WO2022153997A1 - ＡｃｔＲＩＩＡ、ＡｃｔＲＩＩＢ及びＦｎ１４に結合する多重特異性抗体 - Google Patents

ＡｃｔＲＩＩＡ、ＡｃｔＲＩＩＢ及びＦｎ１４に結合する多重特異性抗体 Download PDF

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WO2022153997A1
WO2022153997A1 PCT/JP2022/000661 JP2022000661W WO2022153997A1 WO 2022153997 A1 WO2022153997 A1 WO 2022153997A1 JP 2022000661 W JP2022000661 W JP 2022000661W WO 2022153997 A1 WO2022153997 A1 WO 2022153997A1
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amino acid
seq
multispecific antibody
polynucleotide
polypeptide
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French (fr)
Japanese (ja)
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真司 曽我
健詞 重永
剛 堤
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Astellas Pharma Inc
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Astellas Pharma Inc
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Priority to US18/272,020 priority Critical patent/US20240092916A1/en
Priority to CN202280009978.7A priority patent/CN116802298A/zh
Priority to EP22739403.8A priority patent/EP4279594A4/en
Priority to JP2022575595A priority patent/JPWO2022153997A1/ja
Priority to CA3207850A priority patent/CA3207850A1/en
Priority to MX2023008372A priority patent/MX2023008372A/es
Priority to AU2022207252A priority patent/AU2022207252A1/en
Priority to KR1020237023856A priority patent/KR20230131207A/ko
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Definitions

  • the present invention relates to multispecific antibodies that bind to ActRIIA, ActRIIB and Fn14.
  • Activin receptor type 2A (ActRIIA) and activin receptor type 2B (ActRIIB) are known as receptors for myostatin and activin A.
  • Smad2 and Smad3 are phosphorylated by ligand stimulation to ActRIIA and ActRIIB.
  • Phosphorylated Smad2 / 3 is known to form a complex with Smad4 and bind to various transcription factors to regulate gene expression (Tsuchida K et al., Endocr J. 2008, Vol. 55, p. 11-21).
  • ActRIIA and ActRIIB are known to be involved in proteolysis (Morvan F et al., Proc Natl Acad Sci USA. 2017, vol. 114, p. 12448-12453).
  • Fibroblast growth factor inducible 14 (also referred to as TNFRSF12A) is a member of the tumor necrosis factor receptor superfamily. Fn14 is also known as a receptor for a TNF-like weak apoptosis-inducing factor (TWEAK: TNF-like weak inducer of apoptosis). Fn14 has TWEAK-dependent activation evoked by TWEAK binding and TWEAK-independent activation evoked in the absence of TWEAK.
  • TWEAK TNF-like weak inducer of apoptosis
  • Non-Patent Document 2 Tumor necrosis factor (TNF) ⁇ is involved in the degradation of skeletal muscle proteins via TNF receptors.
  • TNF Tumor necrosis factor
  • Glucocorticoids are also involved in skeletal muscle proteolysis via glucocorticoid receptors.
  • suppression of insulin-like growth factor involved in skeletal muscle anabolic causes skeletal muscle atrophy.
  • IBM Inclusion body myositis
  • sIBM is a chronically progressive muscle disease. Muscle atrophy and weakness are observed mainly in the quadriceps, fingers and wrist flexors. Characteristic findings include the expression of muscle fibers with bordered vacuoles, mononuclear cell invasion and siege into non-necrotic fibers and intramuscular sheath. As it progresses, deterioration of walking function and dysphagia are observed, and the quality of life (QOL: Quality of life) is significantly reduced.
  • QOL Quality of life
  • Bimagrumab which is an ActRIIA and ActRIIB inhibitory antibody, is a drug that has been developed to the highest order, and a phase 2B / phase 3 study was conducted on sIBM. Although an increase in lean body mass was observed in the Phase 2B / Phase 3 study of Bimagramab, no improvement in the 6-minute walking distance, which is the primary endpoint, was observed (Non-Patent Document 4).
  • Non-Patent Document 5 Increased phosphorylation of Smad2 was observed in the skeletal muscle of sIBM patients, suggesting the involvement of ActRIIA and ActRIIB stimulation in muscular atrophy.
  • Non-Patent Document 6 it has been reported that the expression of Fn14 is increased in the skeletal muscle of sIBM patients.
  • no therapeutic agent for sIBM targeting the inhibition of both ActRIIA and ActRIIB and Fn14 pathways has been reported so far.
  • the subject of the present invention is a plurality of cells expected to prevent or treat amyotrophic diseases such as inclusion body myositis by binding to ActRIIA, ActRIIB and Fn14 and inhibiting the muscular atrophic action mediated by ActRIIA, ActRIIB and Fn14.
  • the purpose is to provide a specific antibody.
  • the present inventors have conducted CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 and amino acid number 50 of SEQ ID NO: 2.
  • the first VHH which comprises CDR2 consisting of the amino acid sequences from 1 to 65, and CDR3 consisting of the amino acid sequences of amino acid numbers 98 to 105 of SEQ ID NO: 2, and the amino acid sequences of amino acid numbers 172 to 176 of SEQ ID NO: 2.
  • ActRIIA and ActRIIB containing a second VHH comprising CDR1 consisting of CDR1, CDR2 consisting of the amino acid sequences of amino acids 195 to 210 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequences of amino acids 243 to 251 of SEQ ID NO: 2.
  • a polypeptide that binds to (Example 2) was prepared. Further, a multispecific antibody in which the C-terminal of the polypeptide is linked to the N-terminal of the heavy chain variable region of the antibody that binds to human Fn14 via a peptide linker was prepared (Examples 3 to 4).
  • the multispecific antibody binds to ActRIIA, ActRIIB and Fn14 (Example 5), inhibits the phosphorylation of Smad stimulated by myostatin (Example 6), and inhibits the activation of NF- ⁇ B stimulated by TWEAK. It was found that (Example 7) and that activation of NF- ⁇ B was not induced in the absence of TWEAK (Example 7). As a result, the multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 was provided, and the present invention was completed.
  • a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 (A) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14 or an antigen-binding fragment thereof.
  • the first VHH is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and amino acids 98 to 105 of SEQ ID NO: 2.
  • the second VHH is CDR1 consisting of the amino acid sequences of amino acid numbers 172 to 176 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 195 to 210 of SEQ ID NO: 2, and amino acid numbers 243 to 251 of SEQ ID NO: 2.
  • the C-terminal of the polypeptide of (a) is linked to the N-terminal of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the antibody that binds to Fn14 or an antigen-binding fragment thereof is CDR1 consisting of the amino acid sequences of amino acids 303 to 307 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 322 to 338 of SEQ ID NO: 2, and SEQ ID NO: 2.
  • the antibody that binds to Fn14 or its antigen-binding fragment is a heavy chain variable region consisting of the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 108 of SEQ ID NO: 4.
  • the antibody that binds to Fn14 contains an antibody that binds to Fn14, and the antibody that binds to Fn14 has a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and amino acid mutations of L234A, L235A, M252Y, S254T, and T256E.
  • the multispecific antibody according to any one of the above.
  • An antibody that binds to Fn14 is included, and the antibody that binds to Fn14 includes a heavy chain consisting of the amino acid sequences of amino acids 273 to 720 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence of SEQ ID NO: 4 [1].
  • the multispecific antibody according to any one of [4].
  • a polynucleotide comprising a base sequence encoding the light chain variable region of an antibody that binds to Fn14 of the multispecific antibody according to any one of [5] to [9] or an antigen-binding fragment thereof.
  • a base encoding a polypeptide containing the heavy chain variable region of an antibody that binds to Fn14 or the polypeptide of (a) of the multispecific antibody according to any one of [1] to [9] or an antigen-binding fragment thereof.
  • a polynucleotide containing a sequence [16] A polynucleotide containing a base sequence encoding the polypeptide of the multispecific antibody according to [10].
  • a polynucleotide comprising a base sequence encoding the light chain of the multispecific antibody according to [10].
  • Transformed host cells (I) A poly containing a base sequence encoding a polypeptide containing the light chain variable region of the antibody that binds to Fn14 of the multispecific antibody according to any one of [1] to [9] or an antigen-binding fragment thereof.
  • nucleotide (Ii) A polypeptide containing the polypeptide of (a) of the multispecific antibody according to any one of [1] to [9] and an antibody that binds to Fn14 or a heavy chain variable region of an antigen-binding fragment thereof. A polynucleotide containing the encoding base sequence.
  • Host cells selected from the group consisting of the following (A) to (D): (A) Includes a polynucleotide containing a base sequence encoding the polypeptide of the multispecific antibody according to [10] and a polynucleotide containing a base sequence encoding the light chain of the multispecific antibody according to [10].
  • Host cells transformed with an expression vector (B) An expression vector containing a polynucleotide containing a nucleotide sequence encoding the polypeptide of the multispecific antibody according to [10] and a nucleotide sequence encoding the light chain of the multispecific antibody according to [10].
  • Host cells transformed with an expression vector containing a polynucleotide (C) Host cells transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the polypeptide of the multispecific antibody according to [10]; and the multispecific antibody according to (D) [10].
  • Host cells transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain of an antibody [24] Transformed with an expression vector containing the following (i) and (ii) polynucleotides or transformed with an expression vector containing the following (i) polynucleotide and the following (ii) polynucleotide.
  • a method for producing a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 which comprises the step of culturing the generated host cell and expressing the multispecific antibody: (I) A poly containing a base sequence encoding a polypeptide containing the light chain variable region of the antibody that binds to Fn14 of the multispecific antibody according to any one of [1] to [9] or an antigen-binding fragment thereof. nucleotide; (Ii) A polypeptide containing the polypeptide of (a) of the multispecific antibody according to any one of [1] to [9] and an antibody that binds to Fn14 or a heavy chain variable region of an antigen-binding fragment thereof.
  • a polynucleotide containing the encoding base sequence [25] A method for producing a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14, which comprises a step of culturing a host cell selected from the group consisting of the following (E) to (G) to express a multispecific antibody. : (E) Containing a polynucleotide containing a base sequence encoding the polypeptide of the multispecific antibody according to [10] and a polynucleotide containing a base sequence encoding the light chain of the multispecific antibody according to [10].
  • Host cells transformed with an expression vector (F) Contains an expression vector containing a polynucleotide containing a nucleotide sequence encoding the polypeptide of the multispecific antibody according to [10] and a base sequence encoding the light chain of the multispecific antibody according to [10].
  • the pharmaceutical composition according to [28] which is a pharmaceutical composition for preventing or treating inclusion body myositis.
  • a method for preventing or treating inclusion body myositis which comprises the step of administering a therapeutically effective amount of the multispecific antibody according to any one of [1] to [11], [26] and [27].
  • the multispecific antibody of the present invention is expected to have a muscular atrophy inhibitory effect by inhibiting the phosphorylation of Smad stimulated by myostatin and further inhibiting the activation of Fn14 stimulated by TWEAK without showing agonistic activity. It may be used as a prophylactic or therapeutic agent for muscular atrophic diseases such as inclusion body myositis.
  • the vertical axis shows the inhibition rate of Smad3 phosphorylation by myostatin 100 ng / mL (measured value under myostatin 100 ng / mL stimulation condition is 0% inhibition, and measurement value in the absence of myostatin is 100% inhibition).
  • the horizontal axis shows the logarithm of the antibody concentration (nmol / L). The data represents the average value of two trials (each trial was performed in duplicate).
  • the vertical axis is (A) Inhibition rate against NF- ⁇ B activation by TWEAK 100 ng / mL (measured value under TWEAK 100 ng / mL stimulation condition is 0% inhibition, measurement value in the absence of TWEAK is 100% inhibition), and ( B) The relative activation rate by the evaluation antibody in the absence of TWEAK when the measured value under the stimulation condition of TWEAK 100 ng / mL is 100% and the measured value in the absence of TWEAK is 0% is shown.
  • the horizontal axis shows the logarithm of the antibody concentration (nmol / L). The data represents the average value of two trials (each trial was performed in duplicate).
  • the thigh fat-free weight 4 weeks after the start of administration of the steroid and the test substance in the steroid-induced myopathy model monkey is shown.
  • the vertical axis shows the lean body mass (g) of the thigh measured by a bone mineral density measuring device by the dual energy X-ray absorption measurement method.
  • the data of the normal group represents the measured values and the average value of 2 cases
  • the data of each group of the steroid-induced myopathy model represents the measured values and the average value ⁇ SEM of 3 cases in each group.
  • IgG There are five classes of antibodies: IgG, IgM, IgA, IgD and IgE.
  • the basic structure of an antibody molecule is common to each class and is composed of a heavy chain having a molecular weight of 50,000 to 70,000 and a light chain having a molecular weight of 20,000 to 30,000.
  • a heavy chain usually consists of a polypeptide chain containing about 440 amino acids, has a characteristic structure for each class, and corresponds to IgG, IgM, IgA, IgD, IgE, Ig ⁇ , Ig ⁇ , Ig ⁇ , Ig ⁇ . It is called.
  • IgG has subclasses of IgG1, IgG2, IgG3, and IgG4, and the heavy chains corresponding to each are called Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, and Ig ⁇ 4.
  • the light chain usually consists of a polypeptide chain containing about 220 amino acids, and two types, ⁇ type and ⁇ type, are known and are called Ig ⁇ and Ig ⁇ , respectively.
  • the peptide composition of the basic structure of an antibody molecule is that two heavy chains and two light chains that are homologous to each other are bonded by disulfide bonds (SS bonds) and non-covalent bonds, and have a molecular weight of 150,000 to 190,000. ..
  • the two light chains can be paired with any heavy chain.
  • Each antibody molecule is always made up of two identical light chains and two identical heavy chains.
  • variable region The amino acid sequence on the C-terminal side of the variable region is almost constant for each class or subclass and is called a constant region (each domain of the constant region is CH1, CH2, from the N-terminal side of the heavy chain constant region, respectively. Represented as CH3 and the light chain constant region as CL).
  • the antigen-binding site of an antibody is composed of VH and VL, and the specificity of binding depends on the amino acid sequence of this site.
  • biological activities such as binding to complement and various Fc receptor-expressing cells reflect the difference in the structure of the constant region of each class Ig.
  • CDRs complementarity determining regions
  • the rest of the variable region is called the framework region (FR) and is relatively constant.
  • scFv single-chain variable region fragments
  • Fab single-chain variable region fragments
  • Fab' single-chain variable region fragments
  • F (ab') 2 single-chain variable region fragments
  • scFv is a monovalent antibody fragment composed of linker-linked VH and VL
  • Fab is composed of a light chain and a heavy chain fragment containing a VH, CH1 domain and part of the hinge region. It is a monovalent antibody fragment.
  • Fab' is a monovalent antibody fragment composed of a light chain and a heavy chain fragment containing a VH, CH1 domain and a part of a hinge region, and the part of this hinge region is an inter-heavy chain SS. It contains the cysteine residues that made up the bond.
  • the F (ab') 2 fragment is a divalent antibody fragment in which two Fab'fragments are bound by an inter-heavy chain SS bond in the hinge region.
  • variable region of a heavy chain antibody is called a variable domain of healthy chain of heavy chain antibody (VHH), and the antigen-binding site of the heavy chain antibody is composed of only VHH.
  • VHH contains three CDRs, and the specificity of binding to the antigen depends on the amino acid sequence of the CDRs.
  • VHH in the present specification also includes a humanized form (humanized VHH) in which a part, most, or all of the amino acid sequence of FR is replaced with an amino acid sequence derived from a human immunoglobulin molecule.
  • the method of humanization is not particularly limited, and for example, a humanized antibody can be prepared with reference to International Publication No. 2006/122825, US Pat. No. 5,225,539, US Pat. No. 6,180,370, and the like.
  • multispecific antibody means an antibody that binds to two or more different antigens.
  • peptide linker means a polypeptide consisting of one or more arbitrary amino acid sequences that can be introduced by genetic engineering techniques for linking variable regions.
  • the present invention provides the following multispecific antibodies (hereinafter, also referred to as "multispecific antibodies of the present invention"): A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14.
  • a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 (A) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14 or an antigen-binding fragment thereof.
  • the first VHH is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and amino acids 98 to 105 of SEQ ID NO: 2.
  • the second VHH is CDR1 consisting of the amino acid sequences of amino acid numbers 172 to 176 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 195 to 210 of SEQ ID NO: 2, and amino acid numbers 243 to 251 of SEQ ID NO: 2.
  • the C-terminal of the polypeptide of (a) is linked to the N-terminal of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the first VHH and the second VHH are humanized VHH.
  • the first VHH comprises the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acid numbers 142 of SEQ ID NO: 2. It consists of amino acid sequences up to 262.
  • the C-terminus of the first VHH may be linked to the N-terminus of the second VHH, and the C-terminus of the second VHH is the first. It may be connected to the N-terminus of VHH. In one embodiment of the multispecific antibody of the invention, the C-terminus of the first VHH is linked to the N-terminus of the second VHH.
  • the first VHH and the second VHH may be directly linked or may be linked via a peptide linker. In one embodiment of the multispecific antibody of the invention, the first VHH and the second VHH are linked via a peptide linker.
  • the C-terminus of the first VHH is linked to the N-terminus of the second VHH via a peptide linker.
  • the first VHH and the second VHH are humanized VHH, with the C-terminus of the first VHH mediated by a peptide linker at the N-terminus of the second VHH. Are connected.
  • the first VHH comprises the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acid numbers 142 of SEQ ID NO: 2. It consists of amino acid sequences up to 262, and the C-terminus of the first VHH is linked to the N-terminus of the second VHH via a peptide linker.
  • the length of the peptide linker linking the first VHH and the second VHH is not particularly limited, and can be appropriately selected by those skilled in the art according to the purpose.
  • the first VHH and the second VHH are linked by a peptide linker of 5 amino acids or more, and in one embodiment, the first VHH and the second VHH are 5 amino acids or more and 35 amino acids or less. It is linked with a peptide linker of 5, amino acids or more and 30 amino acids or less, 5 amino acids or more and 25 amino acids or less, 10 amino acids or more and 25 amino acids or less, or 15 amino acids or more and 25 amino acids or less.
  • the peptide linker linking the first VHH and the second VHH is (Gly ⁇ Gly ⁇ Gly ⁇ Gly ⁇ Ser (SEQ ID NO: 7)) n [n is an integer of 1-5]. In one embodiment, the peptide linker linking the first VHH and the second VHH is (Gly ⁇ Gly ⁇ Gly ⁇ Gly ⁇ Ser) 5 (amino acids 117 to 141 of SEQ ID NO: 2). ..
  • the first VHH and the second VHH are humanized VHH, with the C-terminus of the first VHH mediated by a peptide linker at the N-terminus of the second VHH.
  • the peptide linker that links the first VHH and the second VHH is (Gly, Gly, Gly, Gly, Ser) 5 (amino acid numbers 117 to 141 of SEQ ID NO: 2).
  • the polypeptide (a) comprises the amino acid sequences of amino acids 1 to 262 of SEQ ID NO: 2.
  • the antibody that binds to Fn14 or an antigen-binding fragment thereof is CDR1 consisting of the amino acid sequences of amino acid numbers 303 to 307 of SEQ ID NO: 2, and amino acid number 322 of SEQ ID NO: 2. Consists of CDR2 consisting of amino acid sequences from 1 to 338, a heavy chain variable region containing CDR3 consisting of amino acid sequences of amino acid numbers 371 to 379 of SEQ ID NO: 2, and amino acid sequences of amino acid numbers 24 to 34 of SEQ ID NO: 4.
  • CDR1 consisting of the amino acid sequences of amino acids 50 to 56 of SEQ ID NO: 4
  • CDR3 consisting of the amino acid sequences of amino acids 89 to 97 of SEQ ID NO: 4.
  • the antibody that binds to Fn14 or its antigen-binding fragment is a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and SEQ ID NO: 4. It contains a light chain variable region consisting of the amino acid sequences of amino acids 1 to 108.
  • the antigen-binding fragment of the antibody that binds to Fn14 is an antigen-binding fragment that is scFv, Fab, Fab', or F (ab') 2 .
  • any subclass constant region for example, Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3 or Ig ⁇ 4 as a heavy chain, and Ig ⁇ or Ig ⁇ as a light chain
  • the antibody that binds to Fn14 comprises a human Ig ⁇ 1 constant region as a heavy chain constant region and a human Ig ⁇ constant region as a light chain constant region.
  • L234A is the substitution of leucine at amino acid position 234 with alanine according to the EU index of Kabat et al. In the human Ig ⁇ 1 constant region.
  • L235A is the substitution of leucine at amino acid position 235 with alanine according to the EU index of Kabat et al. In the human Ig ⁇ 1 constant region.
  • M252Y is a tyrosine substitution of methionine at amino acid 252 according to the EU index of Kabat et al.
  • S254T is the replacement of serine at amino acid position 254 with threonine according to the EU index of Kabat et al.
  • T256E is the substitution of threonine at amino acid position 256 with glutamic acid according to the EU index of Kabat et al.
  • human Ig ⁇ 1 constant region is the substitution of threonine at amino acid position 256 with glutamic acid according to the EU index of Kabat et al.
  • the multispecific antibody of the invention comprises an antibody that binds to Fn14, a heavy chain constant region in which the antibody that binds to Fn14 has amino acid mutations of L234A, L235A, M252Y, S254T, and T256E. including.
  • the constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E include the human Ig ⁇ 1 constant region consisting of the amino acid sequences of amino acid numbers 391 to 720 of SEQ ID NO: 2.
  • the multispecific antibody of the invention comprises an antibody that binds to Fn14, and the antibody that binds to Fn14 is a heavy chain variable region consisting of the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2.
  • a heavy chain containing a heavy chain constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E, and a light chain variable region and a light chain constant consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4. Includes a light chain containing a region.
  • the multispecific antibody of the invention comprises an antibody that binds to Fn14, wherein the antibody that binds to Fn14 is a heavy chain consisting of the amino acid sequences set forth in amino acid numbers 273 to 720 of SEQ ID NO: 2. Includes a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.
  • the C-terminal of the polypeptide (a) is linked to the N-terminal of the heavy chain variable region of the antibody that binds to Fn14 or its antigen-binding fragment via a peptide linker.
  • the length of the peptide linker used is not particularly limited and can be appropriately selected by those skilled in the art depending on the intended purpose.
  • the polypeptide (a) and the antibody that binds to Fn14 or an antigen-binding fragment thereof are linked by a peptide linker of 5 amino acids or more, and in one embodiment, the polypeptide of (a) and Fn14 are linked.
  • the binding antibody or its antigen-binding fragment is linked with a peptide linker of 5 amino acids or more and 35 amino acids or less, 5 amino acids or more and 30 amino acids or less, 5 amino acids or more and 25 amino acids or less, 5 amino acids or more and 20 amino acids or less, or 10 amino acids or more and 20 amino acids or less. Will be done.
  • the peptide linker that links the polypeptide of (a) to an antibody that binds to Fn14 or an antigen-binding fragment thereof is (Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 7)) n [n. It is an integer of 1 to 5], and in one embodiment, the peptide linker that links the polypeptide of (a) with an antibody that binds to Fn14 or an antigen-binding fragment thereof is (Gly, Gly, Gly, Gly. Ser) 2 (amino acid numbers 263 to 272 of SEQ ID NO: 2).
  • the peptide linker linking the first VHH and the second VHH and the peptide linker linking the polypeptide (a) and the antibody binding to Fn14 or its antigen-binding fragment are the same peptides. It may be a linker or a different peptide linker.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14.
  • a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 (A) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14 or an antigen-binding fragment thereof.
  • the first VHH consists of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • the antibody that binds to Fn14 or its antigen-binding fragment is a heavy chain variable region consisting of the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 108 of SEQ ID NO: 4. Including area The C-terminal of the polypeptide of (a) is linked to the N-terminal of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the first VHH consists of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • the antibody that binds to Fn14 is a heavy chain containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a heavy chain constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E. , And a light chain comprising a light chain variable region and a light chain constant region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4.
  • the C-terminus of the polypeptide (a) is linked to the N-terminus of the heavy chain variable region of the antibody that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the first VHH consists of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • the antibody that binds to Fn14 comprises a heavy chain consisting of the amino acid sequences shown in Amino Acid Nos.
  • the C-terminus of the polypeptide (a) is linked to the N-terminus of the heavy chain variable region of the antibody that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14.
  • a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 (A) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14 or an antigen-binding fragment thereof.
  • the first VHH consists of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2
  • the C of the first VHH is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14.
  • the terminus is linked to the N-terminus of the second VHH via a peptide linker
  • the antibody that binds to Fn14 or its antigen-binding fragment is a heavy chain variable region consisting of the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 108 of SEQ ID NO: 4.
  • Including area The C-terminal of the polypeptide of (a) is linked to the N-terminal of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the first VHH consists of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2
  • the C of the first VHH is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the first VHH consists of the amino acid sequences of
  • the terminus is linked to the N-terminus of the second VHH via a peptide linker
  • the antibody that binds to Fn14 is a heavy chain containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a heavy chain constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E.
  • a light chain comprising a light chain variable region and a light chain constant region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4.
  • the C-terminus of the polypeptide (a) is linked to the N-terminus of the heavy chain variable region of the antibody that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the first VHH consists of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2
  • the C of the first VHH is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the first VHH consists of the amino acid sequences of
  • the terminus is linked to the N-terminus of the second VHH via a peptide linker
  • the antibody that binds to Fn14 comprises a heavy chain consisting of the amino acid sequences shown in Amino Acid Nos. 273 to 720 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the C-terminus of the polypeptide (a) is linked to the N-terminus of the heavy chain variable region of the antibody that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14.
  • a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 (A) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14 or an antigen-binding fragment thereof.
  • the polypeptide (a) comprises the amino acid sequences of amino acid numbers 1 to 262 of SEQ ID NO: 2.
  • the antibody that binds to Fn14 or its antigen-binding fragment is a heavy chain variable region consisting of the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 108 of SEQ ID NO: 4. Including area The C-terminal of the polypeptide of (a) is linked to the N-terminal of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the polypeptide (a) comprises the amino acid sequences of amino acid numbers 1 to 262 of SEQ ID NO: 2.
  • the antibody that binds to Fn14 is a heavy chain containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a heavy chain constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E. , And a light chain comprising a light chain variable region and a light chain constant region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4.
  • the C-terminus of the polypeptide (a) is linked to the N-terminus of the heavy chain variable region of the antibody that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the invention is the following multispecific antibody: A multispecific antibody that binds to ActRIIA, ActRIIB and Fn14. It comprises (a) a polypeptide containing a first VHH and a second VHH that binds to ActRIIA and ActRIIB, and (b) an antibody that binds to Fn14.
  • the polypeptide (a) comprises the amino acid sequences of amino acid numbers 1 to 262 of SEQ ID NO: 2.
  • the antibody that binds to Fn14 comprises a heavy chain consisting of the amino acid sequences shown in Amino Acid Nos. 273 to 720 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the C-terminus of the polypeptide (a) is linked to the N-terminus of the heavy chain variable region of the antibody that binds to Fn14 via a peptide linker. Multispecific antibody.
  • the multispecific antibody of the present invention is a multispecific antibody comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • post-translational modifications include deletion of lysine at the C-terminal of the heavy chain by carboxypeptidase, modification of glutamine or glutamic acid at the N-terminal of the heavy and light chains to pyroglutamylation, glycosylation, oxidation, and deamidation. It is known that such post-translational modifications occur in various antibodies, such as glycosylation and glycosylation (Liu H et al., J Pharm Sci. 2008, Vol. 97, p. 2426-2447). ..
  • the multispecific antibody of the present invention also includes a posttranslationally modified multispecific antibody.
  • the multispecific antibody of the invention is the heavy chain C of the antibody that binds to Fn14 and / or N-terminal polyglutamylation of the polypeptide of (a) of the multispecific antibody of the invention described above. It is a multispecific antibody that has undergone a terminal lysine deletion. It is known in the art that such post-translational modification by N-terminal pyroglutamylation or C-terminal lysine deletion does not affect the activity of the antibody (Lyubarskaya Y et al., Anal Biochem. 2006). , Vol. 348, p. 24-39).
  • a person skilled in the art can prepare a fusion of the multispecific antibody of the present invention with another peptide or protein based on the present invention, or prepare a modified product to which a modifying agent is bound. Yes, these forms of multispecific antibodies are also included in the multispecific antibodies of the present invention.
  • Other peptides and proteins used for fusion are not particularly limited as long as the fusion binds to ActRIIA, ActRIIB and Fn14, and for example, human serum albumin, various tag peptides, artificial helix motif peptides, maltose-binding proteins, glutathione S transferase. , Various toxins, and other peptides or proteins that can promote multimerization.
  • the modifier used for the modification is not particularly limited as long as the modified product binds to ActRIIA, ActRIIB and Fn14, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, and small molecule compounds.
  • the modifier used to modify the multispecific antibody of the invention is polyethylene glycol.
  • the multispecific antibody of the present invention includes a first VHH and a second VHH that bind to ActRIIA and ActRIIB, and an antibody or antigen-binding fragment that binds to Fn14.
  • the multispecific antibody of the present invention binds to ActRIIA, ActRIIB and Fn14. Whether or not it binds to ActRIIA, ActRIIB or Fn14 can be confirmed by using a known binding activity measuring method. Examples of the method for measuring the binding activity include a method such as Enzyme-Linked ImmunoSorbent Assay (ELISA).
  • ELISA Enzyme-Linked ImmunoSorbent Assay
  • ELISA When ELISA is used, for example, ActRIIA, ActRIIB or Fn14 protein is immobilized on an ELISA plate, a test antibody is added thereto and reacted, and then an enzyme labeled with an enzyme such as horseradish peroxidase (HRP) is used. React a secondary antibody such as an IgG antibody. After the reaction, after washing, binding of the secondary antibody by activity measurement using a reagent for detecting the activity (for example, in the case of HRP labeling, TMB Microcell Peroxidase Substrate (Kirkegard & Perry Laboratories, 50-76-03)) or the like. By identifying, it is possible to confirm whether or not the test antibody binds to ActRIIA, ActRIIB or Fn14. As a specific evaluation method, a method as described in Example 5 described later can be used.
  • the multispecific antibody of the present invention includes not only human ActRIIA, human ActRIIB and human Fn14, but also multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 derived from other animals (mouse, monkey, etc.). In one embodiment, the multispecific antibody of the invention binds to human ActRIIA, human ActRIIB and human Fn14.
  • the multispecific antibody of the present invention can be easily prepared by a person skilled in the art using a method known in the art based on the sequence information of the multispecific antibody of the present invention disclosed herein. ..
  • the multispecific antibody of the present invention is not particularly limited, but can be produced, for example, according to the method described in ⁇ Method for producing a multispecific antibody of the present invention> described later.
  • the multispecific antibody of the present invention is further purified if necessary, and then formulated according to a conventional method, and can be used for the prevention or treatment of amyotrophic diseases such as inclusion body myositis.
  • the present invention also provides the following polynucleotides (1) to (4) (also referred to as “polynucleotides of the present invention"): (1) A polynucleotide containing a base sequence encoding a polypeptide containing the first VHH and the second VHH of the multispecific antibody of the present invention (polypeptide of (a)), (2) A polynucleotide containing a base sequence encoding a heavy chain variable region of an antibody that binds to Fn14 of the multispecific antibody of the present invention or an antigen-binding fragment thereof.
  • (4) Includes a polypeptide containing the first VHH and the second VHH of the multispecific antibody of the present invention (polypeptide of (a)) and a heavy chain variable region of an antibody that binds to Fn14 or an antigen-binding fragment thereof.
  • the polynucleotide (1) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2. It is a polynucleotide containing a base sequence encoding a polypeptide containing a second VHH. In one embodiment, the polynucleotide (1) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • polynucleotide comprising a base sequence encoding a polypeptide, comprising two VHHs, the C-terminal of the first VHH linked to the N-terminal of the second VHH via a peptide linker.
  • the polynucleotide (1) is a polynucleotide comprising a base sequence encoding a polypeptide consisting of the amino acid sequences of amino acid numbers 1 to 262 of SEQ ID NO: 2.
  • the polynucleotide (2) is a polynucleotide containing a base sequence encoding a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2. In one embodiment, the polynucleotide (2) has a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a weight having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E. A polynucleotide containing a base sequence encoding a heavy chain containing a chain constant region. In one embodiment, the polynucleotide (2) is a polynucleotide comprising a base sequence encoding a heavy chain consisting of the amino acid sequences set forth in amino acid numbers 273 to 720 of SEQ ID NO: 2.
  • the polynucleotide (3) is a polynucleotide containing a base sequence encoding a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4. In one embodiment, the polynucleotide (3) is a poly comprising a base sequence encoding a light chain containing a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4 and a light chain constant region. It is a nucleotide. In one embodiment, the polynucleotide (3) is a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the polynucleotide (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2. It is a polynucleotide containing a polypeptide containing two VHHs and a base sequence encoding a polypeptide containing a heavy chain variable region of an antibody that binds to Fn14 or an antigen-binding fragment thereof.
  • the polypeptide of (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • a polypeptide comprising two VHHs, the C-terminal of the first VHH being linked to the N-terminal of the second VHH via a peptide linker, and the heavy chain of an antibody or antigen-binding fragment thereof that binds to Fn14.
  • the polynucleotide (4) is a polypeptide consisting of the amino acid sequences of amino acids 1 to 262 of SEQ ID NO: 2 and a poly comprising a heavy chain variable region of an antibody or antigen-binding fragment thereof that binds to Fn14.
  • the polynucleotide (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2. It is a polynucleotide containing a base sequence encoding a polypeptide containing a heavy chain variable region consisting of a polypeptide containing the second VHH and an amino acid sequence of amino acid numbers 273 to 390 of SEQ ID NO: 2.
  • the polypeptide (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • a polypeptide comprising two VHHs, the C-terminal of the first VHH linked to the N-terminal of the second VHH via a peptide linker, and the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2. It is a polynucleotide containing a base sequence encoding a polypeptide containing a heavy chain variable region consisting of.
  • the polynucleotide (4) is a polypeptide consisting of the amino acid sequences of amino acids 1 to 262 of SEQ ID NO: 2 and a heavy chain variable consisting of the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2.
  • the polynucleotide (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • a heavy chain variable region consisting of a polypeptide containing two VHHs and an amino acid sequence of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a heavy chain constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E. It is a polynucleotide containing a base sequence encoding a polypeptide containing a heavy chain containing.
  • the polypeptide (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • a polypeptide comprising two VHHs, the C-terminal of the first VHH linked to the N-terminal of the second VHH via a peptide linker, and the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2.
  • a polynucleotide comprising a base sequence encoding a heavy chain comprising a heavy chain variable region comprising a heavy chain constant region comprising a heavy chain variable region comprising an amino acid mutation of L234A, L235A, M252Y, S254T, and T256E.
  • the polynucleotide (4) is a polypeptide consisting of the amino acid sequences of amino acids 1 to 262 of SEQ ID NO: 2 and a heavy chain variable consisting of the amino acid sequences of amino acids 273 to 390 of SEQ ID NO: 2.
  • a polynucleotide comprising a nucleotide sequence encoding a heavy chain comprising a region and a heavy chain constant region comprising amino acid mutations in L234A, L235A, M252Y, S254T, and T256E.
  • the polynucleotide (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2. It is a polynucleotide containing a base sequence encoding a polypeptide containing a heavy chain consisting of the amino acid sequence shown in amino acid numbers 273 to 720 of SEQ ID NO: 2 and a polypeptide containing the second VHH.
  • the polypeptide (4) comprises a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a first VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • a polypeptide comprising two VHHs, the C-terminal of the first VHH linked to the N-terminal of the second VHH via a peptide linker, and the amino acids set forth in amino acids 273 to 720 of SEQ ID NO: 2. It is a polynucleotide containing a base sequence encoding a polypeptide containing a heavy chain consisting of a sequence.
  • the polynucleotide (4) is a polypeptide consisting of the amino acid sequences of amino acids 1 to 262 of SEQ ID NO: 2 and a heavy chain consisting of the amino acid sequences of amino acids 273 to 720 of SEQ ID NO: 2. It is a polynucleotide containing a base sequence encoding a polypeptide containing.
  • the polynucleotide (4) is a polynucleotide containing a base sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide of the present invention can be easily prepared by those skilled in the art based on its base sequence using a method known in the art.
  • the polynucleotide of the present invention can be synthesized by using a gene synthesis method known in the art.
  • a gene synthesis method various methods known to those skilled in the art such as the method for synthesizing an antibody gene described in International Publication No. 90/07861 can be used.
  • the present invention also provides an expression vector containing the polynucleotide of the present invention (also referred to as "expression vector of the present invention").
  • the expression vector of the present invention comprises any of the polynucleotides (1)-(4) described in the section ⁇ Polynucleotides of the Invention>. Examples of specific embodiments of the polynucleotide contained in the expression vector of the present invention include those described for the polynucleotides (1) to (4) described in the section ⁇ Polynucleotide of the present invention>. Be done.
  • the expression vector of the invention is (i) a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of an antibody or antigen-binding fragment thereof that binds to Fn14 of the multispecific antibody of the invention.
  • a polypeptide containing the first VHH and the second VHH of the multispecific antibody of the present invention polypeptide of (a)) and the heavy chain variable region of the antibody or its antigen-binding fragment that binds to Fn14.
  • Examples of specific embodiments of the polynucleotides (i) and (ii) contained in the expression vector of the present invention include (3) and (4) described in the section ⁇ Polynucleotide of the present invention>. Examples include those described for polynucleotides.
  • the expression vector of the present invention encodes an expression vector containing a nucleotide containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • Expression vectors used to express the polynucleotides of the invention include in various host cells of eukaryotic cells (eg, animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells (eg, Escherichia coli).
  • eukaryotic cells eg, animal cells, insect cells, plant cells, yeast
  • prokaryotic cells eg, Escherichia coli
  • a polynucleotide containing a base sequence encoding the first VHH of the multispecific antibody of the present invention and a polynucleotide containing a base sequence encoding the second VHH of the multispecific antibody are expressed and encoded by these.
  • it is not particularly limited.
  • an expression vector examples include a plasmid vector and a viral vector (for example, adenovirus and retrovirus), and for example, a plasmid vector such as pEE6.4 and pEE12.4 can be used. It is also possible to express an antibody gene by introducing a variable region gene fragment into an expression vector having a human Ig constant region gene in advance, such as AG- ⁇ 1 or AG- ⁇ (see, for example, International Publication No. 94/20632). ..
  • the expression vector of the present invention may contain a promoter operably linked to the polynucleotide of the present invention.
  • promoters for expressing the polynucleotide of the present invention in animal cells include virus-derived promoters such as CMV, RSV, and SV40, actin promoters, EF (elongation factor) 1 ⁇ promoters, and heat shock promoters.
  • promoters for expression in bacteria include trp promoter, lac promoter, ⁇ PL promoter, tac promoter and the like.
  • promoters for expression in yeast include GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like.
  • the expression vector of the present invention may contain a start codon and a stop codon.
  • the expression vector of the present invention is an enhancer sequence, a multispecific antibody of the present invention or an untranslated region on the 5'and 3'side of a gene encoding a heavy chain variable region or a light chain variable region thereof, and a secretory signal. It may include sequences, splicing junctions, polyadenylation sites, replicable units, and the like.
  • the expression vector of the present invention may contain a start codon, a stop codon, a terminator region, and a replicable unit.
  • the expression vector of the present invention may contain a selection marker usually used depending on the purpose (for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene). ..
  • the present invention also provides host cells transformed with the expression vector of the present invention (also referred to as "transformed host cells of the present invention").
  • the host cell of the present invention is a host cell transformed with an expression vector containing any of the polynucleotides (1) to (4) described in the section ⁇ Polynucleotides of the present invention>. be. Examples of specific embodiments of the polynucleotide contained in the expression vector include those described for the polynucleotides (1) to (4) described in the section ⁇ Polynucleotide of the present invention>.
  • the host cell of the invention is transformed with an expression vector containing the polynucleotides (i) and / or (ii) below, or an expression vector containing the polynucleotides (i) below. / Or a host cell transformed with an expression vector containing the following polynucleotide (ii): (I) A polynucleotide containing a base sequence encoding the light chain variable region of an antibody that binds to Fn14 of the multispecific antibody of the present invention or an antigen-binding fragment thereof; (Ii) A polypeptide containing the first VHH and a second VHH of the multispecific antibody of the present invention (polypeptide of (a)) and a heavy chain variable region of an antibody that binds to Fn14 or an antigen-binding fragment thereof. A polynucleotide containing a base sequence encoding a polypeptide.
  • the polynucleotides (3) and (4) described in the section ⁇ Polynucleotide of the present invention> are mentioned.
  • the transformed host cell of the present invention includes a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (A) to (D). .. (A) Transformed with an expression vector containing a nucleotide containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • Host cell (B) An expression vector containing a polynucleotide containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and an expression vector containing a polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • Transformed host cells in (C) A host cell transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; and (D) encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • the host cell to be transformed is not particularly limited as long as it is compatible with the expression vector to be used and can be transformed with the expression vector to express a multispecific antibody.
  • Examples of the host cell to be transformed include various cells (for example, animal cells (for example, CHO-K1SV cells) and insect cells) such as natural cells or artificially established cells usually used in the technical field of the present invention. (For example, Sf9), bacteria (Escherichia spp., Etc.), yeast (Saccharomyces spp., Pikia spp., Etc.), etc.), for example, CHO cells (CHO-K1SV cells, CHO-DG44 cells, etc.), 293 cells, NS0. Cultured cells such as cells can be used.
  • the method for transforming the host cell is not particularly limited, but for example, a calcium phosphate method, an electroporation method, or the like can be used.
  • the present invention also comprises the step of culturing the transformed host cells of the present invention to express a multispecific antibody, a method for producing a multispecific antibody that binds to ActRIIA, ActRIIB and Fn14 ((“ (Also referred to as "the production method of the invention”)).
  • the production method of the present invention is transformed with an expression vector containing the following polynucleotides (i) and (ii) or an expression vector containing the following polynucleotide (i) and the following.
  • the polynucleotides (3) and (4) described in the section ⁇ Polynucleotide of the present invention> are mentioned.
  • the production method of the present invention comprises the steps of culturing a host cell selected from the group consisting of the following (A) to (C) to express a multispecific antibody, ActRIIA, Included are methods of producing multispecific antibodies that bind to ActRIIB and Fn14.
  • A Transformed with an expression vector containing a nucleotide containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • Host cell (B) An expression vector containing a polynucleotide containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and an expression vector containing a polynucleotide containing a nucleotide sequence encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • Transformed host cells in (C) A host cell transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a host cell.
  • the transformed host cell can be cultured by a known method. Culturing conditions, such as temperature, medium pH, and culturing time, are appropriately selected.
  • the medium is, for example, MEM medium (Eagle H, Science. 1959, Vol. 130, p. 432-437) containing about 5 to 20% fetal bovine serum, DMEM medium (Dulvecco). R and Freeman G, Virology. 1959, Vol. 8, p. 396-397), RPMI 1640 medium (Moore GE et al., JAMA. 1967, Vol. 199, p. 519-524), 199 medium (Morgan JF et). Al., Proc Soc Exp Biol Med. 1950, Vol.
  • the pH of the medium is preferably about 6-8, and the culture is usually carried out at about 30-40 ° C. for about 15-336 hours with aeration and stirring as needed.
  • the medium may be, for example, Grace's medium containing fetal bovine serum (Mith GE et al., Proc Natl Acad Sci USA. 1985, Vol. 82, p. 8404-8408). Can be used.
  • the pH of the medium is preferably about 5-8, and the culture is usually carried out at about 20-40 ° C. for about 15-100 hours with aeration and stirring as needed.
  • the medium is, for example, a liquid medium containing a nutrient source.
  • the nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the transformed host cell.
  • carbon sources include glucose, dextran, soluble starch, sucrose, etc.
  • inorganic nitrogen sources or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, and meat extract. , Soybean starch, potato extract, etc.
  • the medium may contain other nutrients (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc.), antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.). good.
  • the pH of the medium is preferably about 5-8.
  • LB medium, M9 medium Mol. Clo., Cold Spring Harbor Laboratory, 2001, Vol. 3, A2.2
  • Culturing is usually carried out at about 14-39 ° C. for about 3-24 hours with aeration and stirring as needed.
  • the host cell is yeast
  • the medium for example, Burkholder's minimum medium (Bostian KA et al., Proc Natl Acad Sci USA. 1980, Vol. 77, p. 4504-4508) and the like can be used. Culturing is usually carried out at about 20-35 ° C. for about 14-144 hours with aeration and stirring as needed.
  • the multispecific antibody of the present invention can be expressed by the culture as described above.
  • the method for producing a multispecific antibody of the present invention includes the step of culturing the transformed host cell of the present invention to express the multispecific antibody, and further, the transformed host cell and /.
  • a step of recovering, for example, isolating or purifying the multispecific antibody from the culture supernatant can be included.
  • the isolation or purification method include a method using solubility such as salting out and solvent precipitation, a method using difference in molecular weight such as dialysis, ultrafiltration, and gel filtration, ion exchange chromatography, and hydroxylapatite chromatography.
  • Method using charge such as imaging, method using specific affinity such as affinity chromatography, method using difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric point such as isoelectric point electrophoresis
  • isoelectric point such as isoelectric point electrophoresis
  • the antibody accumulated in the culture supernatant can be purified by various types of chromatography, for example, column chromatography using a protein A column or a protein G column.
  • the multispecific antibody of the present invention also includes a multispecific antibody produced by the method for producing the multispecific antibody of the present invention.
  • the present invention also provides a pharmaceutical composition (also referred to as "the pharmaceutical composition of the present invention") comprising the multispecific antibody of the present invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition of the present invention can be prepared by a commonly used method using excipients usually used in the art, that is, pharmaceutical excipients, pharmaceutical carriers and the like. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions, which can be administered by intravenous administration, subcutaneous administration, intramuscular administration, or the like. In the formulation, excipients, carriers, additives and the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range.
  • the pharmaceutical composition of the present invention may contain a multispecific antibody produced by post-translational modification of the multispecific antibody of the present invention.
  • the pharmaceutical composition of the present invention may contain a plurality of multispecific antibodies of the present invention.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and a multispecific antibody produced by post-translational modification of the multispecific antibody.
  • the multispecific antibody produced by post-translational modification is the weight of the antibody (also simply referred to as "Fn14 antibody”) that binds to the N-terminal pyroglutamylation and / or Fn14 of the polypeptide of (a). Contains deletion of chain C-terminal lysine.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide containing a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a second VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2, and (B) An antibody that binds to Fn14 containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4. Its antigen binding fragment.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide containing a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a second VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2, and (B) A heavy chain containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a heavy chain constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E, and a sequence.
  • an antibody that binds to Fn14 which comprises a light chain comprising a light chain variable region and a light chain constant region consisting of the amino acid sequences of amino acids Nos. 1 to 108 of No. 4.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide containing a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a second VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2, and (B) An antibody that binds to Fn14, which comprises a heavy chain consisting of the amino acid sequences shown in Amino Acid Nos. 273 to 720 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide containing a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a second VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • a polypeptide in which the C-terminus of the first VHH is linked to the N-terminus of the second VHH via a peptide linker as well as (B) An antibody that binds to Fn14 containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4. Its antigen binding fragment.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide containing a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a second VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • An antibody that binds to Fn14 which comprises a light chain comprising a light chain variable region and a light chain constant region consisting of the amino acid sequences of amino acids Nos. 1 to 108 of No. 4.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide containing a first VHH consisting of the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and a second VHH consisting of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • a polypeptide in which the C-terminus of the first VHH is linked to the N-terminus of the second VHH via a peptide linker as well as (B) An antibody that binds to Fn14, which comprises a heavy chain consisting of the amino acid sequences shown in Amino Acid Nos. 273 to 720 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide consisting of the amino acid sequences of amino acid numbers 1 to 262 of SEQ ID NO: 2, and (B) An antibody that binds to Fn14 containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 108 of SEQ ID NO: 4. An antibody that binds to Fn14 containing the antigen-binding fragment.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide consisting of the amino acid sequences of amino acid numbers 1 to 262 of SEQ ID NO: 2, and (B) A heavy chain containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2 and a heavy chain constant region having amino acid mutations of L234A, L235A, M252Y, S254T, and T256E, and a sequence.
  • an antibody that binds to Fn14 which comprises a light chain comprising a light chain variable region and a light chain constant region consisting of the amino acid sequences of amino acids Nos. 1 to 108 of No. 4.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and the multispecific of the invention.
  • Antibodies include: (A) A polypeptide consisting of the amino acid sequences of amino acid numbers 1 to 262 of SEQ ID NO: 2, and (B) An antibody that binds to Fn14, which comprises a heavy chain consisting of the amino acid sequences shown in Amino Acid Nos. 273 to 720 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the invention comprises a multispecific antibody of the invention and / or a multispecific antibody produced by post-translational modification of the multispecific antibody and is of the multispecificity of the invention.
  • An antibody is a pharmaceutical composition comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence of SEQ ID NO: 4.
  • the post-translational modification is N-terminal polyglutamylation of the polypeptide of (a) and / or deletion of the heavy chain C-terminal lysine of the Fn14 antibody.
  • the pharmaceutical composition of the present invention contains one or more of the following (c)-(f) multispecific antibodies:
  • Multispecific antibodies including.
  • a multispecific antibody comprising a polypeptide consisting of a sequence.
  • F A multispecific antibody comprising a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the amount of the multispecific antibody of the present invention added in the above formulation varies depending on the degree and age of the patient's symptoms, the dosage form of the formulation used, the binding titer of the antibody, etc., but for example, 0.001 mg at the time of administration. It can be used so as to be about / kg to 100 mg / kg.
  • the pharmaceutical composition of the present invention can be used as a prophylactic or therapeutic agent for diseases in which ActRIIA, ActRIIB, and Fn14 are involved in pathogenesis, for example, inclusion body myositis.
  • the present invention includes a pharmaceutical composition for preventing or treating inclusion body myositis, which comprises the multispecific antibody of the present invention.
  • the present invention also includes a method for preventing or treating inclusion body myositis, which comprises the step of administering a therapeutically effective amount of the multispecific antibody of the present invention.
  • the present invention also includes the multispecific antibody of the present invention for use in the prevention or treatment of inclusion body myositis.
  • the present invention includes the use of the multispecific antibody of the present invention in the manufacture of a pharmaceutical composition for the prevention or treatment of inclusion body myositis.
  • the present invention also provides the following polypeptides that bind to ActRIIA and ActRIIB (hereinafter, also referred to as "ActRIIA / B-binding peptides of the present invention"): A polypeptide that binds to ActRIIA and ActRIIB, including a first VHH and a second VHH that bind to ActRIIA and ActRIIB.
  • the first VHH is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2
  • CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2
  • CDR3 consisting of the amino acid sequence of The second VHH is CDR1 consisting of the amino acid sequences of amino acid numbers 172 to 176 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 195 to 210 of SEQ ID NO: 2, and amino acid numbers 243 to 251 of SEQ ID NO: 2.
  • the first VHH and the second VHH are humanized VHH.
  • the first VHH comprises the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2 and the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • the C-terminus of the first VHH may be linked to the N-terminus of the second VHH, and the C-terminus of the second VHH may be linked to the N-terminus of the first VHH. It may have been done. In one embodiment, the C-terminus of the first VHH is linked to the N-terminus of the second VHH.
  • the first VHH and the second VHH may be directly linked or may be linked via a peptide linker. In one embodiment, the first VHH and the second VHH are linked via a peptide linker.
  • the C-terminus of the first VHH is linked to the N-terminus of the second VHH via a peptide linker.
  • the first VHH and the second VHH are humanized VHHs, with the C-terminus of the first VHH linked to the N-terminus of the second VHH via a peptide linker.
  • the first VHH comprises the amino acid sequences of amino acids 1 to 116 of SEQ ID NO: 2
  • the second VHH consists of the amino acid sequences of amino acids 142 to 262 of SEQ ID NO: 2.
  • the C-terminus of the first VHH is linked to the N-terminus of the second VHH via a peptide linker.
  • the peptide linker linking the first VHH and the second VHH is not particularly limited, and is exemplified as a peptide linker linking the first VHH and the second VHH in the section ⁇ Multispecific antibody of the present invention>.
  • Peptide linkers that have been prepared can be used.
  • the peptide linker linking the first VHH and the second VHH is (Gly ⁇ Gly ⁇ Gly ⁇ Gly ⁇ Ser (SEQ ID NO: 7)) n [n is an integer of 1-5].
  • the peptide linker linking the first VHH and the second VHH is (Gly ⁇ Gly ⁇ Gly ⁇ Gly ⁇ Ser) 5 (amino acids 117 to 141 of SEQ ID NO: 2). ..
  • the first VHH and the second VHH are humanized VHHs, the C-terminus of the first VHH is linked to the N-terminus of the second VHH via a peptide linker, said first.
  • the peptide linker linking one VHH and the second VHH is (Gly, Gly, Gly, Gly, Ser) 5 (amino acids 117 to 141 of SEQ ID NO: 2).
  • the ActRIIA / B-binding peptide of the present invention comprises the amino acid sequence of amino acid numbers 1 to 262 of SEQ ID NO: 2.
  • a person skilled in the art can prepare a fusion of the ActRIIA / B-binding peptide of the present invention with another peptide or protein based on the present invention, or prepare a modified product to which a modifier is bound. These fusions and modifications are also included in the ActRIIA / B-binding peptide of the present invention.
  • Other peptides and proteins used for fusion are not particularly limited as long as the fusion binds to ActRIIA and ActRIIB, and for example, an antibody or an antigen-binding fragment thereof, human serum albumin, various tag peptides, artificial helix motif peptides, maltose binding.
  • the modifier used for the modification is not particularly limited as long as the modified product binds to ActRIIA and ActRIIB, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, and small molecule compounds.
  • the modifier used to modify the ActRIIA / B-binding peptide of the present invention is polyethylene glycol.
  • the ActRIIA / B-binding peptide of the present invention, a fusion thereof and a modified product thereof are based on the sequence information of VHH, other peptides or proteins (eg, antibodies) used in the fusion product, and information on the modifier used in the modified product. It can be readily made by one of ordinary skill in the art using methods known in the art.
  • the ActRIIA / B-binding peptide of the present invention is not particularly limited, but can be produced, for example, according to the method described in the section ⁇ Method for producing the multispecific antibody of the present invention>.
  • Example 1 Acquisition of Single VHH We immunized alpaca multiple times with a protein containing the extracellular domain of ActRIIB in order to acquire VHH that targets the extracellular domain of ActRIIB. After completion of immunization, blood is collected from alpaca, peripheral blood lymphocytes of alpaca are isolated from the collected sample, and a phage display is performed according to a known method (Miyazaki Net al., J Biochem. 2015, Vol. 158, p. 205-215). Immune library was constructed.
  • Biopanning for the extracellular domain of ActRIIB using a phage display is a stable expression cell line in which a human ActRIIB gene (NCBI accession number: NM_001106.4) is introduced into CHO cells, a protein containing the extracellular domain of ActRIIB (hereinafter referred to as “stable expression cell line”). This was performed on ActRIIB / CHO cells) and stable expression cell lines (hereinafter referred to as ActRIIB / HEK293 cells) in which the human ActRIIB gene (NCBI accession number: NM_001106.4) was introduced into HEK293 cells.
  • Biopanning of proteins containing the extracellular domain of ActRIIB was performed according to known methods (Miyazaki Net al., J Biochem. 2015, Vol. 158, p. 205-215).
  • biopanning for ActRIIB / CHO cells or ActRIIB / HEK293 cells the phage library and each cell are mixed, the washing operation is repeated several times, and then the phage bound to the cell is eluted from the phage / cell complex with acid. , Phage bound to each cell was obtained.
  • a plurality of phages that bind to the extracellular domain of ActRIIB were obtained.
  • the gene encoding VHH was cloned from the acquired phage and sequenced.
  • Example 2 Acquisition of tandem VHH Among the VHHs obtained in Example 1, genes encoding any two VHHs are encoded by peptide linkers (also referred to as GS linkers) of various lengths composed of Gly and Ser. A plurality of libraries for phage display linked with the gene to be used were constructed, and biopanning was performed in the same manner as in Example 1. Biopanning for the extracellular domain of ActRIIA and the extracellular domain of ActRIIB using a phage display was performed on proteins containing the extracellular domain of ActRIIA, proteins containing the extracellular domain of ActRIIB, and the human ActRIIA gene (NCBI accession number) in CHO cells.
  • peptide linkers also referred to as GS linkers
  • AB529011.1 was introduced into a stable expression cell line (hereinafter referred to as ActRIIA / CHO cells), and ActRIIB / CHO cells were used as subjects.
  • ActRIIA / CHO cells stable expression cell line
  • ActRIIB / CHO cells were used as subjects.
  • This tandem VHH is referred to as 75E9.
  • the tandem VHH75E9 was humanized according to the example of International Publication No. 2006/122825. This is referred to as humanized tandem VHH75E9.
  • the amino acid sequence of humanized tandem VHH75E9 is shown in amino acids 1 to 262 of SEQ ID NO: 2.
  • the first VHH in the humanized tandem VHH75E9 shown in amino acid numbers 1 to 262 of SEQ ID NO: 2 consists of the amino acid sequences of amino acid numbers 1 to 116 of SEQ ID NO: 2, and the second VHH is the amino acid number of SEQ ID NO: 2. It consists of an amino acid sequence from 142 to 262.
  • the first VHH CDR1, CDR2, and CDR3 of the humanized tandem VHH75E9 consist of amino acid sequences of amino acids 31 to 35, 50 to 65, and 98 to 105 of SEQ ID NO: 2, respectively.
  • the second VHH CDR1, CDR2, and CDR3 of the humanized tandem VHH75E9 consist of the amino acid sequences of amino acids 172 to 176, 195 to 210, and 243 to 251 of SEQ ID NO: 2, respectively.
  • Example 3 Acquisition of anti-Fn14 antibody
  • the present inventors according to Example 1 of International Publication No. 2020/090892 and extracellularly human Fn14.
  • Preparation of a human Fc fusion protein containing a domain, and construction of a stable expression cell line (hereinafter referred to as Fn14 / Jurkat cell) in which a human Fn14 gene (NCBI accession number: NM_016639) was introduced into Jurkat cells were performed, and immunization and screening were performed. Used for.
  • an antibody was prepared using a human monoclonal antibody development technology "Velosimune" (VelocImmune antibody technology: Regeneron (US Patent No.
  • the antibody obtained by the belosimune technique is an antibody having a variable region of a human antibody and a constant region of a mouse antibody.
  • Fn14 protein and Fn14 / Jurkat cells were alternately immunized.
  • lymphocytes collected from the lymph nodes of immunized mice were fused with mouse-derived myeloma cells SP2 / 0-Ag14 (ATCC: CRL-1581) to prepare a hybridoma and monocloned.
  • Hybridomas that bind to human Fn14 and Fn14 / Jurkat cells and produce antibodies that suppress NF- ⁇ B activation by TWEAK stimulation (hereinafter referred to as TWEAK-induced NF- ⁇ B activation in the examples below) are selected.
  • TWEAK-induced NF- ⁇ B activation hereinafter referred to as TWEAK-induced NF- ⁇ B activation in the examples below
  • a gene encoding a human Ig ⁇ 1 constant region having amino acid mutations of L234A and L235A on the 3'side of the gene encoding the heavy chain variable region of this antibody (corresponding to the base sequence encoding amino acids 273 to 390 of SEQ ID NO: 2).
  • SEQ ID NO: 13 A gene encoding the gene encoding the signal sequence (White Net al., Protein Eng. 1987, Vol. 1, p. 499-505) was ligated on the 5'side, and then the GS vector pEE6. It was inserted into .4 (Lonza).
  • the sequence obtained by translating SEQ ID NO: 13 into amino acids is SEQ ID NO: 14.
  • the gene encoding the signal sequence is placed on the 5'side of the gene encoding the light chain variable region of this antibody (corresponding to the base sequence encoding amino acids 1 to 108 of SEQ ID NO: 4), and the gene encoding the signal sequence is placed on the 3'side.
  • the genes encoding the constant region of the human kappa chain were ligated and inserted into the GS vector pEE12.4 (Lonza). From these GS vectors, a Double-Gene vector (hereinafter referred to as DGV) in which both heavy chain and light chain genes were inserted was constructed.
  • DGV Double-Gene vector
  • Antibodies were purified from the culture supernatant of CHO-K1SV cells transfected with this vector according to a conventional method. This antibody is referred to as STF8-1.
  • the heavy chain variable region of STF8-1 consists of the amino acid sequences of amino acid numbers 273 to 390 of SEQ ID NO: 2.
  • the light chain variable region of STF8-1 consists of the amino acid sequences of amino acids 1 to 108 of SEQ ID NO: 4.
  • CDR1, CDR2, and CDR3 of the heavy chain variable region of STF8-1 consist of amino acid sequences of amino acids 303 to 307, 322 to 338, and 371 to 379 of SEQ ID NO: 2, respectively.
  • the light chain variable regions CDR1, CDR2, and CDR3 of STF8-1 consist of amino acid sequences of amino acids 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 4, respectively.
  • Example 4 Preparation of humanized 75E9STF8-1
  • a gene encoding a human Ig ⁇ 1 constant region (corresponding to base numbers 1171 to 2160 of SEQ ID NO: 1) is linked in this order to GS vector pEE6.4 ( It was inserted into Lonza).
  • the gene encoding the signal sequence is located on the 5'side of the light chain variable region gene of STF8-1 (corresponding to base numbers 1 to 324 of SEQ ID NO: 3), and the constant region of the human kappa chain is located on the 3'side. (Corresponding to base numbers 325 to 642 of SEQ ID NO: 3) were ligated and inserted into the GS vector pEE12.4 (Lonza).
  • DGV in which both heavy chain and light chain genes were inserted was constructed.
  • Antibodies were purified from the culture supernatant of CHO-K1SV cells transfected with this vector according to a conventional method. This antibody is referred to as humanized 75E9STF8-1.
  • Example 5 Evaluation of binding activity of humanized 75E9STF8-1 The binding activity of humanized 75E9STF8-1 obtained in Example 4 to ActRIIA, ActRIIB and Fn14 was evaluated.
  • Phosphate buffers containing the extracellular domains of human Fn14 prepared in ActRIIA-His (LifeSpan Biosciences, LS-G39063), ActRIIB-His (LifeSpan Biosciences, LS-G38834) and Example 3, respectively. It was diluted to 1 ⁇ g / mL with saline (PBS), 15 ⁇ L of each was added to a maxi soap 384-well transparent plate (Nunc, 464718) per well, and the mixture was immobilized by incubating at 4 ° C.
  • PBS saline
  • TBS-T Tris-buffered saline
  • PBS containing 20% Blocking One Nacalai Tesque, 03953-95
  • a dilution series was prepared by diluting humanized 75E9STF8-1 from a maximum concentration of 30 ⁇ g / mL in 12 steps with a 4-fold common ratio, and 20 ⁇ L was added per well.
  • TBS-T containing 5% Blocking One was used as the diluent.
  • TBS-T As a detection antibody, 20 ⁇ L of Goat Anti-Human Kappa and Mouse ads-HRP (Southern Biotech, 2061-05) diluted 4000-fold with a diluent (TBS-T containing 5% Blocking One) was added per well. After incubating at room temperature for 1.5 hours, the mixture was washed with TBS-T, TMB + Substrate-Chromogen (DAKO, S159985) was added and allowed to stand, and then 1 M sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was defined as Infinite. (Registered trademark) Measured with 200Pro (TECAN).
  • DAKO Substrate-Chromogen
  • Example 6 Evaluation of Smad Phosphorylation Inhibition of Humanized 75E9STF8-1 The inhibitory effect of humanized 75E9STF8-1 on Myostatin-induced Smad3 phosphorylation in ActRIIA and ActRIIB was evaluated.
  • a stable expression cell line (hereinafter referred to as ActRIIA / HEK293 cells) in which a human ActRIIA gene (NCBI accession number: AB529011.1) was introduced into HEK293 cells, and ActRIIB / HEK293 cells were used for evaluation.
  • ActRIIA / HEK293 cells or ActRIIB / HEK293 cells were suspended in 2 ⁇ 10 5 cells / mL with 10% fetal bovine serum-containing DMEM (Sigma, D6429) and placed on a collagen I-coated 96-well plate (IWAKI, 4860-010). 100 ⁇ L was sown per well. It was cultured overnight in a CO 2 incubator set at 37 ° C. and 5% CO 2 . After culturing, the medium was removed by centrifugation, and 80 ⁇ L of DMEM containing 2% fetal bovine serum was added to each well. It was cultured overnight in a CO 2 incubator under the same conditions as above.
  • dilution series were prepared from the maximum concentration of 300 nmol / L for ActRIIA / HEK293 cells and from the maximum concentration of 1000 nmol / L for ActRIIB / HEK293 cells in 8 steps, each with a dilution ratio of about 3 times. 10 ⁇ L was added per well.
  • Bimagrumab of the same dilution series was added as a control.
  • DMEM containing 2% fetal bovine serum was used as the diluting solvent. It was cultured for 15 minutes in a CO 2 incubator under the same conditions as above.
  • myostatin (PEPROTECH, 120-00) prepared in DMEM containing 2% fetal bovine serum was added per well to a final concentration of 100 ng / mL. After culturing in a CO 2 incubator under the same conditions as above for 1 hour, the medium was centrifuged, and AlphaLISA (registered trademark) SureFire (registered trademark) Ultra TM SMAD3 (p-Ser423 / 425) assay kit (PerkinElmer, ALSU-PSM3). ), 50 ⁇ L of Lysis buffer was added per well, and the cells were lysed by stirring at room temperature for 10 minutes. Phosphorylated Smad3 in cytolysis was detected using the kit.
  • the results were shown with an inhibition rate of 0% inhibition of the measured value under myostatin 100 ng / mL stimulation conditions and 100% inhibition of the measured value in the absence of myostatin. Measurements in the absence of myostatin were performed by adding medium instead of myostatin. The data represents the average value of two trials (each trial was performed in duplicate).
  • humanized 75E9STF8-1 inhibited myostatin-induced phosphorylation of Smad3 in ActRIIA / HEK293 cells and ActRIIB / HEK293 cells (Fig. 1).
  • Example 7 Antagonist activity evaluation and agonist activity evaluation of humanized 75E9STF8-1
  • Fn14 When Fn14 is activated in the presence or absence of TWEAK, NF- ⁇ B is activated as a downstream signal.
  • the antagonist activity of humanized 75E9STF8-1 the inhibitory effect of TWEAK-induced NF- ⁇ B activation in human Fn14 was evaluated using this signal as an index.
  • the NF- ⁇ B activating effect in the absence of TWEAK was evaluated.
  • the effect of humanized 75E9STF8-1 on NF- ⁇ B activation in the presence or absence of TWEAK was evaluated by a reporter assay.
  • HEK293 cells (hereinafter referred to as NF- ⁇ B / HEK293 cells) into which the luciferase reporter vector pGL4.32 (Promega, E8941) incorporating the NF- ⁇ B transcription response sequence was stably introduced were prepared and used for evaluation. ..
  • NF- ⁇ B / HEK293 cells were suspended in DMEM (Sigma, D6429) containing 10% fetal bovine serum at 1.25x10 5 cells / mL, and 80 ⁇ L per well was seeded on a clear bottom white 96-well plate (Corning, 3610). did. It was cultured for 2 hours in a CO 2 incubator set at 37 ° C. and 5% CO 2 . A dilution series of humanized 75E9STF8-1 was prepared in the above medium, and then 10 ⁇ L was added per well.
  • the final concentration in each well was set to 11 steps (about 3 times common ratio) from the maximum concentration of 100 nmol / L in the antagonist activity evaluation, and 11 steps (about 3 times common ratio) from the maximum concentration of 300 nmol / L in the agonist activity evaluation.
  • STF8-1 of the same dilution series was added as a control.
  • 10 ⁇ L of TWEAK (PEPROTECH, 310-06) prepared in the above medium was added to each well to a final concentration of 100 ng / mL.
  • 10 ⁇ L of the above medium was added to each well. After culturing overnight at 37 ° C.
  • NF- ⁇ B activation was quantified by measuring the expression level of luciferase using the luciferase measurement reagent ONE-Glo TM Luciferase Assay System (Promega, E6120). It became. The results are shown by the inhibition rate of 0% inhibition of the measured value under TWEAK 100 ng / mL stimulation condition and 100% inhibition of the measured value in the absence of TWEAK in the antagonist activity evaluation, and TWEAK 100 ng / mL stimulation condition in the agonist activity evaluation. The measured value was 100%, and the measured value in the absence of TWEAK was 0%. The measurement in the absence of TWEAK was carried out by adding medium instead of TWEAK. The data represents the average value of two trials (each trial was performed in duplicate).
  • humanized 75E9STF8-1 completely inhibited Fn14-mediated NF- ⁇ B activation by 100 ng / mL TWEAK.
  • humanized 75E9STF8-1 did not induce NF- ⁇ B activation in the absence of TWEAK (Fig. 2). Therefore, it was found that humanized 75E9STF8-1 has antagonistic activity but not agonistic activity.
  • Example 8 Preparation and functional evaluation of antibody used in mouse in vivo drug efficacy evaluation
  • 75E9 based on the amino acid sequences of humanized tandem VHH75E9, STF8-1, and humanized 75E9STF8-1.
  • MFc mouse in vivo drug efficacy evaluation
  • STF8-1 mFc
  • 75E9STF8-1 mFc
  • 75E9 (mFc) is an antibody in which Hinge, CH2, and CH3 of the mouse IgG1 heavy chain constant region are linked to the C-terminal of 75E9 of tandem VHH in Example 2 (SEQ ID NO: 15).
  • STF8-1 (mFc) is an antibody in which the constant region of STF8-1 of Example 3 is replaced with a mouse constant region for both heavy and light chains (the heavy chain of STF8-1 (mFc) is shown in SEQ ID NO: 16 and STF8).
  • the light chain of -1 (mFc) is shown in SEQ ID NO: 17).
  • 75E9STF8-1 (mFc) the N-terminal of the STF8-1 (mFc) heavy chain is linked to the C-terminal of 75E9 in Example 2 with a peptide linker (Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 7)) 2 .
  • It is an antibody consisting of the peptide (SEQ ID NO: 18) and the light chain of STF8-1 (mFc).
  • the amino acid sequences of the first VHH CDR1, CDR2, CDR3 of 75E9STF8-1 (mFc) and the second VHH CDR1, CDR2, CDR3 of 75E9STF8-1 (mFc) are the first VHH of humanized 75E9STF8-1, respectively. It is the same as the amino acid sequence of CDR1, CDR2, CDR3 of the second VHH of CDR1, CDR2, CDR3 and humanized 75E9STF8-1.
  • the heavy chain variable region and the light chain variable region of STF8-1 (mFc) contained in 75E9STF8-1 (mFc) are the heavy chain variable region and the light chain variable region of STF8-1 contained in humanized 75E9STF8-1, respectively. Is the same as.
  • 75E9 (mFc) and 75E9STF8-1 (mFc) were evaluated as described in Example 6 and confirmed to have Smad phosphorylation inhibitory activity. Further, the functional evaluation described in Example 7 was performed, and STF8-1 (mFc) and 75E9STF8-1 (mFc) had antagonistic activity, and 75E9STF8-1 (mFc) had no agonistic activity. It was confirmed.
  • Example 9 Evaluation of in vivo drug efficacy against steroid-induced myopathy model mice
  • in vivo drug efficacy on muscle atrophy and muscle function was evaluated using muscle weight and grip strength as indicators.
  • a muscular atrophy model animal a steroid-induced myopathy model (SIM model) by continuous administration of steroids was used, and the limb grip force and the excised quadriceps muscle weight were measured on the 14th day after continuous administration of the test substance.
  • SIM model steroid-induced myopathy model
  • the body weight and grip strength of the mice were measured, and 10 cases were assigned to each group so that the body weight and grip strength were even among the groups.
  • the group composition is a total of 6 groups, 1 group for the normal group and 5 groups for the SIM model group, and 5 groups of the SIM model are the solvent administration group, 75E9 (mFc) group, STF8-1 (mFc) group, and 75E9 (mFc).
  • the + STF8-1 (mFc) group and the 75E9STF8-1 (mFc) group were used.
  • Corticosterone dissolved in filtered water mixed with 1% ethanol at a concentration of 100 ⁇ g / mL was continuously administered as free drinking water for 14 days to the SIM model group to induce muscle atrophy.
  • test substance 1 and test substance 2 were started on the same day as the start of administration of corticosterone. Details of administration of the test substance in each group are shown in Table 1.
  • the dose of the 75E9STF8-1 (mFc) group was set so as to have a molar concentration similar to the dose of each single administration group.
  • the grip strength was measured with a small animal grip strength measuring device (MELQUEST, GPM-100). Then, the mice were exsanguinated under anesthesia, and the quadriceps muscle was removed and weighed.
  • 75E9 (mFc) or STF8-1 (mFc) partially suppressed skeletal muscle atrophy and weakness in the SIM model. Furthermore, the mixed treatment of 75E9 (mFc) and STF8-1 (mFc) showed a significantly stronger skeletal muscle atrophy inhibitory effect and grip strength decrease inhibitory effect than the respective single administrations. In addition, 75E9STF8-1 (mFc) showed a significantly stronger skeletal muscle atrophy inhibitory effect and grip strength reduction inhibitory effect as compared with the mixed treatment of 75E9 (mFc) and STF8-1 (mFc) (FIG. 3).
  • Example 10 Evaluation of in vivo drug efficacy against steroid-induced myopathy model monkeys
  • the in vivo drug efficacy was evaluated for thigh fat-free weight 4 weeks after administration of the test substance.
  • the non-fat weight is the total weight of muscle, bone, blood, etc. excluding fat, and is known to correlate with skeletal muscle mass (Wowski CO et al., Nutrients. 2020, Vol. 12, 755). ).
  • the lean body mass of the thigh of the cynomolgus monkey was measured with a bone mineral density measuring device (Holographic, Discovery C) by the dual energy X-ray absorption measurement method, and the cynomolgus monkeys were assigned to 4 groups so as to be uniform among the groups.
  • Prednisolone (30 mg / 3 mL / kg) was administered subcutaneously to the back of the SIM model group twice daily 4-5 days / week to induce muscular atrophy.
  • Physiological saline was similarly administered subcutaneously to the back of the normal group.
  • the test substances were PBS for the normal group and the solvent-administered group, humanized 75E9STF8-1 for the humanized 75E9STF8-1 group 40 mg / 2.4 mL / kg, and Bimagrumab 30 mg / 2.4 mL / kg for the Bimagrumab group.
  • Prednisolone was administered intravenously on the first day of administration.
  • the administration dose was set so that the number of moles of administration of humanized 75E9STF8-1 and Bimagrumab was about the same.
  • Thigh lean body mass was measured 4 weeks after administration of the test substance.
  • Lean body mass was measured on each of the left and right thighs, and the average value was used as the measured value of the individual.
  • the thigh fat-free weight of the humanized 75E9STF8-1 group was significantly heavier than the thigh fat-free weight of the solvent-administered group. No significant difference was observed in the Bimagramab group as compared with the solvent-administered group (Fig. 4).
  • the multispecific antibody of the present invention is useful for the prevention or treatment of various diseases in which human ActRIIA, human ActRIIB and human Fn14 are involved in pathogenesis.
  • the method for producing a polynucleotide, an expression vector, a transformed host cell and an antibody of the present invention is expected to be useful for producing the multispecific antibody.
  • the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing binds to the first VHH of the multispecific antibody, the second VHH of the multispecific antibody, and Fn14 of the multispecific antibody. It is a base sequence encoding a polypeptide containing a heavy chain of an antibody, and the base sequence represented by SEQ ID NO: 3 is a base sequence encoding the light chain of an antibody that binds to Fn14 of the multispecific antibody.
  • the amino acid sequence represented by SEQ ID NO: 2 binds to the first VHH of the multispecific antibody encoded by SEQ ID NO: 1, the second VHH of the multispecific antibody, and Fn14 of the multispecific antibody.
  • the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of the light chain of the antibody that binds to Fn14 of the multispecific antibody encoded by SEQ ID NO: 3.
  • SEQ ID NOs: 5-12 are the amino acid sequences of exemplary peptide linkers.
  • the base sequence represented by SEQ ID NO: 13 is a base sequence encoding the heavy chain of an antibody that binds to Fn14
  • the amino acid sequence represented by SEQ ID NO: 14 is an amino acid sequence encoded by the base sequence of SEQ ID NO: 13. Is.
  • the amino acid sequence represented by SEQ ID NO: 15 is the amino acid sequence of an antibody in which a mouse constant region is linked to the C-terminal of tandem VHH.
  • the amino acid sequence represented by SEQ ID NO: 16 is the amino acid sequence of the heavy chain of the antibody that binds to Fn14
  • the amino acid sequence represented by SEQ ID NO: 17 is the amino acid sequence of the light chain of the antibody that binds to Fn14.
  • the amino acid sequence represented by SEQ ID NO: 18 is a polypeptide containing a heavy chain of an antibody that binds to tandem VHH and Fn14.

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CN116802298A (zh) 2023-09-22
MX2023008372A (es) 2023-07-26
JPWO2022153997A1 (https=) 2022-07-21

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