Multispecific antibodies, compositions containing the same, and vectors, as well as their uses

Abstract

The present disclosure provides multispecific antibodies with increased in vivo sustainability, which comprise one or more biologically active effector moieties linked to one or both of the N-terminus and C-terminus of an antigen-binding fragment, Fab, that binds human serum albumin. TIFF2023511963000159.tif51170

Description

[Technical Field] 【0001】 Cross-reference of related applications This application claims priority to Korean Patent Application No. 10-2020-0009565 filed on 24 January 2020 and U.S. Patent Application No. 16 / 878,255 filed on 19 May 2020, the entirety of which disclosures are incorporated herein by reference. 【0002】 Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entire listing of which is incorporated herein by reference. The ASCII copy was created on January 19, 2021, as 2662-0001WO01_Sequence Listing_ST25.txt and is 68 KB in size. 【0003】 field This disclosure relates to a fusion construct comprising an antigen-binding fragment and a bioactive effector moiety. More specifically, this disclosure relates to a multispecific antibody comprising two or more bioactive effector moieties ligated to one or both of the N-terminus and C-terminus of an antigen-binding fragment that binds to human serum albumin. [Background technology] 【0004】 background The CD40-CD40L interaction is essential to the development of antigen-specific antibody immune responses, and autoantibodies are associated with the development of various autoimmune diseases. To effectively treat these diseases, various CD40L-specific or CD40-specific antibodies that can inhibit and / or suppress the CD40-CD40L interaction have been studied. For example, anti-CD40L monoclonal antibodies such as hu5c8 IgG1 (BG-9588, luprizumab, Antova®, Biogen, Cambridge, Massachusetts) and IDEC-131 (E6040, IDEC Pharmaceuticals, San Diego, California) have been studied for the treatment of various autoimmune diseases, including systemic lupus erythematosus (SLE) and idiopathic thrombocytopenic purpura (ITP), but further development of such antibodies has been interrupted by the occurrence of side effects such as thromboembolism. Therefore, many research groups are attempting approaches to address the problem of thromboembolic side effects through Fc engineering, and several reports have been submitted, including, for example, PEGylated anti-CD40L Fab, CDP7657 (dapyrolizumab pegol, Biogen), and the TN3-HSA fusion protein VIB4920 (VIELABIO, Gaithersburg, Maryland) designed to link to CD40L. In connection with this, other research groups are developing therapeutic agents that target CD40 rather than CD40L, such as the BI655064 antibody with weakened Fc function (Boehringer Ingelheim, Germany) and the human IgG4 type bleselumab antibody (Kyowa Kirin Pharmaceutical Development, La Jolla, California). [Overview of the project] 【0005】 overview This disclosure provides a multispecific antibody having a long in vivo retention time. This disclosure also provides a pharmaceutical composition comprising the multispecific antibody. This disclosure also provides a method for producing the multispecific antibody. 【0006】 For example, this specification discloses a novel autoimmune disease treatment that suppresses CD40-CD40L signaling while eliminating the Fc-based thromboembolic side effect of anti-CD40L antibodies. To achieve this, the variable region gene V of the CD40L-binding luprizumab antibody hu5c8 is disclosed. H and V L By ligating a single-stranded variable fragment (scFv) consisting of the above to the N-terminus of SL335 Fab, a recombinant bispecific antibody represented as (anti-CD40L scFv)2-(anti-HSA Fab)-(anti-TNF-α Fv) has been developed that can maintain serum sustainability without an Fc region. Furthermore, this specification discloses the identified functions and characteristics of a multispecific antibody represented as (anti-CD40L scFv)2-(anti-HSA Fab)-(anti-TNF-α Fv), and the antibody protein produced, by ligating an Fv or dsFv containing the variable region gene of a TNF-α-binding certolizumab pegol antibody to the C-terminus of SL335 Fab of the bispecific antibody using a peptide linker. 【0007】 In this specification, structural formula A multispecific antibody containing TIFF0007875806000001.tif16128, In the formula, the antigen-binding fragment (Fab) is serum albumin Fab; R 1 and R 2 These are bioactive effector regions ligated to the N-terminus of Fab, each ligated to either the heavy-chain variable domain or the light-chain variable domain of Fab; R 3 and R 4 These are bioactive effector regions ligated to the C-terminus of Fab, each ligated to either the heavy-chain variable domain or the light-chain variable domain of Fab; m is an integer greater than or equal to 0 or 1; n is an integer greater than or equal to 0 or 1. A multispecific antibody is provided. In some embodiments, R 1 and R 2are the same or different single-chain variable fragments (scFvs). In some embodiments, R 3 and R 4 are the same or different Fv fragments or disulfide-stabilized Fv (dsFv) fragments. 【0008】 In some embodiments, R 1 , R 2 , R 3 , and R 4 can each be linked to the Fab by one or more linkers. Each linker can comprise 1 to 20 amino acids. Each linker can comprise an amino acid sequence having at least 90% identity to SEQ ID NO:3 or SEQ ID NO:4. Each linker can comprise the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. 【0009】 In some embodiments, the Fab (a) a heavy-chain complementarity-determining region 1 (CDR1) comprising the amino acid sequence SYGIS (SEQ ID NO:61), an amino acid sequence TIFF0007875806000002.tif4128 comprising the heavy-chain CDR2, and an amino acid sequence TIFF0007875806000003.tif4128 comprising the heavy-chain CDR3; (b) a heavy-chain complementarity-determining region 1 (CDR1) comprising the amino acid sequence SYGIS (SEQ ID NO:61), an amino acid sequence TIFF0007875806000004.tif4128 comprising the heavy-chain CDR2, and an amino acid sequence TIFF0007875806000005.tif4128 comprising the heavy-chain CDR3; (c) a heavy-chain complementarity-determining region 1 (CDR1) comprising the amino acid sequence NYGIH (SEQ ID NO:65), an amino acid sequence TIFF0007875806000006.tif4128 comprising the heavy-chain CDR2, and an amino acid sequence TIFF0007875806000007.tif4128 comprising the heavy-chain CDR3; (d) Amino acid sequence SYAMS (SEQ ID NO:68) containing heavy chain complementarity determination domain 1 (CDR1), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000008.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000009.tif4128; (e) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYWIA (SEQ ID NO: 71), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000010.tif4128, and heavy chain CDR3 containing amino acid sequence LYSGSYSP (SEQ ID NO:73); or (f) Amino acid sequence AYSMN (SEQ ID NO: 74) containing heavy chain complementarity-determining domain 1 (CDR1), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000011.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000012.tif4128 It includes heavy chain variable domains. 【0010】 In some embodiments, Fab is (g) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000013.tif4128, light chain CDR2 containing amino acid sequence GASRLES (SEQ ID NO: 78), and light chain CDR3 containing amino acid sequence QQSDSVPVT (SEQ ID NO: 79); (h) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000014.tif4128, light chain CDR2 containing amino acid sequence AASSLQS (SEQ ID NO:81), and amino acid sequence Light chain CDR3 containing TIFF0007875806000015.tif4128; (i) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000016.tif4128, light chain CDR2 containing amino acid sequence DASNRAT (SEQ ID NO:84), and amino acid sequence Light chain CDR3 containing TIFF0007875806000017.tif4128; (j) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000018.tif4128, light chain CDR2 containing amino acid sequence GASSRAT (SEQ ID NO: 87), and light chain CDR3 containing amino acid sequence QQYGSSPRT (SEQ ID NO: 88); (k) amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000019.tif4128, light chain CDR2 containing amino acid sequence GASSRAT (SEQ ID NO: 87), and light chain CDR3 containing amino acid sequence QKYSSYPLT (SEQ ID NO: 90); or (l) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000020.tif4128, light chain CDR2 containing amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence Light chain CDR3 containing TIFF0007875806000021.tif4128 Includes light chain variable domains. 【0011】 In some embodiments, Fab is Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000022.tif4128, and amino acid sequence Heavy-chain CDR3 including TIFF0007875806000023.tif4128, and amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000024.tif4128, light chain CDR2 containing amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence Light chain CDR3 containing TIFF0007875806000025.tif4128 Includes. 【0012】 In some embodiments, the Fab includes a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99. 【0013】 In some embodiments, the Fab includes a light chain variable domain having an amino acid sequence having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. 【0014】 In some embodiments, the Fab comprises a heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99, and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. 【0015】 In some embodiments, Fab comprises a heavy chain domain (V) containing the amino acid sequence of SEQ ID NO:45. H -C H1 The domain, and the light chain domain (V) containing the amino acid sequence of SEQ ID NO:46 L -C L Includes domains. 【0016】 In some embodiments, R 1 and R 2 These are, respectively, anti-CD40L hu5c8 scFv. 1 and R 2These may each be anti-CD40L hu5c8 scFv containing an amino acid sequence having at least 80% identity with SEQ ID NO:47 or SEQ ID NO:48. 1 and R 2 These could each be an anti-CD40L hu5c8 scFv containing the amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48, respectively. 【0017】 In some embodiments, R 3 and R 4 Each of these is one or more bioactive effector moieties, including anti-TNF-α Fv, anti-TNF-α disulfide-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and / or anti-IFNAR1 dsFv. 3 and R 4 These each contain an anti-TNF-α Fv containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:49 and a light chain amino acid sequence with 80% identity to SEQ ID NO:50, an anti-TNF-α disulfide-stabilized Fv (dsFv) containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:51 and a light chain amino acid sequence with 80% identity to SEQ ID NO:52, an anti-IL-23 Fv containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:53 and a light chain amino acid sequence with 80% identity to SEQ ID NO:54, an anti-IL-23 dsFv containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:55 and a light chain amino acid sequence with 80% identity to SEQ ID NO:56, and a heavy chain amino acid sequence with 80% identity to SEQ ID NO:57 and SEQ ID It may be one or more bioactive effector moieties comprising anti-IFNAR1 containing a light chain amino acid sequence having 80% identity with NO:58, and / or anti-IFNAR1 dsFv containing a heavy chain amino acid sequence having 80% identity with SEQ ID NO:59 and a light chain amino acid sequence having 80% identity with SEQ ID NO:60. 3 and R 4Each of these may comprise one or more bioactive effector moieties, including an anti-TNF-α Fv containing the heavy chain of SEQ ID NO:49 and the light chain of SEQ ID NO:50, an anti-TNF-α disulfide-stabilized Fv (dsFv) containing the heavy chain of SEQ ID NO:51 and the light chain of SEQ ID NO:52, an anti-IL-23 Fv containing the heavy chain of SEQ ID NO:53 and the light chain of SEQ ID NO:54, an anti-IL-23 dsFv containing the heavy chain of SEQ ID NO:55 and the light chain of SEQ ID NO:56, an anti-IFNAR1 containing the heavy chain of SEQ ID NO:57 and the light chain of SEQ ID NO:58, and / or an anti-IFNAR1 dsFv containing the heavy chain of SEQ ID NO:59 and the light chain of SEQ ID NO:60. 【0018】 This specification discloses compositions comprising the multispecific antibodies and excipients disclosed herein. This specification also discloses pharmaceutical compositions comprising the multispecific antibodies and pharmaceutically acceptable excipients disclosed herein. 【0019】 This specification also discloses a method for treating autoimmune diseases in subjects where such treatment is needed, the method comprising the step of administering a pharmaceutical composition disclosed herein to the subject. 【0020】 This specification further states, (a) promoter, (b) A first nucleic acid molecule encoding an antigen-binding fragment (Fab) that binds to serum albumin, and (c) A second nucleic acid molecule encoding the bioactive effector moiety and linker. Expression vectors including are further disclosed, The promoter, the first nucleic acid sequence, and the second nucleic acid molecule are functionally linked. The second nucleic acid molecule may encode two or more bioactive effector moieties and linkers. 【0021】 In some embodiments, the first nucleic acid molecule is (a) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000026.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000027.tif4128; (b) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000028.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000029.tif4128; (c) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence NYGIH (SEQ ID NO:65), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000030.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000031.tif4128; (d) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 68), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000032.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000033.tif4128; (e) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYWIA (SEQ ID NO: 71), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000034.tif4128, and Heavy chain CDR3 containing amino acid sequence LYSGSYSP (SEQ ID NO:73); or (f) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000035.tif4128, and Heavy chain CDR3 containing amino acid sequence ETVMAGKALDY (SEQ ID NO:76) It contains a nucleic acid sequence encoding Fab, which includes a heavy chain variable domain. 【0022】 In some embodiments, the first nucleic acid molecule is (g) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000036.tif4128, Light chain CDR2 containing the amino acid sequence GASRLES (SEQ ID NO: 78), and Light chain CDR3 containing amino acid sequence QQSDSVPVT (SEQ ID NO: 79); (h) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000037.tif4128, Light chain CDR2 containing amino acid sequence AASSLQS (SEQ ID NO:81), and amino acid sequence Light chain CDR3 containing TIFF0007875806000038.tif4128; (i) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000039.tif4128, Light chain CDR2 containing amino acid sequence DASNRAT (SEQ ID NO: 84), and amino acid sequence Light chain CDR3 containing TIFF0007875806000040.tif4128; (j) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000041.tif4128, Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QQYGSSPRT (SEQ ID NO: 88); (k) amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000042.tif4128, Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QKYSSYPLT (SEQ ID NO:90); or (l) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000043.tif4128, Light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence Light chain CDR3 containing TIFF0007875806000044.tif4128 It contains a nucleic acid sequence encoding Fab, which includes a light chain variable domain. 【0023】 In some embodiments, the first nucleic acid molecule is Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000045.tif4128, and amino acid sequence Heavy-chain CDR3 including TIFF0007875806000046.tif4128, and amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000047.tif4128, light chain CDR2 containing amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence Light chain CDR3 containing TIFF0007875806000048.tif4128 Includes a nucleic acid sequence that codes for Fab. 【0024】 In other embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab which includes a heavy chain variable domain having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99. 【0025】 In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab which includes a light chain variable domain having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. 【0026】 In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding Fab, each comprising a heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99, and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. In some embodiments, the first nucleic acid molecule comprises a heavy chain domain (V) containing the amino acid sequence of SEQ ID NO: 45. H -C H1 The domain, and the light chain domain (V) containing the amino acid sequence of SEQ ID NO:46 L -C L Contains a nucleic acid sequence encoding a Fab, including the domain. 【0027】 In some embodiments, the bioactive effector moiety is anti-TNF-α Fv, anti-TNF-α dsFv, anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1 Fv, and / or anti-IFNAR1 dsFv. The second nucleic acid molecule may contain a nucleotide sequence encoding one or more amino acid sequences from SEQ ID NO: 49-60. 【0028】 This disclosure provides host cells, such as animal cells, that contain expression vectors, for example, CHO cell lines. [Invention 1001] Structural formula TIFF0007875806000049.tif21128 A multispecific antibody containing, In the formula, the antigen-binding fragment (Fab) is serum albumin Fab; R 1 and R 2 These are bioactive effector regions ligated to the N-terminus of Fab, and are linked to either the heavy-chain variable domain or the light-chain variable domain of Fab, respectively; R 3 and R 4 These are bioactive effector regions ligated to the C-terminus of Fab, and are linked to either the heavy-chain variable domain or the light-chain variable domain of Fab, respectively; m is an integer greater than or equal to 0 or 1; n is an integer greater than or equal to 0 or 1. The multispecific antibody. [Invention 1002] R 1 and R 2 The antibody of the present invention 1001, which is the same or different single-stranded variable fragment (scFv). [Invention 1003] R 3 and R 4 The antibody of Invention 1001 or 1002, wherein the antibody is the same or a different Fv fragment or disulfide-stabilized Fv (dsFv) fragment. [Invention 1004] R 1 、R 2 、R 3 , and R 4 Each antibody according to one of the invention 1001 to 1003 is linked to the Fab by one or more linkers. [Invention 1005] Fab, (a) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence TIFF0007875806000050.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000051.tif4128 Heavy chain CDR3 containing; (b) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence TIFF0007875806000052.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000053.tif4128 Heavy chain CDR3 containing; (c) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence NYGIH (SEQ ID NO:65), amino acid sequence TIFF0007875806000054.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000055.tif4128 Heavy chain CDR3 containing; (d) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 68), amino acid sequence TIFF0007875806000056.tif5128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000057.tif4128 Heavy chain CDR3 containing; (e) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYWIA (SEQ ID NO: 71), amino acid sequence TIFF0007875806000058.tif4128 Heavy chain CDR2 including, and Heavy chain CDR3 containing amino acid sequence LYSGSYSP (SEQ ID NO:73); or (f) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence TIFF0007875806000059.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000060.tif4128 heavy chain CDR3 An antibody according to any of the present invention 1001 to 1004, comprising a heavy chain variable domain. [Invention 1006] Fab, (g) Amino acid sequence TIFF0007875806000061.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASRLES (SEQ ID NO: 78), and Light chain CDR3 containing amino acid sequence QQSDSVPVT (SEQ ID NO: 79); (h) Amino acid sequence TIFF0007875806000062.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing amino acid sequence AASSLQS (SEQ ID NO:81), and amino acid sequence TIFF0007875806000063.tif4128 Light chain CDR3 containing; (i) Amino acid sequence TIFF0007875806000064.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing amino acid sequence DASNRAT (SEQ ID NO: 84), and amino acid sequence TIFF0007875806000065.tif4128 Light chain CDR3 containing; (j) Amino acid sequence TIFF0007875806000066.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QQYGSSPRT (SEQ ID NO: 88); (k) amino acid sequence TIFF0007875806000067.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QKYSSYPLT (SEQ ID NO:90); or (l) Amino acid sequence TIFF0007875806000068.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence TIFF0007875806000069.tif4128 Light chain CDR3 An antibody according to any of the invention 1001 to 1005, comprising a light chain variable domain. [Invention 1007] Fab, Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence TIFF0007875806000070.tif4128 Heavy chain CDR2 containing, and amino acid sequence TIFF0007875806000071.tif4128 Heavy chain CDR3 including, amino acid sequence TIFF0007875806000072.tif4128 Light chain complementarity determination domain 1 (CDR1) containing, light chain CDR2 containing amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence TIFF0007875806000073.tif4128 Light chain CDR3 An antibody according to any of the present invention 1001 to 1006, including the above. [Invention 1008] An antibody according to any of Invention 1001 to 1007, wherein Fab comprises a heavy chain variable domain having an amino acid sequence having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99. [Invention 1009] An antibody according to any of Invention 1001 to 1008, wherein Fab contains a light chain variable domain having an amino acid sequence having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. [Invention 1010] An antibody according to any of Invention 1001 to 1009, wherein Fab comprises a heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99, and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. [Invention 1011] Fab contains the heavy chain domain (V) containing the amino acid sequence of SEQ ID NO:45. H -C H1 The domain, and the light chain domain (V) containing the amino acid sequence of SEQ ID NO:46 L -C L An antibody according to any of the present invention 1001 to 1010, including the domain. [Invention 1012] Each linker contains 1 to 20 amino acids, and each antibody is one of the present inventions 1001 to 1011. [Invention 1013] An antibody according to any of Invention 1001 to 1012, wherein each linker contains an amino acid sequence having at least 90% identity with SEQ ID NO:3 or SEQ ID NO:4. [Invention 1014] An antibody according to any of Invention 1001 to 1013, wherein each linker contains the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. [Invention 1015] R 1 and R 2 Each of these is an antibody according to one of the present invention 1001 to 1014, which is anti-CD40L hu5c8 scFv. [Invention 1016] R 1 and R 2 An antibody according to any of the present invention 1001 to 1015, wherein each of these is an anti-CD40L hu5c8 scFv containing an amino acid sequence having at least 80% identity with SEQ ID NO:47 or SEQ ID NO:48. [Invention 1017] R 1 and R 2 The antibodies according to any of the present invention 1001 to 1016, wherein each of these is an anti-CD40L hu5c8 scFv containing the amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48. [Invention 1018] R 3 and R 4 An antibody according to any of the present invention 1001 to 1017, wherein each of these is one or more biologically active effector moieties selected from the group consisting of anti-TNF-α Fv, anti-TNF-α disulfide-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and anti-IFNAR1 dsFv. [Invention 1019] R 3 and R 4 Each of these includes an anti-TNF-α Fv containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:49 and a light chain amino acid sequence with 80% identity to SEQ ID NO:50, an anti-TNF-α disulfide-stabilized Fv (dsFv) containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:51 and a light chain amino acid sequence with 80% identity to SEQ ID NO:52, an anti-IL-23 Fv containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:53 and a light chain amino acid sequence with 80% identity to SEQ ID NO:54, an anti-IL-23 dsFv containing a heavy chain amino acid sequence with 80% identity to SEQ ID NO:55 and a light chain amino acid sequence with 80% identity to SEQ ID NO:56, and a heavy chain amino acid sequence with 80% identity to SEQ ID NO:57 and SEQ ID An antibody according to any of the inventions 1001 to 1018, comprising one or more bioactive effector moieties, including anti-IFNAR1 containing a light chain amino acid sequence having 80% identity with NO:58, and / or anti-IFNAR1 dsFv containing a heavy chain amino acid sequence having 80% identity with SEQ ID NO:59 and a light chain amino acid sequence having 80% identity with SEQ ID NO:60. [Invention 1020] R 3 and R 4 An antibody according to any of the Invention 1001 to 1019, wherein each of these is one or more bioactive effector moieties comprising: anti-TNF-α Fv containing a heavy chain of SEQ ID NO:49 and a light chain of SEQ ID NO:50; anti-TNF-α disulfide-stabilized Fv (dsFv) containing a heavy chain of SEQ ID NO:51 and a light chain of SEQ ID NO:52; anti-IL-23 Fv containing a heavy chain of SEQ ID NO:53 and a light chain of SEQ ID NO:54; anti-IL-23 dsFv containing a heavy chain of SEQ ID NO:55 and a light chain of SEQ ID NO:56; anti-IFNAR1 containing a heavy chain of SEQ ID NO:57 and a light chain of SEQ ID NO:58; and / or anti-IFNAR1 dsFv containing a heavy chain of SEQ ID NO:59 and a light chain of SEQ ID NO:60. [Invention 1021] A composition comprising any of the multispecific antibodies specified in Invention 1001 to 1020, and an excipient. [Invention 1022] A pharmaceutical composition comprising any of the multispecific antibodies according to invention 1001 to 1020, and a pharmaceutically acceptable excipient. [Invention 1023] A method for treating an autoimmune disease in a subject in need thereof, comprising the step of administering a pharmaceutical composition of the present invention 1022 to the subject. [Invention 1024] (a) promoter, (b) A first nucleic acid molecule encoding an antigen-binding fragment (Fab) that binds to serum albumin, and (c) A second nucleic acid molecule encoding the bioactive effector moiety and linker. An expression vector comprising, A vector in which the promoter, the first nucleic acid sequence, and the second nucleic acid molecule are functionally linked. [Invention 1025] A vector according to the present invention 1024, wherein a second nucleic acid molecule encodes two or more bioactive effector moieties and a linker. [Invention 1026] The first nucleic acid molecule, (a) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence TIFF0007875806000074.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000075.tif4128 Heavy chain CDR3 containing; (b) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence TIFF0007875806000076.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000077.tif4128 Heavy chain CDR3 containing; (c) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence NYGIH (SEQ ID NO:65), amino acid sequence TIFF0007875806000078.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000079.tif4128 Heavy chain CDR3 containing; (d) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 68), amino acid sequence TIFF0007875806000080.tif4128 Heavy chain CDR2 including, and Heavy chain CDR3 containing amino acid sequence AGWLRQYGMDV (SEQ ID NO: 70); (e) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYWIA (SEQ ID NO: 71), amino acid sequence TIFF0007875806000081.tif4128 Heavy chain CDR2 including, and Heavy chain CDR3 containing amino acid sequence LYSGSYSP (SEQ ID NO:73); or (f) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence TIFF0007875806000082.tif4128 Heavy chain CDR2 including, and amino acid sequence TIFF0007875806000083.tif4128 heavy chain CDR3 A vector according to the present invention 1024 or 1025, comprising a nucleic acid sequence encoding Fab, which includes a heavy chain variable domain. [Invention 1027] The first nucleic acid molecule, (g) Amino acid sequence TIFF0007875806000084.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASRLES (SEQ ID NO: 78), and Light chain CDR3 containing amino acid sequence QQSDSVPVT (SEQ ID NO: 79); (h) Amino acid sequence TIFF0007875806000085.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing amino acid sequence AASSLQS (SEQ ID NO:81), and amino acid sequence TIFF0007875806000086.tif4128 Light chain CDR3 containing; (i) Amino acid sequence TIFF0007875806000087.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing amino acid sequence DASNRAT (SEQ ID NO: 84), and amino acid sequence TIFF0007875806000088.tif4128 Light chain CDR3 containing; (j) Amino acid sequence TIFF0007875806000089.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QQYGSSPRT (SEQ ID NO: 88); (k) amino acid sequence TIFF0007875806000090.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QKYSSYPLT (SEQ ID NO:90); or (l) Amino acid sequence TIFF0007875806000091.tif4128 Light chain complementarity determination domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence TIFF0007875806000092.tif4128 Light chain CDR3 A vector according to any one of the invention 1024 to 1026, comprising a nucleic acid sequence encoding Fab, which includes a light chain variable domain. [Invention 1028] The first nucleic acid molecule, Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence TIFF0007875806000093.tif4128 Heavy chain CDR2 containing, and amino acid sequence TIFF0007875806000094.tif4128 Heavy chain CDR3 including, amino acid sequence TIFF0007875806000095.tif4128 Light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence GASTGAT (SEQ ID NO:92), light chain CDR2 containing the amino acid sequence QQYYSFLAKT (SEQ ID NO:93), and light chain CDR3 containing the amino acid sequence QQYYSFLAKT (SEQ ID NO:93). A vector comprising a nucleic acid sequence encoding Fab, any of invention 1024 to 1027. [Invention 1029] A vector according to any one of Invention 1024 to 1028, wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab which includes a heavy chain variable domain having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99. [Invention 1030] A vector according to any one of the invention 1024 to 1029, wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab which includes a light chain variable domain having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. [Invention 1031] A vector according to any of Invention 1024 to 1030, wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding Fab, each comprising a heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99, and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. [Invention 1032] The first nucleic acid molecule contains a heavy chain domain (V) with the amino acid sequence of SEQ ID NO:45. H -C H1 The domain, and the light chain domain (V) containing the amino acid sequence of SEQ ID NO:46 L -C L A vector according to any of invention 1024 to 1031, comprising a nucleic acid sequence encoding a Fab including a domain. [Invention 1033] A vector according to any of Invention 1024-1032, wherein the bioactive effector portion is anti-TNF-α Fv, anti-TNF-α dsFv, anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1 Fv, and / or anti-IFNAR1 dsFv. [Invention 1034] A vector according to any of Invention 1023 to 1033, wherein the second nucleic acid molecule contains a nucleotide sequence encoding one or more amino acid sequences from SEQ ID NO: 49 to 60. 【Brief Description of the Drawings】 【0029】 The above-mentioned and other aspects, features, and advantages of the specific embodiments of the present disclosure will become more apparent from the following description in conjunction with the accompanying drawings. 【0030】 [Figure 1A] Figures 1A and 1B show the vector map and amino acid sequence of APB-A1. [Figure 1B] Refer to the description of Figure 1A. [Figure 2A] Figures 2A and 2B show the results of HPLC analysis and SDS-PAGE of APB-A1. [Figure 2B] Refer to the description of Figure 2A. [Figure 3] Figure 3 shows the results of mass spectrometry of APB-A1. [Figure 4A]Figures 4A and 4B show the PI values ​​of APB-A1. [Figure 4B] See the explanation in Figure 4A. [Figure 5] Figure 5 shows the change in the charge of APB-A1. [Figure 6] Figure 6 shows the simultaneous binding of rhCD40L-APB-A1-HSA. [Figure 7] Figure 7 shows the results of the flow cytometry analysis. [Figure 8A] Figures 8A-8D show the results of the in vitro analysis of APB-A1. [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 9A] Figures 9A-9D show the effects of various ICs on platelet aggregation. [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 9D] See the explanation in Figure 9A. [Figure 10] Figure 10 shows the effects of various ICs on serotonin levels. [Figure 11] Figure 11 shows the PK values ​​of APB-A1 concentrations measured using cynomolgus monkeys. [Figure 12A] Figures 12A and 12B show the results of pharmacokinetic analysis using cynomolgus monkeys. [Figure 12B] See the explanation in Figure 12A. [Figure 13A] Figures 13A-13C show SAFA-based bispecific antibodies and mammalian expression vectors. [Figure 13B] See the explanation in Figure 13A. [Figure 13C] See the explanation in Figure 13A. [Figure 14] Figure 14 shows the amino acid sequences of the APB-B1 heavy and light chains. [Figure 15A] Figures 15A-15D show bispecific antibody constructs purified by CaptureSelect IgG-CH1 affinity chromatography. [Figure 15B] See the description of Figure 15A. [Figure 15C] See the description of Figure 15A. [Figure 15D] See the description of Figure 15A. [Figure 16A] Figures 16A and 16B show the APB-B1 constructs purified by two-step chromatography. [Figure 16B] See the description of Figure 16A. [Figure 17] Figure 17 shows heat stability shift assays under various pH and buffer conditions. [Figure 18A] Figures 18A-18C show the determination results of the binding specificities of the APB-B1 constructs to three different antigens compared to the parental antibody, determined by ELISA. [Figure 18B] See the description of Figure 18A. [Figure 18C] See the description of Figure 18A. [Figure 19] Figure 19 shows the results of the simultaneous binding of APB-B1a to three different antigens analyzed by biolayer interferometry. [Figure 20] Figure 20 shows the binding of the SAFA-based construct to the cell membrane of D1.1 cells identified by flow cytometry. [Figure 21] Figure 21 shows the inhibition of TNF-α-mediated cytotoxicity by the SAFA-based bispecific antibody identified in L929 mouse cells. [Figure 22A] Figures 22A-22C show the determination of the ability of APB-B1 to inhibit one or both of the CD40L-CD40 interaction and the TNFα-TNFαR interaction identified in HEK-blue™ reporter cells. [Figure 22B] See the description of Figure 22A. [Figure 22C]See the explanation in Figure 22A. [Figure 23] Monkey PK data. Plasma APB-A1 concentration was measured using PK ELISA. The data represents the average of the experiments performed. [Figure 24] Figures 24A and 24B: Immunophenotyping (dividing B cells). Peripheral blood immunophenotyping showed a decrease in dividing B cells compared to the control group in the 30, 10, and / or 3 mg / kg groups on day 1 (Figure 24A) and day 11 (Figure 24B). [Figure 25] Serum anti-KLH IgG titer. Measurement of anti-KLH antibodies showed decreased IgG antibody titers in the 10 and 30 mg / kg groups compared to the control group from days 8 to 29 (* P<0.05, ** P<0.01: statistically significant difference from control). [Figure 26] Ki67-positive cells in axillary lymph nodes (immunohistochemical examination). Pathological and immunohistochemical examinations on day 29 revealed a decrease in anti-Ki67-positive cells in germinal centers of axillary lymph nodes in the 10 and / or 30 mg / kg groups. [Figure 27] Serum APB-A1 concentrations in the PD trial. In the PK group, Cmax and AUC0-t values ​​increased almost proportionally with the first and second doses in the 3-30 mg / kg groups. [Modes for carrying out the invention] 【0031】 Detailed explanation The various embodiments will be discussed in detail below, examples of which are illustrated in the accompanying drawings, and similar reference numerals throughout the drawings refer to similar elements. In this regard, the embodiments may take various forms and should be interpreted as not being limited to the descriptions shown herein. Accordingly, the embodiments described below are provided solely for the purpose of illustrating the aspects of this specification with reference to the drawings. As used herein, the term "and / or" includes one or more of the relevant enumerated items and any combination thereof. Expressions such as "at least one (of)" modify the entire list of elements, not the individual elements of the list, when relating to a list of elements. 【0032】 In this specification, structural formula A multispecific antibody containing TIFF0007875806000096.tif16128, In the formula, the antigen-binding fragment (Fab) is serum albumin Fab; R 1 and R 2 These are bioactive effector regions ligated to the N-terminus of Fab, each ligated to either the heavy-chain variable domain or the light-chain variable domain of Fab; R 3 and R 4 These are bioactive effector regions ligated to the C-terminus of Fab, each ligated to either the heavy-chain variable domain or the light-chain variable domain of Fab; m is an integer greater than or equal to 0 or 1; n is an integer greater than or equal to 0 or 1. Multispecific antibodies are provided. 【0033】 Isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA), encoding such antibodies are also provided. Furthermore, vectors (e.g., expression vectors) and cells (e.g., host cells) containing nucleic acids (polynucleotides) encoding such antibodies are provided. Methods for producing such antibodies are also provided. In other parts, methods and uses for inducing, increasing, or enhancing multispecific activity and for treating specific pathological conditions such as autoimmune diseases are provided. Related compositions (e.g., pharmaceutical compositions), kits, and detection methods are also provided. 【0034】 terminology As used herein, when the terms “about” and “approximately” are used to modify a numerical value or range, they indicate that a deviation of 5% to 10% above and 5% to 10% below that value or range falls within the intended meaning of the stated value or range. 【0035】 As used herein, the term "antibody" is a technical term that may be used interchangeably herein and refers to a molecule having an antigen-binding site that specifically binds to an antigen. 【0036】 Examples of antibodies include monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies containing two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain / antibody heavy chain pairs, intracellular antibodies, heterocompound antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies. 【0037】 As used herein, the term “multispecific antibody” is a technical term and may be used to refer to a molecule having two or more biologically active effector moieties or antigen-binding sites, each of which specifically binds to an antigen. The multispecific antibodies disclosed herein may have two, three, four, five, six, seven, eight, or more biologically active effector moieties linked thereto. 【0038】 Antibodies can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a Or IgG 2b ) may be. In certain embodiments, the antibody described herein is an IgG antibody, or a class thereof (e.g., human IgG1, IgG2, or IgG4) or a subclass thereof. In some embodiments, the antibody is a humanized monoclonal antibody. In other embodiments, the antibody is a human monoclonal antibody, which is, for example, an immunoglobulin. 【0039】 As used herein, the terms “bioeffector moiety,” “antigen-binding domain,” “antigen-binding region,” “antigen-binding site,” and similar terms refer to a portion of a multispecific antibody molecule that contains amino acid residues (e.g., complementarity-determining regions (CDRs)) that confer its specificity to an antigen on the antibody molecule. Antigen-binding regions may originate from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans. 【0040】 As used herein, the terms “variable region” or “variable domain” are interchangeable and common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically the approximately 110–120 amino acids at the amino terminus of the mature heavy chain and approximately 90–115 amino acids of the mature light chain. Its sequence varies considerably from antibody to antibody and is used for the binding and specificity of a particular antibody to a particular antigen. Sequence diversity is concentrated in a region called the complementarity-determining region (CDR), while a more highly conserved region within the variable domain is called the framework region (FR). While not bound by any specific mechanism or theory, the CDRs of the light and heavy chains are thought to be primarily responsible for the antibody's interaction with and specificity to the antigen. In certain embodiments, the variable region is the human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is the primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes a rodent or mouse CDR and a primate (e.g., non-human primate) framework region (FR). 【0041】 The terms "VL" and "VL domain" are interchangeable to refer to the variable region of the antibody light chain. 【0042】 The terms "VH" and "VH domain" are interchangeable to refer to the variable region of the antibody's heavy chain. 【0043】 The term "Kabat numbering," and similar terms, are recognized in the art and refer to a system of numbering of amino acid residues in the heavy and light chain variable regions of antibodies, or the antigen-binding region of an antibody. In certain contexts, the CDR of an antibody may be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31–35, but potentially including one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50–65 (CDR2), and amino acid positions 95–102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24–34 (CDR1), amino acid positions 50–56 (CDR2), and amino acid positions 89–97 (CDR3). In some embodiments, the CDRs of the antibodies described herein were determined according to the Kabat numbering scheme. 【0044】 As used herein, the terms “constant region” or “constant domain” are interchangeable and their meanings are widely known in the art. The constant region is a part of an antibody, such as the carboxyl-terminal portion of the light and / or heavy chain, which is not directly involved in the antibody’s binding to an antigen but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to the immunoglobulin variable domain. 【0045】 As used herein, the term “heavy chain,” when used in reference to antibodies, may refer to any distinct type based on the amino acid sequence of the constant domain, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which result in antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4, respectively. 【0046】 As used herein, the term “light chain,” when used in reference to antibodies, may refer to any distinct type, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain. 【0047】 "Binding affinity" generally refers to the sum of the strengths of the non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D It can be expressed as (K). Affinity is not limited, but the equilibrium dissociation constant (K D ) and equilibrium association constant (K A ) can be measured and / or expressed in several ways known in the art, including K. D is, k off / k on It is calculated from the quotient of K A is, k on / k off It is calculated from the quotient of k. on k refers to, for example, the binding rate constant of an antibody to an antigen. off This refers, for example, to the dissociation of an antibody from an antigen. on and k off BIAcore (登録商標) Alternatively, it can be determined by techniques known to those skilled in the art, such as KinExA. 【0048】 As used herein, “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within the CDR or framework region of an antibody may be replaced by amino acid residues having a similar side chain. 【0049】 As used herein, “epitope” is a term used in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, an adjacent amino acid of a polypeptide (linear or adjacent epitope), or it may be, for example, a collection of two or more non-adjacent regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-adjacent epitope). In certain embodiments, the epitope to which an antibody binds may be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, a combination of hydrogen / deuterium exchange and mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenicity mapping (e.g., site-directed mutagenicity mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).Antibody: Antigen crystals can be investigated using well-known X-ray diffraction techniques and can also be improved using computer software such as X-PLOR (Yale University, 1992, sold by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; US 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenicity mapping can be achieved using any method known to those skilled in the art. For descriptions of mutagenicity techniques, including the alanine scanning mutagenicity technique, see, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085. In some embodiments, the epitopes of antibodies are determined by alanine scanning mutagenicity investigations. 【0050】 As used herein, the terms “immunely binding,” “immunely recognizing,” “specifically binding,” and “specifically recognizing” are synonymous in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope, an immune complex, or a binding partner of an antigen-binding site), and such binding will be understood by those skilled in the art. For example, a molecule that specifically binds to one antigen may also bind to other peptides or polypeptides, e.g., in immunoassays, BIAcore (登録商標)As determined by the KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho), or other assays known in the art, it can generally bind with lower affinity. In some embodiments, a molecule that binds immunospecifically to one antigen may have a lower affinity than the K2 when the molecule binds to another antigen. A At least 2 logs, 2.5 logs, 3 logs, 4 logs, or more than K A They are joined together. 【0051】 In other embodiments, a molecule that immunospecifically binds to a certain antigen does not cross-react with other proteins under similar binding conditions. In some embodiments, a molecule that immunospecifically binds to a certain antigen does not cross-react with other proteins. In some embodiments, multispecific antibodies are provided that bind to a particular antigen with a higher affinity than they bind to another unrelated antigen. In certain embodiments, multispecific antibodies are provided that bind to a particular antigen (e.g., human serum albumin) with an affinity 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more higher than they bind to another unrelated antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion assay. In some embodiments, as measured, for example by radioimmunoassay, the degree of binding of the multispecific antibodies described herein to unrelated proteins is less than 10%, 15%, or 20% of the binding of the antibodies to specific antigens. 【0052】 In some embodiments, the Specified provides multispecific antibodies that bind to a human antigen with a higher affinity than they bind to another species of the antigen. In certain embodiments, the Specified provides multispecific antibodies that bind to a human antigen with an affinity of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more than they bind to another species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion assay. In some embodiments, the multispecific antibodies described herein that bind to a human antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion assay, bind to another species of the antigen protein at a rate less than 10%, 15%, or 20% of the binding of the antibody to the human antigen protein. 【0053】 As used herein, the term “host cell” may refer to any type of cell, e.g., primary cells, cultured cells, or cells derived from a cell line. In some embodiments, the term “host cell” refers to a cell that has been genetically modified using nucleic acid molecules, or to the offspring or potential offspring of such a cell. Such offspring may not be identical to the parent cell genetically modified using nucleic acid molecules, for example, due to mutations or environmental influences that may occur in later generations, or due to the incorporation of nucleic acid molecules into the host cell genome. 【0054】 As used herein, the term “effective dose” refers to the amount of therapy administered to a subject that produces the desired preventive or therapeutic effect. 【0055】 As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be an animal. In some embodiments, the subject is a mammal such as a non - primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human), or a human. In some embodiments, the subject is a cynomolgus monkey. In certain embodiments, such terms refer to non - human animals (e.g., non - human animals such as pigs, horses, cows, cats, or dogs). In some embodiments, such terms refer to pet or livestock animals. In certain embodiments, such terms refer to humans. 【0056】 Bispecific antibody As used herein, the structural formula A bispecific antibody comprising TIFF0007875806000097.tif19128, wherein the antigen - binding fragment (Fab) is a serum albumin Fab; R 1 and R 2 are bioactive effector moieties linked to the N - terminus of the Fab, each being linked to the heavy - chain variable domain or the light - chain variable domain of the Fab; R 3 and R 4 are bioactive effector moieties linked to the C - terminus of the Fab, each being linked to the heavy - chain variable domain or the light - chain variable domain of the Fab; m is 0, or an integer of 1, 2, 3, or more; n is 0, or an integer of 1, 2, 3, or more, A bispecific antibody is disclosed. 【0057】 In some embodiments, R 1 and R 2 are the same or different single - chain variable fragments (scFv), or the same or different Fv fragments or disulfide - stabilized Fv (dsFv) fragments. In some embodiments, R 3 and R 4These are the same or different scFv or Fv fragment or dsFv fragment. 【0058】 In some embodiments, R 1 , R 2 , R 3 , and R 4 Each can be linked to the Fab by one or more linkers. Each linker may contain, but is not limited to, 1 to 20 amino acids of any length or range in between, e.g., 2, 3, 4, or other amino acids. Each linker may contain an amino acid sequence having at least 90% identity with SEQ ID NO:3 or SEQ ID NO:4. Each linker may contain the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. 【0059】 In some embodiments, Fab is (a) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000098.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000099.tif4128; (b) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000100.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000101.tif4128; (c) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence NYGIH (SEQ ID NO:65), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000102.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000103.tif4128; (d) Amino acid sequence SYAMS (SEQ ID NO:68) containing heavy chain complementarity determination domain 1 (CDR1), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000104.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000105.tif4128; (e) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYWIA (SEQ ID NO: 71), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000106.tif4128, and heavy chain CDR3 containing amino acid sequence LYSGSYSP (SEQ ID NO:73); or (f) Amino acid sequence AYSMN (SEQ ID NO: 74) containing heavy chain complementarity-determining domain 1 (CDR1), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000107.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000108.tif4128 It includes heavy chain variable domains. 【0060】 In some embodiments, Fab is (g) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000109.tif4128, light chain CDR2 containing amino acid sequence GASRLES (SEQ ID NO: 78), and light chain CDR3 containing amino acid sequence QQSDSVPVT (SEQ ID NO: 79); (h) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000110.tif4128, light chain CDR2 containing amino acid sequence AASSLQS (SEQ ID NO: 81), and light chain CDR3 containing amino acid sequence QQSYSTPPYT (SEQ ID NO: 82); (i) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000111.tif5128, light chain CDR2 containing amino acid sequence DASNRAT (SEQ ID NO:84), and amino acid sequence Light chain CDR3 containing TIFF0007875806000112.tif4128; (j) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000113.tif5128, light chain CDR2 containing amino acid sequence GASSRAT (SEQ ID NO: 87), and light chain CDR3 containing amino acid sequence QQYGSSPRT (SEQ ID NO: 88); (k) Light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSVSSSSLA (SEQ ID NO: 89), light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and light chain CDR3 containing the amino acid sequence QKYSSYPLT (SEQ ID NO: 90); or (l) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000114.tif4128, light chain CDR2 containing amino acid sequence GASTGAT (SEQ ID NO:92), and light chain CDR3 containing amino acid sequence QQYYSFLAKT (SEQ ID NO:93). Includes light chain variable domains. 【0061】 In some embodiments, Fab is Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000115.tif4128, and amino acid sequence Heavy-chain CDR3 including TIFF0007875806000116.tif4128, and amino acid sequence A light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000117.tif4128, a light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO:92), and a light chain CDR3 containing the amino acid sequence QQYYSFLAKT (SEQ ID NO:93), or any combination of the heavy chains CDR1, CDR2, and CDR3 disclosed above, and the light chains CDR1, CDR2, and CDR3. Includes. 【0062】 In some embodiments, the Fab includes a heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 94, 95, 96, 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 94, 95, 96, 97%, at least 98%, at least 99%, or 100% identity. 【0063】 In some embodiments, the Fab includes a light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. 【0064】 In some embodiments, Fab includes a heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 94, 95, 96, 97%, at least 98%, at least 99%, or 100%, and a light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105, or any combination of heavy chain variable domains and light chain variable domains disclosed herein. For example, Fab may include a heavy chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:99, and a light chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:105. 【0065】 In some embodiments, Fab comprises a heavy chain domain (V) containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:45. H -C H1 A light chain domain (V) containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:46. L -CL Includes domains. 【0066】 In some embodiments, R 1 , R 2 , R 3 , and R 4 These could each be a bioactive effector portion such as anti-CD40L hu5c8 scFv. For example, R 1 , R 2 , R 3 , and R 4 These could each be an anti-CD40L hu5c8 scFv containing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:47 or SEQ ID NO:48. 1 , R 2 , R 3 , and R 4 These could each be an anti-CD40L hu5c8 scFv containing the amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48, respectively. 【0067】 In some embodiments, R 1 and R 2 These could each be a bioactive effector portion such as anti-CD40L hu5c8 scFv. For example, R 1 and R 2 These could each be an anti-CD40L hu5c8 scFv containing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:47 or SEQ ID NO:48. 1 and R 2 These could each be an anti-CD40L hu5c8 scFv containing the amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48, respectively. 【0068】 In some embodiments, R 1, R 2 , R 3 , and R 4 Each of these is one or more bioactive effector moieties, including, for example, anti-TNF-α Fv, anti-TNF-α disulfide-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and / or anti-IFNAR1 dsFv. 【0069】 In some embodiments, R 3 and R 4 Each of these is one or more bioactive effector moieties, including, for example, anti-TNF-α Fv, anti-TNF-α disulfide-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and / or anti-IFNAR1 dsFv. 【0070】 In some embodiments, R 1 , R 2 , R 3 , and R 4Each of these includes an anti-TNF-α Fv containing a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:49, and a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:50, and a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:51, and SEQ ID Anti-TNF-α disulfide-stabilized Fv (dsFv) containing a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:52, SEQ ID NO:53 and a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:54, anti-IL-23 Fv, SEQ ID A heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:55, and SEQ IDAnti-IL-23 dsFv containing a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:56, a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:57, and anti-IFNAR1 containing a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:58, and / or SEQ ID It may be one or more bioactive effector moieties containing anti-IFNAR1 dsFv, which includes a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:59, and a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:60. 1 , R 2 , R 3 , and R 4Each of these may comprise one or more bioactive effector moieties, including an anti-TNF-α Fv containing the heavy chain of SEQ ID NO:49 and the light chain of SEQ ID NO:50, an anti-TNF-α disulfide-stabilized Fv (dsFv) containing the heavy chain of SEQ ID NO:51 and the light chain of SEQ ID NO:52, an anti-IL-23 Fv containing the heavy chain of SEQ ID NO:53 and the light chain of SEQ ID NO:54, an anti-IL-23 dsFv containing the heavy chain of SEQ ID NO:55 and the light chain of SEQ ID NO:56, an anti-IFNAR1 containing the heavy chain of SEQ ID NO:57 and the light chain of SEQ ID NO:58, and / or an anti-IFNAR1 dsFv containing the heavy chain of SEQ ID NO:59 and the light chain of SEQ ID NO:60. 【0071】 In some embodiments, R 3 and R 4Each of these includes an anti-TNF-α Fv containing a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:49, and a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:50, and a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:51, and SEQ ID Anti-TNF-α disulfide-stabilized Fv (dsFv) containing a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:52, SEQ ID NO:53 and a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:54, anti-IL-23 Fv, SEQ ID A heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:55, and SEQ IDAnti-IL-23 dsFv containing a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:56, a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:57, and anti-IFNAR1 containing a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:58, and / or SEQ ID It may be one or more bioactive effector moieties containing anti-IFNAR1 dsFv, which includes a heavy chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:59, and a light chain amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:60. 3 is R 4Each of these may consist of one or more bioactive effector moieties, including an anti-TNF-α Fv containing the heavy chain of SEQ ID NO:49 and the light chain of SEQ ID NO:50, an anti-TNF-α disulfide-stabilized Fv (dsFv) containing the heavy chain of SEQ ID NO:51 and the light chain of SEQ ID NO:52, an anti-IL-23 Fv containing the heavy chain of SEQ ID NO:53 and the light chain of SEQ ID NO:54, an anti-IL-23 dsFv containing the heavy chain of SEQ ID NO:55 and the light chain of SEQ ID NO:56, an anti-IFNAR1 containing the heavy chain of SEQ ID NO:57 and the light chain of SEQ ID NO:58, and / or an anti-IFNAR1 dsFv containing the heavy chain of SEQ ID NO:59 and the light chain of SEQ ID NO:60. 【0072】 In some embodiments, the multispecific antibodies disclosed herein are anti-HSA Fab (SL335) and anti-CD40L IgG (luprizumab), respectively. 1 and R 2 , as well as R, which is an anti-TNF-α IgG (adalimumab) 3 and R 4 , and / or anti-TNF-α Fab' (certolizumab). In some embodiments, the multispecific antibodies disclosed herein are anti-HSA Fab (SL335), anti-CD40L scFv (hu5c8), and R 1 and R 2 , and R, each of which is 0. 3 m and R 4 Includes m and n of n 【0073】 In certain contexts, the multispecific antibodies described herein may be described by their VL domain only, or their VH domain only, or their three VL CDRs only, or their three VH CDRs only. For example, see Rader C et al., (1998) PNAS 95: 8910-8915 (entirely incorporated herein by reference), which describes the humanization of a mouse anti-αvβ3 antibody by identifying complementary light or heavy chains from a human light or heavy chain library, resulting in a humanized antibody variant with the same or higher affinity as the original antibody. See also Clackson T et al., (1991) Nature 352: 624-628 (entirely incorporated herein by reference), which describes a method for producing antibodies that bind to a specific antigen by using a specific VL domain (or VH domain) and screening a library for complementary variable domains. The screening yielded 14 novel partners for specific VH domains and 13 novel partners for specific VL domains, which were determined by ELISA to be potent binders. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577 (the whole is incorporated herein by reference), which describes a method for producing antibodies that bind to specific antigens by using specific VH domains and screening a library (e.g., a human VL library) for complementary VL domains, and the selected VL domains can be used as a guide for the selection of additional complementary (e.g., human) VH domains. 【0074】 In certain contexts, the CDR of an antibody may be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loop (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, using the Kabat numbering system, the Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34; the Chothia CDR-H2 loop is located at heavy chain amino acids 52-56; the Chothia CDR-H3 loop is located at heavy chain amino acids 95-102; the Chothia CDR-L1 loop is located at light chain amino acids 24-34; the Chothia CDR-L2 loop is located at light chain amino acids 50-56; and the Chothia CDR-L3 loop is located at light chain amino acids 89-97. The ends of the Chothia CDR-H1 loop differ from H32 to H34 depending on the loop length, according to the Kabat numbering scheme (this is because the Kabat numbering scheme has insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; and if both 35A and 35B exist, the loop ends at 34). 【0075】 In certain aspects, this specification provides a multispecific antibody that specifically binds to serum albumin (e.g., human serum albumin) and includes the VL of Chothia VL CDR. In certain aspects, this specification provides an antibody that specifically binds to serum albumin (e.g., human serum albumin) and includes the VH of Chothia VH CDR. In certain aspects, this specification provides an antibody that specifically binds to serum albumin (e.g., human serum albumin) and includes the VL of Chothia VL CDR and the VH of Chothia VH CDR. In certain embodiments, an antibody that specifically binds to serum albumin (e.g., human serum albumin) comprises one or more CDRs, where both Chothia CDR and Kabat CDR have the same amino acid sequence. In certain embodiments, this specification provides an antibody that specifically binds to serum albumin (e.g., human serum albumin) and includes a combination of Kabat CDR and Chothia CDR. 【0076】 In certain contexts, the CDR of an antibody can be determined according to the IMGT numbering system described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212. In the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 at positions 51-57, VH-CDR3 at positions 93-102, VL-CDR1 at positions 27-32, VL-CDR2 at positions 50-52, and VL-CDR3 at positions 89-97. 【0077】 In certain situations, the CDR of an antibody may be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, for example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains” in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). 【0078】 In certain situations, the CDR of an antibody can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region, a compromise between the Kabat CDR and the Chothia structural loop, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). 【0079】 In some embodiments, one or more positions of the CDRs on the VH region (e.g., CDR1, CDR2, or CDR3) and / or on the VL region (e.g., CDR1, CDR2, or CDR3) of the antibodies described herein may differ by only 1, 2, 3, 4, 5, or 6 amino acid positions, provided that immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, the position defining the CDR of an antibody described herein may differ by shifting the N-terminal and / or C-terminal boundary of the CDR by 1, 2, 3, 4, 5, or 6 amino acids relative to the CDR position of a multispecific antibody described herein, insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the lengths of one or more CDRs on the VH region (e.g., CDR1, CDR2, or CDR3) and / or on the VL region (e.g., CDR1, CDR2, or CDR3) of the antibodies described herein may differ by only one, two, three, four, five, or more amino acids (e.g., shorter or longer), insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). 【0080】 In some embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be one, two, three, four, five, or more amino acids shorter than one or more of the CDRs described herein, insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be 1, 2, 3, 4, 5, or more amino acids longer than one or more of the CDRs described herein, insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino terminus of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be elongated by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein, insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the carboxyl terminus of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be elongated by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein, insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In other embodiments, the amino terminus of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be shortened by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein, insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxyl terminus of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be shortened by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein, insofar as immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art, such as the binding assays and conditions described in the "Examples" section herein, may be used to determine whether immunospecific binding to the antigen is maintained. 【0081】 The determination of the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can also be achieved using mathematical algorithms. A specific non-principled example of a mathematical algorithm used to compare two sequences is a modified version of the algorithm in Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, as shown in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs in Altschul SF et al., (1990) J Mol Biol 215: 403. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, a BLAST nucleotide search may be performed using, for example, a set of NBLAST nucleotide program parameters with a score of 100 and a word length of 12. To obtain amino acid sequences homologous to the protein molecules described herein, a BLAST protein search may be performed using, for example, a set of XBLAST program parameters with a score of 50 and a word length of 3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389 3402. Alternatively, PSI BLAST can be used to perform iterative searches to detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another specific non-resolved example of a mathematical algorithm used for sequence comparison is the algorithm in Myers and Miller, 1988, CABIOS 4:11 17. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When comparing amino acid sequences using the ALIGN program, you can use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. 【0082】 The percentage of identity between two sequences can be determined, with or without gaps, using techniques similar to those described above. Typically, only exact matches are counted in the calculation of the percentage of identity. 【0083】 Multispecific antibodies can be fused or conjugated (e.g., covalently or noncovalently) with detectable labels or substances. Examples of detectable labels or substances include enzyme labels, e.g., glucose oxidase; radioactive isotopes, e.g., iodine. 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 Examples include Tc; luminescence labels, such as luminol; and fluorescent labels, such as fluorescent and rhodamine, as well as biotin. Such labeled antibodies can be used to detect antigen proteins. 【0084】 For example, monoclonal antibodies targeting CD40 or CD40L have been developed to suppress the CD40-CD40L interaction, a major pathway in autoimmune diseases or allograft rejection. However, further development of these antibodies has stalled due to the occurrence of side effects such as thromboembolism induced by the Fc of IgG1 antibodies. To eliminate or reduce these side effects, a recombinant antibody (anti-hu5c8 scFv)2-SL335 (named APB-A1) has been developed, produced by combining a Fab with anti-CD40L scFv. SL335 is an antigen-binding fragment (Fab) that specifically binds to human serum albumin, thereby increasing its in vivo sustainability. See U.S. Patent No. 9,879,077 (the entire patent is incorporated herein by reference). 【0085】 To identify the binding ability of APB-A1 and its efficacy in suppressing thromboembolism, binding ability and cell-based inhibitory efficacy were evaluated. The binding affinity of APB-A1 to HSA and rhCD40L was determined by bilayer interferometry, and the results showed a tendency for the dissociation constant (KD) of APB-A1 to decrease compared to each control group. In the evaluation of cell-based inhibitory efficacy, there was no significant difference in the inhibitory efficacy of hu5c8 IgG1 when HSA was added, but the levels of APB-A1's binding inhibitory efficacy to rhCD40L antigen and D1.1 cells increased by approximately 1.6-fold and 3-fold, respectively. This suggests that the size change due to the binding of SL335 to HSA results in a similar level of efficacy as in the positive control group. In other words, inhibitory efficacy increased in the presence of HSA even at lower affinity than hu5c8 IgG1. 【0086】 To investigate whether the removal of the Fc region of IgG1 resolves thromboembolic side effects, platelet aggregation rates and serotonin secretion levels of APB-A1 were measured and analyzed. Platelet aggregation was not observed from the immune complex (IC) formed by APB-A1 and rhCD40L, even at a high concentration of 400 ng / ml. However, the aggregation response to IC between hu5c8 IgG1 and rhCD40L initiated at a relatively low concentration of 60 ng / ml, as identified by permeability and platelet aggregation rates. Analysis of serotonin release levels in platelet dense granules revealed that serotonin release levels from APB-A1 IC were statistically significantly lower than those from hu5c8 IgG1 IC. These results suggest that the anti-CD40L antibody disclosed herein can effectively resolve thromboembolic-related impairments in vivo. 【0087】 Pharmacokinetic analysis was performed to evaluate the half-life of APB-A1. APB-A1 was administered to two experimental groups of cynomolgus monkeys by a single intravenous injection at a dose of 5 mg / kg (Group 1) or 20 mg / kg (Group 2). As a result, assuming that the two groups had equal renal clearance rates, the in vivo half-life of Group 2 was determined to be 9.59 ± 0.79 days, which is 1.38 times higher than the in vivo half-life of Group 1, which was 6.94 ± 4.6 days. 【0088】 In another example, pharmacodynamic analysis was performed to evaluate the immune response to anti-TT IgG antibodies induced by tetanus toxoid (TT) injection. Cynomolgus monkeys were used as test animals, and APB-A1 was administered by a single intravenous injection at doses of 5 mg / kg (Group 1) or 20 mg / kg (Group 2) to two test groups of cynomolgus monkeys. As a result, when APB-A1 was administered intravenously at a high concentration (20 mg / kg), the inhibitory effect on the IgG antibody immune response was much higher than when DXT was administered to the positive control group, and this inhibitory effect was maintained for up to 30-40 days. Furthermore, although the CD40-CD40L interaction is also acted upon by memory B cells, a significant inhibitory effect with APB-A1 was identified on day 27 after TT boosting (day 20). Thus, it was confirmed that APB-A1 of this disclosure significantly improved thromboembolic-related disorders compared to hu5c8 IgG1. 【0089】 In another embodiment, novel bispecific antibodies named APB-B1a and APB-B1b, respectively, are used against the single-strand variable fragment (scFv) (V) of CD154 (CD40 ligand; CD40L). H -[peptide linker]-V L It was produced by ligating tumor necrosis factor alpha (anti-TNF-α) variable fragment (Fv) or disulfide-stabilized Fv (dsFv). Experiments using biolayer interferometry (BLI) confirmed that APB-B1 had the ability to simultaneously bind to three targets, namely recombinant human CD40L, recombinant human TNF-α, and human serum albumin (HSA) protein, and possessed antigen-binding affinity levels similar to anti-CD40L IgG or anti-TNF-αFab' parental antibodies. 【0090】 In another aspect of this, the melting temperature (Tm) measured in a buffered state without excipients was 62°C regardless of the presence or absence of interchain disulfide bonds in anti-TNF-α Fv, confirming that interchain disulfide bonds did not contribute to structural stability. 【0091】 In yet another aspect, in vitro cell-based assays showed that APB-B1's CD40L inhibitory ability was similar to that of the parental antibody anti-CD40L IgG1, and that its TNF-α inhibitory ability was slightly lower than that of the parental antibody anti-TNF-α Fab'. Nevertheless, APB-B1 demonstrated higher inhibitory activity against both CD40L and TNF-α, as well as higher inhibitory activity than that of the parental antibody, anti-CD40L IgG1, and anti-TNF-α Fab', respectively. 【0092】 Antibody production The multispecific antibodies disclosed herein can be produced by any antibody synthesis method known in the art, for example, by chemical synthesis and by recombinant expression techniques. Unless otherwise specified, the methods described herein utilize conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of the art. These techniques are described and fully explained in the references cited herein, for example.For example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Please refer to Cold Spring Harbor Laboratory Press. 【0093】 In some embodiments, the multispecific antibodies described herein are antibodies (e.g., recombinant antibodies) prepared, expressed, produced, or isolated by any means, such as synthesis or genetic manipulation of DNA sequences. In certain embodiments, such antibodies contain sequences (e.g., DNA sequences or amino acid sequences) that are not naturally present in the in vivo antibody germline repertoire of animals or mammals (e.g., humans). 【0094】 In some aspects, this specification provides a method for producing multispecific antibodies disclosed herein, comprising culturing cells or host cells described herein. In some aspects, this specification provides a method for producing multispecific antibodies, comprising expressing (e.g., recombinantly expressing) the antibody using cells or host cells described herein (e.g., cells or host cells containing polynucleotides encoding the antibody described herein). In some embodiments, the cells are isolated cells. In some embodiments, exogenous polynucleotides are introduced into the cells. In some embodiments, the method further comprises a step of purifying the antibody obtained from the cells or host cells. 【0095】 Methods for producing polyclonal antibodies are well known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York). 【0096】 Multispecific antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, monoclonal antibodies are known in the art and can be produced using hybridoma techniques, such as those taught by Hammerling GJ et al. in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). The term “monoclonal antibody” as used herein is not limited to antibodies produced by hybridoma techniques. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing the antibodies described herein. 【0097】 As used herein, a “monoclonal antibody” is an antibody produced by a single cell (e.g., a hybridoma or host cell producing recombinant antibodies) that immunospecifically binds to an antigen (e.g., human serum albumin), which is determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or in the examples provided herein. In certain embodiments, a monoclonal antibody may be a chimeric antibody or a humanized antibody. In certain embodiments, a monoclonal antibody may be a monovalent or polyvalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody may be a Fab fragment or an F(ab')2 fragment. Monoclonal antibodies described herein can be produced, for example, by a hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495, or they can be isolated from a phage library using, for example, the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., as mentioned above). 【0098】 Methods for producing and selecting specific antibodies using hybridoma technology are routine and well-known in this art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaque monkeys, are immunized to induce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the antigen used for immunization (e.g., human serum albumin). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). In addition, animals can be immunized using the RIMMS (Repeated Multiple Site Immunization) technique (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, the entire text is incorporated by reference). 【0099】 In some embodiments, a mouse (or other animal, e.g., rat, monkey, donkey, pig, sheep, hamster, or dog) may be immunized with an antigen (e.g., human serum albumin), and once an immune response is detected, for example, when antigen-specific antibodies are detected in the mouse serum, the mouse spleen is collected and splenocytes are isolated. The splenocytes are then converted into any suitable myeloma cells, e.g., American Type Culture Collection (ATCC). (登録商標) Cells from the SP20 cell line, available from Manassas, Virginia, are fused with NS0 myeloma cells using a known technique to form hybridomas. The hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes from immunized mice are harvested and fused with NS0 myeloma cells. 【0100】 The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that may contain one or more substances that inhibit the proliferation or survival of asexual parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma culture medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances will inhibit the proliferation of HGPRT-deficient cells. 【0101】 Certain embodiments utilize myeloma cells that efficiently fuse, support stable, high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. These myeloma cell lines include mouse myeloma cell lines, such as the NS0 cell line, or cell lines derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, as well as SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection in Rockville, Maryland, USA. Human myeloma and mouse-human heterozygous myeloma cell lines for human monoclonal antibody production have also been described (Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). 【0102】 The culture medium in which hybridoma cells are proliferating is assayed for the production of monoclonal antibodies against the antigen. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, such as immunoprecipitation, or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). 【0103】 After hybridoma cells are identified as producing antibodies of desired specificity, affinity, and / or activity, their clones can be subcloned using limiting dilution procedures and proliferated using standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, cited above). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals. 【0104】 Monoclonal antibodies secreted by subclones are preferably isolated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. 【0105】 The antibodies described herein may be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein may be produced by protein cleavage of immunoglobulin molecules using enzymes such as papain (which produces the Fab fragment) or pepsin (which produces the F(ab')2 fragment). The Fab fragment corresponds to one of the two identical arms of the tetramer antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of the tetramer antibody molecule linked by a disulfide bond at the hinge region. 【0106】 Furthermore, the antibodies described herein may also be generated using various phage display methods known in the art. In phage display methods, proteins are presented on the surface of phage particles carrying the polynucleotide sequence they encode. Specifically, DNA sequences encoding the VH domain and the VL domain are amplified from an animal cDNA library (e.g., a cDNA library of human or mouse diseased tissue). The DNA encoding the VH domain and the DNA encoding the VL domain are recombined with each other by PCR using an scFv linker and then cloned into a phagemide vector. The vector is electroporated into Escherichia coli (E. coli) to infect the E. coli with helper phages. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are usually recombinate-fused with either phage gene III or gene VIII. Phages expressing antibodies that bind to a specific antigen may be selected or identified using the antigen, e.g., a labeled antigen, or an antigen bound to or captured on a solid surface or beads.Examples of phage display methods that may be used to produce the antibodies described herein include: Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT / GB91 / 001134; WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; Also included are those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108. 【0107】 As described in the references above, after phage selection, the antibody-coding region derived from the phage can be isolated and used to produce antibodies, including human antibodies, which can then be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, such as WO92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043. Techniques for recombinant production of antibodies such as Fab, Fab', and F(ab')2 fragments can also be used, employing methods known in the art, such as those disclosed in these publications. 【0108】 In some cases, to generate antibodies, a VH or VL sequence, such as an scFv clone, can be amplified from a template using PCR primers containing a VH or VL nucleotide sequence, a restriction site, and a flanking sequence protecting the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing the VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing the VL constant region, such as the human kappa or lambda constant region. The VH and VL domains can also be cloned into a single vector expressing the required constant region. Then, using techniques known to those skilled in the art, the heavy chain conversion vector and the light chain conversion vector can be simultaneously introduced into a cell line to generate a stable or transient cell line expressing an antibody, such as IgG. 【0109】 Chimeric antibodies are molecules in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain a variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Patents No. 5,807,715, No. 4,816,567, No. 4,816,397, and No. 6,331,415. 【0110】 A humanized antibody comprises a framework region capable of binding to a given antigen and substantially having the amino acid sequence of a human immunoglobulin, and a CDR substantially having the amino acid sequence of a non-human immunoglobulin (e.g., mouse immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of the constant region (Fc) of the immunoglobulin, typically the constant region of a human immunoglobulin. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies include, but are not limited to, CDR implantation (EP 239400; WO91 / 09967; and U.S. Patents 5,225,539, 5,530,101, and 5,585,089), coating (veneering), or surface modification (EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7(6): 805-814; and Roguska MA et al., (1994) PNAS 91: 969-973), chain shuffle (US Pat. No. 5,565,332), and e.g. US Pat. No. 6,407,213, US Pat. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 It can be produced using various techniques known in the art, including the techniques disclosed in (23 Supp): 5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al., (1994) J Mol Biol 235(3): 959-73. See also US 2005 / 0042664 A1 (February 24, 2005) (the whole is incorporated herein by reference). 【0111】 Single-domain antibodies, such as antibodies without light chains, can be produced by methods well known in the art. See Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Patent No. 6,005,079; and WO94 / 04678, WO94 / 25591, and WO01 / 44301. 【0112】 Furthermore, using antibodies that bind immunospecifically to an antigen, anti-idiotype antibodies that "mimic" the antigen can be generated using techniques well known to those skilled in the art. (See, for example, Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438). 【0113】 In certain embodiments, a multispecific antibody described herein that binds to the same antigen of interest epitope as the antibody described herein (e.g., human serum albumin) is a human antibody. In certain embodiments, an antibody described herein that competitively (e.g., dose-dependently) blocks any one of the antibodies described herein from binding to serum albumin (e.g., human serum albumin) is a human antibody. Human antibodies can be produced using any method known in the art. For example, a transgenic mouse can be used that can express human immunoglobulin genes but cannot express functional exogenous immunoglobulins. Specifically, human heavy-light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy-light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. The mouse heavy-light chain immunoglobulin genes can be made non-functional, either separately from or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. Specifically, J HHomozygous deletion of the region prevents endogenous antibody production. Modified embryonic stem cells are proliferated and microinjected into blastocysts to produce chimeric mice. These chimeric mice are then bred to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized using a select antigen, e.g., all or part of the antigen, in the usual manner. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes in transgenic mice are rearranged during B cell differentiation, followed by class transmutation and somatic mutation. Therefore, such techniques make it possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technique for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed description of this technique for producing human antibodies and human monoclonal antibodies, and the protocols for producing such antibodies, see, for example, WO98 / 24893, WO96 / 34096, and WO96 / 33735; and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. An example of a mouse capable of producing human antibodies is Xenomouse. (商標) (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse (商標) (Mederex, Inc. / Gen Pharm; U.S. Patent No. 5,545,806 and No. 5,569,825), Trans Chromo Mouse (商標) (Kirin), and KM Mouse (商標) (Medarex / Kirin) is one example. 【0114】 Human antibodies that specifically bind to antigens can be produced by various methods known in the art, including the phage display method described above, which uses antibody libraries derived from human immunoglobulin sequences. See also U.S. Patents 4,444,887, 4,716,111, and 5,885,793; as well as WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741. 【0115】 In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies. These mouse-human hybridomas can then be screened to determine which secrete human monoclonal antibodies that bind immunospecifically to a target antigen. Such methods are known and described in the art; see, for example, Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31. 【0116】 Polynucleotides, vectors, and cells In certain contexts, this specification provides polynucleotides comprising nucleotide sequences encoding antibodies or fragments thereof (e.g., variable light chain regions and / or variable heavy chain regions) described herein that bind immunospecifically to an antigen, as well as vectors, for example, vectors containing such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). This specification provides polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, as well as vectors containing such polynucleotide sequences, for example, expression vectors for efficiently expressing such polynucleotides in host cells, for example, mammalian cells. 【0117】 As used herein, “isolated” polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule that has been isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., mouse or human). Furthermore, “isolated” nucleic acid molecules, such as cDNA molecules, may substantially contain other cellular material or culture media if produced by recombinant techniques, or substantially contain chemical precursors or other chemicals if chemically synthesized. For example, the phrase “substantially free of” includes preparations of polynucleotide or nucleic acid molecules that contain less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (especially less than about 10%) of other materials, e.g., cellular material, culture media, other nucleic acid molecules, chemical precursors, and / or other chemicals. In some embodiments, the nucleic acid molecules encoding the antibodies described herein are isolated or purified. 【0118】 In certain contexts, this specification provides for antibodies that immunospecifically bind to an antigen polypeptide (e.g., human serum albumin) and include an amino acid sequence described herein, as well as polynucleotides that include a nucleotide sequence encoding an antibody that competes with such an antibody (e.g., in a dose-dependent manner) for binding to the antigen polypeptide, or an antibody that binds to the same epitope to which such an antibody binds. 【0119】 In certain contexts, this specification provides polynucleotides comprising nucleotide sequences encoding the light chain or heavy chain of an antibody described herein. A polynucleotide may comprise a nucleotide sequence encoding the light chain, comprising the VL FR and CDR of an antibody described herein. A polynucleotide may comprise a nucleotide sequence encoding the heavy chain, comprising the VH FR and CDR of an antibody described herein. 【0120】 In certain embodiments, the Specified herein provides a polynucleotide comprising a nucleotide sequence encoding a multispecific antibody comprising a Fab comprising three VH chain CDRs, including, for example, VL CDR1, VL CDR2, and VL CDR3 of an antibody against human serum albumin as described herein, and three VH chain CDRs, including VH CDR1, VH CDR2, and VH CDR3 of an antibody against human serum albumin as described herein. 【0121】 In certain embodiments, this specification provides polynucleotides comprising a nucleotide sequence encoding a multispecific antibody or a fragment thereof containing a VL domain. 【0122】 In certain embodiments, the polynucleotides described herein include a nucleotide sequence encoding a multispecific antibody provided herein, comprising a light chain variable region containing an amino acid sequence described herein (e.g., SEQ ID NO: 46), the antibody immunospecifically conjugates to serum albumin (e.g., human serum albumin). 【0123】 In certain embodiments, the polynucleotides described herein include a nucleotide sequence encoding an antibody provided herein, comprising a heavy chain variable region containing an amino acid sequence described herein (e.g., SEQ ID NO: 45), the antibody immunospecifically conjugates to serum albumin (e.g., human serum albumin). 【0124】 In certain aspects, this specification provides polynucleotides comprising nucleotide sequences encoding antibodies comprising light and heavy chains, for example, separate light and heavy chains. With respect to the light chain, in some embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding kappa light chains. In other embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding lambda light chains. In yet another embodiment, the polynucleotides provided herein comprise nucleotide sequences encoding antibodies described herein, comprising human kappa light chains or human lambda light chains. In some embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding antibodies that immunospecifically bind to serum albumin (e.g., human serum albumin), where the antibody comprises a light chain, the amino acid sequence of the VL domain may comprise the amino acid sequence shown in SEQ ID NO:46, and the constant region of the light chain comprises the amino acid sequence of the human kappa light chain constant region. For example, the human constant region sequence may be the sequence described in U.S. Patent No. 5,693,780. 【0125】 This specification also provides polynucleotides encoding multispecific antibodies or fragments thereof that have been optimized, for example, by codon / RNA optimization, substitution with heterologous signal sequences, and removal of mRNA instability elements. Methods for generating optimized nucleic acids encoding multispecific antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and / or removing inhibitory regions in mRNA can be carried out using, for example, the optimization methods described in U.S. Patents 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. For example, potential splice sites and instability elements in RNA (e.g., A / T or A / U rich elements) can be mutated without changing the amino acids encoded in the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These modifications utilize the degeneracy of the genetic code, for example, by using alternative codons for the same amino acids. In some embodiments, it may be desirable to change one or more codons to encode a conservative mutation, such as a similar amino acid having a similar chemical structure and properties and / or function to the original amino acid. 【0126】 In certain embodiments, an optimized polynucleotide sequence encoding a multispecific antibody or fragment thereof (e.g., a VL domain or a VH domain) described herein may hybridize to an antisense (e.g., complementary) polynucleotide of an unoptimized polynucleotide sequence encoding a multispecific antibody or fragment thereof (e.g., a VL domain or a VH domain) described herein. In certain embodiments, an optimized nucleotide sequence encoding a multispecific antibody or fragment thereof may hybridize to an antisense polynucleotide of an unoptimized polynucleotide sequence encoding a multispecific antibody or fragment thereof under high stringency conditions. In some embodiments, an optimized nucleotide sequence encoding a multispecific antibody or fragment thereof may hybridize to an antisense polynucleotide of an unoptimized polynucleotide sequence encoding a multispecific antibody or fragment thereof under high stringency, intermediate, or lower stringency hybridization conditions. Information regarding hybridization conditions is provided, for example, in US 2005 / 0048549 (e.g., paragraphs 72-73) (incorporated herein by reference), see therefor. 【0127】 Polynucleotides can be obtained and their nucleotide sequences determined by any method known in the art. The nucleotide sequences encoding the antibodies described herein, and modified versions of these antibodies, can be determined using methods known in the art, i.e., by assembling nucleotide codons known to encode specific amino acids to generate nucleic acids encoding antibodies. Such antibody-encoding polynucleotides can be assembled from chemically synthesized oligonucleotides (as described, for example, Kutmeier G et al., (1994), BioTechniques 17: 242-246), which, in brief, involves the synthesis of overlapping oligonucleotides containing a portion of the antibody-encoding sequence, annealing and ligation of these oligonucleotides, and PCR amplification of the ligated oligonucleotides. 【0128】 Alternatively, the polynucleotides encoding the antibodies or fragments thereof described herein may be generated from nucleic acids derived from a suitable source (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of known sequences may be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Using such a PCR amplification method, nucleic acids containing sequences encoding the light and / or heavy chains of the antibody can be obtained. Using such a PCR amplification method, nucleic acids containing sequences encoding the variable light and / or variable heavy chain regions of the antibody can be obtained. The amplified nucleic acids can then be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies. 【0129】 If a clone containing nucleic acid encoding a specific antibody or fragment is unavailable, but the sequence of the antibody molecule or fragment is known, the nucleic acid encoding the immunoglobulin or fragment can be obtained by chemical synthesis or by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence from a suitable source (e.g., an antibody cDNA library, or any tissue or cell expressing the antibody, such as a cDNA library made from selected hybridoma cells expressing the antibody described herein, or nucleic acids such as poly A+ RNA isolated therefrom), or by cloning to identify, for example, an antibody-encoding cDNA clone from a cDNA library using oligonucleotide probes specific to a particular gene sequence. The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art. 【0130】 The DNA encoding the multispecific antibodies described herein can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the multispecific antibodies). Hybridoma cells can be a source of such DNA. After isolation, the DNA can be placed in an expression vector, and the vector can then be introduced into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System® (Lonza)), or myeloma cells that do not separately produce immunoglobulin proteins, to obtain synthesis of multispecific antibodies in recombinant host cells. 【0131】 To generate antibodies, the VH or VL sequence of an scFv clone can be amplified using PCR primers containing the VH or VL nucleotide sequence, a restriction site, and a flanking sequence protecting the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as the human gamma-4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as the human kappa or lambda constant region. In certain embodiments, the vector expressing the VH or VL domain includes an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the required constant region. Then, using techniques known to those skilled in the art, the heavy chain conversion vector and the light chain conversion vector are co-introduced into a cell line to generate a stable or transient cell line expressing a full-length antibody, such as IgG. 【0132】 DNA can also be modified, for example, by replacing the coding sequences of human heavy and light chain constant domains with mouse sequences, or by covalently joining all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. 【0133】 Polynucleotides are also provided that hybridize to polynucleotides encoding the antibodies described herein under high-stringency, intermediate, or lower-stringency hybridization conditions. In certain embodiments, the polynucleotides described herein hybridize to polynucleotides encoding the VH domain and / or VL domains provided herein under high-stringency, intermediate, or lower-stringency hybridization conditions. 【0134】 Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridizing the filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50–65°C, while hybridization under high stringent conditions may involve hybridizing the filter-bound nucleic acid in 6x SSC at about 45°C, followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art, and is described, for example, on pages 6.3.1–6.3.6 and 2.10.3 of Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York. 【0135】 In this specification, (a) promoter, (b) A first nucleic acid molecule encoding an antigen-binding fragment (Fab) that binds to serum albumin, and (c) A second nucleic acid molecule encoding the bioactive effector moiety and linker. Expression vectors including are further disclosed, The promoter, the first nucleic acid sequence, and the second nucleic acid molecule are functionally linked. The second nucleic acid molecule may encode two, three, four, five, six, or more bioactive effector moieties and linkers. 【0136】 In some embodiments, the first nucleic acid molecule is (a) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000118.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000119.tif4128; (b) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence SYGIS (SEQ ID NO:61), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000120.tif5128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000121.tif4128; (c) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence NYGIH (SEQ ID NO:65), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000122.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000123.tif4128; (d) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 68), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000124.tif4128, and Heavy chain CDR3 containing amino acid sequence AGWLRQYGMDV (SEQ ID NO: 70); (e) Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYWIA (SEQ ID NO: 71), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000125.tif4128, and Heavy chain CDR3 containing amino acid sequence LYSGSYSP (SEQ ID NO:73); or (f) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence Heavy-chain CDR2 including TIFF0007875806000126.tif4128, and amino acid sequence Heavy-chain CDR3 containing TIFF0007875806000127.tif4128 It contains a nucleic acid sequence encoding Fab, which includes a heavy chain variable domain. 【0137】 In some embodiments, the first nucleic acid molecule is (g) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000128.tif4128, Light chain CDR2 containing the amino acid sequence GASRLES (SEQ ID NO: 78), and Light chain CDR3 containing amino acid sequence QQSDSVPVT (SEQ ID NO: 79); (h) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000129.tif4128, Light chain CDR2 containing amino acid sequence AASSLQS (SEQ ID NO:81), and amino acid sequence Light chain CDR3 containing TIFF0007875806000130.tif4128; (i) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000131.tif4128, Light chain CDR2 containing amino acid sequence DASNRAT (SEQ ID NO: 84), and amino acid sequence Light chain CDR3 containing TIFF0007875806000132.tif4128; (j) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000133.tif4128, Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QQYGSSPRT (SEQ ID NO: 88); (k) amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000134.tif4128, Light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 87), and Light chain CDR3 containing amino acid sequence QKYSSYPLT (SEQ ID NO:90); or (l) Amino acid sequence Light chain complementarity determination domain 1 (CDR1) including TIFF0007875806000135.tif4128, Light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence Light chain CDR3 containing TIFF0007875806000136.tif4128 It contains a nucleic acid sequence encoding Fab, which includes a light chain variable domain. 【0138】 For example, the first nucleic acid molecule may include a nucleic acid sequence encoding Fab, comprising a heavy chain variable domain including (a) and a light chain variable domain including (g); a heavy chain variable domain including (b) and a light chain variable domain including (h); a heavy chain variable domain including (c) and a light chain variable domain including (i); a heavy chain variable domain including (d) and a light chain variable domain including (j); a heavy chain variable domain including (e) and a light chain variable domain including (k); a heavy chain variable domain including (f) and a light chain variable domain including (l); or any combination of the heavy chain variable domains and the light chain variable domains. In some embodiments, the first nucleic acid molecule is Heavy chain complementarity-determining domain 1 (CDR1) containing amino acid sequence AYSMN (SEQ ID NO: 74), amino acid sequence Heavy chain CDR2 containing TIFF0007875806000137.tif4128, and amino acid sequence Heavy-chain CDR3 including TIFF0007875806000138.tif4128, and amino acid sequence Light chain complementarity determination domain 1 (CDR1) containing TIFF0007875806000139.tif4128, light chain CDR2 containing amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence Light chain CDR3 containing TIFF0007875806000140.tif4128 It contains a nucleic acid sequence encoding Fab(SL335) which includes [the specified part of the sequence]. 【0139】 In other embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab which includes a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 94, 95, 96, 97, 98, at least 99%, or 100% identity with an amino acid sequence. 【0140】 In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab which includes a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. 【0141】 In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab, each comprising a heavy chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 94, 95, 96, 97, 98, or 99, and a light chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 100, 101, 102, 103, 104, or 105. 【0142】 In some embodiments, the first nucleic acid molecule has a heavy chain domain (V) containing the amino acid sequence of SEQ ID NO:45. H -C H1 The domain, and the light chain domain (V) containing the amino acid sequence of SEQ ID NO:46 L -C L Contains a nucleic acid sequence encoding Fab(SL335) including the domain. 【0143】 In some embodiments, the bioactive effector moiety is anti-TNF-α Fv, anti-TNF-α dsFv, anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1 Fv, and / or anti-IFNAR1 dsFv. For example, the second nucleic acid molecule may contain a nucleotide sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with one or more SEQ ID NO:49-60. In some embodiments, the second nucleic acid molecule may contain nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with one or more of SEQ ID NO: 6-15, 39, and 40. 【0144】 In certain contexts, the Specified Publication provides cells (e.g., host cells) that express (e.g., recombinantly) multispecific antibodies described herein that specifically bind to serum albumin (e.g., human serum albumin), as well as related polynucleotides and expression vectors. The Specified Publication provides vectors (e.g., expression vectors) comprising polynucleotides containing nucleotide sequences encoding multispecific antibodies or fragments that are recombinantly expressed in host cells, such as mammalian cells. The Specified Publication also provides host cells containing such vectors that recombinantly express multispecific antibodies described herein (e.g., human or humanized antibodies). The Specified Publication also provides methods for producing antibodies described herein, which include expressing such antibodies in host cells. 【0145】 Recombinant expression of antibodies or fragments thereof described herein (e.g., the heavy or light chain of an antibody described herein) that specifically bind involves the construction of an expression vector containing a polynucleotide encoding the antibody or fragment. Once a polynucleotide encoding an antibody or fragment thereof described herein (e.g., a heavy chain variable domain or a light chain variable domain) is obtained, a vector for producing the antibody molecule can be produced by recombinant DNA techniques using techniques well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antibody fragment (e.g., a light or heavy chain) is described herein. An expression vector containing an antibody or antibody fragment (e.g., a light or heavy chain) coding sequence and appropriate transcription and translation control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. A replicable vector is also provided, containing a nucleotide sequence encoding an antibody molecule, an antibody heavy or light chain, an antibody heavy chain variable domain or light chain variable domain, or a fragment thereof, or a heavy chain CDR or light chain CDR, functionally linked to a promoter, as described herein. Such vectors may, for example, contain a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody can be cloned into such a vector to express the entire heavy chain, the entire light chain, or both the heavy and light chains. 【0146】 The expression vector can be introduced into cells (e.g., host cells) using conventional techniques, and the resulting cells can be cultured using conventional techniques to produce the antibodies described herein. 【0147】 The antibody molecules described herein can be expressed using various host-expression vector systems. Such host-expression systems can serve as vehicles, thereby producing and purifying the coding sequence of interest, and can also become cells capable of expressing the antibody molecules described herein in situ, if transformed or transgenerated with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell lines infected with recombinant virus expression vectors containing antibody coding sequences (e.g., baculovirus); and plant cell lines (e.g., Chlamydomonas) infected with recombinant virus expression vectors containing antibody coding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid). Examples include green algae such as reinhardtii); or mammalian cell lines having recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter; vaccinia virus 7.5K promoter) (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells). In some embodiments, cells expressing the antibodies described herein (e.g., antibodies containing either the CDR of antibody pab1949 or pab2044) are CHO cells, e.g., CHO GS System® (Lonza) cells.In some embodiments, the cells expressing the antibodies described herein are human cells, e.g., human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC® or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), are used for the expression of recombinant antibody molecules, particularly for the expression of recombinant whole antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, when combined with a vector such as a major early gene promoter element derived from human cytomegalovirus, constitute an effective antibody expression system (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In some embodiments, the expression of the nucleotide sequence encoding the antibodies described herein is controlled by a constitutive promoter, an inductive promoter, or a tissue-specific promoter. 【0148】 In bacterial systems, several expression vectors may be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when producing large quantities of such antibodies to create pharmaceutical compositions of antibody molecules, a vector that directs the expression of a readily purifiable fusion protein product at high levels may be desirable. Examples of such vectors, though not limited to them, include the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794) (where the antibody coding sequence can be individually ligated within the vector, aligning the frame with the lac Z coding region, resulting in the production of a fusion protein); and the pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509). For example, a pGEX vector can be used to express an exogenous polypeptide as a fusion protein with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from thawed cells by elution in the presence of free glutathione after adsorption and binding to matrix glutathione agarose beads. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST portion. 【0149】 In insects, for example, the Autographa californica nuclear polyhedron disease virus (AcNPV) can be used as a vector to express foreign genes. This virus replicates in armyworm (Spodoptera frugiperda) cells. Antibody coding sequences can be individually cloned into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter). 【0150】 Several virus-based expression systems can be used in mammalian host cells. When using adenovirus as an expression vector, the antibody-coding sequence of interest can be ligated to the adenovirus transcription / translation regulatory complex, such as the late promoter and tri-element reader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region El or E3) results in a recombinant virus that is viable in an infected host and can express the antibody molecule (see, e.g., Logan J & Shenk T (1984) PNAS 81: 3655-3659). Specific start signals may also be required for efficient translation of the inserted antibody-coding sequence. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must be aligned with the read frame of the desired coding sequence to ensure that the entire insert is translated. These exogenous translation regulatory signals and start codons can be of various origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcriptional enhancers, transcriptional terminators, and other elements (see, for example, Bitter G et al., (1987) Methods Enzymol 153: 516-544). 【0151】 In addition, host cell lines can be selected that regulate the expression of the inserted sequence or modify and process the gene product as desired. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of a protein product may be important for the function of the protein. Various host cells have characteristic and specific mechanisms with respect to the post-translational processing and modification of protein and gene products. An appropriate cell line or host system can be selected to ensure that the expressed foreign protein is properly modified and processed. To achieve this, eukaryotic host cells with cellular mechanisms for proper processing of the primary transcript, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not exogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the multispecific antibodies described herein (e.g., antibodies containing CDR) are produced in mammalian cells such as CHO cells. 【0152】 In some embodiments, the antibodies described herein have a low fucose content or are fucose-free. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies may be expressed in cells that have insufficient or deficient ability to fucosylate. In a specific example, a low fucose-content antibody can be produced using a cell line having knockout of both alleles of α1,6-fucosyltransferase. Potelligent (登録商標) The Lonza system is an example of such a system and can be used to produce antibodies with low fucose content. 【0153】 To produce recombinant proteins in high yield over long periods, stable expression cells can be generated. For example, cell lines that stably express multispecific antibodies can be created. In certain embodiments, the cells provided herein stably express light chain / light chain variable domains and heavy chain / heavy chain variable domains, which associate to form antibodies described herein (e.g., antibodies containing CDRs). 【0154】 In certain contexts, instead of using an expression vector containing a viral replication origin, host cells may be transformed with DNA controlled by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcriptional terminators, polyadenylation sites, etc.) and a selection marker. After introducing the foreign DNA / polynucleotide, the engineered cells may be grown in concentrated medium for 1-2 days before being switched to a selection medium. The selection marker in the recombinant plasmid provides resistance to selection and allows the cell to stably incorporate the plasmid into its chromosome and grow until it forms a focus, which can then be cloned and grown into a cell line. This method can be advantageously used to create cell lines expressing the multispecific antibodies or fragments thereof described herein. Such engineered cell lines may be particularly useful for sorting and evaluating compositions that interact directly or indirectly with antibody molecules. 【0155】 Several select systems can be used, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-232), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-823) genes, which can be used in tk- cells, hgprt- cells, or aprt- cells, respectively. Furthermore, the following genes can be used as a basis for selection based on resistance to antimetabolites: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-3570; O'Hare K et al., (1981) PNAS 78: 1527-1531); gpt, which confers resistance to mycophenolate (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-2076); neo, which confers resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-215); and hygro (Santerre RF et al., (1984) Gene 30(1-3): 147-156), which provides resistance to hygromycin.Methods of recombinant DNA techniques generally known in the art can be routinely applied to the selection of desired recombinant clones, such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13 of Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); and Colbere-Garapin F et al., (1981) J Mol Biol 150: 1-14, all of which are incorporated herein by reference. 【0156】 The expression level of antibody molecules can be increased by vector amplification (see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987) for an overview). If a marker in an antibody-expressing vector system is amplified, an increase in the level of the inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66). 【0157】 Host cells may be co-introduced with two or more expression vectors described herein, the first vector encoding a heavy-chain polypeptide and the second vector encoding a light-chain polypeptide. The two vectors may contain identical selection markers that enable equal expression of the heavy-chain polypeptide and the light-chain polypeptide. Host cells may be co-introduced using two or more expression vectors in different amounts. For example, host cells may be gene-transduced using the first and second expression vectors in any one of the following ratios: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. 【0158】 Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used. In such situations, the light chain should be placed before the heavy chain to avoid excessive toxic free heavy chains (Proudfoot NJ (1986) Nature 322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199). The coding sequences for the heavy and light chains may include cDNA or genomic DNA. Expression vectors can be monocistronic or multicistronic. Multicistronic nucleic acid constructs may encode 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20. For example, a bicistronic nucleic acid construct may include a promoter, a first gene (e.g., the heavy chain of the antibody described herein), and a second gene (e.g., the light chain of the antibody described herein), in the order listed. In such an expression vector, the transcription of both genes may be driven by the promoter, but the translation of the mRNA of the first gene may be obtained by a cap-dependent scanning mechanism, while the translation of the mRNA of the second gene may be obtained by a cap-independent mechanism, such as IRES. 【0159】 The vector may include a first nucleic acid molecule encoding an antigen-binding fragment (Fab) that binds to serum albumin, a second nucleic acid molecule encoding a bioactive effector moiety, and a linker. 【0160】 The antibody molecules described herein, once produced by recombinant expression, can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography for certain antigens below protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Furthermore, to facilitate purification, the antibodies described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art. 【0161】 In certain embodiments, the antibodies described herein are isolated or purified. Generally, an isolated antibody is an antibody that substantially does not contain other antibodies having different antigen specificity from the isolated antibody. For example, in some embodiments, the antibody preparations described herein are substantially free of cellular material and / or chemical precursors. The phrase “substantially free of cellular material” includes antibody preparations in which the antibody has been isolated from the cellular components of the cell from which it is isolated or recombinantly produced. Thus, an antibody substantially free of cellular material includes antibody preparations that contain less than about 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% (by dry weight) of heterologous proteins (also referred herein as “contaminating proteins”) and / or antibody variants, e.g., antibodies with different post-translational modification forms. When antibodies or fragments are produced recombinantly, they generally contain substantially no culture medium; that is, the culture medium accounts for less than about 20%, less than 10%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the volume of the protein preparation. When antibodies or fragments are produced by chemical synthesis, they generally contain substantially no chemical precursors or other chemicals; that is, they are separated from chemical precursors or other chemicals involved in protein synthesis. Thus, such preparations of antibodies or fragments contain less than about 30%, less than 20%, less than 10%, or less than 5% (by dry weight) of chemical precursors or compounds that are not the antibody or fragment of interest. In some embodiments, the antibodies described herein are isolated or purified. 【0162】 composition This specification provides compositions comprising a multispecific antibody described herein having a desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). This specification also discloses pharmaceutical compositions comprising the multispecific antibody described herein and a pharmaceutically acceptable excipient. The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration used. 【0163】 The pharmaceutical compositions of this disclosure may provide rapid, sustained, or delayed release of the active ingredient after administration to a subject and may be formulated using methods well known to those skilled in the art. Formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injections, sterile powders, etc. Examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, arginate, gelatin, calcium phosphate, calcium silicate, cellulose, crystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, formulations may additionally contain fillers, anti-agglutinating agents, lubricants, wetting agents, favoring agents, emulsifiers, preservatives, etc. 【0164】 The pharmaceutical compositions described herein may be useful for enhancing, inducing, or activating the activity of multispecific antibodies, and for treating diseases or conditions such as autoimmune conditions or diseases. 【0165】 Compositions used for in vivo administration can be sterile. This can be easily achieved, for example, by filtering through a sterile filtration membrane. 【0166】 Usage and Method This specification discloses methods for treating autoimmune diseases or conditions in subjects where such treatment is needed, the methods comprising the step of administering a multispecific antibody or pharmaceutical composition disclosed herein to the subject. Autoimmune diseases or conditions that can be treated include, but are not limited to, neuromyelitis optica spectrum disorder, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, systemic lupus erythematosus, ANCA-associated vasculitis, ulcerative colitis, and Crohn's disease. 【0167】 In some aspects, this specification presents methods for modulating one or more immune functions or responses in a subject, the methods comprising administering a multispecific antibody or composition thereof to the subject in need. This specification discloses methods for activating, enhancing, or inducing one or more immune functions or responses in a subject, the methods comprising administering a multispecific antibody or composition thereof to the subject in need. In some aspects, this specification presents methods for preventing and / or treating diseases in which it is desirable to activate or enhance one or more immune functions or responses, the methods comprising administering a multispecific antibody or composition thereof to the subject in need. In certain aspects, this specification presents methods for treating autoimmune diseases or conditions, the methods comprising administering a multispecific antibody or composition thereof to the subject in need. 【0168】 This specification also discloses the use of multispecific antibodies or compositions disclosed herein for the treatment of autoimmune diseases or conditions, modulation of one or more immune functions or responses, activation, enhancement, or induction of one or more immune functions or responses, or prevention and / or treatment of diseases in which activation or enhancement of one or more immune functions or responses is desirable. This specification also discloses multispecific antibodies or compositions disclosed herein for use in the manufacture of pharmaceuticals for the treatment of autoimmune diseases or conditions, modulation of one or more immune functions or responses, activation, enhancement, or induction of one or more immune functions or responses, or prevention and / or treatment of diseases in which activation or enhancement of one or more immune functions or responses is desirable. 【0169】 In some embodiments, using assays well known in the art, such as ELISPOT, ELISA, and cell proliferation assays, the multispecific antibodies described herein activate, enhance, or induce one or more immune functions or responses in a subject by at least 99%, at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10%, or by only 10% to 25%, 25% to 50%, 50% to 75%, or 75% to 95%, relative to the immune function of a subject not administered with the multispecific antibodies described herein. 【0170】 Route of administration and dosage The pharmaceutical compositions of this disclosure may be administered to subjects through a variety of routes of administration, including oral, transdermal, subcutaneous, intravenous, and intramuscular routes. 【0171】 The amount of antibody or composition effective in treating and / or preventing a disease depends on the nature of the disease and can be determined by standard clinical techniques. 【0172】 In this disclosure, the actual amount of multispecific antibodies administered is determined in light of various relevant factors, including the disease being treated, the chosen route of administration, the patient's age, sex, and weight, as well as the severity of the disease, and the type of bioactive polypeptide used as the active ingredient. Because the multispecific antibodies in this disclosure have excellent blood persistence, the number and frequency of administrations of peptide preparations containing the fusion proteins in this disclosure can be significantly reduced. 【0173】 The precise dosage used in a composition depends on the route of administration and the severity of the disease, and should be determined according to the physician's judgment and the specific circumstances of each patient. For example, the effective dose may vary depending on the means of administration, the target site, the patient's physiological state (including age, weight, and health status), whether the patient is human or animal, other drugs being administered, or whether the treatment is preventive or therapeutic. While patients are usually human, non-human mammals, including transgenic mammals, may also be treated. The therapeutic dose should be optimally titrated to optimize safety and efficacy. 【0174】 In some embodiments, the dosage of the multispecific antibodies disclosed herein is 0.1 mg / kg to 100 mg / kg (body weight of the subject), 0.1 mg / kg to 80 mg / kg (body weight of the subject), 0.1 mg / kg to 60 mg / kg (body weight of the subject), 0.1 mg / kg to 50 mg / kg (body weight of the subject), 0.1 mg / kg to 40 mg / kg (body weight of the subject), 0.1 mg / kg to 30 mg / kg (body weight of the subject), 0.1 mg / kg to 20 mg / kg (body weight of the subject), 0.1 mg / kg to 10 mg / kg (body weight of the subject), 1 mg / kg to 100 mg / kg (body weight of the subject), 1 mg / kg to 80 mg / kg (body weight of the subject), 1 mg / kg to 60 mg / kg (body weight of the subject), 1 mg / kg to 50 mg / kg (body weight of the subject), 1 mg / kg to 40 mg / kg (body weight of the subject), 1 mg / kg to 30 mg / kg (body weight of the subject), 1 The dosage ranges are mg / kg to 20 mg / kg (body weight of the subject), 1 mg / kg to 10 mg / kg (body weight of the subject), 5 mg / kg to 100 mg / kg (body weight of the subject), 5 mg / kg to 80 mg / kg (body weight of the subject), 5 mg / kg to 60 mg / kg (body weight of the subject), 5 mg / kg to 50 mg / kg (body weight of the subject), 5 mg / kg to 40 mg / kg (body weight of the subject), 5 mg / kg to 30 mg / kg (body weight of the subject), 5 mg / kg to 20 mg / kg (body weight of the subject), 5 mg / kg to 10 mg / kg (body weight of the subject), or any dosage or range included in this specification, for example, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg (body weight of the subject). 【0175】 In certain embodiments, in vitro assays are used to aid in determining the optimal dose range. The effective dose can be estimated from dose-response curves derived from in vitro or animal model test systems. 【0176】 Generally, human antibodies have a longer half-life in the human body than antibodies from other species, which is due to the immune response to foreign polypeptides. Therefore, lower doses and lower administration frequencies of human antibodies are often possible. 【0177】 kit This specification provides kits comprising one or more antibodies or conjugates thereof as described herein. In some embodiments, this specification provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more antibodies provided herein. In some embodiments, the kit comprises the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, e.g., those described herein. Such containers may be accompanied by a cautionary statement in a format prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, the cautionary statement reflecting that the manufacture, use, or sale for human administration has been approved by such agency. This specification also provides kits that can be used in the methods described above. In some embodiments, the kit comprises one or more containers of antibodies described herein, e.g., purified antibodies. In some embodiments, the kit described herein comprises a substantially isolated antigen (e.g., human serum albumin) that can be used as a control. In other embodiments, the kit described herein further comprises a control antibody that does not react with serum albumin antigen. In other embodiments, the kits described herein include one or more elements for detecting the binding of an antibody to serum albumin antigen (for example, the antibody may be bound to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes a first antibody may be bound to a detectable substrate). In certain embodiments, the kits provided herein may include recombinantly produced or chemically synthesized serum albumin antigen. The serum albumin antigen prepared in the kit may also be attached to a solid support. In some embodiments, the detection means of the above kits include a solid support to which the serum albumin antigen is attached. Such kits may also include an unattached, reporter-labeled anti-human antibody or anti-mouse / rat antibody. The binding of an antibody to serum albumin antigen can be detected by binding to the reporter-labeled antibody. 【0178】 From this point forward, the present disclosure will be described with reference to several embodiments and accompanying drawings. The following embodiments and drawings are provided for illustrative purposes only and are not intended to limit the present disclosure as set forth in the accompanying claims. [Examples] 【0179】 Example 1. Materials and Methods 1. Gene cloning 1-1. Cloning of APB-A1 ((anti-CD40L scFv)2-anti-HSA Fab structure) Gene cloning was performed using standard recombination methods. hu5c8 scFv (SEQ ID NO: 5) was synthesized using codon optimization suitable for mammalian cells (Cosmo Genetech, South Korea). Cloning was performed using commercially available primers from Macrogen (Seoul, South Korea). The initial cloning was carried out via polymerase chain reaction (PCR) by ligating the synthesized hu5c8 scFv to the N-terminuses of the SL335 heavy and light chains using a flexible linker in pcDNA3.3 and pOptiVEC vectors (Thermo Fisher Scientific). Subsequently, the cloning was confirmed by protein expression in the ExpiCHO-S® cell line (Thermo Fisher Scientific, Waltham, Massachusetts). Next, to establish a production cell line from the GS NullCHO K1 cell line (Horizon Discovery, Cambridge, UK), cloning was performed using animal cell expression vectors, specifically pd2535nt (Horizon Discovery) for the heavy chain and pd2539 (Horizon Discovery) for the light chain. PCR was performed for a total of 25 cycles, with each cycle conducted at 95°C for 30 seconds, 61°C for 30 seconds, and 72°C for 1 minute, followed by a 5-minute extension period, and then a temperature reduction to 4°C. Table 1 shows the primer sets for producing recombinant vectors by cloning the APB-A1 heavy chain and light chain genes inserted into the pcDNA3.3 vector and pOptiVEC vector into the pd2535nt vector and pd2539 vector, respectively. In addition, the pGL3c(1b)(Satorius) plasmid vector was used as an additional vector. 【0180】 (Table 1) TIFF0007875806000141.tif67159 【0181】 In detail, PCR was performed as described above to obtain PCR products of the APB-A1 heavy and light chains, each approximately 1600 base pairs (bp) in length. Both ends of the heavy chain and the pd2535nt vector of the PCR product were treated with BbsI (Thermo Fisher Scientific), the 5' end of the light chain and the pd2539 vector were treated with BsrGI (Thermo Fisher Scientific), and the 3' end of the light chain and the pd2539 vector were treated with restriction enzyme BbsI, followed by T4 DNA ligase (Takara, Japan). Subsequently, E. coli strain DH5-alpha (RBC, Canada) was transformed with the produced plasmid by applying heat shock, and then purified using the midiprep kit (Macherey Nagel®, Germany) according to the manufacturer's protocol, followed by elution with nuclease-free water. The heavy and light chains of APB-A1 can have amino acid sequences with SEQ ID NO:41 and SEQ ID NO:42, respectively, where the 104th amino acid from the N-terminus of these sequences corresponds to glycine (G) or glutamine (Q). 【0182】 1-2. Cloning of APB-B1((anti-CD40L scFv)2-(anti-HSA Fab)-(anti-TNF-α Fv)) (1) The Fab gene SL335 that binds to human serum albumin, the luprizumab scFv gene that binds to CD40L, and a bioactive effector gene were synthesized. The bioactive effector may be anti-TNF-α Fv (certolizumab with heavy chain SEQ ID NO: 6 and light chain SEQ ID NO: 7) or anti-TNF-α dsFv (certolizumab with heavy chain SEQ ID NO: 8 and light chain SEQ ID NO: 9), anti-IL-23 Fv (ustekinumab with heavy chain SEQ ID NO: 10 and light chain SEQ ID NO: 11) or anti-IL-23 dsFv (ustekinumab with heavy chain SEQ ID NO: 12 and light chain SEQ ID NO: 13), or anti-IFNAR1 Fv (aniflorumab with heavy chain SEQ ID NO: 14 and light chain SEQ ID NO: 15) or anti-IFNAR1 dsFv (aniflorumab with heavy chain SEQ ID NO: 39 and light chain SEQ ID NO: 40). Tables 2, 3, and 4 provide PCR primer sets for producing APB-B1 (Macrogen, Korea). 【0183】 (Table 2) TIFF0007875806000142.tif215159 【0184】 (Table 3) TIFF0007875806000143.tif222161 【0185】 (Table 4) TIFF0007875806000144.tif222161 【0186】 To amplify the scFv, Fab, dsFv, and Fv genes, 30 cycles of PCR were performed using a T100® thermal cycler (Bio-Rad, Hercules, California) with Taq DNA polymerase (Takara, Japan), with each cycle performed at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. Next, to assemble each chain reaction product in (scFv)2-Fab-Fv or (scFv)2-Fab-dsFv format, assembly PCR was performed under cycling conditions of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute 30 seconds. Heavy chain assembly products and the pD2535NT vector (Horizon Discovery, UK) obtained by PCR were treated with BbsI restriction enzyme (Thermo Fisher Scientific, Waltham, Massachusetts), and light chain assembly products and the pd2539 vector (Horizon Discovery) were treated with Bbs I and Bsr GI restriction enzymes (New England Biolabs, Ipswich, Massachusetts). The chain reaction assembly products treated with each restriction enzyme and the plasmid vectors were assembled together using T4 DNA ligase (Takara). The assembled products were placed in soluble competent cells, treated with CaCl2, and then transformed by heat shock. Next, these transformed clones were selected using culture medium containing the antibiotic kanamycin. 【0187】 Furthermore, a cloning assay was performed in the same manner as described above to produce recombinant human CD40L (rhCD40L-his). The recombinant human CD40L gene (SEQ ID NO:38) synthesized by Cosmo Genetech was cloned into the pcDNA3.3™ vector (Thermo Fisher Scientific) using restriction enzymes Xba I (Takara) and Not I (Takara). 【0188】 (2) The protein was produced by transient expression using CHO cells. To produce SAFA-based bispecific antibodies and recombinant human CD40L protein samples, ExpiCHO cells (Thermo Fisher Scientific) were incubated in a shaking incubator at 37°C, 140 rpm, 5% CO2, and 80% humidity using ExpiCHO expression medium (Thermo Fisher Scientific). To produce transient expression cells, the cells were expressed at a concentration of 6.0 × 10⁶ 6 Cells were seeded in 125 ml culture flasks at a rate of cells / ml. These seeded cells were then transfused with plasmid vectors pD2535NT and pD2539, which contained the heavy and light chains of three sequenced genes (certolizumab, ustekinumab, and aniflorumab), and vector pcDNA3.3-TOP®, which contained the recombinant human CD40L gene, using the ExpiFectamine CHO gene transduction kit (Thermo Fisher Scientific). After incubating the cells in a shaking incubator for 16 hours, they were treated with ExpiCHO feed and enhancer, and then incubated in the same conditions for 3 days. On the third day of incubation, an additional ExpiCHO feed was added, and the cells were incubated at 32°C, 140 rpm, 5% CO2, and humidity above 80%. On day 9 of incubation, the culture medium was collected and centrifuged at 4000 rpm for 15 minutes at 4°C to isolate the cells from the culture medium. The isolated culture medium was then filtered through a 0.2 μm filter sheet to remove impurities. 【0189】 2. Production of cell lines 2-1. GS Null CHO K1 Cell Line in the Case of APB-A1 Glutamine synthesis (GS)-null CHO K1 cell line (Horizon Discovery) was used. CDfortiCHO (Thermo Fisher Scientific) medium supplemented with 4 mM L-glutamine (Gibco, Thermo Fisher Scientific) was used, and the culture medium was incubated in a 125 rpm shaking incubator at 80% humidity, 5% CO2, and 37°C. Gene transfer was performed using Freestyle™ Max reagent (Invitrogen, Thermo Fisher Scientific) in a modified version of the standard protocol provided by Horizon Discovery. Gene transfer was performed by simultaneously introducing a total of 37.6 μg of plasmid vector, with the light chain and heavy chain in a ratio of 1:1 to 1:3 (pd2539:pd2535nt). Two days after gene transfer, incubated cells were removed, transferred to a 50 ml conical tube (Nunc, Denmark), centrifuged, and then dissolved in L-glutamine-free CDfortiCHO culture medium. Cell concentration and viability were determined using a COUNTESS II automated cell counter (Invitrogen, Thermo Fisher Scientific). Partial selection was performed by adding 50 μM methionine sulfoximine (MSX) (Sigma-Aldrich, St. Louis, Missouri). Two days later, 10 μg / ml puromycin (Gibco) was added to the MSX-treated cells, and they were incubated for 48 hours. Next, the incubated cells were separated by a precipitate cell concentration of 0.5 × 10⁶. 6 To avoid a cell / ml concentration, the cells were dissolved in a culture medium that did not contain L-glutamine, and then MSX and puromycin were added to perform total selection. Subsequently, 2.0 × 10⁻⁶ cells were collected. 6 Cell concentrations were maintained so as not to exceed cells / ml, and incubation was carried out until the cell viability reached over 90%, thereby producing cell lines. 【0190】 2-2. GS null CHO-K1 cell line in the case of APB-B1 HD-BIOP3 GS-null CHO-K1 cells (Horizon Discovery) seeded in CD FortiCHO (Thermo Fisher Scientific) culture medium supplemented with 4 mM L-glutamine were subjected to a concentration of 3.0 × 10⁻⁶. 5 The cells were prepared at a concentration of cells / ml, and seed culture was carried out for one day in a shaking incubator at 37°C, 5% CO2, and humidity above 80%. For gene transfer, the cells were introduced at a concentration of 4.8 × 10⁶. 5 Seed cells at a concentration of 1.0 × 10⁶ / ml, incubated for another day, and finally reached a concentration of 1.0 × 10⁶. 6 The cells were prepared as cells / ml. Seeded cells were transfected with plasmid vectors (pD2535NT and pD2539) containing the heavy and light chain genes of sequenced certolizumab-associated SAFA-based bispecific antibodies using OptiPRO SFM culture medium and Freestyle max reagent (Invitrogen, Carlsbad, California), and incubated for 2 days at 37°C, 5% CO2, and humidity ≥80%. All incubated cells were transferred to CD FortiCHO culture medium without L-glutamine and treated with 50 μm methionine sulfoximine (MSX) (Sigma-Aldrich, St. Louis, Missouri) and 10 μg / ml puromycin (Thermo Fisher Scientific) at 2-day intervals to remove cells that did not contain the vector. Next, the existing culture medium was removed using a centrifuge, and then replaced with CD FortiCHO culture medium containing both MSX and puromycin at intervals of 7 to 10 days. Incubation was then performed for 21 days, and the cell count was increased to 5.0 × 10⁶. 5 The cell count was maintained at cells / ml. After 21 days, when the cell viability rate exceeded 70%, the cell count was increased to 3.0 × 10⁶. 5 The culture was maintained at a cell / ml level, and when the cell viability exceeded 90%, subculture production was started to obtain subculture 0 (zero) stock, which was continued until stock 3 was produced. 【0191】 3. Isolation, purification, and analysis of proteins 3-1. APB-A1 protein (1) ELISA Recombinant hCD40L antigen, rhCD40L (AprilBio, Chuncheon, South Korea), prepared according to this disclosure, was coated onto a 96-well MaxiSorp ELISA plate (Nunc) at a concentration of 100 ng / well overnight at 4°C using carbonate coating buffer (pH 9.6). The plate was blocked by treating it with blocking buffer (Starting Block (brand name) (PBS) (Thermo Fisher Scientific)) at room temperature for 3 hours. After washing with washing buffer (phosphate-buffered saline + 0.1% Tween 20; 0.1% PBST), the supernatant of a recombinant antibody called APB-A1, having the structure anti-CD40L scFv)2-anti-HSA Fab, produced from GS null CHOK1 cells, was serially diluted with dilution buffer (0.1% PBST + 0.3% BSA; 0.3% PBA) and reacted at room temperature for 1 hour. A horseradish peroxidase (HRP)-conjugated goat-anti-human Fd antibody (Southern Biotechnology, Birmingham, Alabama) was used as the secondary antibody, and luminescence was induced using a tetramethylbenzidine (TMB) substrate (BD Science, Franklin Lakes, New Jersey). Absorbance was measured at 450 nm using an ELISA reader (BMG Labtech, Germany). For the PK ELISA, rhCD40L antigen was diluted to a concentration of 1 μg / ml in PBS (Roman Industries, Japan), and 100 μl of this solution was coated onto an ELISA plate overnight at 4°C. The following day, blocking buffer (0.3% BSA in PBS, 300 μl) was added to each well, and blocking was performed at 25°C for 3 hours. Then, 100 μl of standard and QC samples were transferred to each well and reacted at 25°C for 1.5 hours. After washing three times with washing buffer (300 μl / well), anti-human light chain goat IgG-biotin (monkey absorbed; Immuno-Biological Laboratories, Japan) was added to each well at a concentration of 100 μl / well, and the mixture was reacted at 25°C for 1 hour.After washing four times, the substrates were reacted with Pierce-sensitive streptavidin-HRP (Thermo Fisher Scientific) under the same volume and time conditions, followed by another wash. Next, 100 μl of 1-step ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was transferred to each well and allowed to react at room temperature for 5 minutes. Subsequently, 1 mol / L sulfuric acid (Wako Pure Chemical, Japan; 100 μl / well) was added as a stop solution, and the mixture was mixed using a microplate mixer at 600 rpm for 10 seconds. The absorbance was then measured at 450–650 nm. 【0192】 (2) Protein purification GS null CHO K1 cell lines expressing the produced APB-A1 were incubated in CDfortiCHO culture medium for 11 days using a WAVE bioreactor (GE Healthcare). The resulting supernatant and cell pellet were centrifuged at 4°C and 4000 rpm for 20 minutes, and the culture supernatant was filtered through a 0.2 μm filter. The APB-A1 protein was purified by a three-step chromatography process. First, CaptureSelect IgG-C H1Affinity chromatography was performed using affinity matrix resin (Life Technologies). After washing the matrix with 5 column volumes (CV) of PBS, sample binding was performed at a flow rate of 20 ml / min. To remove proteins other than the target protein, washing was performed using 4 CV of high-salt washing buffer (PBS, 500 mM NaCl, pH 7.4) and 2 CV of low-salt washing buffer (25 mM sodium phosphate, pH 7.6) at a flow rate of 25 ml / min. Elution buffer (20 mM citrate, pH 3.0, 150 mM sodium chloride) was passed through the matrix at a flow rate of 20 ml / min, and the protein solution with a UV peak of 50 mAU or higher was collected, transferred to a 250 ml container, and refrigerated for 1 hour. The collected protein solution was neutralized by adding 1 M Tris-HCl (pH 8.0), and impurities were removed using a 0.2 μm filter, thereby eluting APB-A1. Next, cation exchange purification was performed using Capto® SP ImpRes resin equilibrated with a 25 mM sodium phosphate (pH 7.6) solution. Affinity chromatography eluted samples, diluted 4x with sterile distilled water, were coupled to the column, and the flow rate was set to 5 ml / min. Elution steps of 30%~50%~100% were performed using elution buffer (25 mM sodium phosphate, pH 7.6, 1 M sodium chloride). The elutes from each step were collected, and impurities were removed using a 0.2 μm filter. Subsequently, anion exchange purification was performed using POROS-based anion exchange 50 HQ resin. First, the resin column was washed with washing buffer (2 M sodium chloride) at a flow rate of 3 ml / min, and then equilibrated by passing 5 CV of 20 mM sodium phosphate (pH 6.5) solution through the resin column. To bind impurities to the resin, the sample was dialyzed with 20 mM sodium phosphate pH 6.5 buffer to adjust the pH level and salt concentration. The sample was passed through a resin column at a flow rate of 5 ml / min to collect the protein solution. Impurities were then removed using a 0.2 μm filter, and the resulting protein was quantified and analyzed. 【0193】 (3) Protein analysis - SDS-PAGE Purified APB-A1 protein was analyzed by SDS-PAGE. The sample buffers used for the analysis were LDS non-reducing sample buffer (4x; Thermo Fisher Scientific) and reducing sample buffer (4x) prepared by adding 5% mercaptoethanol to the non-reducing sample buffer. Each sample buffer was mixed with the sample, placed in a water bath, boiled for 5 minutes, and then analyzed under non-reducing boiling conditions and reducing conditions. Furthermore, the non-reducing sample buffer (4x) and the sample were mixed in a 1:4 ratio and analyzed under non-boiling conditions without heat treatment. Protein samples were loaded at a concentration of 1 μg / well onto 4-15% gradient gels (Bio-Rad, Hercules, California), and electrophoresis was performed at 150 V for 50 minutes, thereby performing SDS-PAGE analysis. After electrophoresis, the separated gels were stained with Ez-Gel staining solution (DoGenBio, Korea) for 1 hour and then destained with water. 【0194】 (4) Protein analysis - High-performance liquid chromatography (HPLC) To evaluate the size and purity of the purified proteins, SE-HPLC (high-performance liquid chromatography) was performed using prominence HPLC (Shimadzu, Japan) and a TSK gel Ultra SW aggregate column (Tosoh Bioscience, Japan). Samples were diluted with 100 mM Na2HPO4, 100 mM Na2SO4, and 0.05% (w / v)NaN3 (pH 6.7). 50 μg of the diluted sample was injected at 15°C using an automated sample injector, and then eluted using a mobile phase (200 mM phosphate, pH 6.7, 0.05% (w / v)NaN3 (flow rate: 0.5 ml / min)). UV absorbance was measured at a wavelength of 280 nm. 【0195】 (5) Protein analysis - Mass spectrometry The molecular weights of reduced and unreduced APB-A1 were measured using LC-ESI MS spectroscopy, and then analyzed in combination with Dionex UHPLC (Thermo Fisher Scientific) and Q-TOF 5600+ MS / MS system (AB SCIEX, California, USA). Using an Acquity UPLC® BEH1 30 C4 1.7 μm column, the masses of the heavy chain (H) and light chain (L) of APB-A1 were measured by passing the mobile phase [acetonitrile (ACN; JT Baker)] through the column at a flow rate of 300 μl / min. 【0196】 (6) Protein analysis - Isoelectric focusing (IEF) (4) To evaluate the isoelectric point of the purified protein, the pl value of the isolated protein was measured using an isoelectric focusing gel (pH 3-10). After loading 1 μg, 3 μg, and 5 μg of the sample onto the gel at a density of 1 mg / ml, isoelectric focusing was performed at 100 V for 1 hour, 200 V for 1 hour, and 500 V for 2 hours. After staining with 12% trichloroacetic acid (TCA) and Coomassie brilliant blue (CBB), the samples were analyzed using ImageMaster® 2D Platinum (GE healthcare, ver 5.0). 【0197】 (7) Protein analysis - Charge variant analysis To analyze the charge variant of APB-A1, ion-exchange chromatography was performed using Protein-Pak HiRes CM. A 20 μg protein sample was injected at 30°C using an automated sample injector, and then eluted over 30 minutes using a mobile phase with a gradient of 0–40% [25 mM 2-(N-morpholino)ethanesulfonic (MES), 500 nM NaCl, pH 6.5]. The flow rate was 0.3 ml / min, and UV absorbance was measured at a wavelength of 280 nm. 【0198】 3-2. APB-B1 protein (1) Isolation and purification To purify bispecific antibody protein samples present in CHO cell culture medium, affinity chromatography (AC) is used with CaptureSelect IgG-C H1The experiment was conducted using affinity matrix resin (Life Technologies, Carlsbad, California) and an AKTA pure 150 L instrument (GE Healthcare, Chicago, Illinois) as follows: The resin-packed column was equilibrated with phosphate-buffered saline (PBS) (pH 7.4) buffer, and the culture medium containing proteins expressed from transient expression cells was isolated and passed through the resin column at a flow rate of 1.5 ml / min. Subsequently, the column was passed through PBS pH 7.4 buffer containing 500 mM NaCl to wash any material that had nonspecifically bound to the resin. The equilibration and washing steps for each material were performed in 10 column volumes (CV). To elute the SAFA-based bispecific antibody from the resin, a 20 mM citrate pH 3.0 buffer containing 150 mM NaCl was used. The eluted buffer was neutralized with 1 M Tris-HCl (pH 8.0) to a neutral pH level, and the concentration of the purified protein was measured at a wavelength of A280 nm using a microplate spectrophotometer (BMG LABTECH, Germany). To purify the bispecific antibody protein sample, anion exchange chromatography (AEX) was performed after affinity chromatography using Q Sepharose HP resin (GE Healthcare) as follows: After equilibration, approximately 10 CV of 20 mM citrate pH 6.0 buffer without added NaCl was passed through the Q Sepharose HP resin. The protein initially purified by affinity chromatography was then passed through this resin, and the protein that did not bind to the resin was recovered. The concentration of the recovered protein was measured at a wavelength of A280 nm using a microplate spectrophotometer. Cation exchange chromatography (CEX) was performed by packing a column with CM Sepharose FF resin (GE Healthcare) to isolate the dimer-sized (scFv)2-Fab-dsFv, i.e., the protein corresponding to the SAFA-based bispecific antibody containing a disulfide bond, and the monomer-sized (intact) protein.Prior to purification, the protein purified by affinity chromatography was pre-treated by dialyzing with 20 mM citrate pH 6.0 binding buffer without added NaCl. Approximately 10 CV of binding buffer was passed through a resin-packed column at a flow rate of 1.0 ml / min to equilibrate, and then the pre-treated protein was added at the same flow rate and reacted with the resin. Next, 5 CV of binding buffer was passed through the column at the same flow rate, and then 3 CV of binding buffer with 100 mM NaCl was added to wash and remove any non-specifically bound material. Next, the bispecific antibody protein, existing as a monomer, was eluted from the resin by adding binding buffer with 120 mM NaCl. The concentration of the purified protein was measured at A280 nm. To purify recombinant human CD40L protein samples, a Profinity® IMAC (Bio-Rad), Hitrap Q HP, 5 ml (GE Healthcare), Hitrap SP HP, 5 ml (GE Healthcare) resin, and an AKTA pure 150 L instrument were used for three-step chromatography. First, 20 mM sodium phosphate pH 7.2 buffer was used to equilibrate and wash the three resins. To elute the protein samples, 20 mM sodium phosphate pH 7.2 buffer (Sigma) containing 500 mM imidazole was used for affinity chromatography. The protein samples were purified by anionic and cation chromatography using 15 mM sodium phosphate pH 7.4 buffer containing 1 M NaCl. The concentration of the purified protein was measured at A280 nm using a microplate spectrophotometer. 【0199】 (2)SDS-PAGE analysis First, purified bispecific antibody protein samples were diluted with non-reducing 4x SDS sample buffer (Thermo Fisher Scientific) and 2-mercaptoethanol-containing reducing sample buffer. Under non-reducing conditions, samples heated at 100°C for 5 minutes and unheated samples were prepared to compare the heat-dependent protein type and size. For size comparison, protein size markers (SMOBio, Taiwan) and (scFv)2-Fab protein samples were processed together. The prepared protein samples were loaded at a density of 2 μg / well onto 4-15% 15-well Miniprotein TGX precast gels (Bio-Rad), and electrophoresis was performed in Tris-glycine SDS electrophoresis buffer at 150 V for 1 hour. After electrophoresis, the SDS-PAGE gels were stained with EZ-Gel staining solution (DoGenBio, Korea) for 1 hour and destained in distilled water for 1 day. 【0200】 (3) Analysis of protein melting temperature To evaluate the thermal stability of bispecific antibody protein samples, the protein melting temperature was analyzed using a hydrophobic dye (5000x, SYPRO Orange) and a Light Cycler 480 II (Roche, Switzerland) as a real-time PCR instrument. Protein samples were diluted to 300 μg / ml in sodium phosphate buffer (pH 7.0), and then 5x reagent and SYPRO Orange dye were placed on an ultraAmp PCR plate (Sorenson Bioscience, Salt Lake City, Utah) until the final concentration in each well reached 5.4 μg / well. Next, the excitation filter and fluorescence filter were set to 465 nm and 580 nm, respectively, and protein denaturation was evaluated as the temperature increased at a rate of 1°C / min within the range of 20°C to 85°C. 【0201】 (4) High-performance liquid chromatography using molecular sieves (SE-HPLC) The purity of the purified bispecific antibodies was analyzed using a TSKgel UltraSW Aggregate 7.8 x 300 mm column (Tosoh Bioscience, Japan) and a 1260 infinity II LC system (Agilent Technologies, Santa Clara, California) as the HPLC instrument. Before analyzing the samples, the column and HPLC instrument were equilibrated with 100 mM 20 mM citrate (pH 5.5) buffer. The samples to be analyzed were diluted in 20 mM citrate pH 5.5 buffer and loaded onto the column at a maximum density of 25 μg. SE-HPLC analysis was performed over 30 minutes at a flow rate of 0.7 ml / min and a maximum pressure limit of 120 bar, and absorbance was measured at A280 nm. 【0202】 (5) Enzyme-linked immunosorbent assay (ELISA) ELISA was performed to evaluate the binding reaction of purified bispecific antibody samples to human serum albumin (Sigma-Aldrich), recombinant human CD40L protein, and recombinant human TNF-α protein (BioLegend, San Diego, California). Human serum albumin, CD40L protein, and TNF-α protein were diluted to a concentration of 1 μg / ml in sodium carbonate pH 9.6 buffer, and 100 μl of each was seeded into each well of a 96-well maxisoap plate (Nunc, Denmark), followed by coating at 4°C for 1 day. Uncoated proteins and buffer were completely removed, and then blocking was performed by adding 300 μl each of PBS pH 7.4 buffer containing 3% calf serum albumin (BSA) (Sigma-Aldrich) and 0.1% Tween-20 to each well. After blocking for 2 hours, washing was performed by adding 300 μl each of buffer (PBS-T) (pH 7.4) containing 0.1% Tween-20 PBS, and then removing the buffer a total of three times, repeating the process to completely remove the added buffer. After removing the remaining water after washing, each antibody was tested in PBS pH 7.4 buffer (0.3% PBA) containing 0.3% calf serum albumin and 0.1% Tween-20 at concentrations ranging from 100 nM to 1.0 × 10⁶. -4 The antibody samples were sequentially diluted to nM to a 10-fold decrease, and 100 μl of each diluted antibody sample was added to each well and reacted at room temperature for 2 hours. After washing in the same manner as above, HRP-conjugated goat anti-human Fd antibody (Southern Biotechnology, Birmingham, Alabama) was diluted 1:4000 in 0.3% PBA buffer, and 100 μl of each diluted antibody sample was added to each well and reacted at room temperature for 1 hour. To evaluate the antigen-specific binding of the antibody reacting after washing, TMB substrate (BD Bioscience, Franklin Lakes, New Jersey) was added and the reaction was carried out, and the absorbance was measured at A450 nm using a microplate spectrophotometer. 【0203】 (6) Biolayer Interference (BLI) Assay The antigen affinities of bispecific antibodies and individual monospecific antibodies against human serum albumin, CD40L, and TNF-α were evaluated using biolayer interferometry (BLI) with an Octet Red instrument (Forte Bio, Fremont, California). First, TNF-α protein (30 μg / ml), CD40L protein (10 μg / ml), and human serum albumin (20 μg / ml) were immobilized on an amine-reactive second-generation (AR2G) biosensor (Forte Bio) using sodium acetate buffer at pH 5.0. Unimmobilized material was removed with 1 M ethanolamine (pH 8.5), and then bispecific antibodies at serial dilution concentrations were reacted. The binding and dissociation constants with each antigen were then measured. Furthermore, to evaluate the simultaneous binding ability of the bispecific antibody to three antigens, CD40L protein (10 μg / ml) was immobilized on an AR2G biosensor using sodium acetate buffer at pH 5.0, and the binding ability was measured in the following order: bispecific antibody (3.2 μg / ml), human serum albumin (13.2 μg / ml), human serum albumin (13.2 μg / ml), and TNF-α (2 μg / ml). The evaluation results were analyzed using DataAnalysis8 software. 【0204】 (7) Flow cytometry analysis To identify the binding of the bispecific antibody to the cell membrane CD40L, flow cytometry analysis was performed using a FACSVerse instrument (BD Biosciences, Franklin Lakes, New Jersey). D1.1 cells expressing cell membrane CD40L on their cell surface (CRL-10915, ATCC, Manassas, Virginia) were incubated in RPMI1640 (Thermo Fisher Scientific) containing 10% fetal bovine serum (Thermo Fisher Scientific) and measured 3.0 × 10⁶ 5Cells / test tubes were prepared and washed twice with 0.3% PBA buffer. SAFA-based bispecific antibodies and control antibodies were reacted with the washed cells, respectively, for 30 minutes at 4°C, washed twice, and then a 1:1000 dilution of fluorescent isothiocyanate (FITC)-conjugated goat anti-human kappa antibody (LifeSpan BioSciences, Inc., Seattle, Washington) was added, followed by reaction at 4°C for 30 minutes. The washing step was then repeated twice, and the antibody binding reaction to the cell membrane CD40L was evaluated using a FACSVerse instrument. 【0205】 Example 2: APB-A1 1-1: Binding assay using biolayer interferometry Real-time binding assays of human serum albumin (HSA) (Sigma-Aldrich) to SL335 and rhCD40L antigen to APB-A1 were performed using biolayer interferometry with the Octet RED system. To evaluate HSA binding affinity, 20 μg / ml of HSA and 10 μg / ml of rhCD40L were immobilized on an AR2G biosensor (pH 5.0), and unbound molecules were removed from the biosensor surface using kinetics buffer (1 M ethanolamine, pH 8.5). Experiments were conducted in the concentration range of 10 nM to 0.3125 nM to identify the affinity of APB-A1 binding to HSA and rhCD40L. To identify bispecific binding, the rhCD40L antigen was immobilized on an AR2G biosensor, reacted first with a predetermined concentration of APB-A1, and then bound to HSA. Binding and dissociation kinetics were obtained using Octet QK software. The bond rate constants were calculated so that the observed bond curves fit a 1:1 bond model. 【0206】 1-2: Determination of binding or non-binding of APB-A1 protein to CD40L-expressing cells Flow cytometry was performed at the College of Pharmacy, Kangwon National University to confirm that the APB-A1 protein binds to D1.1 cells expressing CD40L cells. The D1.1 cells were centrifuged and the supernatant was removed, then filtered in MACS buffer (1x PBS containing 0.5% BSA and 2 mM EDTA, 0.22 μm filtration) at a concentration of 1.0 × 10⁶. 6 The cells were resuspended at a concentration of cells / ml. 100 μl of cells at each concentration (1.0 10 5 Cells (or test cells) were seeded into 1.5 ml test tubes and centrifuged at 4°C and 500x g for 5 minutes, thereby removing the supernatant. APB-A1, hu5c8 IgG1, and SL335 were sequentially diluted by 1 / 10 at five time points, starting from 1 μg / ml each. 100 μl of the initial diluted antibody was pipetteed into the cell pellet in a 1.5 ml test tube and incubated at 4°C for 30 minutes. 500 μl of MACS buffer was added to each test tube, and the cells were washed by centrifuging at 4°C and 500x g for 5 minutes. After removing the supernatant, the cell pellet was lysed by adding a 1:1000 dilution of goat-anti-human kappa-FITC sample (Lifespan Biosciences, Washington, Seattle) in 50 μl of MACS buffer, incubated at 4°C for 30 minutes, and the washing process was repeated. After removing the supernatant, the cell pellet was lysed by adding 200 μl of 0.4% paraformaldehyde (PFA in PBS) buffer, then stored and fixed at 4°C, and the cells were analyzed using a BD FACSverse instrument. 【0207】 1-3: Analysis of in vitro CD40-CD40L inhibition To analyze the efficacy of APB-A1 in inhibiting the CD40-CD40L interaction, HEKBlue® CD40L reporter cells (InvivoGen, San Diego, California) were used, and D1.1 cells expressing mCD40L and rhCD40L were used as CD40L donors. APB-A1, hu5c8 IgG1, and SL335 were diluted by 1 / 3 starting from a concentration of 200 nM using Dulbecco's PBS buffer (Corning) with or without 20 M HSA. After seeding 20 μl of each diluted sample into a 96-well cell culture plate (Corning), 1 × 10⁶ wells of the same volume were added. 4 D1.1 cells were added at a cell / well ratio and incubated in an incubator at 37°C and 5% CO2 for 3 hours. Next, HEKBlue® CD40L cells were added in 5 × 10⁶ wells. 4 Cells were added at the specified cell / well density and incubated in an incubator under the same conditions for 21 hours. After 24 hours, 40 μl of the supernatant from each plate was transferred to another 96-well EIA / RIA plate (Corning) using a multipipette. 160 μl of QUANTI-Blue® solution (InvivoGen) was added to each well, each well was wrapped in foil to block light, and the incubators were placed in a 37°C CO2 incubator for 1 hour. The absorbance was then measured at 655 nm using a spectrophotometer. 70 μl of 300 ng / ml rhCD40L antigen was added to test tubes containing 70 μl of diluted samples obtained by diluting three samples to the same concentration by 1 / 3, starting from 200 nM, and the mixture was reacted in a CO2 incubator at 37°C for 30 minutes. Next, HEKBlue® CD40L cells were added to a final concentration of 3.125 × 10⁶. 5 After diluting to a cell / ml concentration, 560 μl of diluted cells were added to a test tube containing the sample mixed with rhCD40L antigen, then inverted, and the cells were then seeded into a 96-well cell culture plate at a concentration of 200 μl / well. Incubation was performed for 21 hours in the same manner as for D1.1 cells, and the resulting cells were reacted with the substrate, thereby evaluating the absorbance. 【0208】 1-4: Platelet Aggregation Assay Human platelet-rich plasma (PRP) was collected from healthy volunteers with prior consent and provided by the Korean Red Cross Blood Center (KRBC) (Republic of Korea). Anticoagulated PRP was centrifuged in dextrose citrate solution (0.8% citrate, 2.2% sodium citrate, 2.45% glucose) to remove red blood cells, and then centrifuged again at 360x g for 15 minutes to obtain plate pellets. Platelets were then added to platelet-poor plasma (PPP) at a final concentration of 5.0 × 10⁶. 8 The PRP was dissolved in / ml and the entire procedure was carried out at room temperature (23±2℃). To evaluate platelet aggregation, transmitted light platelet aggregation was performed using a Chrono-log agglutinator (CHRONO-LOG®, Havertown, Pennsylvania). Immune complexes (ICs), i.e., rhCD40L + hu5c8 IgG1 (30 μg / ml + 60 ng / ml), rhCD40L + APB-A1 (30 μg / ml + 40 ng / ml), or rhCD40L, hu5c8 IgG1, and APB-A1 respectively, were added to 5-10 mM CaCl2 at 37℃ for 2 minutes, and then the PRP was stimulated for 5 minutes under continuous stirring conditions at a suboptimal ADP concentration (CHRONO-LOG®). Once the agglutination reaction was complete, the platelet mixture was centrifuged, and serotonin release was measured from the supernatant using a serotonin EIA kit (Labor DiagnostikaNord, Germany) as instructed by the manufacturer. 【0209】 1-5: Pharmacokinetic (PK) analysis and pharmacodynamic (PD) analysis (1)PK analysis To evaluate the serum half-life of APB-A1, pharmacokinetic analyses were performed using a cynomolgus monkey model [Shin Nippon Biomedical Laboratories (SNBL, Japan)]. APB-A1 protein was administered to three male cynomolgus monkeys in each group via a single intravenous injection at a dose of 5 mg / kg (Group 1) or 20 mg / kg (Group 2). Blood samples were collected at 17 time points after administration: one time point was pre-administration, and 16 time points were at 0.25 hours, 1 hour, 2 hours, 6 hours, and 24 hours after administration, as well as at 4, 7, 10, 13, 16, 19, 22, 25, 28, 34, and 40 days. The concentration of APB-A1 in the platelets of each cynomolgus monkey was measured by ELISA. 【0210】 (2)PD analysis The efficacy of APB-A1 in suppressing the anti-tetanus toxoid (TT) antibody response was analyzed (Southern Research, Birmingham, Alabama). Four sample groups—vehicle (negative control: 20 mmol / L sodium phosphate, pH 6.5), dexamethasone (DXT) (positive control), and APB-A1 (5 mg / kg and 20 mg / kg)—were administered intravenously to cynomolgus monkeys (female; n=3 / group). First, to induce the anti-TT antibody response, TT (5 Lf) was administered intramuscularly for the first time on day 1, and a second intramuscular dose for a boost was administered on day 20. DXT was administered in four doses of 1 mg / kg each: two days before the first TT injection, and on days 1, 5, and 8 after the first TT injection. APB-A1 was administered once at the time of the first TT injection (day 1). Blood samples for analysis were collected before injection, and on days 10, 12, 14, 16, 20, 27, 30, and 40. Anti-TT IgG antibody levels were measured by ELISA. For a second analysis of the antibody response, various B-cell immunophenotypes were examined using immunophenotyping. Blood samples were collected a total of five times: approximately 2 hours after TT injection, and on days 20, 27, 30, and 40 after TT injection. The collected blood samples were stored in test tubes containing K2-EDTA to prevent blood coagulation from the non-injected portion. Immunophenotyping was performed using an antibody panel of markers such as CD45, CD20, CD27, Ki67, and IgD. Predetermined portions of four cell groups, including CD45+ / 20+, CD45+ / 20+ / Ki67+, CD45+ / 20- / 27hi / IgD-, and CD45+20+ / 27+IgD- / Ki67+, were used for immunophenotyping. 【0211】 Example 3: APB-B1 1-1: Testing of the inhibitory effect on TNF-α-mediated cytotoxicity using mouse L929 cells. To compare the soluble TNF-α protein inhibitory abilities of a bispecific antibody and the parent antibody (certolizumab Fab'), L929 mouse cells expressing the TNF receptor on the cell surface and recombinant soluble TNF-α protein were used. L929 cells (Korean Cell Line Bank) were incubated in an incubator at 37°C, 5% CO2, and over 80% humidity with RPMI1640 culture medium containing 10% fetal bovine serum. L929 cells were incubated at a concentration of 5.0 × 10⁶. 4 Cells were seeded into a 96-well cell culture plate (Corning Inc., New York City, NY) at a cell / well rate, and then incubated in an incubator under the same conditions for 24 hours. After 24 hours of incubation, the existing culture medium was removed, and 1 μg / ml of actinomycin D (Sigma-Aldrich), diluted in RPMI1640 culture medium containing 10% fetal bovine serum, was added to each well. The mixture was then incubated in an incubator at 37°C, 5% CO2, and over 80% humidity for 30 minutes. Next, antibodies diluted in series according to various concentrations were added to each well, followed by 10 ng / ml of recombinant soluble TNF-α protein. The mixture was then incubated in an incubator at 37°C, 5% CO2, and over 80% humidity for 24 hours. After 24 hours of reaction, 10 μl of CCK-8 (Dojindo, Japan) was added to each well containing the reaction product by transferring it using a multichannel pipette. After a 2-hour reaction, the supernatant was transferred to another plate using a pipette, and the absorbance was measured at a wavelength of A450 nm. 【0212】 1-2: Inhibition of CD40L and TNF-α using CD40L HEK-blue® reporter cells The co-inhibitory ability of bispecific antibodies and parental antibodies (luprizumab IgG1 and certolizumab Fab') against one or both of the cell membrane CD40L and soluble TNF-α proteins was analyzed. To achieve this, CD40L HEK-Blue® reporter cells expressing secreted embryonic alkaline phosphatase (SEAP) (InvivoGen, San Diego, California) were used to analyze CD40L and TNF-α, D1.1 cells expressing cell membrane CD40L on their cell surface, and recombinant soluble TNF-α protein. CD40L HEK-Blue® cells were incubated in an incubator at 37°C, 5% CO2, and over 80% humidity with DMEM (Thermo Fisher Scientific) culture medium containing 10% fetal bovine serum and antibiotics (normocin, blastocydin, and zeosin). D1.1 cells were incubated in an incubator under the same conditions using RPMI1640 culture medium containing 10% fetal bovine serum. Next, the D1.1 cells were incubated at a concentration of 5.0 × 10⁶. 4 Cells were seeded in 96-well cell culture plates at a concentration of cells / well, or recombinant soluble TNF-α protein was seeded in 96-well cell culture plates at a concentration of 10 ng / ml. To evaluate the simultaneous binding reaction to the two samples, D1.1 cells and recombinant soluble TNF-α protein were seeded together in 96-well cell culture plates. After adding serial dilutions (pre-treated with human serum albumin) to the 96-well cell culture plates seeded with either or both cells and / or recombinant protein, the samples were incubated at 37°C, 5% CO2, and over 80% humidity for 3 hours. After 3 hours of incubation, CD40L HEK-Blue® cells were added to all wells of the plate at a concentration of 5 × 10⁶. 4After adding the reagent to each well, the cells were incubated under the same conditions for 21 hours. Next, 160 μl of QUANTI-Blue reagent (SEAP detection reagent, InvivoGen), which had been reacted in 37°C water for 30 minutes, was seeded into all wells of a new 96-well cell culture plate. 40 μl of the reaction solution, which had been reacted in the incubator for 21 hours, was added to each well containing the QUANTI-Blue reagent, and the reaction was then initiated. After incubating in a 37°C incubator for 1 hour, the absorbance was measured at a wavelength of 655 nm. 【0213】 Example 4. Results 1. Experimental results of APB-A1 (1) Expression and production of APB-A1 To produce APB-A1, hu5c8 scFv(V L + V H )- Flexible linker (SEQ ID NO:3 - TIFF0007875806000145.tif3128; Linker 1)-SL335 Fd(V H + C H1 The gene (named APB-A1 heavy chain) and hu5c8 scFv(V L + V H )- Flexible linker (SEQ ID NO: 4- TIFF0007875806000146.tif4128; Linker 2)-SL335 Kappa (V LThe APB-A1 H (CL) (named APB-A1 L light chain) gene was linked by linking PCR. After cloning them into pcDNA3.3 and pOptiVEC vectors, respectively, these genes were introduced into the ExpiCHO-S® cell line for transient expression, and Western blotting was used to determine whether 101.7 kDa-APBA1 (APB-A1 H chain; 50.7 kDa-483 amino acids and APB-A1 L chain; 50.9 kDa-478 amino acids) was normally expressed. Next, for stable expression in CHO cells, the APB-A1 H and APB-A1 L genes were cloned into pd2535nt and pd2539 vectors, respectively, to produce recombinant vectors (Figure 1A), and amino acid sequencing confirmed that there were no abnormalities (Figure 1B). Figure 1A shows the APB-A1 heavy and light chains inserted into the pd2535nt and pd2539 vectors. Figure 1B shows the amino acid sequences of APB-A1 H (483 amino acids total) and APB-A1 L (478 amino acids total), which are SL335 H and SL335 L with hu5c8 scFv linked by linker 1 and linker 2, respectively. The recombinant vector was introduced into GS null CHO K1 cells before use and then sorted using MSX and puromycin to construct a stable CHO cell line. To produce APB-A1 protein for evaluation of in vitro and in vivo effects, the stable CHO cell line was cultured in a bioreactor, the supernatant was obtained, and purification was performed using a total of three steps including affinity chromatography, cation exchange chromatography, and anion exchange chromatography. Affinity chromatography, as the first step, yielded APB-A1 protein in yield of over 95%, and APB-A1 samples were obtained in yield of 82% using cation and anion exchange chromatography, the second and third steps, to remove impurities and endotoxins. Next, to evaluate the purity of the protein, SE-HPLC was performed under native conditions. The results of three replicate experiments confirmed that the perfect APB-A1 sample had a purity of over 95%, and Figure 2A shows one of these replicate experiments.To determine the purity and molecular weight of the samples obtained by the purification process, the obtained samples were analyzed by SDS-PAGE under reducing, non-reducing (boiling), and non-reducing (non-boiling) conditions (Figure 2B). Therefore, Figure 2A shows the analysis results of the characteristics of APB-A1 identified by HPLC, and Figure 2B shows the analysis results of the characteristics of APB-A1 identified by SDS-PAGE. The characteristics of APB-A1 purified from the supernatant of purified GS null CHO K1 cell culture medium were identified by (A) HPLC and (B) SDS-PAGE on a 4-15% gradient gel under reducing (R), non-reducing (boiling) (NR(B)), and non-reducing (non-boiling) (NR(NB)) conditions. In the HPLC analysis in Figure 2A, the protein sample was analyzed by SE-HPLC under negative conditions (no DTT). The purity was 95% or higher. In the SDS-PAGE gel shown in Figure 2B, the protein bands were visualized using Ez-gel staining solution. As mentioned above, the theoretical molecular weights of APB-A1 H (50.7 kDa) and APB-A1 L polypeptide (50.9 kDa) were 50.7 kDa and 50.9 kDa, respectively. Under reducing (R) conditions, two protein bands were identified around a molecular weight size of 50 kDa, but because the molecular weights of the APB-A1 H polypeptide and the APB-A1 L polypeptide are very similar, it was extremely difficult to accurately distinguish between the two bands (lane R in Figure 2B). Under non-reducing (boiling) conditions, the two bands were identified at slightly smaller positions than under reducing conditions [lane NR(B) in Figure 2B], and Western blotting confirmed that the APB-A1 H band was located slightly higher than the APB-A1 L band (data not shown). Under non-reducing (non-boiling) conditions, the identified APB-A1 was found to have a molecular weight smaller than its theoretical molecular weight (101.6 kDa) [Lane NR(NB) in Figure 2B]. 【0214】 (2) Molecular characteristics of APB-A1 To accurately measure the mass of the APB-A1 protein, Q-TOF analysis was performed under reducing and non-reducing conditions. The measured masses of the heavy chain (H) and light chain (L) of APB-A1 were 50.77 kDa and 50.98 kDa, respectively (Figure 3). In Figure 3, the characteristics of the purified APB-A1 protein were identified using a mass spectrometer (ProteomeTech, South Korea). In mass spectrometry, the protein sample was analyzed under reducing and non-reducing conditions. The theoretical molecular weights of the APB-A1 heavy and light chains were 50,777 Da and 50,994 Da, respectively, which were substantially the same as the values ​​measured by mass spectrometry in this case. Considering that APB-A1 did not have an N-linked glycosylation site as confirmed by glycosylation prediction software, and that no particular peaks were observed in Q-TOF analysis other than the H and L peaks of APB-A1, it was predicted that APB-A1 would not contain N-linked glycosylation. Analysis using pI analysis software confirmed that APB-A1 has a theoretical pI value of 8.65, while the measured values ​​from isoelectric focusing at pH 3-10 and capillary isoelectric focusing (cIEF) were 9.16 and 9.2, respectively (Figures 4A and 4B). In Figures 4A and 4B, pI analysis of purified APB-A1 protein was performed using ProteomeTech. The pI values ​​identified by isoelectric focusing (IEF) gels at pH 3-10 shown in Figure 4A and by capillary isoelectric focusing (cIEF) shown in Figure 4B were 9.16 and 9.2, respectively. To accurately analyze the charge variants of APB-A1, charge variant experiments were repeatedly performed using ultra-high-performance liquid chromatography (UPLC). The UPLC assays yielded peak profiles showing that 76.3% of the samples had a main peak with a constant charge, 4.6% had an acidic peak, and 19.1% had a basic peak (Figure 5). 【0215】 (3) Functional characteristics of APB-A1 in vitro To evaluate the binding affinity of APB-A1 to HSA and rhCD40L antigens, biolayer interferometry was performed using the Octet Red instrument. The results confirmed that the dissociation constants (KD) of APB-A1 to HSA and rhCD40L antigens are 748 pM and 127 pM, respectively. Therefore, the equilibrium dissociation constant (KD) of APB-A1-HSA is calculated as follows: D ) is the K of SL335-HSA D It is approximately 2.6 times larger (748 pM and 286 pM respectively), and the dissociation constant of APB-A1-rhCD40L is the same as that of hu5c8 IgG1-rhCD40L. D It was confirmed that these values ​​were approximately 2.6 times larger (127 pM and 49.6 pM, respectively). 【0216】 To confirm the binding affinity of APB-A1 to HSA and rhCD40L antigens, biolayer interferometry was performed again using the Octet Red instrument. From the evaluation results, the equilibrium dissociation constant (K) of APB-A1 for HSA and rhCD40L antigens was determined. D The concentrations were confirmed to be 628 pM and 186 pM, respectively. The average of the two results is provided in Table 5. 【0217】 (Table 5) TIFF0007875806000147.tif23159 【0218】 In addition, to identify that APB-A1 co-binds to HSA and rhCD40L antigens, rhCD40L antigen was immobilized on an AR2G biosensor, and biolayer interference was performed by sequentially reacting it with APB-A1 and HSA. As a result, it was confirmed that APB-A1 has the ability to co-bind to HSA and rhCD40L antigens (Figure 6). For cell-based in vitro evaluation, D1.1 cells expressing mCD40L were used, and as a preliminary experiment, flow cytometry analysis was used to determine whether APBA1 and hu5c8 IgG1, as control groups, bound to mCD40L expressed by D1.1 cells. The analysis results showed that SL335, as a negative control group, did not bind to mCD40L on D1.1 cells, but APB-A1 and hu5c8 IgG1 did (Figure 7). In Figure 7, APB-A1 (a) and hu5c8 IgG1 (b) were bound to D1.1 cells expressing mCD40L, and SL335 (c), which does not bind to D1.1 cells, was used as a negative control group. Next, to identify the efficacy of APB-A1 in suppressing CD40-CD40L interaction, HEKBlue® CD40L reporter cells were combined with D1.1 cells or rhCD40L antigen with or without HSA, and then reacted by adding APB-A1, hu5c8 IgG1, and SL335 (concentrations ranging from 0.01 to 22.2 nM). The alkaline phosphatase (AP) response of the reporter cells was then measured (Figures 8A-8D). In Figures 8A-8D, the ability of APB-A1 and hu5c8 IgG1 to inhibit the CD40L-CD40 interaction in the absence of HSA was 0.9907 nM and 0.289 nM (Figure 8B), and in the presence of HSA it was 1.031 nM and 0.4729 nM (Figure 8A). IC50 of the ability of APB-A1 and hu5c8 IgG1 to inhibit the interaction between soluble CD40L and CD40 in the absence of HSA 50The values ​​were 1.031 nM and 0.4729 nM (Figure 8D), and 0.6371 nM and 0.501 nM in the presence of HSA (Figure 8C). In experiments using reporter cells and D1.1 cells, APB-A1 demonstrated low inhibitory efficacy, approximately three times lower than hu5c8 IgG1 in the absence of HSA, but in the presence of HSA, APB-A1 and hu5c8 IgG1 demonstrated substantially the same inhibitory efficacy (Figures 8A and 8B). Similarly, in experiments using reporter cells and rhCD40L antigen, the inhibitory efficacy of APB-A1 was approximately 2.1 times lower than hu5c8 IgG1 in the absence of HSA, but in the presence of HSA, APB-A1 and hu5c8 IgG1 demonstrated substantially the same inhibitory efficacy (Figures 8C and 8D). Furthermore, SL335, used as a negative control, did not demonstrate inhibitory efficacy under any conditions (Figures 8A-8D). When using D1.1 cells, the IC (Impression Captivity) obtained from the results shown in Figures 8A and 8B demonstrates the CD40L-CD40 interaction inhibitory ability. 50 The values ​​were 0.9907 nM in the absence of HSA and 0.2988 nM in the presence of HSA, respectively. IC of hu5c8 IgG1 as a positive control. 50 The value was identified as approximately 0.2-0.3 nM, and the presence or absence of HSA was determined by the IC of hu5c8 IgG1. 50 It was suggested that there was no effect on the values ​​(Figures 8A and 8B). When using rhCD40L antigen, IC 50 The IC5c8 IgG1 IC5c8 is 1.031 nM in the absence of HSA and 0.6371 nM in the presence of HSA. 50 The value was determined to be approximately 0.47–0.5 nM, which was substantially the same as the results of the experiment described above (Figures 8C and 8D). 【0219】 (4) Analysis of the effect of APB-A1 on platelet aggregation We determined whether platelet aggregation, a typical side effect of conventional anti-CD40L IgG antibodies, was induced by APB-A1. To achieve this, we produced rhCD40L hu5c8 IgG1 IC and rhCD40L + APB-A1 IC, and then reacted them with ADP-stimulated platelets. The occurrence of platelet aggregation was determined by measuring the transmittance. The results showed that only rhCD40L hu5c8 IgG1 IC strongly stimulated platelet aggregation (Figures 9A-9D). In Figures 9A-9D, PRP was pre-cultured at 37°C for 2 minutes in the presence of 5-10 mM CaCl2 with hCD40L (30 μg / ml) and different concentrations of hu5c8 IgG1 (6 ng / ml, 60 ng / ml, and 600 ng / ml) or different concentrations of APB-A1 (4 ng / ml, 40 ng / ml, and 400 ng / ml). Next, platelets were further stimulated with ADP at a concentration below the optimal level, while continuous stirring was performed. Platelet aggregation occurred in the hu5c8 IgG1 + rhCD40L IC sample (Figures 9A and 9B), while the platelet aggregation rate was low in the rhCD40L + APB-A1 sample (Figures 9C and 9D). The data show the standard deviation (SD) of experiments using at least six different donors. To quantitatively analyze the results shown in Figures 9A-9D, the platelet aggregation percentage was calculated. According to these calculations, the rhCD40L + hu5c8 IgG1 IC sample demonstrated a response of over 80% at concentrations of 60 ng / ml and 600 ng / ml, while the rhCD40L + APB-A1 sample demonstrated less than 10% platelet aggregation at a concentration of 400 ng / ml (Figure 9D). In addition, the concentration of serotonin released when platelets were activated was measured, and the results showed that serotonin release levels did not increase with activation using rhCD40L + APB-A1 and other sample groups, but serotonin release levels increased to approximately 300 pg / ml with activation using rhCD40L + hu5c8 IgG1 IC (Figure 10). In Figure 10, the amount of released serotonin increased in the case of recombinant rhCD40L + hu5c8 IgG1, but no significant difference was observed in the other groups.The data show the mean ± standard deviation (SD) of four or more independent experiments (***p<0.001, compared to IC (recombinant hCD40L + hu5c8 IgG1) control). 【0220】 (5) Pharmacokinetic study of APB-A1 To evaluate the in vivo half-life of APB-A1, pharmacokinetic assays were performed using a cynomolgus monkey model (n=3 / group). APB-A1 was administered by a single intravenous injection at two doses: 5 mg / kg and 20 mg / kg. Blood samples were collected at the same time points as described in the Materials and Methods section, and the concentration of APB-A1 in plasma was measured using PK ELISA (Figure 11). In Figure 11, purified APB-A1 antibody was injected into male cynomolgus monkeys (n=3) at various concentrations (5 mg / kg and 20 mg / kg). Experiments were performed using SNBL. The APB-A1 concentration in the samples at each time point was measured by ELISA, and the data represent the mean of the experiments performed. The half-lives were calculated using Phoenix WinNonlin software (ver 6.4; Certara LP, Princeton, New Jersey, USA) based on ELISA results, and the half-lives of 5 mg / kg APB-A1 and 20 mg / kg APB-A1 were determined to be approximately 7 days and 9.6 days, respectively. Therefore, it was understood that the half-life of APB-A1 was approximately 1.4 times longer at a dose of 20 mg / kg than at a dose of 5 mg / kg. The Cmax values ​​were 143 μg / ml and 509 μg / ml at doses of 5 mg / kg and 20 mg / kg, respectively, and the rectal clearance (CL) rates were at the same level regardless of dose, at 4.44 ml / day / kg and 4.72 ml / day / kg (Table 6). 【0221】 (Table 6) TIFF0007875806000148.tif34164 【0222】 (6) APB-A1 Pharmacodynamic Assay To evaluate the in vivo efficacy of APB-A1, a pharmacodynamic study was conducted using cynomolgus monkeys (n=3 / group). First, TT was intramuscularly injected twice into the animals to induce initial and memory anti-TT antibody immune responses. Then, each animal was intravenously administered a vehicle (negative control, single-dose injection), a positive control (DXT; 1 mg / kg, 4-dose injections), and APBA1 (5 mg / kg or 20 mg / kg, single-dose injection), and the concentration of anti-TT IgG antibodies produced in the serum was measured by ELISA. As a result, the initial anti-TT IgG immune response induced by single-dose TT injection was not statistically significant, but the vehicle control induced a normal initial anti-TT IgG immune response, while no initial anti-TT IgG immune response was observed in the DXT and APB-A1 injection groups (Figure 12A). In Figures 12A and 12B, female cynomolgus monkeys (n=3) were injected with tetanus toxoid (TT) along with vehicle, DXT, and APB-A1 (5 mg / kg and 20 mg / kg) to analyze the inhibition of the memory antibody response. These experiments were performed using SNBL. (A) Anti-TT antibody levels were measured by ELISA. (B) The percentage of memory B cells was significantly reduced in both the 5 mg / kg APB-A1 group and the 20 mg / kg APB-A1 group (t-test against vehicle control, *p<0.03). Twenty days after the initial TT injection, a second TT injection was administered to induce a memory anti-TT IgG immune response, and the concentration of anti-TT IgG antibodies produced in the serum was then measured. As a result, after the second TT injection, the vehicle control group and the DXT group exhibited a normal anti-TT IgG immune response, while the APB-A1 injection group demonstrated a statistically significant dose-dependent inhibition of the second anti-TT IgG immune response on day 27. The suppressive effect of APB-A1 on the initial CD40-CD40L response was confirmed, and the population percentages in each group were compared by immunophenotyping. Memory B cells (CD45+20+ / 27+IgD- / Ki67+) and the mitotic population of B cells (CD45+ / 20+ / Ki67+) demonstrated a statistically significant suppressive effect of APB-A1 up to day 27 after the second TT injection (Figure 12B).Assays on plasma cells (CD45+20- / 27hi / IgD-) and all B cells (CD45+ / 20+) showed no significant differences between groups, and the overall inhibitory effect of the vehicle control gradually decreased on days 30 and 40 (data not shown). 【0223】 2. Experimental results of APB-B1 (1) Production of SAFA-based bispecific antibodies To produce SAFA-based bispecific antibodies, the heavy chain C H1 The cysteine ​​forming the interchain disulfide bond between the hinge region (EPKSC-) and the light chain CL(NRGEC-) was replaced with serine (Ser), and the resulting SL335 Fab variants, EPKSS- and NRGES-, in which the two interchain disulfide bonds were removed, were used. The scFv antibody fragment that binds to human CD40L [derived from luprizumab (or hu5c8)] H Gene sequence and V L [Having a gene sequence], (GGGGS)3 or The peptide linker TIFF0007875806000149.tif3128 was used for fusion, and then the Fv antibody fragment (derived from certolizumab pegol) that binds to TNF-α was used. H Gene sequence and V L (containing a gene sequence), Fv antibody fragment that binds to IL-23 (derived from ustekinumab) H Gene sequence and V L (having a gene sequence), and an antibody fragment that binds to INFAR1 (derived from aniflorumab V H Gene sequence and V L The gene sequences were fused to the C-terminus of SL335 Fab using a peptide linker. In the case of Fv fusion, the C-terminal heavy chain V H Fragment and C-terminal light chain V L The fragments were fused together. V of Fv H Fragments and V LTo compare antibody function with or without the presence of artificial interchain disulfide bonds formed between the fragments, the (scFv)2-Fab-Fv format without disulfide bonds was used. H G44C (G542C, Figure 14) and V L Substitution with Q100C (Q593C, Figure 13A) was performed, thereby forming (scFv)2-Fab-dsFv formats having disulfide bonds, respectively. In Figures 13A and 13B, the APB-B1a(a), (scFv)2-Fab-Fv construct and the APB-B1b(b), (scFv)2-Fab-dsFv construct, having anti-CD40L scFv, anti-HSA Fab, and anti-TNF-α Fv (with or without disulfide bonds), are shown, along with (GGGGS)3 (SEQ ID NO:3) and TIFF0007875806000150.tif3128 is linked by a peptide linker. (scFv)2-Fab-dsFv is a variable light chain (V) of the anti-TNF-α dsFv fragment. L ) and variable heavy chain (V H (scFv)2-Fab-Fv contains an interchain disulfide bond (ss) between the two proteins, but (scFv)2-Fab-Fv does not contain an interchain disulfide bond. Figure 13C shows recombinant pD2539 and recombinant pD2535NT after DNA cloning. Here, the bispecific antibodies fused with Fv and dsFv derived from certolizumab were named APB-B1a and APB-B1b, respectively (Figures 13A and 13B). 【0224】 In Figure 14, the G542C residue in the heavy chain and the Q593C residue in the light chain are shown in bold as interchain disulfide bonds between the heavy and light chains of APB-B1b. The peptide linker residues [(GGGGS)3 and The underlined part is [TIFF0007875806000151.tif3128]. The SAFA-based bispecific antibody protein thus produced has a theoretical size of up to 128 kDa and, in order to utilize the CHO cell expression system, two polypeptide coding genes (N'-anti-CD40L scFv-SL335 H chain-anti-TNF-α V) are used to construct APB-B1a or APB-B1b. H -C' and N'-anti-CD40L scFv-SL335 L-chain-anti-TNF-α V L -C') was cloned into the mammalian expression vectors pD2535NT and pD2539, respectively, thereby producing recombinant pD2535NT and recombinant pD2539 vectors (Figure 13C). SAFA-based bispecific antibodies derived from the ustekinumab and aniflorumab genes were cloned in the same manner as above, thereby producing recombinant pD2535NT and recombinant pD2539 vectors (data not shown). 【0225】 To produce SAFA-based bispecific antibody proteins, the produced recombinant pD2535NT vector and recombinant pD2539 vector, ExpiCHO cells, and HD-BIOP3 GS null CHO-K1 cells were used for transient expression and stable pool production. Recombinant CHO cells were cultured in flasks for 7-9 days and then centrifuged to obtain culture medium with 90% cell viability. In terms of transient expression levels, for all three SAFA-based bispecific antibodies derived from the certolizumab, ustekinumab, and aniflorumab genes, the expression level of the (scFv)2-Fab-Fv construct without disulfide bonds was 1.5-3 times higher than that of the (scFv)2-Fab-dsFv construct with disulfide bonds. The APB-B1a fragment produced in the stable pool was cultured in flasks for 7 days to obtain approximately 150 mg / L. 【0226】 (2) Production of SAFA-based bispecific antibodies To compare the size and pattern of SAFA-based bispecific antibodies purified by affinity chromatography, SDS-PAGE analysis was performed under reducing and non-reducing conditions as shown in Figure 15. Under reducing conditions, a protein band was observed at the 60-75 kDa position, which is the heavy and light chains of the (scFv)2-Fab-Fv and (scFv)2-Fab-dsFv fragments, scFv-Fab H chain-Fv V. H and (scFv-Fab L chain-Fv V L) were consistent with their respective theoretical sizes, i.e., 64 kDa (Figure 15A), and the heavy and light chain bands of (scFv)2-Fab were located at positions of 45–60 kDa (Figure 15A). Furthermore, under non-reducing (boiling) conditions, two protein bands corresponding to the heavy and light chains of (scFv)2-Fab-Fv, where no interchain disulfide bond is formed between the heavy and light chains, were observed at positions of 60–75 kDa, as in the case of reducing conditions. In the case of (scFv)2-Fab-dsFv, where an interchain disulfide bond is formed between the heavy and light chains, one protein band was observed at positions of 100–140 kDa, which was within the theoretical size range of 128 kDa (Figure 15B). Under non-reducing (non-boiling) conditions, for (scFv)2-Fab-Fv, one band was observed at a size of approximately 130 kDa, corresponding to the sum of the heavy and light chain sizes. Unexpectedly, two protein bands for the heavy and light chains, located separately, were also observed in the 60-75 kDa range, as in the case of non-reducing (boiling) conditions (Figure 15C). Figures 15A, 15B, and 15C show the results of SDS-PAGE analysis performed under reducing, non-reducing, and non-reducing (non-boiling) conditions, respectively. Four SAFA-based samples, including (1) (scFv)2-Fab, (2) certolizumab-related BsAb (APB-B1), (3) ustekinumab-related BsAb, and (4) aniflorumab-related BsAb, were loaded into each well in varying amounts up to 2 μg. Figure 15D shows the molecular sieve HPLC analysis of the purified (scFv)2-Fab-Fv (up to 25 μg) construct. The (scFv)2-Fab-dsFv dimer is indicated by an arrow. In the case of the (scFv)2-Fab-dsFv construct, the separately located heavy and light chain bands were not visualized, but two bands were observed in the 100–140 kDa range, and one protein band was identified at the same or higher position as 245 kD, which was thought to likely correspond to the position of the (scFv)2-Fab-dsFv dimer.To identify the SDS-PAGE results under non-reducing (non-boiling) conditions, the results for (scFv)2-Fab-Fv protein and (scFv)2-Fab-dsFv protein under native conditions were analyzed by SE-HPLC. The analysis results showed that the (scFv)2-Fab-dsFv protein contained a small amount of (scFv)2-Fab-dsFv dimer, as shown in the SDS-PAGE (indicated by the arrow). In the case of (scFv)2-Fab-Fv, only one peak appeared at the monomer position, and no peaks corresponding to the heavy and light chains, which were identified as being located separately in SDS-PAGE, were observed (Figure 15D). Therefore, the separately located heavy and light chains of the polypeptide shown in the (scFv)2-Fab-Fv protein sample under non-reducing (non-boiling) conditions in the SDS-PAGE experiment are thought to have been observed as two bands due to partial cleavage of non-covalent bonds present between the heavy and light chains, which is due to the SDS present in the SDS-PAGE experiment or the heat transferred to the protein during the experiment. 【0227】 (3) Purification of SAFA-based bispecific antibody proteins Prior to the purification of APB-B1a and APB-B1b proteins isolated by affinity chromatography, protein samples were analyzed using SDS-PAGE and SE-HPLC (Figures 15A-15D). Cation exchange chromatography was performed using CM Sepharose FF resin to remove the protein identified at the dimer position of APB-B1b (up to 245 kDa) (Figures 15C and 15D). Anion exchange chromatography was performed on the APB-B1a protein sample, which was identified with high purity only by affinity chromatography, using Q Sepharose HP. Each purification step was performed using an AKTA pure 150 L system, and the final purified product was determined by SE-HPLC. The remaining APB-B1b protein with the dimer position after affinity chromatography was further removed by cation exchange chromatography (Figure 16B), and a peak similar to that of APB-B1a was identified at one retention time (Figure 16A). In Figures 16A and 16B, SAFA-based constructs were analyzed at 280 nm under native conditions on a TSKgel UltraSW aggregation column (in 20 mM citrate pH 5.5 buffer). APB-B1a(a) was analyzed using CaptureSelect IgG-C H1 APB-B1b(b) was purified by affinity chromatography and Q Sepharose-HP anion exchange chromatography. H1 Purified by affinity chromatography and CM Sepharose FF cation exchange chromatography. 【0228】 (4) Comparison of protein stability with the presence of disulfide bonds for protein stability and with an optimal pH buffer. To compare the stability of (scFv)2-Fab-Fv and (scFv)2-Fab-dsFv proteins with or without interchain disulfide bonds in Fv, three types of SAFA-based bispecific antibodies derived from certolizumab, ustekinumab, and aniflorumab genes were gradually heat-treated in sodium phosphate buffer (pH 7.0) from 20°C to 90°C. Protein denaturation temperatures were measured using SYPRO Orange dye conjugated to hydrophobic amino acids via a real-time PCR process. The results showed that the protein denaturation temperatures differed depending on the Fv clone of the three SAFA-based bispecific antibodies, but (scFv)2-Fab-Fv and (scFv)2-Fab-dsFv denatured at the same temperature regardless of the presence of disulfide bonds (Table 7a). To identify the optimal buffer for the storage stability of APB-B1 protein, the protein denaturation temperature was measured by heat-treating the protein at melting temperatures ranging from 20°C to 90°C, gradually increasing as described above, under pH conditions of 3.0–11.0 (Table 7b and Figure 17). In Figure 17, purified APB-B1a and APB-B1b proteins were incubated in various buffers at 4°C for 1 day, and 1 mg / ml of each was taken and analyzed using a LightCycler 480 II (RT-PCR) and SYPRO Orange dye. As a result, all proteins demonstrated the same denaturation temperature under various pH conditions, regardless of the presence or absence of disulfide bonds in Fv. In addition, protein denaturation began at relatively low temperatures under low pH levels (3.0–4.0) and high pH levels (9.0–11.0). However, under extremely acidic or basic conditions such as pH levels of 3.0 or 11.0, protein denaturation began at a temperature of 4°C. The pH level of the APB-B1 protein sample that exhibited the highest thermal stability was 5.0 to 7.0. This APB-B1 protein showed the best thermal stability at pH 6.0 in citrate buffer, histidine buffer, and sodium phosphate buffer (Tm = 61°C) (Table 7b). 【0229】 (Table 7a) TIFF0007875806000152.tif45128 【0230】 (Table 7b) TIFF0007875806000153.tif102128 【0231】 (5) Determination of antigen specificity and affinity of SAFA-based bispecific antibodies The binding affinities of purified APB-B1a and APB-B1b bispecific antibodies to three different antigens—HSA, CD40L, and TNF-α—were determined by ELISA and BLI (Figures 18A–18C, and Table 8). In Figures 18A–18C, the three target proteins—human serum albumin (Figure 18A), CD40L (Figure 18B), and TNF-α (Figure 18C)—were coated at a density of 1 μg / ml on 96-well maxisoape plates. APB-B1a, APB-B1b, and the parent antibody were conjugated to each target at pH 7.4. HRP-conjugated goat anti-human Fd antibody was used as the secondary antibody. Data were analyzed at 450 nm using an ELISA reader. *Parent antibodies: anti-HSA Fab (SL335), anti-CD40L IgG (luprizumab), anti-TNF-α IgG (adalimumab), and anti-TNF-α Fab (certolizumab). ELISA results showed that the binding strength of APB-B1a or APB-B1b to human serum albumin was approximately 2-3 times lower than that of SL335 Fab used as a control (Figure 18A). Affinity evaluation using BLI measured the equilibrium dissociation constant (KD) of APBB1a, APB-B1b, and SL335 Fab to human serum albumin, with results of 765 pM, 809 pM, and 286 pM, respectively (Table 8). In the evaluation of the CD40L binding ability of APB-B1a and APB-B1b antibodies, these two bispecific antibodies demonstrated approximately 1.5 times lower binding ability than the parent antibody, anti-human CD40L IgG1 (luprizumab) (Figure 18B). The binding affinity of APB-B1a, APB-B1b, and the parent antibody was measured, and the KD values ​​were 192 pM, 167 pM, and 49 pM, respectively (Table 8). When measured by ELISA, the TNF-α antigen affinity of APB-B1a and APB-B1b was approximately 1.5 to 2 times lower than that of the parent antibody α Fab' (Figure 18C). When measured by BLI, the binding affinities of APB-B1a, APB-B1b, and the parent antibody were KD values ​​of 164 pM, 446 pM, and 157 pM, respectively (Table 8), which are similar in pattern to the evaluation results by ELISA and BLI described above. 【0232】 In addition, BLI was performed to identify the ability of APB-B1a to simultaneously bind to three types of antigens (Table 8). 500 nM CD40L was reacted with an AR2G biosensor and immobilized on the sensor, and then 25 nM APB-B1a was reacted with the immobilized CD40L and associated with it. Next, human serum albumin was reacted with APB-1a at concentrations up to 10 times higher than the concentration at which APB-1a saturates the albumin-binding site of APB-B1a, and the same concentrations of human serum albumin were reacted with 50 nM TNF-α. The results are shown in Figure 19. In Figure 19, recombinant human CD40L was immobilized on an AR2G biosensor in pH 5.0, 10 μg / ml sodium acetate buffer. APB-B1a was loaded at a concentration of 3.2 μg / ml (association 1). HSA was loaded at a concentration of 13.2 μg / ml (Association 2), and HSA and TNF-α were loaded at concentrations of 13.2 μg / ml and 2 μg / ml, respectively (Association 3). Each association step was performed for 900 seconds. Data were analyzed using Octet DataAnalysis 8 software. 【0233】 (Table 8) TIFF0007875806000154.tif33160 【0234】 Table 8 shows the binding affinity of purified APB-B1a and APB-B1b analyzed using the Octet RED instrument. Each antigen, HSA, CD40L, and TNF-α were immobilized on amine-reactive second-generation (AR2G) biosensors at concentrations of 20 μg / ml, 10 μg / ml, and 30 μg / ml in pH 5.0 sodium acetate buffer. Purified antibodies were diluted 2-fold by serial processing in pH 7.4 1× kinetic buffer. Data were analyzed using Octet Data Analysis 8 software. *Parent antibodies: anti-HSA Fab (SL335), anti-CD40L IgG1 (luprizumab), and anti-TNF-α Fab (certolizumab). 【0235】 (6) Binding of SAFA-based bispecific antibodies to the cell membrane CD40L protein We used FACSVerse to determine whether the purified SAFA-based bispecific antibody anti-CD40L scFv specifically binds not only to soluble CD40L but also to cell membrane CD40L expressed on the cell surface. The results confirmed that the SAFA-based bispecific antibody had the ability to bind to cell membrane CD40L, similar to the parent antibody anti-human CD40L IgG1 (Figure 20). Figure 20 shows D1.1 cells at a concentration of 3.0 × 10⁶. 5 Cells / reactions were prepared, and FITC-conjugated goat anti-human kappa antibody was used as a secondary antibody to detect SL335-based constructs, SL335 Fab (negative control), and anti-CD40L IgG (positive control). Binding signals, indicated by cell counts, were measured using a FASCVerse flow cytometer. 【0236】 (7) Evaluation of cell-based inhibitory activity of SAFA-based bispecific antibodies To determine whether APB-B1a and APB-B1b have the ability to inhibit the biological function of TNF-α, their in vitro inhibitory capacity was evaluated using L929 mouse cells that express the cell membrane TNF receptor and exhibit TNF-α-dependent cytotoxicity in the presence of actinomycin D (Figure 21). In Figure 21, the concentration of the TNF-α sample was reduced threefold from 20 nM to 0.24 nM by serial dilution, and then reacted with L929 mouse cells expressing the TNF receptor and TNF-α (10 ng / ml) on the cell membrane in the presence of actinomycin D. Anti-TNF-α-Fab' (certolizumab) was used as a positive control group. Cell viability was measured using the cell counting kit-8. Inhibitory activity was evaluated by adding soluble TNF-α (10 ng / ml) and serially diluted antibodies, namely SL335 Fab, APB-B1a, APB-B1b, and the parent antibody anti-TNFα Fab'. As a result, in the presence of human serum albumin (HSA), the semi-maximal inhibitory concentration (IC) of the parent antibody was evaluated. 50 The value is 0.6259 nM, and the ICs of APB-B1a and APB-B1b50 The values ​​were 5.3 nM and 12.7 nM, respectively, which are 8 to 20 times lower than the parent antibody. However, SL335 did not demonstrate inhibitory activity (Figure 21). Therefore, it was confirmed that APB-B1a and APB-B1b retained their ability to inhibit TNFα-TNFR interaction. To determine whether APB-B1a and APB-B1b have the ability to co-bind cell membrane CD40L and soluble TNF-α expressed on the cell surface in the presence of human serum albumin, and thus co-inhibit the CD40L-CD40 and TNFα-TNFR interaction pathways, co-inhibitory activity was observed using HEK-blue™ CD40L reporter cells expressing both cell membrane CD40 and cell membrane TNF receptor. In Figures 22A-22C, antibody constructs against CD40L or TNF-α were serially diluted fourfold from a concentration of 50 nM to 0.0122 nM. Both anti-CD40L IgG1 and anti-TNF-α were used as control groups for their respective target molecules. Secretory embryonic alkaline phosphatase (SEAP) expressed in response to the reaction of HEK-blue® reporter cells with CD40L or TNF-α was measured using QUANTIBlue reagent, and the signal was measured at A655 nm. The inhibitory ability of APB-B1a and APB-B1 was identified by measuring the response of HEK-blue® reporter cells to various interactions, including (a) the interaction between CD40L-expressing D1.1 cells and CD40-expressing HEK-blue® cells (Figure 22A), (b) the interaction between TNF receptor-expressing HEK-blue® cells and soluble TNF-α (Figure 22B), and (c) the interaction between D1.1 cells and HEK-blue® cells, and the interaction between HEK-blue® cells and soluble TNF-α (Figure 22C). IC2C of APB-B1a and APB-B1b against the cell membrane CD40L 50 The value is 0.15-0.18 nM, IC of the parent antibody (luprizumab, IgG1) 50The value was identified as 0.30 nM (Figure 22A), and the inhibitory abilities of APB-B1a and APB-B1b against soluble TNF-α (10 ng / ml) were 3.05 nM and 4.13 nM, respectively, which are 6-8 times lower than the parent antibody (certolizumab, Fab'), i.e., 0.56 nM, and are similar to those observed in experiments using L929 mouse cells (Figure 22B). In assays co-inhibiting both cell membrane CD40L and soluble TNF-α antigen, IC50 of APB-B1a and APB-B1b 50 The values ​​were 1.98 nM and 3.79 nM, respectively, meaning that the antigen was completely (100%) inhibited by APB-B1a and APB-B1b. However, the parent antibody, anti-TNF-α Fab', could only inhibit the TNF-α antigen, meaning that only about 60% of the overall response was inhibited. In the case of CD40L IgG1, CD40L IgG1 was able to inhibit CD40L, but anti-CD40L IgG1 could not inhibit the response at all due to the extremely high SEAP activity of the reporter cell against uninhibited TNF-α, which is similar to the case of SL335 Fab (negative control). Furthermore, when the two parent antibodies, anti-TNF-α Fab' and anti-CD40L IgG1, were administered simultaneously (combination therapy), the two target species were completely (100%) inhibited by these two parent antibodies, as in the case of bispecific antibodies, and the measured IC50 was not significantly reduced. 50 The value was 0.47 nM, which was 3 to 8 times higher than that of APB-B1. Therefore, although these two antibodies have similar levels of inhibitory ability against CD40L, it is thought that they had lower inhibitory ability than in combination therapy due to the difference in their inhibitory abilities against TNF-α (Figure 22C). 【0237】 Example 5. Additional pharmacokinetic and pharmacodynamic analyses (1)PK analysis To further evaluate the serum half-life of APB-A1, pharmacokinetic analyses were performed again in a cynomolgus monkey model (SNBL, Japan). APB-A1 protein was administered to three male cynomolgus monkeys in each group via a single intravenous injection at a dose of 10 mg / kg (Group 1) or 30 mg / kg (Group 2). Blood samples were collected at 14 time points after administration: 1 was pre-administration, followed by 13 time points at 0.25 hours, 1 hour, 6 hours, 12 hours, and 24 hours post-administration, as well as at 4, 8, 13, 19, 26, 33, 40, and 47 days. The concentration of APB-A1 in the serum of each cynomolgus monkey was measured by ELISA (SNBL, Japan). 【0238】 (2)PD analysis The anti-keyhole limpet hemocyanin (KLH) antibody response suppressed by the efficacy of APB-A1 was analyzed (SNBL, Japan). Four sample groups, including a vehicle (negative control group: 20 mmol / L L-histidine, HCl pH 5.8, 5% sucrose) and APB-A1 (3 mg / kg, 10 mg / kg, and 30 mg / kg), were administered intravenously to cynomolgus monkeys (male; n=5 / group) once weekly (2 times in total). First, to induce the anti-KLH antibody response, the first subcutaneous injection of KLH (2 mg / kg) was administered on day minus 28, and a second subcutaneous injection was administered on day 1 (approximately 1 hour after the end of administration of the test substance and control substance) for boosting. APB-A1 was injected twice in total: at the time of the second KLH injection (day 1) and one week later (day 8). Blood samples were collected before the first KLH injection (day minus 28), day minus 21, day minus 14, day minus 7, day 1 (approximately 30 minutes before administration of the test substance and control substance), day 4, day 8 (approximately 30 minutes before administration of the test substance and control substance), day 11, day 15, day 22, and day 29. Anti-KLH IgG antibody titers and anti-KLH IgM antibody titers were measured by ELISA. For peripheral blood immunophenotyping analysis, blood samples were collected a total of six times: before the first KLH injection (day minus 28), day 1 (approximately 30 minutes before administration of the test substance and control substance), day 4, day 11, day 22, and day 29. Immunophenotyping was performed using antibody panels for markers such as CD45, CD20, CD27, CD38, CD3, CD4, CD8, and Ki-67. For PK analysis, blood samples were collected on days 1, 4, 8, 11, 15, 22, and 29 (a total of 7 time points). The concentration of APB-A1 in the serum of each cynomolgus monkey was measured by ELISA. Organs for immunohistochemical examination were excised the day after necropsy and processed in an automated embedding system two days after macroscopic examination. Three serial sections were prepared. The first section was stained with hematoxylin-eosin (HE), and the second and third sections were used for immunohistochemical (IHC) examination.In IHC, thin sections were stained using immunoenzymatic methods with the following antibodies: mouse anti-CD20 antibody (L26, Leica Biosystems, Germany) and mouse anti-Ki67 antibody (MIB-1, Dako Denmark A / S, Denmark). For statistical analysis, the data was analyzed for homovariance using Barrett's test. If the variances were homovariant, Dunnett's test was used to perform multiple comparisons between the control group and each test substance group. If the variances were heterogeneous according to Barrett's test, Dunnett's type test (Miller's test) was used to perform multiple comparisons between the control group and each test substance group. 【0239】 (3) Repeated dose-to-toxicology study over 2 weeks To determine the potential toxicity of APB-A1 for the treatment of autoimmune diseases when administered to cynomolgus monkeys by intravenous (slow bolus) injection twice, one week apart. In this study, the following parameters and endpoints were evaluated: clinical observations, body weight, clinicopathological parameters (hematology, coagulation, clinical chemistry, and urinalysis), bioanalysis and gross autopsy findings, and organ weights. 【0240】 (4) Results (1) Additional PK testing of APB-A1 To evaluate the in vivo half-life of APB-A1, pharmacokinetic assays were performed using a cynomolgus monkey model (male, n=3 / group). APB-A1 was administered by a single intravenous injection at two doses: 10 mg / kg and 30 mg / kg. Blood samples were collected at the same time points described in the Materials and Methods section, and the concentration of APB-A1 in plasma was measured using PK ELISA (Figure 23). The data represent the mean of the experiments performed. Based on the ELISA results, the half-lives were calculated using Phoenix WinNonlin software (ver 6.4; Certara LP, Princeton, New Jersey, USA), and the half-lives of 10 mg / kg APB-A1 and 30 mg / kg APB-A1 were identified as approximately 9.33 ± 1.54 days and 10.1 ± 1.8 days, respectively. The Cmax values ​​were approximately 347 μg / ml and 1230 μg / ml for doses of 10 mg / kg and 30 mg / kg, respectively, and the clearance (CL) rates were 3.48 ml / day / kg and 3.44 ml / day / kg, respectively, which were at the same level regardless of the dose (Table 9). 【0241】 (Table 9) Additional monkey PK data TIFF0007875806000155.tif41164 【0242】 (2) Additional PD trials for APB-A1 None of the animals in any group were found dead or euthanized due to being near death. No test substance-related changes were observed in any group in terms of clinical signs, body weight, or macroscopic findings. Peripheral blood immunophenotyping showed a decrease in dividing B cells in the 30, 10, and / or 3 mg / kg groups compared to the control group (Figures 24A and 24B). Measurement of anti-KLH antibodies showed a decrease in IgG antibody titer in the 10 and 30 mg / kg groups compared to the control group between days 8 and 29 (* P<0.05, ** P<0.01: statistically significant difference from control) (Figure 25), but no test substance-related changes were observed in IgM antibody titer (data not shown). Histopathological and immunohistochemical examinations revealed a decrease in germinal center cell type (cellularity), anti-CD20-positive cells, and anti-Ki67-positive cells in the axillary and submandibular lymph nodes in the 10 and / or 30 mg / kg groups on day 29 (Figure 26 and Table 10). In PK, Cmax and AUC0-t values ​​increased almost proportionally to the dose between the first and second doses in the 3–30 mg / kg groups (Figure 27 and Table 11). 【0243】 (Table 10) Histopathological and immunohistochemical examinations TIFF0007875806000156.tif73162-: No abnormal change, ±: Very small, +: Small, 2+: Medium, 3+: Large 【0244】 (Table 11) PK results of the PD trial TIFF0007875806000157.tif49159 【0245】 2-week repeated dose-to-toxicology study Neither male nor female monkeys treated with APB-A1 showed premature death, or any clinical observations or changes in body weight, hematology, coagulation, clinical chemistry, or urinalysis, nor any significant macroscopic findings. Administration of APB-A1 by intravenous (slow bolus) injection two times one week apart, at a maximum dose level of 100 mg / kg / dose, was well tolerated in cynomolgus monkeys. 【0246】 This disclosure demonstrates the successful production of a novel format of multispecific antibody constructs with increased in vivo sustainability based on existing bispecific antibody techniques, which were developed by the inventors. In particular, multispecific antibodies capable of binding to CD40L, TNF-α, or other physiologically active effectors can be conveniently applied as therapeutic agents for various autoimmune diseases. 【0247】 The descriptions of the specific embodiments described above fully illustrate the overall characteristics of the invention, so that others, by applying knowledge of the scope of the art, can easily modify and / or apply it to various uses without departing from the overall concept of the invention. Such applications and modifications are therefore considered to be within the meaning and scope of the equivalents of the embodiments disclosed herein, based on the teachings and guidance provided herein. It should be understood that the terms and descriptions herein are for illustrative purposes only, not limitation, and therefore, to those skilled in the art, they are to be interpreted in light of the teachings and guidance provided herein. 【0248】 The breadth and scope of the present invention should not be limited to any of the exemplary embodiments described above, but should be defined solely by the appended claims and their equivalents. 【0249】 All the various aspects, embodiments, and options described herein can be combined in a wide variety of ways. 【0250】 All publications, patents, and patent applications referenced herein are incorporated by reference in the same manner as each publication, patent, or patent application is specifically and individually indicated as being incorporated by reference herein. 【0251】 Industrial applicability The multispecific antibodies disclosed herein can be used in the development of autoimmune disease treatments that have long in vivo retention periods while reducing side effects such as thromboembolism.

Claims

[Claim 1] Structural formula A multispecific antibody containing, In the formula, the antigen-binding fragment (Fab) is serum albumin Fab; R 1 and R 2 This is a bioactive effector portion linked to the N-terminus of Fab, and is linked to either the heavy chain variable domain or the light chain variable domain of Fab, and R 1 and R 2 These are, respectively, anti-CD40L hu5c8 scFv; R 3 and R 4 These are bioactive effector regions ligated to the C-terminus of Fab, and are linked to either the heavy-chain variable domain or the light-chain variable domain of Fab, respectively; m is 0; n is 0, Fab, Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence AYSMN (SEQ ID NO:74), amino acid sequence Heavy chain CDR2 including, and amino acid sequence heavy chain CDR3 It includes a heavy chain variable domain, and amino acid sequence Light chain complementarity-determining domain 1 (CDR1), Light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO:92), and amino acid sequence Light chain CDR3 Including a light chain variable domain, The multispecific antibody. [Claim 2] R 1 、 R 2 、 R 3 、 and R 4 are each linked to the Fab by one or more linkers, the multispecific antibody according to claim 1. [Claim 3] A multispecific antibody according to any one of claims 1 to 2, wherein Fab comprises a heavy chain variable domain having an amino acid sequence having at least 98% identity with SEQ ID NO:

99. [Claim 4] A multispecific antibody according to any one of claims 1 to 3, wherein Fab comprises a light chain variable domain having an amino acid sequence having at least 98% identity with SEQ ID NO:

105. [Claim 5] A multispecific antibody according to any one of claims 1 to 4, wherein Fab comprises a heavy chain variable domain containing an amino acid sequence having at least 98% identity with SEQ ID NO:99, and a light chain variable domain containing an amino acid sequence having at least 98% identity with SEQ ID NO:

105. [Claim 6] Fab contains the heavy chain domain (V) containing the amino acid sequence of SEQ ID NO:

45. H -C H1 The domain), and the light chain domain (V) containing the amino acid sequence of SEQ ID NO:46 L -C L A multispecific antibody according to any one of claims 1 to 5, comprising a domain. [Claim 7] A multispecific antibody according to any one of claims 2 to 6, wherein each linker contains 1 to 20 amino acids. [Claim 8] A multispecific antibody according to any one of claims 2 to 7, wherein each linker comprises an amino acid sequence having at least 90% identity with SEQ ID NO:3 or SEQ ID NO:

4. [Claim 9] A multispecific antibody according to any one of claims 2 to 8, wherein each linker contains the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4. [Claim 10] R 1 and R 2 The multispecific antibody according to any one of claims 1 to 9, wherein each of these is an anti-CD40L hu5c8 scFv containing the amino acid sequence of SEQ ID NO:47 or SEQ ID NO:

48. [Claim 11] R 1 and R 2 The multispecific antibody according to any one of claims 1 to 10, wherein each of these is an anti-CD40L hu5c8 scFv containing the amino acid sequence of SEQ ID NO:

48. [Claim 12] A multispecific antibody according to any one of claims 1 to 11, comprising a heavy chain containing the amino acid sequence of SEQ ID NO:41 and a light chain containing the amino acid sequence of SEQ ID NO:

42. [Claim 13] A composition comprising a multispecific antibody according to any one of claims 1 to 12, and an excipient. [Claim 14] A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 12, and a pharmaceutically acceptable excipient.

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