBiol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085 (each of these references is incorporated herein by reference in its entirety) to describe mutagenesis techniques, including alanine scanning mutagenesis.

[0049] As used herein, the term “treatment” refers to the therapeutic or preventative measures described herein. “Treatment” methods include administering antibodies to a subject who has a disease or disorder, or is predisposed to developing such a disease or disorder, in order to prevent, cure, delay, reduce the severity of the disease or disorder or recurrent disease or disorder, or improve one or more symptoms of the disease or disorder or recurrent disease or disorder, or in order to prolong the subject’s expected survival in the absence of such treatment.

[0050] As used herein, in the context of administering therapy to a subject, the term "effective amount" refers to the amount of therapy that achieves the desired preventive or therapeutic effect.

[0051] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In another embodiment, the subject is a human.

[0052] As used herein with respect to peptides, antibodies, or polynucleotides, the term "isolated" means a peptide, antibody, or polynucleotide separated from one or more contaminants (e.g., peptides, polynucleotides, lipids, or carbohydrates) present in the natural source of the peptide, antibody, or polynucleotide. All examples of "isolated peptides" described herein are also considered as peptides that may, but are not required to, be isolated. All examples of "isolated antibodies" described herein are also considered as antibodies that may, but are not required to, be isolated. All examples of "isolated polynucleotides" described herein are also considered as polynucleotides that may, but are not required to, be isolated. All examples of "peptides" described herein are also considered as antibodies that may, but are not required to, be isolated. All examples of "antibodies" described herein are also considered as antibodies that may, but are not required to, be isolated. All examples of "polynucleotides" described herein are also considered as polynucleotides that may, but are not required to, be isolated.

[0053] The determination of the "percentage of identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. A specific, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin S and Altschul SF (1990) PNAS 87: 2264-2268, modified by Karlin S and Altschul SF (1993) PNAS 90: 5873-5877, each of which is incorporated herein by reference in its entirety. This algorithm is also incorporated in the NBLAST and XBLAST procedures of Altschul SF et al., (1990) J Mol Biol 215: 403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, for example, setting score = 100 and word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameter set, for example, with a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules described herein. For gapped alignments for comparative purposes, Gapped BLAST, as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, is used, which is incorporated herein by reference in its entirety. Alternatively, iterative searches can be performed using PSI BLAST to detect distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) website on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. This algorithm was incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, a 12-bit gap length penalty, and a 4-bit gap penalty can be used.

[0054] The percentage of identity between two sequences can be determined using techniques similar to those described above, regardless of whether gaps are allowed. When calculating the percentage of identity, typically only exact matches are counted.

[0055] TGFβRII polypeptide This disclosure provides a polypeptide comprising the extracellular region of a variant human TGFβRII. The amino acid sequences of the extracellular region of wild-type human TGFβRII and exemplary variant human TGFβRII are shown in Table 1.

[0056] Table 1: Exemplary amino acid sequences of the extracellular region of wild-type human TGFβRII and its variants .

[0057]

[0058]

[0059] In some embodiments, this disclosure provides a polypeptide comprising the extracellular region of a variant human TGFβRII, wherein the amino acid sequence of the extracellular region of the variant human TGFβRII is: lacking amino acid sequences corresponding to amino acids 1-15 of SEQ ID NO: 1; lacking amino acid sequences corresponding to amino acids 1-22 of SEQ ID NO: 1; comprising threonine and proline at amino acid positions corresponding to amino acids 5 and 9 of SEQ ID NO: 1, respectively; comprising isoleucine and alanine at amino acid positions corresponding to amino acids 33 and 35 of SEQ ID NO: 1, respectively; comprising isoleucine, alanine, and alanine at amino acid positions corresponding to amino acids 33, 35, and 36 of SEQ ID NO: 1, respectively; comprising threonine, proline, isoleucine, and alanine at amino acid positions corresponding to amino acids 5, 9, 33, and 35 of SEQ ID NO: 1, respectively; comprising threonine, proline, isoleucine, alanine, and alanine at amino acid positions corresponding to amino acids 5, 9, 33, 35, and 36 of SEQ ID NO: 1, respectively; comprising isoleuc ... The amino acids 1, 1, contain isoleucine and alanine at positions corresponding to amino acids 33 and 35 of SEQ ID NO: 1, and lack amino acid sequences corresponding to amino acids 1-15 of SEQ ID NO: 1; contain isoleucine and alanine at positions corresponding to amino acids 33 and 35 of SEQ ID NO: 1, and lack amino acid sequences corresponding to amino acids 1-22 of SEQ ID NO: 1; contain isoleucine, alanine, and alanine at positions corresponding to amino acids 33, 35, and 36 of SEQ ID NO: 1, and lack amino acid sequences corresponding to amino acids 1-15 of SEQ ID NO: 1; or contain isoleucine, alanine, and alanine at positions corresponding to amino acids 33, 35, and 36 of SEQ ID NO: 1, and lack amino acid sequences corresponding to amino acids 1-22 of SEQ ID NO: 1.

[0060] In some embodiments, this disclosure provides a polypeptide comprising a variant human TGFβRII extracellular region, wherein the variant human TGFβRII extracellular region is at least 75% identical to SEQ ID NO: 1 in its full length (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical). In some embodiments, the polypeptide comprises a variant human TGFβRII extracellular region, wherein the variant human TGFβRII extracellular region is at least 75% identical to SEQ ID NO: 1 along its full length (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical).

[0061] In some embodiments, this disclosure provides a polypeptide comprising a variant human TGFβRII extracellular region, wherein the amino acid sequence of the variant TGFβRII extracellular region comprises the amino acid sequence shown in any of SEQ ID NO: 2-12.

[0062] In some embodiments, this disclosure provides a polypeptide comprising a variant human TGFβRII extracellular region, wherein the polypeptide includes an antigen-binding region. In some embodiments, the antigen-binding region comprises scFv, VH, VL, VHH, an antibody heavy chain, or an antibody light chain.

[0063] In some embodiments, this disclosure provides a polypeptide comprising a variant of the human TGFβRII extracellular region, wherein the polypeptide comprises an antibody constant region. Any antibody constant region may be used in the fusion molecules disclosed herein. In some embodiments, the antibody constant region is human immunoglobulin (Ig) of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) immunoglobulin molecule. In one embodiment, the antibody constant region is an IgG constant region (e.g., a human IgG constant region). In one embodiment, the antibody constant region is an IgG1 constant region (e.g., a human IgG1 constant region). In one embodiment, the antibody constant region is a chimeric constant region comprising portions of several different constant regions. Suitable examples of chimeric constant regions are shown in US 2011 / 0243966A1, which is incorporated herein by reference in its entirety. Various constant region gene sequences (e.g., human constant region gene sequences) can be obtained in the form of publicly accessible deposits.

[0064] In some embodiments, the extracellular region of the variant human TGFβRII is directly linked to the antigen-binding region or antibody constant region. In some embodiments, the extracellular region of the variant human TGFβRII is linked to the antigen-binding region or antibody constant region via a linker sequence. In some embodiments, the linker is a peptide linker. Examples of peptide linkers are well known, and those skilled in the art can select suitable peptide linkers for linking the extracellular region of the variant human TGFβRII to the antigen-binding region or antibody constant region. Exemplary linker sequences are described herein.

[0065] The linker sequence can be of any length. In some embodiments, the length and amino acid composition of the linker sequence can be optimized to alter the orientation and / or proximity of these parts to achieve a desired activity. In some embodiments, the linker sequence is about 1 to about 100 amino acids long, about 8 to about 40 amino acids long, or about 15 to about 25 amino acids long. In some embodiments, the linker sequence is approximately 8 amino acids long, approximately 9 amino acids long, approximately 10 amino acids long, approximately 11 amino acids long, approximately 12 amino acids long, approximately 13 amino acids long, approximately 14 amino acids long, approximately 15 amino acids long, approximately 16 amino acids long, approximately 17 amino acids long, approximately 18 amino acids long, approximately 19 amino acids long, approximately 20 amino acids long, approximately 21 amino acids long, approximately 22 amino acids long, approximately 23 amino acids long, approximately 24 amino acids long, approximately 25 amino acids long, approximately 26 amino acids long, approximately 27 amino acids long, approximately 28 amino acids long, approximately 29 amino acids long, approximately 30 amino acids long, approximately 31 amino acids long, approximately 32 amino acids long, approximately 33 amino acids long, approximately 34 amino acids long, approximately 35 amino acids long, approximately 36 amino acids long, approximately 37 amino acids long, approximately 38 amino acids long, approximately 39 amino acids long, or approximately 40 amino acids long. In some implementations, the linker sequence is 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, 15 amino acids long, 16 amino acids long, 17 amino acids long, 18 amino acids long, 19 amino acids long, 20 amino acids long, 21 amino acids long, 22 amino acids long, 23 amino acids long, 24 amino acids long, 25 amino acids long, 26 amino acids long, 27 amino acids long, 28 amino acids long, 29 amino acids long, 30 amino acids long, 31 amino acids long, 32 amino acids long, 33 amino acids long, 34 amino acids long, 35 amino acids long, 36 amino acids long, 37 amino acids long, 38 amino acids long, 39 amino acids long, or 40 amino acids long.

[0066] In some embodiments, the adapter sequence contains only glycine and / or serine residues (e.g., a glycine-serine adapter or a GS adapter). In some embodiments, the adapter sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and 14, or a variant thereof comprising 1-5 amino acid variations. In some embodiments, the adapter sequence comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the adapter sequence comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the adapter sequence consists of the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the adapter sequence consists of the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the adapter sequence is a GS adapter of approximately 21 amino acids in length. In some embodiments, the adapter sequence is a GS adapter of 21 amino acids in length.

[0067] In some embodiments, this disclosure provides a polypeptide comprising a variant human TGFβRII extracellular region, wherein the polypeptide comprises, from its N-terminus to its C-terminus: an antibody heavy chain or its Fc fragment; and the variant human TGFβRII extracellular region. In some embodiments, the polypeptide comprises, from its N-terminus to its C-terminus: an antibody heavy chain or its Fc fragment; and the variant human TGFβRII extracellular region. In some embodiments, the polypeptide comprises, from its N-terminus to its C-terminus: an antibody heavy chain or its Fc fragment; a linker sequence; and the variant human TGFβRII extracellular region. In some embodiments, the polypeptide comprises, from its N-terminus to its C-terminus: an antibody heavy chain or its Fc fragment; a linker sequence; and the variant human TGFβRII extracellular region.

[0068] In some embodiments, this disclosure provides a polypeptide comprising the extracellular region of a variant human TGFβRII, wherein the polypeptide comprises the amino acid sequence shown in any of SEQ ID NO: 15-25. In some embodiments, the polypeptide consists of the amino acid sequence shown in any of SEQ ID NO: 15-25.

[0069] In one aspect, this disclosure provides an antibody molecule comprising the extracellular region of a variant human TGFβRII disclosed herein. In some embodiments, the antibody molecule comprises two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from its N-terminus to its C-terminus: a variable region; a constant region; a linker sequence; and a variant human TGFβRII extracellular region comprising the amino acid sequence shown in any of SEQ ID NO: 2-12. In some embodiments, the antibody molecule comprises two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from its N-terminus to its C-terminus: a variable region; a constant region; a linker sequence; and a variant human TGFβRII extracellular region comprising the amino acid sequence shown in any of SEQ ID NO: 2-12. In some embodiments, the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the antibody molecule comprises the amino acid sequence shown in any of SEQ ID NO: 15-25.

[0070] In some embodiments, the antibody molecule is a full-length heterotetrameric antibody molecule comprising two heavy chain polypeptides and two light chain polypeptides. In some embodiments, the antibody molecule is a full-length heavy chain antibody molecule comprising two heavy chain polypeptides and no light chain polypeptides. In some embodiments, each heavy chain polypeptide comprises a variant human TGFβRII extracellular region consisting of an amino acid sequence shown in any of SEQ ID NO: 2-12. In some embodiments, each heavy chain polypeptide comprises an amino acid sequence shown in any of SEQ ID NO: 15-25.

[0071] In some embodiments, this disclosure provides an antibody molecule comprising the extracellular region of the variant human TGFβRII disclosed herein, wherein the constant region of the antibody molecule is a human heavy chain constant region. In some embodiments, the antibody molecule comprises two heavy chain polypeptides, wherein each heavy chain polypeptide comprises a constant region. In some embodiments, each constant region of the antibody molecule is a human heavy chain constant region. In some embodiments, the human heavy chain constant region is an isotype of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM. In some embodiments, each constant region of the antibody molecule is a human heavy chain constant region of an isotype of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM.

[0072] Polynucleotides, vectors and production methods This disclosure also provides polynucleotides encoding any of the polypeptides, antibody molecules, or portions thereof described herein. In some embodiments, the polynucleotide encodes a polypeptide comprising the extracellular region of a variant human TGFβRII of this disclosure. In some embodiments, the polynucleotide encodes an antibody molecule of this disclosure. In some embodiments, the polynucleotide encodes a polypeptide comprising the extracellular region of a variant human TGFβRII and an antigen-binding region or antibody constant region, and optionally a linker. In some embodiments, the polynucleotide encodes a polypeptide comprising the extracellular region of a variant human TGFβRII and an antibody heavy chain or its Fc fragment, and optionally a linker. In some embodiments, the polynucleotide described herein is a DNA molecule. In some embodiments, the polynucleotide described herein is an RNA molecule.

[0073] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising or consisting of a variant human TGFβRII extracellular region, the variant human TGFβRII extracellular region being at least 75% identical in length to SEQ ID NO: 1 (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising or consisting of a variant human TGFβRII extracellular region, the variant human TGFβRII extracellular region comprising the amino acid sequence shown in any of SEQ ID NO: 2-12. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising or consisting of a variant human TGFβRII extracellular region, the variant human TGFβRII extracellular region consisting of an amino acid sequence shown in any of SEQ ID NO: 2-12.

[0074] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising a variant human TGFβRII extracellular region and a linker sequence or thereof. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a variant human TGFβRII extracellular region that is at least 75% identical to SEQ ID NO: 1 (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in its full length), and the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 13 or 14. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising a variant human TGFβRII extracellular region and a linker sequence or thereof, wherein the variant human TGFβRII extracellular region comprises the amino acid sequence shown in any of SEQ ID NO: 2-12, and the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 13 or 14. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising a variant human TGFβRII extracellular region and a linker sequence or thereof, wherein the variant human TGFβRII extracellular region comprises the amino acid sequence shown in any of SEQ ID NO: 2-12, and the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 13 or 14. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in any of SEQ ID NO: 15-25.

[0075] In some embodiments, the polynucleotide comprises a first nucleotide sequence encoding a polypeptide comprising or consisting of a variant human TGFβRII extracellular region and a second nucleotide sequence encoding an antigen-binding region and a polypeptide comprising or consisting of an antigen-binding region. In some embodiments, the polynucleotide comprises a first nucleotide sequence encoding a polypeptide comprising or consisting of a variant human TGFβRII extracellular region and a second nucleotide sequence encoding an antibody-constant region and a polypeptide comprising or consisting of an antibody-constant region. In some embodiments, the first nucleotide sequence encodes a polypeptide comprising or consisting of a variant human TGFβRII extracellular region composed of an amino acid sequence that is at least 75% identical (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of the amino acid sequences in SEQ ID NO: 2-12. In some embodiments, the first nucleotide sequence encodes an amino acid sequence comprising any of SEQ ID NO: 2-12 or a variant thereof, the extracellular region of human TGFβRII.

[0076] In some embodiments, the first or second nucleotide sequence further encodes a linker. In some embodiments, the first nucleotide sequence encodes the variant human TGFβRII extracellular region and the linker, and the second nucleotide sequence encodes the antigen-binding region. In some embodiments, the first nucleotide sequence encodes the variant human TGFβRII extracellular region, and the second nucleotide sequence encodes the antigen-binding region and the linker. In some embodiments, the first nucleotide sequence encodes the variant human TGFβRII extracellular region and the linker, and the second nucleotide sequence encodes the antibody constant region. In some embodiments, the first nucleotide sequence encodes the variant human TGFβRII extracellular region, and the second nucleotide sequence encodes the antibody constant region and the linker. The linker can be any linker described herein, including peptide linkers or linkers comprising the amino acid sequence shown in SEQ ID NO: 13 or 14. In some embodiments, the linker consists of the amino acid sequence shown in SEQ ID NO: 13 or 14.

[0077] In some embodiments, the first nucleotide sequence encodes a polypeptide comprising the amino acid sequence shown in any of SEQ ID NO: 15-25, and the second nucleotide sequence encodes an antigen-binding region. In some embodiments, the first nucleotide sequence encodes a variant of the human TGFβRII extracellular region shown in any of SEQ ID NO: 2-12, and the second nucleotide sequence encodes an antigen-binding region and a linker. In some embodiments, the first nucleotide sequence encodes a polypeptide comprising the amino acid sequence shown in any of SEQ ID NO: 15-25, and the second nucleotide sequence encodes an antibody constant region. In some embodiments, the first nucleotide sequence encodes a variant of the human TGFβRII extracellular region shown in any of SEQ ID NO: 2-12, and the second nucleotide sequence encodes an antibody constant region and a linker.

[0078] This document also provides polynucleotides encoding polypeptide or antibody molecules as described above, which are optimized, for example, through codon / RNA optimization, substitution with heterologous signal sequences, and elimination of unstable elements in mRNA. Methods for generating optimized nucleic acids for recombinant expression by introducing codon changes and / or eliminating repressive regions in mRNA can be performed by adapting optimization methods described, for example, in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splicing sites and unstable elements within RNA (e.g., A / T- or A / U-rich elements) can be mutated to increase the stability of RNA for recombinant expression without altering the amino acids encoded by the nucleic acid sequence. Alterations can utilize the degeneracy of the genetic code, for example, by using alternative codons with the same amino acids. In one embodiment, it may be necessary to alter one or more codons to encode conserved mutations, for example, similar amino acids having similar chemical structures and properties and / or functions to the original amino acid.

[0079] Polynucleotides can be obtained by any method known in the art, and their nucleotide sequences can be determined. The nucleotide sequences encoding the polypeptides, antibody molecules, or portions thereof, as well as modifications thereof, described herein can be determined using methods well known in the art, i.e., assembling in such a manner as to produce nucleic acids encoding polypeptides, antibody molecules, or portions thereof. Such polynucleotides encoding proteins can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, which is incorporated herein by reference in its entirety). Briefly, this involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding polypeptides, antibody molecules, or portions thereof, annealing and linking those oligonucleotides, and then amplifying the linked oligonucleotides by PCR.

[0080] Alternatively, polynucleotides encoding the polypeptides, antibody molecules, or portions thereof described herein can be produced from nucleic acids from suitable sources using methods well known in the art, such as PCR and other molecular cloning methods. For example, PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the polypeptide or antibody molecule of interest. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding polypeptides, antibody molecules, or portions thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning.

[0081] If a clone of a nucleic acid encoding a specific polypeptide is unavailable, but the sequence of the polypeptide is known, the nucleic acid encoding the polypeptide can be chemically synthesized or amplified by PCR using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or cloned using oligonucleotide probes specific to the specific gene sequence to identify, for example, cDNA clones from a cDNA library encoding the polypeptide obtained from a suitable source (e.g., a cDNA library generated from any tissue or cell expressing the polypeptide described herein, or nucleic acid isolated from any tissue or cell expressing the polypeptide described herein, preferably polyA+RNA). The amplified nucleic acid produced by PCR can then be cloned into a reproducible cloning vector using any method well known in the art.

[0082] DNA encoding the polypeptides, antibody molecules, or portions thereof described herein can be readily isolated and sequenced using standard procedures. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells (such as Escherichia coli). E. coli Cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., from CHO GSSystem) ™In (Lonza) CHO cells or myeloma cells that do not otherwise produce the molecules described herein.

[0083] Also provided are polynucleotides that hybridize with polynucleotides encoding polypeptides, antibody molecules, or portions thereof described herein under high-strictness, medium-strictness, or low-strictness hybridization conditions.

[0084] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization of DNA bound to a filter membrane in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50°C–65°C; hybridization under highly stringent conditions may involve hybridization of nucleic acids bound to a filter membrane in 6x SSC at about 45°C, followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent conditions is known to those skilled in the art and has been described, see, for example, Ausubel FM et al. (eds.), (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Group and John Wiley & Son Publishing, New York, pp. 6.3.1–6.3.6 and 2.10.3, which is incorporated herein by reference in its entirety.

[0085] In one aspect, this document provides (e.g., recombinant) cells (e.g., host cells) for expressing the polypeptides or portions thereof described herein, along with associated polynucleotides and expression vectors. This document provides vectors (e.g., expression vectors) comprising polynucleotides containing nucleotide sequences encoding the polypeptides or portions thereof described herein for recombinant expression in host cells, preferably mammalian cells (e.g., CHO cells). This document also provides host cells comprising such vectors for recombinant expression of the polypeptides or portions thereof described herein. In one aspect, this document provides methods for generating the polypeptides, antibody molecules, or portions thereof described herein, including methods comprising expressing the polypeptides, antibody molecules, or portions thereof by host cells.

[0086] The recombinant expression of proteins described herein typically involves constructing expression vectors containing polynucleotides encoding polypeptide or antibody molecules. Once a polynucleotide encoding the polypeptide or antibody molecule described herein is obtained, a vector for producing the polypeptide or antibody molecule can be generated using recombinant DNA techniques well known in the art. Therefore, methods for preparing polypeptides, antibody molecules, or portions thereof by expressing polynucleotides containing nucleotide sequences encoding polypeptide or antibody molecules are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing polypeptide-coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Reproducible vectors comprising nucleotide sequences operatively linked to a promoter and encoding polypeptides, antibody molecules, or portions thereof described herein are also provided. Such vectors may, for example, comprise nucleotide sequences encoding the Fc region of a polypeptide (see, for example, International Publications WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464, the entire contents of which are incorporated herein by reference).

[0087] Expression vectors can be transferred to cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce the polypeptides, antibody molecules, or portions thereof described herein. Therefore, this document provides host cells containing polynucleotides encoding the polypeptides or antibody molecules, or portions thereof, described herein.

[0088] In some embodiments, the vector is a non-viral vector. Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episome plasmids, microcircles, microstrings, and oligonucleotides (e.g., mRNA, naked DNA). In some embodiments, the vector is a DNA plasmid vector.

[0089] In some implementations, the vector is a viral vector. The viral vector may be replicative or non-replicative. The viral vector may be integrated or non-integrated. Many virus-based systems have been developed for transferring genes into mammalian cells, and those skilled in the art can choose a suitable viral vector. Exemplary viral vectors include, but are not limited to, adenovirus vectors (e.g., adenovirus 5), adeno-associated virus (AAV) vectors (e.g., AAV2, 3, 5, 6, 8, 9), retroviral vectors (MMSV, MSCV), lentiviral vectors (e.g., HIV-1, HIV-2), gamma retroviral vectors, herpesvirus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flaviviruses (e.g., Kunzin virus, West Nile virus, dengue virus), rod-shaped virus vectors (e.g., rabies virus, VSV), measles virus vectors (e.g., MV-Edm), Newcastle disease virus vectors, poxvirus vectors (e.g., VV), measles virus, and piconemavirus vectors (e.g., Coxsackie virus).

[0090] In some implementations, the vector or expression cassette contains one or more additional elements. These additional elements include, but are not limited to, promoters, enhancers, polyadenylated (polyA) sequences, and selection genes.

[0091] In some embodiments, the vector comprises a polynucleotide sequence encoding an amino acid sequence that is at least 75% identical (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the amino acid sequence described in Table 1. In some embodiments, the vector comprises a polynucleotide sequence encoding the amino acid sequence described in Table 1.

[0092] Usage and Purpose On the other hand, this disclosure provides a method for treating a subject using the peptide or antibody molecules disclosed herein. The peptide or antibody molecules disclosed herein can be used to treat any disease or disorder in a subject that would benefit from reduced human TGFβ function and / or TGFβ-related signaling.

[0093] In another aspect, this disclosure provides a method for inhibiting the activity of human TGFβ, the method comprising contacting human TGFβ with a peptide or antibody molecule disclosed herein. In yet another aspect, this disclosure provides a method for inhibiting the activity of human TGFβ in a subject, the method comprising administering to the subject a peptide or antibody molecule disclosed herein.

[0094] The peptides or peptide molecules provided in this article can be used to treat diseases characterized by human TGFβ expression, particularly abnormal expression of human TGFβ compared to normal tissues of the same cell type (e.g., overexpression, or expression in a different pattern in cells). The peptides or antibody molecules provided in this article can be used to treat any tumor or cancer expressing human TGFβ. Specific malignancies that can be treated with the peptides or antibody molecules provided in this article include, for example, lung cancer, colon cancer, gastric cancer, breast cancer, head and neck cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer, and skeletal muscle cancer.

[0095] The peptides or antibody molecules disclosed herein can be used to inhibit tumor growth or kill tumor cells. For example, peptides or antibody molecules can bind to human TGFβ on the membrane or cell surface of cancer cells (tumor cells or tumor stromal cells) and trigger cancer cell killing mediated by, for example, ADCC or other effectors.

[0096] Alternatively, peptide or antibody molecules can be used to block the function of human TGFβ, particularly by physically interfering with its binding to another compound. In one aspect, a peptide or antibody molecule is provided for use as a medicament. In another aspect, a peptide or antibody molecule is provided for treating diseases characterized by the expression of human TGFβ. In some embodiments, a peptide or antibody molecule is provided for use in a treatment method. In some embodiments, the invention provides a peptide or antibody molecule in a method for treating a subject suffering from a disease characterized by the expression of human TGFβ, the method comprising administering an effective amount of the peptide or antibody molecule to the subject. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is an anticancer agent, such as a chemotherapy agent, a tumor cell proliferation inhibitor, or a tumor cell apoptosis activator.

[0097] On the other hand, the use of the peptide or antibody molecules disclosed herein in the manufacture or preparation of medicaments is provided. In one embodiment, the medicament is used to treat a disease characterized by the expression of human TGFβ. In another embodiment, a method of using the medicament to treat a disease characterized by the expression of human TGFβ includes administering an effective amount of the medicament to a subject suffering from a disease characterized by the expression of human TGFβ. In such an embodiment, the method further includes administering an effective amount of at least one additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is an anticancer agent, such as a chemotherapy agent, a tumor cell proliferation inhibitor, or a tumor cell apoptosis activator.

[0098] For the prevention or treatment of disease, the appropriate dose of the polypeptide or antibody molecule of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody molecule, the severity and course of the disease, whether the polypeptide or antibody molecule is administered for preventive or therapeutic purposes, prior therapy, the patient’s clinical history and response to the polypeptide or antibody molecule, and the judgment of the attending physician.

[0099] In one embodiment, the present invention relates to the polypeptide or antibody molecule of the present invention used in a method of the present invention, wherein the method further includes administering an additional therapeutic agent to a subject. In one embodiment, the present invention relates to (a) the polypeptide or antibody molecule of the present invention and (b) an additional therapeutic agent used as a medicament. In one embodiment, the present invention relates to (a) the polypeptide or antibody molecule of the present invention and (b) an additional therapeutic agent used in a method of treating cancer. In another embodiment, the present invention relates to a pharmaceutical composition, kit, or kit comprising (a) the polypeptide or antibody molecule of the present invention and (b) an additional therapeutic agent. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapy agent, or a checkpoint targeting agent.

[0100] The polypeptide or antibody molecules described herein can be delivered to subjects via a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, local, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intraarterial, and subcutaneous routes. Lung administration can also be employed, for example, by using an inhaler or nebulizer and formulations containing a nebulizing agent for use as a nebulizer. In some embodiments, the polypeptide or antibody molecules described herein are delivered subcutaneously or intravenously. In some embodiments, the polypeptide or antibody molecules described herein are delivered intraarterially. In some embodiments, the polypeptide or antibody molecules described herein are delivered intratumorally. In some embodiments, the polypeptide or antibody molecules described herein are delivered to tumor-draining lymph nodes.

[0101] The amount of peptide or antibody molecules that will be effective in treating and / or preventing symptoms will depend on the nature of the disease and can be determined using standard clinical techniques.

[0102] The precise dosage employed will also depend on the route of administration and the severity of the infection or disease it causes, and should be determined based on the physician's judgment and the individual subject's circumstances. For example, the effective dosage can also vary depending on the method of administration, target site, patient's physiological state (including age, weight, and health status), whether the patient is human or animal, and whether other medications or treatments administered are prophylactic or therapeutic. Typically, the patient is human, but treatment can also be given to non-human mammals, including genetically modified mammals. Optimally titrating the therapeutic dose optimizes safety and efficacy.

[0103] The peptides or antibody molecules described herein can also be used to determine human TGFβ protein levels in biological samples using classical immunohistochemical methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay markers are known in the art and include enzyme markers such as glucose oxidase; radioactive isotopes such as iodine (… 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, as well as biotin. Such labels can be used to label the peptide or antibody molecules described herein. Alternatively, a second antibody that recognizes the peptide or antibody molecule described herein can be labeled and used in combination with the peptide or antibody molecule to detect human TGFβ protein levels. Thus, in one embodiment, the present invention relates to the use of the peptide or antibody molecule of the present invention for in vitro detection of human TGFβ protein in biological samples. In another embodiment, the present invention relates to the use of the peptide or antibody molecule of the present invention for in vitro determination and / or detection of human TGFβ protein levels in biological samples, optionally wherein the peptide or antibody molecule is conjugated with a radionuclide or a detectable label, and / or carries a label described herein, and / or wherein an immunohistochemical method is used.

[0104] Determining the expression level of human TGFβ protein aims to include qualitatively or quantitatively measuring or estimating the human TGFβ level in a first biological sample, either directly (e.g., by determining or estimating an absolute protein level) or relatively (e.g., by comparing it with disease-related protein levels in a second biological sample). The human TGFβ expression level in the first biological sample can be measured or estimated and compared with a standard human TGFβ level, which can be determined, for example, from a second biological sample obtained from individuals who have never had the disorder, or by averaging the levels of a population of individuals who have never had the disorder. As understood in the art, once a “standard” human TGFβ level is known, that level can be repeatedly used as a standard for comparison. Therefore, in another embodiment, the present invention relates to an in vitro method for determining and / or detecting the human TGFβ protein level in a biological sample, the method comprising qualitatively or quantitatively measuring or estimating the human TGFβ protein level in the biological sample by immunohistochemical methods.

[0105] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cellular source that potentially expresses human TGFβ. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral blood mononuclear cells.

[0106] In one embodiment, the peptide or antibody molecule can be used in the immunohistochemistry of a biopsy sample. In one embodiment, the method is an in vitro method. In another embodiment, the peptide or antibody molecule can be used to detect levels of human TGFβ, or levels of cells containing human TGFβ on their membrane surface, which can then be associated with certain disease symptoms. The peptide or antibody molecules described herein may carry a detectable or functional label and / or may be conjugated to a radionuclide or a detectable label. When fluorescent labeling is used, the specifically binding member can be identified and quantified using currently available microscopy and fluorescence activated cell sorter analysis (FACS) or a combination of two methods known in the art. The peptide or antibody molecules described herein may carry a fluorescent label or may be conjugated to it. Exemplary fluorescent labels include, for example, reactive probes and conjugated probes, such as aminocoumarins, fluorescein and Texas Red, Alexa Fluor dyes, Cy dyes and DyLight dyes. Anti-FAP (e.g., human, mouse or cynomolgus monkey FAP) antibodies may carry a radiolabel or a radionuclide (such as an isotope). 3 H, 14 C 32 P, 35 S, 36 C1, 51 Cr 57 Co、 58 Co、 59 Fe、 67 Cu、 90 Y、 99 Tc, 111 In、 117 Lu、 121 I, 124 I, 125 I, 131 I, 198 Au、 211 At、 213 Bi、 225 Ac and 186(Re) or may be conjugated thereto. When using radiolabeling, the specific binding of peptide or antibody molecules to human TGFβ can be identified and quantified using counting procedures known in the art. In the case that the label is an enzyme, detection can be performed by any of the colorimetric, spectrophotometric, fluorescence spectrophotometric, galvanic, or gas analysis techniques known in the art. This can be achieved by contacting a sample or control sample with the peptide or antibody molecule under conditions that allow the formation of a complex between the peptide or antibody molecule and human TGFβ. Any complexes formed between the peptide or antibody molecule and human TGFβ are detected and compared in the sample and control. This document also includes an assay system that can be prepared in the form of a test kit, reagent kit, or kit kit for the quantitative analysis of, for example, the presence of human TGFβ or human TGFβRII / human TGFβ complexes. The system, test kit, reagent kit, or kit kit may contain labeled components (e.g., labeled antibodies) and one or more additional immunochemical reagents.

[0107] Reagent test kit Any composition described herein may be included in a kit. In a non-limiting example, the kit contains one or more of the peptide or antibody molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the peptide or antibody molecule, or a host cell expressing the peptide or antibody molecule.

[0108] The kit may also contain reagents or instructions for use in subjects with the peptide or antibody molecule described herein, a nucleic acid molecule encoding the peptide or antibody molecule (e.g., an expression vector), or host cells expressing the peptide or antibody molecule. The kit may also contain one or more buffers.

[0109] The kit components may be packaged in an aqueous medium or in lyophilized form. The kit's container devices will typically include at least one vial, test tube, flask, bottle, syringe, or other container device into which the components can be placed, and preferably appropriately aliquoted. Where more than one component is present in the kit (labeling reagents and labels may be packaged together), the kit will typically also contain a second, third, or additional container into which additional components can be placed individually. The kit may also include a second container device for containing sterile, pharmaceutically acceptable buffers and / or other diluents. However, various combinations of components may be contained in vials. The kits disclosed herein typically also include devices for containing the polypeptide or antibody molecules described herein, nucleic acid molecules encoding polypeptide or antibody molecules (e.g., expression vectors), or host cells expressing polypeptide or antibody molecules, as well as any other reagent containers with closed-loop constraints for commercial sale.

[0110] When the kit components are provided as one or more liquid solutions, the liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly preferred. However, the kit components may also be provided in dry powder form. When reagents and / or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is envisioned that the solvent may also be provided in a separate container.

[0111] Example The embodiments disclosed herein are provided by way of illustration and explanation and are not intended to limit the scope of this disclosure.

[0112] Example 1: Excision of the extracellular region of variant human TGFβRII This example describes the characterization of the cleavage (i.e., proteolytic cleavage) of the extracellular region of the variant human TGFβRII when fused to the C-terminus of the anti-FAP antibody heavy chain (an anti-FAP-TGFβRII bispecific variant). Two different anti-FAP antibodies, named AF1 and AF2, were used.

[0113] Residues 24-159 of the human TGFβRII sequence were fused to the C-terminus of the human IgG1 (iso3) heavy chain with the terminal lysine removed via a 4x(G4S)G linker sequence (SEQ ID NO: 14). The extracellular region of human TGFβRII was engineered using a combination of sequence deletions and point mutations to reduce cleavage in the extracellular region during expression. Three potential cleavage sites were identified using internal mass spectrometry data and computer prediction of the relevant molecules. Two truncations were designed based on the cleavage site (site 1) in the unstructured region of the N-terminus of TGFβRII. Two point mutations (V28T and V32P, based on position numbers in WT TGFβRII) were designed to reduce the recognition sequence of the predicted cleavage site in the unstructured region of TGFβRII. Three point mutations (V56I, F58A, and S59A) were designed to reduce the recognition sequence in the structured region (site 3) of TGFβRII. Eleven variants of the human TGFβRII extracellular region were designed using two truncations and five point mutations. The amino acid sequences of the tested variants are shown in Table 1.

[0114] When in 10ml When expressed at a small scale in fed-batch cultures TGFβRII Cut CHO cells were engineered to express each anti-FAP-TGFβRII bispecific variant as a stable cell line. Each sequence was optimized for CHO expression, then cloned into a vector, linearized, and transfected into the cell lines. Selection pressure was used to generate CHO cell pools that stably expressed variants. Once generated, the cell lines were used for library construction. Vials of each variant were thawed and fed in 10 ml batches under standard culture conditions. After 10 days, the cultures were harvested, cells were removed by centrifugation and filtration, and the supernatant was purified. The supernatant was purified by drop-column protein A affinity chromatography, and the concentration was determined by using absorbance and extinction coefficient at 280 nm.

[0115] The antibodies were developed using the PerkinElmer LabChip GX II Touch HT and Protein Express Assay Kit to disrupt all non-covalent interactions and any disulfide bonds, and then molecules were separated based on size using a voltage applied to the sieving matrix.

[0116] Each antibody was diluted to 0.1 mg / ml in Protein Express Sample Buffer containing 40 mM DTT and then heated at 80 °C for 5 min. Each sample was aspirated into the instrument's microfluidic stream via the LabChip, mixed with Protein Express Dye solution, and then a few pL were pulled into the separation channel by an applied voltage. The voltage was used to separate molecules based on size as they were pulled through the sieving matrix. Once separated, the sample was destaining with a destaining solution, and the separated molecules were detected by the fluorescence of the dye as they passed through a laser. Size was determined using the molecular weight standard (ladder) provided with the Protein Express Assay Kit. Data quality was validated using parameters determined by the LabChip used for successful assay runs and by visual inspection. A light chain peak at 28 kDa was identified, as well as a full-length heavy chain that migrated as two broad peaks at 90 kDa–120 kDa due to N-linked glycosylation. The peak between 60 kDa and 90 kDa was identified as the full-length heavy chain that had been cleaved within the extracellular region of TGFβRII.

[0117] As shown in Table 2, the amount of cleaved human TGFβRII extracellular region varied among variants. The area under the curve of heavy chain material was determined using LabChip GXReviewer software, and the cleaved TGFβRII% was calculated as a percentage of the total heavy chain.

[0118] Table 2: cleavage in bispecific variants of anti-FAP-TGFβRII .

[0119]

[0120] When in 7L Expression in a bioreactor TGFβRII Cut The stabilized cell lines described above were thawed, expanded, and used to inoculate cell cultures in a 7L bioreactor under standard culture conditions. After 14 days, the cultures were harvested, and cells and large debris were removed by centrifugation and filtration. The supernatant was purified. Protein A affinity drop columns were loaded with a product density of 25 mg / ml resin (therefore the total volume used for purification depended on the specific titer), and concentrations were determined by using absorbance and extinction coefficient at 280 nm. Briefly, antibodies were unfolded using a Beckman Coulter PA800 plus and a capillary cassette equipped with exposed fused silica capillaries (50 µm × 30.5 ± 0.3 cm) to disrupt all non-covalent interactions and any disulfide bonds, and molecules were then separated based on size using a voltage applied to the sieving gel matrix.

[0121] Each bispecific antibody was diluted to 1 mg / mL in a sample buffer containing sodium dodecyl sulfate (SDS) with 2-mercaptoethanol and a 10 kDa internal standard, and then heated at 60 °C for 10 min. Each sample was injected into a capillary filled with a hydrophilic polymer gel. A voltage was applied based on size to separate molecules as they were pulled along the length of the capillary through the gel sieving matrix. Once separated, the sample passed through a detector window in the capillary, and the separated molecules were detected by ultraviolet (UV) absorbance. Data quality was validated using predefined system suitability requirements. A light chain peak at 28 kDa was identified, as well as a full-length heavy chain that migrated as a broad peak at 90-120 kDa due to N-linked glycosylation. The peak between 60 kDa and 90 kDa was identified as a heavy chain that had been cleaved in TGFβRII.

[0122] As shown in Table 3, the amount of cleaved TGFβRII varied among the anti-FAP-TGFβRII bispecific variants. The area under the curve for the heavy chain was determined using LabChip GX Reviewer software, and the percentage of cleaved TGFβRII was calculated as a proportion of the total heavy chain.

[0123] Table 3: cleavage in bispecific variants of anti-FAP-TGFβRII .

[0124]

[0125] Example 2: Functional resistance to FAP-TGFβRII bispecific variants In this embodiment, the ability of the anti-FAP-TGFβRII bispecific variant to inhibit TGFβ-induced signaling was tested by measuring luciferase activity in HEK293 SMAD reporter cells (BPS Bioscience / 60653). These cells stably express the firefly luciferase reporter gene under the control of SMAD responsive elements.

[0126] HEK293 SMAD luciferase cells were expanded using minimum essential medium (MEM) supplemented with 10% heat-inactivated FBS, sodium pyruvate (1X), 400 µg / mL genistein, and 1% penicillin-streptomycin, containing Earle's balanced salt solution (EBSS) and L-glutamine. During the logarithmic growth phase, cells were trypsinized, counted, and seeded into white 96-well flat-bottomed assay plates at 4 × 10⁻⁶ cells / well. 5 Cells were incubated at a final concentration of 100 cells / mL in a humidified chamber at 37°C with 5% CO2 overnight. The medium was then replaced with MEM containing EBSS and L-glutamine, supplemented with 0.5% heat-inactivated FBS, sodium pyruvate (1X), 400 µg / mL genistein, and 1% penicillin-streptomycin by gently discarding the supernatant. Cells were then incubated in a humidified chamber for 4.5 hours. After incubation, the medium was gently removed and replaced with 50 µL of a dose-titrated (20 nM - 0.003 nM) anti-FAP-TGFβRII bispecific variant and 50 µL of TGFβ1 (2 ng / mL), TGFβ2 (22 ng / mL), or TGFβ3 (1.8 ng / mL) prepared in the indicated medium. To measure luciferase activity, 100 mL of a Bio-Glo luciferase assay system (Promega / G7940) was added to each well. The emission intensity (RLU) was measured using a Tecan Infinite M1000-Pro plate reader. The RLU values ​​were plotted against the concentration of the anti-FAP-TGFβRII bispecific variant using GraphPad Prism software, and the 50% inhibition concentration (IC50) was calculated. 50 )value.

[0127] like Figure 1A As shown, all anti-FAP-TGFβRII bispecific variants effectively neutralized TGFβ1, thereby eliminating SMAD signaling in HEK cells. The IC50 values ​​used to calculate TGFβ1 neutralization were... 50 The values ​​are shown in Table 4.

[0128] Table 4: Calculated IC50 of dose-dependent inhibition of TGFβ1 by the bispecific variant of anti-FAP-TGFβRII 50 value .

[0129]

[0130] The neutralization of TGFβ2 and TGFβ3 by the anti-FAP-TGFβRII bispecific variant was also tested. Although dose-dependent neutralization of TGFβ3 activity was observed ( Figure 1B This is consistent with its low binding affinity for TGFβ2 and the lack of a TGFβRIII co-receptor in the described experimental system (Lopez-Casillas et al., 1993), but does not demonstrate inhibition of TGFβ2-induced signaling (not shown). The calculated IC50 for neutralizing TGFβ3... 50 The values ​​are shown in Table 5.

[0131] Table 5: Calculated IC50 of dose-dependent inhibition of TGFβ3 by the bispecific variant of anti-FAP-TGFβRII 50 value .

[0132]

[0133] Example 3: Neutralization of TGFβ-mediated cancer cell invasion by a bispecific variant of anti-FAP-TGFβRII TGFβ protein can enhance cancer cell invasion / migration, which can promote tumorigenesis and metastasis. To evaluate the ability of the anti-FAP-TGFβRII bispecific variant to inhibit invasive potential by neutralizing TGFβ, a wound healing assay was used as an alternative to migration assay. This assay tested the ability of TGFβ to accelerate migration and the ability of the “Trap” antibody to delay migration.

[0134] Oris Pro 96-well plates containing non-toxic biocompatible gel (BCG) were prepared using 2.5 × 10⁻⁶ microplates. 5 4T1 breast cancer cells or 4T1 cells overexpressing human FAP were seeded at 100 cells / mL and plated using CellTracker. ™ Red (Invitrogen) labeling was performed, and the culture was allowed to adhere as a monolayer at 37°C for 2 hours. Subsequently, "wounds" were formed by BCG dissolution to create a centrally located, temporary cell-free zone on the culture surface. The culture medium was removed and replaced with TGFβ1 or TGFβ3 and test / control antibodies. The plates were then imaged on an ImageXpress confocal microscope (Molecular Devices) to detect CellTrackers in the "wound" areas over 7 days. ™ Invasion of Red-positive cells. In the absence of TGFβ inhibition (control group), the wound closed rapidly, resulting in a low wound area. In the presence of TGFβ inhibition, wound closure was delayed, leading to a higher wound area.

[0135] Table 6 shows the percentage of open wound area on day 4 of treatment. Blocking TGFβ with the anti-FAP-TGFβRII bispecific variant significantly increased wound closure, from 20%–50% to approximately 70%–90%, indicating inhibition of the invasive potential of 4T1 cancer cells due to TGFβ neutralization. V02 and V06 variants showed slightly reduced responses compared to WT TGFβRII or V03 and V07 variants, consistent with reduced potency in HEK-SMAD signal transduction assays (Tables 4 and 5).

[0136] Table 6: Percentage of open wound area on day 4 after treatment with TGFβ and anti-FAP-TGFβRII bispecific variant .

[0137]

[0138] Example 4: Neutralization of TGFβ-mediated mesenchymal antigen expression by a bispecific variant of anti-FAP-TGFβRII To determine the effect of the anti-FAP-TGFβRII bispecific variant on epithelial-mesenchymal transition (EMT) in tumor cell lines, the coding sequence for red fluorescent protein (RFP) was inserted at the 3' end of the endogenous open reading frame (ORF) of the vimentin gene in the lung cancer cell line A549 (ATCC CCL-185 EMT). The A549-vimentin-RFP cell reporter gene line allows for the detection of vimentin (a biomarker of EMT) by measuring RFP fluorescence.

[0139] In summary, seven-point dose dilutions of the anti-FAP-TGFβRII bispecific variant or isotype control, ranging from 100 nM to 0.14 nM, were prepared using F12-K medium incorporating TGFβ1 (25 ng / mL). The mixture of TGFβ1 and the anti-FAP-TGFβRII bispecific variant was pre-incubated at room temperature for 20 min. A549-Vimentin-RFP cells were grown at 37 °C and 5% CO2 in F12-K medium supplemented with 10% FBS and seeded at a density of 20,000 cells / well in 96-well flat-bottom cell culture plates. The mixture of TGFβ1 and the anti-FAP-TGFβRII bispecific variant was then added to the cells and incubated for 48 h. Cells were washed with FACS buffer (PBS, 2 mM EDTA, 5% BSA, pH 7.2) and analyzed using an LSR Tortessa (BD Biosciences). The flow data was analyzed using FlowJo software and plotted using GraphPadPrism software.

[0140] like Figure 2As shown, the anti-FAP-TGFβRII bispecific variants, rather than the isotype control (without TGFβRII), suppressed TGFβ1-induced vimentin expression. Variances V03 and V07 were comparable to WT TGFβRII in neutralizing TGFβ1, while variants V02 and V06 were worse than WT in reducing vimentin expression.

[0141] Example 5: Binding kinetics of the bispecific variant of FAP-TGFβRII This example describes the characterization of the anti-FAP-TGFβRII bispecific variant that binds to human FAP and human TGFβ1 proteins.

[0142] Use Biacore ™ SPR experiments were conducted using a T200 instrument. Biacore was used. ™ The 200 evaluation software calculates the association rate (ka), dissociation rate (KD), and dissociation constant (KD) for each experiment.

[0143] The anti-FAP-TGFβRII bispecific variant was captured onto an S-series CM5 sensor chip (BR100530, Cytiva) immobilized with an anti-human IgG1 (Fc) capture antibody (29234600, Cytiva). To test for human FAP-His and hTGFβ1-His binding, approximately 6 µg / mL of the anti-FAP-TGFβRII bispecific variant, diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20), was captured in a single flow cell of the CM5 chip immobilized with the anti-human Fc antibody, with the single flow cell maintained as a reference. The anti-FAP-TGFβRII bispecific variant was captured using a 30-second injection at a flow rate of 10 µL / min to achieve the appropriate response units (RU) (approximately 100 RU of capture for human FAP binding). Human FAP (diluted to concentrations of 300 nM, 100 nM, 33.3 nM, 11.1 nM, and 0 nM in running buffer) and human TGFβ1 (diluted to concentrations of 3 nM, 1 nM, 0.33 nM, 0.11 nM, and 0 nM in running buffer) were flowed over the chip surface at a flow rate of 30 µL / min, with an association phase of 3 min and a dissociation phase of 5 min. The sensor chip was regenerated between cycles of 30 µL / min injection of 3.0 M MgCl2 for 30 seconds, followed by a 60-second settling period. The sensor map was evaluated using Biacore Evaluation 3.1 software and fitted to a simple Langmuir 1:1 interaction model. Bias and curve fitting were visually examined and R-squared was evaluated. max , Χ 2 The parameters Tc are used to verify data quality.

[0144] The binding kinetics of the anti-FAP-TGFβRII bispecific variant with FAP and TGFβ1 proteins are shown in Table 7.

[0145] Table 7: Binding of anti-FAP-TGFβRII bispecific variants to ECD-His-tagged human FAP and human TGFβ1 proteins kinetic parameters .

[0146]

[0147] NBD = No binding detected.

[0148] anti- FAP-TGFβRII Binding kinetics of bispecific variants This embodiment describes the binding kinetics and simultaneous binding of human FAP and human TGFβ1 to the bispecific variant AF1-TGFβRII (V03).

[0149] Use Biacore ™ SPR experiments were conducted using a T200 instrument. Association rates (kJ / kJ) were calculated from each experiment using a divalent analyte model and BiacoreEvaluation 3.0 software. a ), dissociation rate (k d ) and dissociation constant ( K d ).

[0150] To test binding kinetics, the anti-FAP-TGFβRII bispecific variant was captured using the S-series sensor chip CM7 (28953828, Cytiva) immobilized with an anti-human IgG1 (Fc) capture antibody (29234600, Cytiva). For human FAP, cynomolgus monkey FAP, and mouse FAP, approximately 2 µg / mL of AF1-TGFβRII (V03) diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) and an isotype control were captured in a single flow cell of the CM7 chip immobilized with the anti-human Fc antibody, with the single flow cell maintained as a reference. The capture antibody was injected for 30 seconds at a flow rate of 10 µL / min to achieve an appropriate response unit (RU) (approximately 60 RU of capture for human FAP binding). Human FAP, cynomolgus monkey FAP, and mouse FAP (each diluted in run buffer to concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.56 nM, 0.78 nM, 0.39 nM, 0.195 nM, 0.098 nM, and 0 nM, respectively) were flowed through the chip surface at a flow rate of 30 µL / min for a 3-minute association phase and a 20-minute dissociation phase. For human TGFβ1, human TGFβ2, and human TGFβ3 proteins, approximately 6 µg / mL of AF1-TGFβRII (V03) diluted in run buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) and an isotype control were captured in a single flow cell of a CM7 chip immobilized with anti-human Fc antibody, with each single flow cell serving as a reference. The antibody was captured using a 30-second injection at a flow rate of 10 µL / min to achieve an appropriate response unit (RU) (approximately 120 RU capture for human TGFβ binding). Human TGFβ1 or human TGFβ3 (each diluted in running buffer to concentrations of 8 nM, 4 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM, 0.125 nM, 0.0625 nM, 0.0312 nM, and 0 nM) or human TGFβ2 (diluted in running buffer to concentrations of 32 nM, 16 nM, 8 nM, 4 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM, 0.125 nM, 0.0625 nM, 0.0312 nM, and 0 nM) was flowed over the chip surface at a flow rate of 30 µL / min for an association phase of 3 minutes and a dissociation phase of 20 minutes. The sensor chip was regenerated by cycling through two 30-second injections of 3.0 M MgCl2 at a rate of 30 µL / min, followed by a 60-second stabilization period. (Using Biacore) ™Evaluation 3.1 uses the software's global data analysis options to evaluate the sensor map and fit it to the bivalent analyte model. Bias and curve fitting are visually checked, and data quality is verified by evaluating the parameters Rmax, χ², and Tc.

[0151] The combined dynamics are shown in Table 8.

[0152] Table 8: Bispecific variant of anti-FAP-TGFβRII AF1-DLE-V03 and ECD-His-tagged FAP and TGFβ proteins White binding dynamics .

[0153]

[0154] In another experiment, the simultaneous binding of AF1-TGFβRII(V03) to human FAP and human TGFβ1 was tested. In short, using Biacore... ™ SPR experiments were performed using an 8K+ instrument. The test antibody was captured using an S-series sensor chip CM3 (BR-1005-36, Cytiva) immobilized with anti-human IgG1 (Fc) capture antibody (29234600, Cytiva).

[0155] To evaluate simultaneous binding to human FAP and human TGFβ1, approximately 0.8 µg / mL of the anti-FAP-TGFβRII bispecific variant AF1-TGFβRII (V03), diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20), and an isotype control were captured in a single flow cell of a CM3 chip immobilized with anti-human Fc antibody, with the single flow cell maintained as a reference. These antibodies were captured using 60-second injections at a flow rate of 10 µL / min to achieve approximately 150 RU. Human FAP and human TGFβ1 proteins were injected individually or in various combinations using a double-injection method, with running buffer as a control (Table 9). Each double-injection was performed at a flow rate of 30 µL / min for 180 seconds, followed by a 300-second dissociation phase.

[0156] Table 9: Injection Combinations .

[0157]

[0158] The sensor chip was regenerated between cycles of 45-second injection of 10 mM glycine at pH 1.5 at a rate of 30 µL / min, followed by a 60-second settling period. A Biacore sensor was used for the kinetic assay. ™Data analysis was performed using Insight assessment software version 4.0.8.20368 (GE Healthcare Life Sciences). The software's visualization tools were used to manually evaluate sensor maps to analyze the data and report relevant RU (Reference Unit). Figure 3 ).

[0159]

[0160] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

[0161] All references cited in this article (e.g., publications, patents, or patent applications) are incorporated herein by reference in their entirety, and for all purposes, to the extent that each individual reference (e.g., publications, patents, or patent applications) is specifically and individually indicated to be incorporated herein by reference in its entirety for all purposes.

[0162] Other implementations are within the scope of the following claims.

Claims

1. A polypeptide comprising a variant human TGFβRII extracellular region, wherein the amino acid sequence of the variant human TGFβRII extracellular region is: (a) The amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1 is missing; (b) The amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1 is missing; (c) Contains threonine and proline at the positions of amino acids 5 and 9 of SEQ ID NO: 1, respectively; (d) Contains isoleucine and alanine at the positions corresponding to amino acids 33 and 35 of SEQ ID NO: 1, respectively; (e) Contains isoleucine, alanine and alanine at the positions corresponding to amino acids 33, 35 and 36 of SEQ ID NO: 1, respectively; (f) Contains threonine, proline, isoleucine and alanine at the positions corresponding to amino acids 5, 9, 33 and 35 of SEQ ID NO: 1, respectively; (g) Contains threonine, proline, isoleucine, alanine and alanine at the positions corresponding to amino acids 5, 9, 33, 35 and 36 of SEQ ID NO: 1, respectively; (h) contains isoleucine and alanine at the positions corresponding to amino acids 33 and 35 of SEQ ID NO: 1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-15 of SEQ ID NO: 1; (i) Contains isoleucine and alanine at the positions corresponding to amino acids 33 and 35 of SEQ ID NO: 1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-22 of SEQ ID NO: 1; (j) Contains isoleucine, alanine, and alanine at positions corresponding to amino acids 33, 35, and 36 of SEQ ID NO: 1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-15 of SEQ ID NO: 1; or (k) contains isoleucine, alanine and alanine at the positions corresponding to amino acids 33, 35 and 36 of SEQ ID NO: 1, respectively, and lacks the amino acid sequences corresponding to amino acids 1-22 of SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein the extracellular region of the variant human TGFβRII is at least 75% identical to SEQ ID NO: 1 along its full length (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical).

3. The polypeptide according to any one of the preceding claims, wherein the amino acid sequence of the extracellular region of the variant human TGFβRII consists of the amino acid sequence shown in any one of SEQ ID NO: 2-12.

4. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises an antigen-binding region.

5. The polypeptide according to any one of the preceding claims, wherein the antigen-binding region comprises scFv, VH, VL, VHH, an antibody heavy chain, or an antibody light chain.

6. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises an antibody constant region.

7. The polypeptide according to any one of the preceding claims, wherein the extracellular region of the variant human TGFβRII is linked to the antigen-binding region or the antibody constant region via a linker sequence.

8. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises, from the N-terminus to the C-terminus: an antibody heavy chain or its Fc fragment; and the extracellular region of the variant human TGFβRII.

9. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises, from the N-terminus to the C-terminus: an antibody heavy chain or its Fc fragment; a linker sequence; and the extracellular region of the variant human TGFβRII.

10. The polypeptide according to any one of the preceding claims, wherein the adapter sequence comprises the amino acid sequence shown in SEQ ID NO:

13.

11. The polypeptide according to any one of the preceding claims, wherein the adapter sequence comprises the amino acid sequence shown in SEQ ID NO:

14.

12. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 15-25.

13. An antibody molecule comprising two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from the N-terminus to the C-terminus: a variable region; a constant region; an adapter sequence; and a variant human TGFβRII extracellular region consisting of an amino acid sequence represented by any one of SEQ ID NO: 2-12.

14. The antibody molecule of claim 13, wherein the adapter sequence comprises the amino acid sequence shown in SEQ ID NO:

13.

15. The antibody molecule according to claim 13 or 14, wherein the adapter sequence comprises the amino acid sequence shown in SEQ ID NO:

14.

16. The antibody molecule according to any one of claims 13 to 15, wherein the antibody molecule comprises the amino acid sequence shown in any one of SEQ ID NO: 15-25.

17. The antibody molecule according to any one of claims 13 to 16, wherein the antibody molecule is a full-length heterotetrameric antibody molecule comprising two heavy chain polypeptides and two light chain polypeptides.

18. The antibody molecule according to any one of claims 13 to 16, wherein the antibody molecule is a full-length heavy chain antibody molecule comprising two heavy chain polypeptides and not comprising light chain polypeptides.

19. The antibody molecule according to any one of claims 13 to 18, wherein the constant region is the human heavy chain constant region.

20. The antibody molecule of claim 19, wherein the human heavy chain constant region is an isotype of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE or IgM.

21. A polynucleotide encoding a polypeptide or antibody molecule according to any one of claims 1 to 20.

22. A vector comprising the polynucleotide according to claim 21.

23. An engineered cell comprising the polynucleotide or vector according to claim 21 or 22.

24. A method for producing a polypeptide, the method comprising culturing engineered cells according to claim 23 under conditions that cause the expression of a polynucleotide and the production of the polypeptide.

25. A method for inhibiting the activity of human TGFβ, the method comprising contacting the human TGFβ with a polypeptide or antibody molecule according to any one of claims 1 to 20.

26. A method for inhibiting the activity of human TGFβ in a subject, the method comprising administering to the subject a polypeptide or antibody molecule according to any one of claims 1 to 20.

Images