US20210230285A1 - Multispecific antibodies, compositions comprising the same, and vectors and uses thereof - Google Patents

Multispecific antibodies, compositions comprising the same, and vectors and uses thereof Download PDF

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US20210230285A1
US20210230285A1 US16/878,255 US202016878255A US2021230285A1 US 20210230285 A1 US20210230285 A1 US 20210230285A1 US 202016878255 A US202016878255 A US 202016878255A US 2021230285 A1 US2021230285 A1 US 2021230285A1
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seq
acid sequence
amino acid
light chain
heavy chain
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Sang Hoon Cha
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Aprilbio Co Ltd
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Aprilbio Co Ltd
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Priority to PCT/IB2021/050519 priority Critical patent/WO2021149015A1/en
Priority to CR20220415A priority patent/CR20220415A/es
Priority to CN202180023540.XA priority patent/CN116113642A/zh
Priority to IL294917A priority patent/IL294917A/en
Priority to AU2021211241A priority patent/AU2021211241A1/en
Priority to JP2022545037A priority patent/JP2023511963A/ja
Priority to CA3165029A priority patent/CA3165029A1/en
Priority to EP21744981.8A priority patent/EP4093764A4/en
Priority to KR1020227029379A priority patent/KR20220154097A/ko
Priority to US17/759,313 priority patent/US20230073034A1/en
Priority to MX2022009150A priority patent/MX2022009150A/es
Priority to JOP/2022/0171A priority patent/JOP20220171A1/ar
Publication of US20210230285A1 publication Critical patent/US20210230285A1/en
Assigned to APRILBIO CO., LTD. reassignment APRILBIO CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHA, SANG HOON
Priority to DO2022000150A priority patent/DOP2022000150A/es
Priority to CL2022001993A priority patent/CL2022001993A1/es
Priority to CONC2022/0011885A priority patent/CO2022011885A2/es
Priority to ECSENADI202265625A priority patent/ECSP22065625A/es
Priority to US18/151,227 priority patent/US11773176B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to fusion constructs comprising an antigen binding fragment and bioactive effector moieties. More particularly, the present disclosure relates to a multispecific antibodies comprising two or more bioactive effector moieties linked to either or both of an N-terminal and a C-terminal of an antigen binding fragment that binds to human serum albumin.
  • a CD40-CD40L interaction essentially acts on the creation of antigen-specific antibody immune responses, and autoantibodies involve pathogenesis of various autoimmune diseases.
  • CD40L- or CD40-specific antibodies capable of inhibiting and/or suppressing the CD40-CD40L interaction have been researched.
  • anti-CD40L monoclonal antibodies hu5c8 IgG1 (BG-9588, ruplizumab, AntovaTM, Biogen, Cambridge, Mass.), and IDEC-131 (E6040, IDEC Pharmaceuticals, San Diego, Calif.) have been studied for treatment of various autoimmune diseases, including, for example, systemic lupus erythematosus (SLE) and idiopathic thrombocytopenic purpura (ITP), but additional development of such antibodies has been halted due to incidence of side effects such as thromboembolism.
  • SLE systemic lupus erythematosus
  • ITP idiopathic thrombocytopenic purpura
  • a therapeutic agent targeting CD40 not CD40L
  • a B1655064 antibody having a weakened Fc function Boehringer Ingelheim, Germany
  • a bleselumab antibody of a human IgG4 type Kelowa Kirin Pharmaceutical Development, La Jolla, Calif.
  • the present disclosure provides multispecific antibodies having an extended in vivo retention time.
  • the present disclosure also provides pharmaceutical compositions comprising the multispecific antibody.
  • the present disclosure also provides methods of producing the multispecific antibody.
  • novel autoimmune disease therapeutic agents for suppressing a CD40-CD40L signal while eliminating the Fc-based thromboembolic side effect of an anti-CD40L antibody.
  • a recombinant bispecific antibody has been developed, represented by (anti-CD40L scFv) 2 -(anti-HSA Fab)-(anti-TNF- ⁇ Fv) capable of maintaining serum sustainability without a Fc region by linking a single-chain variable fragment (scFv) consisting of variable region genes V H and V L of hu5c8, a ruplizumab antibody binding to CD40L, to the N-terminal of SL335 Fab.
  • scFv single-chain variable fragment
  • multispecific antibodies represented by (anti-CD40L scFv) 2 -(anti-HSA Fab)-(anti-TNF- ⁇ Fv) by linking Fv or dsFv containing of a variable region gene of a certolizumab pegol antibody binding to TNF- ⁇ to the C-terminal of SL335 Fab of the bispecific antibody using a peptide linker, and identified functions and characteristics of the produced antibody protein.
  • multispecific antibodies comprising a structural formula of:
  • the antigen binding fragment is a serum albumin Fab
  • R 1 and R 2 are bioactive effector moieties linked to an N-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
  • R 3 and R 4 are bioactive effector moieties linked to a C-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
  • m is 0 or an integer of 1 or greater
  • R 1 and R 2 are same or different single-chain variable fragments (scFv).
  • R 3 and R 4 are same or different Fv fragments or disulfide-stabilized Fv (dsFv) fragments.
  • each of R 1 , R 2 , R 3 , and R 4 can 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 an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
  • the Fab comprises a heavy chain variable domain comprising
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61), a heavy chain CDR2 comprising the amino acid sequence of WINTYSGGTKYAQKFQG (SEQ ID NO:62), and a heavy chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO:63);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61), a heavy chain CDR2 comprising the amino acid sequence of RINTYNGNTGYAQRLQG (SEQ ID NO:64), and a heavy chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO:63);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of NYGIH (SEQ ID NO:65), a heavy chain CDR2 comprising the amino acid sequence of SISYDGSNKYYADSVKG (SEQ ID NO:66), and a heavy chain CDR3 comprising the amino acid sequence of DVHYYGSGSYYNAFDI (SEQ ID NO:67);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:68), a heavy chain CDR2 comprising the amino acid sequence of VISHDGGFQYYADSVKG (SEQ ID NO:69), and a heavy chain CDR3 comprising the amino acid sequence of AGWLRQYGMDV (SEQ ID NO:70);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYWIA (SEQ ID NO:71), a heavy chain CDR2 comprising the amino acid sequence of MIWPPDADARYSPSFQG (SEQ ID NO:72), and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:73); or
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76).
  • the Fab comprises a light chain variable domain comprising
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSISRYLN (SEQ ID NO:77), a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:78), and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:79);
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO:80), a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:81), and a light chain CDR3 comprising the amino acid sequence of QQSYSTPPYT (SEQ ID NO:82);
  • CDR1 a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSIFNYVA (SEQ ID NO:83), a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:84), and a light chain CDR3 comprising the amino acid sequence of QQRSKWPPTWT (SEQ ID NO:85);
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASETVSSRQLA (SEQ ID NO:86), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:88);
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSVSSSSLA (SEQ ID NO:89), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:90); or
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
  • the Fab comprises
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76), and
  • CDR1 a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
  • the Fab comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99.
  • the Fab comprises a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105.
  • the Fab comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99, and a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105, respectively.
  • the Fab comprises a heavy chain domain comprising an amino acid sequence of SEQ ID NO:45 (V H -C H1 domain) and a light chain domain comprising an amino acid sequence of SEQ ID NO:46 (V L -C L domain).
  • each of the R 1 and R 2 is an anti-CD40L hu5c8 scFv.
  • Each of the R 1 and R 2 can be an anti-CD40L hu5c8 scFv comprising an amino acid sequence having at least 80% identity to SEQ ID NO:47 or SEQ ID NO:48.
  • Each of the R 1 and R 2 can be an anti-CD40L hu5c8 scFv comprising an amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48.
  • each of R 3 and R 4 is one or more bioactive effector moieties comprising anti-TNF- ⁇ Fv, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and/or anti-IFNAR1 dsFv.
  • bioactive effector moieties comprising anti-TNF- ⁇ Fv, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and/or anti-IFNAR1 dsFv.
  • Each of R 3 and R 4 can be one or more bioactive effector moieties comprising an anti-TNF- ⁇ Fv comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:49 and a light chain amino acid sequence having 80% identity to SEQ ID NO:50, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv) comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:51 and a light chain amino acid sequence having 80% identity to SEQ ID NO:52, anti-IL-23 Fv comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:53 and a light chain amino acid sequence having 80% identity to SEQ ID NO:54, anti-IL-23 dsFv comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:55 and a light chain amino acid sequence having 80% identity to SEQ ID NO:56, anti-IFNAR1 comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:57
  • Each of R 3 and R 4 can be one or more bioactive effector moieties comprising an anti-TNF- ⁇ Fv comprising a heavy chain of SEQ ID NO:49 and a light chain of SEQ ID NO:50, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv) comprising a heavy chain of SEQ ID NO:51 and a light chain of SEQ ID NO:52, anti-IL-23 Fv comprising a heavy chain of SEQ ID NO:53 and a light chain of SEQ ID NO:54, anti-IL-23 dsFv comprising a heavy chain of SEQ ID NO:55 and a light chain of SEQ ID NO:56, anti-IFNAR1 comprising a heavy chain of SEQ ID NO:57 and a light chain of SEQ ID NO:58, and/or anti-IFNAR1 dsFv comprising a heavy chain of SEQ ID NO:59 and a light chain of SEQ ID NO:60.
  • dsFv disulfie
  • compositions comprising multispecific antibodies disclosed herein and an excipient. Also disclosed herein are pharmaceutical compositions comprising multispecific antibodies disclosed herein and a pharmaceutically accepted excipient.
  • Also disclosed here are methods of treating an autoimmune disease in a subject in need thereof, the methods comprising administering a pharmaceutical composition disclosed herein to the subject.
  • expression vectors comprising:
  • a second nucleic acid molecule encoding a bioactive effector moiety and a linker, wherein the promoter, the first nucleic acid sequence, and the second nucleic acid molecules are operably linked.
  • the second nucleic acid molecule can encode 2 or more bioactive effector moieties and linkers.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76), and
  • CDR1 a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99, and a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105, respectively.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain domain comprising an amino acid sequence of SEQ ID NO:45 (V H -C H1 domain) and a light chain domain comprising an amino acid sequence of SEQ ID NO:46 (V L -C L domain).
  • the bioactive effector moieties are anti-TNF- ⁇ Fv, anti-TNF- ⁇ dsFv, anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1 Fv, and/or anti-IFNAR1 dsFv.
  • the second nucleic acid molecule can comprise a nucleotide sequence encoding the amino acid sequence of one or more of SEQ ID NOs: 49-60.
  • the present disclosure provides host cells comprising the expression vector, such as an animal cell, e.g., a CHO cell line.
  • FIGS. 1A and 1B represent the vector maps and amino acid sequences of APB-A1.
  • FIGS. 2A and 2B represent the HPLC analysis and SDS-PAGE results for APB-A1.
  • FIG. 3 represents the mass analysis result for APB-A1.
  • FIGS. 4A and 4B represent PI values of APB-A1.
  • FIG. 5 represents a change in the charge quantity of APB-A1.
  • FIG. 6 represents simultaneous binding of rhCD40L-APB-A1-HSA.
  • FIG. 7 represents the result of flow cytometry analysis.
  • FIGS. 8A to 8D represent the in vitro analysis results for APB-A1.
  • FIGS. 9A to 9D represent various IC effects on platelet aggregation.
  • FIG. 10 represent various IC effects on serotonin levels.
  • FIG. 11 represents PK values for APB-A1 concentrations measured using cynomolgus monkeys.
  • FIGS. 12A and 12B represent the pharmacokinetic analysis results using cynomolgus monkeys.
  • FIGS. 13A to 13C represent SAFA-based bispecific antibodies and mammalian expression vectors.
  • FIG. 14 represents amino acid sequences of APB-B1 heavy and light chains.
  • FIGS. 15A to 15D represent bispecific antibody constructs, purified by CaptureSelect IgG-C H1 affinity chromatography.
  • FIGS. 16A and 16B represent APB-B1 constructs, purified by 2-step chromatography.
  • FIG. 17 represents a thermal stability shift assay under various pH and buffer conditions.
  • FIGS. 18A to 18C represent the determination results of the binding specificities of APB-B1 constructs for three different antigens to be compared with parental antibodies, determined by ELISA.
  • FIG. 19 represents the result of simultaneous binding of APB-B1a to three different antigens, analyzed by bio-layer interferometry.
  • FIG. 20 represents binding of SAFA-based constructs to cellular membranes on D1.1 cells, identified by flow cytometry.
  • FIG. 21 represents the inhibition of TNF- ⁇ mediated cytotoxicity by SAFA-based bispecific antibodies, identified in L929 mouse cells.
  • FIGS. 22A to 22C represent the determination of the capacity of APB-B1 inhibiting either or both of a CD40L-CD40 interaction and a TNF ⁇ -TNF ⁇ R interaction, identified in a HEK-BlueTM reporter cell.
  • multispecific antibodies comprising a structural formula of:
  • the antigen binding fragment is a serum albumin Fab
  • R 1 and R 2 are bioactive effector moieties linked to an N-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
  • R 3 and R 4 are bioactive effector moieties linked to a C-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
  • m is 0 or an integer of 1 or greater
  • n is 0 or an integer of 1 or greater.
  • nucleic acids such as complementary DNA (cDNA)
  • vectors e.g., expression vectors
  • cells e.g., host cells
  • methods of making such antibodies are also provided.
  • methods and uses for inducing, increasing or enhancing multispecific activities, and treating certain conditions, such as autoimmune diseases are also provided.
  • antibody and “antibodies” are terms of art and can be used interchangeably herein and refer to a molecule with an antigen-binding site that specifically binds an antigen.
  • Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab′) 2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies.
  • monoclonal antibodies recombinantly produced antibodies
  • human antibodies humanized antibodies, resurfaced antibodies, chimeric antibodies,
  • multispecific antibody and “multispecific antibodies” are terms of art and can be used to refer to a molecule(s) with more than one bioactive effector moieties or antigen-binding sites, wherein each antigen-binding site specifically binds an antigen.
  • the multispecific antibodies disclosed herein can have 2, 3, 4, 5, 6, 7, 8, or more bioactive effector moieties linked thereto.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , or IgA 2 ), or any subclass (e.g., IgG 2a or IgG 2b ) of immunoglobulin molecule.
  • antibodies described herein are IgG antibodies, or a class (e.g., human IgG 1 , IgG 2 , or IgG 4 ) or subclass thereof.
  • the antibody is a humanized monoclonal antibody.
  • the antibody is a human monoclonal antibody, e.g., that is an immunoglobulin.
  • bioeffector moiety refers to the portions of the multispecific antibody molecules that comprises the amino acid residues that confer on the antibody molecule its specificity for the antigen (e.g., the complementarity determining regions (CDR)).
  • the antigen-binding region can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans.
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • CDRs complementarity determining regions
  • FR framework regions
  • variable region is a human variable region.
  • variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • FRs human framework regions
  • the variable region is a primate (e.g., non-human primate) variable region.
  • the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).
  • VL and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.
  • VH and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.
  • Kabat numbering and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen-binding portion thereof.
  • the CDRs of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat E A & Wu T T (1971) Ann NY Acad Sci 190: 382-391 and Kabat E A et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3).
  • CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).
  • the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.
  • constant region or “constant domain” are interchangeable and have its meaning common in the art.
  • the constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
  • the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ), and mu ( ⁇ ), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG 1 , IgG 2 , IgG 3 , and IgG 4 .
  • the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa ( ⁇ ) or lambda ( ⁇ ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.
  • Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (K D ), and equilibrium association constant (K A ).
  • K D is calculated from the quotient of k off /k on
  • K A is calculated from the quotient of k on /k off
  • k on refers to the association rate constant of, e.g., an antibody to an antigen
  • k off refers to the dissociation of, e.g., an antibody to an antigen.
  • the k on and k off can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind.
  • An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope).
  • the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping).
  • NMR spectroscopy e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping).
  • crystallization can be accomplished using any of the known methods in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).
  • Antibody:antigen crystals can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H W et al.; U.S.
  • the epitope of an antibody is determined using alanine scanning mutagenesis studies.
  • the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope, immune complex, or binding partner of an antigen-binding site) as such binding is understood by one skilled in the art.
  • a molecule that specifically binds to an antigen can bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, Id.), or other assays known in the art.
  • molecules that immunospecifically bind to an antigen bind to the antigen with a K A that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the K A when the molecules bind to another antigen.
  • molecules that immunospecifically bind to an antigen do not cross react with other proteins under similar binding conditions. In some embodiments, molecules that immunospecifically bind to an antigen do not cross react with other proteins. In some embodiments, provided herein is a multispecific antibody that binds to a specified antigen with higher affinity than to another unrelated antigen.
  • a multispecific antibody that binds to a specified antigen (e.g., human serum albumin) with a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity than to another, unrelated antigen as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.
  • the extent of binding of a multispecific antibody described herein to an unrelated, protein is less than 10%, 15%, or 20% of the binding of the antibody to the specified antigen as measured by, e.g., a radioimmunoassay.
  • multispecific antibodies that bind to a human antigen with higher affinity than to another species of the antigen.
  • multispecific antibodies that bind to a human antigen with a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher affinity than to another species as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.
  • the multispecific antibodies described herein which bind to a human antigen, will bind to another species of the antigen protein with less than 10%, 15%, or 20% of the binding of the antibody to the human antigen protein as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.
  • the term “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line.
  • the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell cannot be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.
  • the terms “subject” and “patient” are used interchangeably.
  • the subject can be an animal.
  • 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.
  • the subject is a cynomolgus monkey.
  • such terms refer to a non-human animal (e.g., a non-human animal such as a pig, horse, cow, cat, or dog).
  • such terms refer to a pet or farm animal.
  • such terms refer to a human.
  • multispecific antibodies comprising a structural formula of:
  • the antigen binding fragment is a serum albumin Fab
  • R 1 and R 2 are bioactive effector moieties linked to an N-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
  • R 3 and R 4 are bioactive effector moieties linked to a C-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
  • n is 0 or an integer of 1, 2, 3, or greater;
  • n is 0 or an integer of 1, 2, 3, or greater.
  • R 1 and R 2 are same or different single-chain variable fragments (scFv) or same or different Fv fragments or disulfide-stabilized Fv (dsFv) fragments.
  • R 3 and R 4 are same or different scFv or Fv fragments or dsFv fragments.
  • each of R 1 , R 2 , R 3 , and R 4 can be linked to the Fab by one or more linkers.
  • Each linker can comprise but is not limited to 1 to 20 amino acids or any length or range therein, such as 2, 3, 4, etc.
  • 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 an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
  • the Fab comprises a heavy chain variable domain comprising
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61), a heavy chain CDR2 comprising the amino acid sequence of WINTYSGGTKYAQKFQG (SEQ ID NO:62), and a heavy chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO:63);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61), a heavy chain CDR2 comprising the amino acid sequence of RINTYNGNTGYAQRLQG (SEQ ID NO:64), and a heavy chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO:63);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of NYGIH (SEQ ID NO:65), a heavy chain CDR2 comprising the amino acid sequence of SISYDGSNKYYADSVKG (SEQ ID NO:66), and a heavy chain CDR3 comprising the amino acid sequence of DVHYYGSGSYYNAFDI (SEQ ID NO:67);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:68), a heavy chain CDR2 comprising the amino acid sequence of VISHDGGFQYYADSVKG (SEQ ID NO:69), and a heavy chain CDR3 comprising the amino acid sequence of AGWLRQYGMDV (SEQ ID NO:70);
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYWIA (SEQ ID NO:71), a heavy chain CDR2 comprising the amino acid sequence of MIWPPDADARYSPSFQG (SEQ ID NO:72), and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:73); or
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76).
  • the Fab comprises a light chain variable domain comprising
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSISRYLN (SEQ ID NO:77), a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:78), and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:79);
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO:80), a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:81), and a light chain CDR3 comprising the amino acid sequence of QQSYSTPPYT (SEQ ID NO:82);
  • CDR1 a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSIFNYVA (SEQ ID NO:83), a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:84), and a light chain CDR3 comprising the amino acid sequence of QQRSKWPPTWT (SEQ ID NO:85);
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASETVSSRQLA (SEQ ID NO:86), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:88);
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSVSSSSLA (SEQ ID NO:89), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:90); or
  • a light chain complementarity determining domain 1 comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
  • the Fab comprises
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76), and
  • CDR1 a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93), or any combinations of the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 disclosed above.
  • CDR1 a light chain complementarity determining domain 1
  • RASQSVGSNLA SEQ ID NO:91
  • a light chain CDR2 comprising the amino acid sequence of GASTGAT
  • QQYYSFLAKT SEQ ID NO:93
  • the Fab comprises 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 to SEQ ID NO:94, 95, 96, 97, 98, or 99.
  • the Fab comprises 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 to SEQ ID NO:100, 101, 102, 103, 104, or 105.
  • the Fab comprises 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 to SEQ ID NO:94, 95, 96, 97, 98, or 99, 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 to SEQ ID NO:100, 101, 102, 103, 104, or 105, respectively or any combinations of heavy chain variable domain and light chain variable domain disclosed herein.
  • the Fab can comprise 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 to SEQ ID NO:99 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 to SEQ ID NO:105.
  • the Fab comprises a heavy chain 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 to SEQ ID NO:45 (V H —C f n domain) and a light chain 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 to SEQ ID NO:46 (V L -C L domain).
  • each of the R 1 , R 2 , R 3 and R 4 can be a bioactive effector moiety, such as an anti-CD40L hu5c8 scFv.
  • each of the R 1 , R 2 , R 3 and R 4 can be an anti-CD40L hu5c8 scFv comprising an amino acid sequence 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 to SEQ ID NO:47 or SEQ ID NO:48.
  • Each of the R 1 , R 2 , R 3 and R 4 can be an anti-CD40L hu5c8 scFv comprising an amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48.
  • each of the R 1 and R 2 can be a bioactive effector moiety, such as an anti-CD40L hu5c8 scFv.
  • each of the R 1 and R 2 can be an anti-CD40L hu5c8 scFv comprising an amino acid sequence 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 to SEQ ID NO:47 or SEQ ID NO:48.
  • Each of the R 1 and R 2 can be an anti-CD40L hu5c8 scFv comprising an amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48.
  • each of R 1 , R 2 , R 3 and R 4 is one or more bioactive effector moieties comprising, e.g., anti-TNF- ⁇ Fv, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and/or anti-IFNAR1 dsFv.
  • bioactive effector moieties comprising, e.g., anti-TNF- ⁇ Fv, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and/or anti-IFNAR1 dsFv.
  • each of R 3 and R 4 is one or more bioactive effector moieties comprising, e.g., anti-TNF- ⁇ Fv, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and/or anti-IFNAR1 dsFv.
  • bioactive effector moieties comprising, e.g., anti-TNF- ⁇ Fv, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv), anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1, and/or anti-IFNAR1 dsFv.
  • each of R 1 , R 2 , R 3 and R 4 can be one or more bioactive effector moieties comprising an anti-TNF- ⁇ Fv comprising 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 to 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 to SEQ ID NO:50, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv) comprising 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 to SEQ ID NO:
  • Each of R 1 , R 2 , R 3 and R 4 can be one or more bioactive effector moieties comprising an anti-TNF- ⁇ Fv comprising a heavy chain of SEQ ID NO:49 and a light chain of SEQ ID NO:50, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv) comprising a heavy chain of SEQ ID NO:51 and a light chain of SEQ ID NO:52, anti-IL-23 Fv comprising a heavy chain of SEQ ID NO:53 and a light chain of SEQ ID NO:54, anti-IL-23 dsFv comprising a heavy chain of SEQ ID NO:55 and a light chain of SEQ ID NO:56, anti-IFNAR1 comprising a heavy chain of SEQ ID NO:57 and a light chain of SEQ ID NO:58, and/or anti-IFNAR1 dsFv comprising a heavy chain of SEQ ID NO:59 and a light chain of SEQ ID NO:60.
  • each of R 3 and R 4 can be one or more bioactive effector moieties comprising an anti-TNF- ⁇ Fv comprising 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 to 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 to SEQ ID NO:50, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv) comprising 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 to SEQ ID NO:51 and a light chain amino acid
  • Each of R 3 and R 4 can be one or more bioactive effector moieties comprising an anti-TNF- ⁇ Fv comprising a heavy chain of SEQ ID NO:49 and a light chain of SEQ ID NO:50, anti-TNF- ⁇ disulfied-stabilized Fv (dsFv) comprising a heavy chain of SEQ ID NO:51 and a light chain of SEQ ID NO:52, anti-IL-23 Fv comprising a heavy chain of SEQ ID NO:53 and a light chain of SEQ ID NO:54, anti-IL-23 dsFv comprising a heavy chain of SEQ ID NO:55 and a light chain of SEQ ID NO:56, anti-IFNAR1 comprising a heavy chain of SEQ ID NO:57 and a light chain of SEQ ID NO:58, and/or anti-IFNAR1 dsFv comprising a heavy chain of SEQ ID NO:59 and a light chain of SEQ ID NO:60.
  • dsFv disulfie
  • the Fab is anti-HSA Fab (SL335)
  • R 1 and R 2 are each anti-CD40L IgG (ruplizumab)
  • R 3 and R 4 are each anti-TNF- ⁇ IgG (adalimumab), and/or anti-TNF- ⁇ Fab ‘(certolizumab).
  • a multispecific antibody described herein can be described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95: 8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-av ⁇ 3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody.
  • the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M, (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. Pat. No. 7,709,226).
  • Chothia numbering scheme refers to the location of immunoglobulin structural loops
  • the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34
  • the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56
  • the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102
  • the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34
  • the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56
  • the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97.
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • multispecific antibodies that specifically bind to serum albumin (e.g., human serum albumin) and comprise the Chothia VL CDRs of a VL.
  • antibodies that specifically bind to serum albumin (e.g., human serum albumin) and comprise the Chothia VH CDRs of a VH are antibodies that specifically bind to serum albumin (e.g., human serum albumin) and comprise the Chothia VL CDRs of a VL and comprise the Chothia VH CDRs of a VH.
  • antibodies that specifically bind to serum albumin comprise one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence.
  • antibodies that specifically bind to serum albumin e.g., human serum albumin
  • comprise combinations of Kabat CDRs and Chothia CDRs are provided herein.
  • the CDRs of an antibody can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212.
  • VH-CDR1 is at positions 26 to 35
  • VH-CDR2 is at positions 51 to 57
  • VH-CDR3 is at positions 93 to 102
  • VL-CDR1 is at positions 27 to 32
  • VL-CDR2 is at positions 50 to 52
  • VL-CDR3 is at positions 89 to 97.
  • the CDRs of an antibody can be determined according to MacCallum R M et al., (1996) J Mol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering , Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).
  • the CDRs of an antibody can be determined according to the AbM numbering scheme, which refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).
  • the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein can vary by one, two, three, four, five, or six amino acid positions so long as immunospecific binding to an antigen is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • the position defining a CDR of an antibody described herein can vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the CDR position of a multispecific antibody described herein, so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein can vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described herein so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be one, two, three, four, five or more amino acids longer than one or more of the CDRs described herein so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art can be used to ascertain whether immunospecific binding to the antigen(s) is maintained, for example, the binding assays and conditions described in the “Examples” section herein.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul S F et al., (1990) J Mol Biol 215: 403.
  • Gapped BLAST can be utilized as described in Altschul S F et al., (1997) Nuc Acids Res 25: 3389 3402.
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Gapped BLAST
  • PSI Blast programs the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).
  • NCBI National Center for Biotechnology Information
  • Another specific, nonlimiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • a multispecific antibody can be fused or conjugated (e.g., covalently or noncovalently linked) to a detectable label or substance.
  • detectable labels or substances include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • detectable labels or substances include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine
  • a monoclonal antibody targeting CD40 or CD40L which is a major route of an autoimmune disease or an allograft rejection response
  • a monoclonal antibody targeting CD40 or CD40L but additional development thereof stopped due to incidence of a side effect, such as thromboembolism, induced by the Fc of IgG1 antibody.
  • a recombinant antibody (anti-hu5c8 scFv) 2 -SL335(termed APB-A1) produced by combining Fab with anti-CD40L scFv was developed.
  • SL335 is an antigen-binding fragment (Fab) that has increased in vivo sustainability by specifically binding to the human serum albumin. See U.S. Pat. No. 9,879,077, incorporated herein by reference in its entirety.
  • binding capacity of APB-A1 and its potency of suppressing thromboembolism binding capacity and the cell-based suppressive potency were evaluated.
  • the binding affinities of APB-A1 to HSA and rhCD40L were identified by bilayer interferometry, and the result showed that the dissociation constants (KD) of APB-A1 for HSA and rhCD40L tended to decrease, compared to each control group.
  • KD dissociation constants
  • the cell-based suppressive potency when HSA was added, there was no significant difference in the suppressive potency of hu5c8 IgG1, while the binding suppressive potency levels of APB-A1 for rhCD40L antigen and D1.1 cell were increased about 1.6 times and 3 times, respectively.
  • the rate of platelet aggregation and the level of serotonin secretion 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, an aggregation response to the IC of hu5c8 IgG1 and rhCD40L was initiated at a relatively low concentration of 60 ng/ml, which was identified by the transmittance and the rate of platelet aggregation.
  • APB-A1 For assessment of the half-life of APB-A1, pharmacokinetic analysis was conducted. APB-A1 was administered to two test groups of cynomolgus monkeys at a dose of each 5 mg/kg (group 1) or each 20 mg/kg (group 2) through a single intravenous injection. As a result, on the assumption that the 2 groups have an equal renal clearance rate, it was identified that the in vivo half-life of the group 2 was 9.59 ⁇ 0.79 days, which is 1.38 times higher than that of the group 1, that is, 6.94 ⁇ 4.6 days.
  • the CD40-CD40L interaction is also operated by a memory B cell, and a significant suppressive efficacy was identified with APB-A1 on day 27 with TT boosting (on day 20). Therefore, it was confirmed that the APB-A1 of the present disclosure markedly improved the thromboembolism-related disorder, compared to hu5c8 IgG1.
  • new bispecific antibodies termed APB-B1a and APB-B1b, respectively, were produced by linking an anti-CD154 (CD40 ligand; CD40L) single chain variable fragment (scFv) (V H -[peptide linker]-V L ) and a tumor necrosis factor-alpha (anti-TNF- ⁇ ) variable fragment (Fv) or disulfide-stabilized Fv (dsFv).
  • CD40L single chain variable fragment
  • scFv V H -[peptide linker]-V L
  • anti-TNF- ⁇ tumor necrosis factor-alpha
  • Fv tumor necrosis factor-alpha
  • dsFv disulfide-stabilized Fv
  • APB-B1 possessed the capacity of simultaneously binding to three targets, that is, recombinant human CD40L, recombinant human TNF- ⁇ and human serum albumin (HSA) proteins, and similar level of antigen-binding affinity to that of anti-CD40L IgG or anti-TNF- ⁇ Fab′ parental antibody.
  • targets that is, recombinant human CD40L, recombinant human TNF- ⁇ and human serum albumin (HSA) proteins, and similar level of antigen-binding affinity to that of anti-CD40L IgG or anti-TNF- ⁇ Fab′ parental antibody.
  • the melting temperature (Tm) measured in an excipient-free buffered state was 62° C., regardless of the presence or absence of inter-chain disulfide bond in anti-TNF- ⁇ Fv, confirming that the inter-chain disulfide bond did not contribute to the structural stability.
  • the in vitro cell-based assay showed that the CD40L inhibiting capacity of APB-B1 was similar to that of the parental antibody anti-CD40L IgG1, and the TNF- ⁇ inhibiting capacity was slightly reduced, compared to the parental antibody anti-TNF- ⁇ Fab′. Nevertheless, APB-B1 demonstrated an inhibitory activity to both of CD40L and TNF- ⁇ and a higher inhibitory activity than each of the parental antibodies, anti-CD40L IgG1 and anti-TNF- ⁇ Fab′.
  • Multispecific antibodies disclosed herein can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques.
  • the methods described herein employ, unless otherwise indicated, 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 skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature.
  • a multispecific antibody described herein is an antibody (e.g., recombinant antibody) prepared, expressed, created or isolated by any means that involves creation, e.g., via synthesis, genetic engineering of DNA sequences.
  • an antibody e.g., recombinant antibody
  • such antibody comprises sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.
  • provided herein is a method of making a multispecific antibody disclosed herein comprising culturing a cell or host cell described herein.
  • a method of making a multispecific antibody comprising expressing (e.g., recombinantly expressing) the antibody using a cell or host cell described herein (e.g., a cell or a host cell comprising polynucleotides encoding an antibody described herein).
  • the cell is an isolated cell.
  • the exogenous polynucleotides have been introduced into the cell.
  • the method further comprises the step of purifying the antibody obtained from the cell or host cell.
  • Multispecific antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein.
  • a “monoclonal antibody,” as used herein, is an antibody produced by a single cell (e.g., hybridoma or host cell producing a recombinant antibody), wherein the antibody immunospecifically binds to an antigen (e.g., human serum albumin) as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the Examples provided herein.
  • an antigen e.g., human serum albumin
  • a monoclonal antibody can be a chimeric antibody or a humanized antibody.
  • a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody.
  • a monoclonal antibody can be a Fab fragment or a F(ab′) 2 fragment.
  • Monoclonal antibodies described herein can, for example, be made by the hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phage libraries using the techniques as described herein, for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F M et al., supra).
  • hybridoma Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art.
  • a mouse or other appropriate host animal such as a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen (e.g., human serum albumin)) used for immunization.
  • lymphocytes can be immunized in vitro.
  • Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding J W (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Additionally, a RIMMS (repetitive immunization multiple sites) technique can be used to immunize an animal (Kilpatrick K E et al., (1997) Hybridoma 16:381-9, incorporated by reference in its entirety).
  • a suitable fusing agent such as polyethylene glycol
  • mice or other animals, such as rats, monkeys, donkeys, pigs, sheep, hamster, or dogs
  • an antigen e.g., human serum albumin
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, Va.), to form hybridomas.
  • ATCC® American Type Culture Collection
  • lymph nodes of the immunized mice are harvested and fused with NS0 myeloma cells.
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • myeloma cells that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • myeloma cell lines are murine myeloma lines, such as NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, Md., USA.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against an antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example, immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding J W (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium.
  • the hybridoma cells can be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Antibodies described herein can be generated by any technique known to those of skill in the art.
  • Fab and F(ab′) 2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′) 2 fragments).
  • a Fab fragment corresponds to one of the two identical arms of a tetrameric antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain.
  • a F(ab′) 2 fragment contains the two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.
  • the antibodies described herein can also be generated using various phage display methods known in the art.
  • phage display methods proteins are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues).
  • the DNA encoding the VH and VL domains are recombined together with a scFv linker by PCR and cloned into a phagemid vector.
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and M13, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antibody that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184: 177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F (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; and U.S.
  • the antibody coding regions from the phage can be isolated and used to generate antibodies, including human antibodies, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences from a template, e.g., scFv clones.
  • a template e.g., scFv clones.
  • the PCR amplified VH domains can be cloned into vectors expressing a VH constant region
  • the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions.
  • VH and VL domains can also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules.
  • a chimeric antibody can contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L (1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J Immunol Methods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.
  • a humanized antibody is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin).
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the antibody also can include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG 1 , IgG 2 , IgG 3 and IgG 4 .
  • Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (EP 239400; WO91/09967; and U.S. Pat. Nos.
  • Single domain antibodies for example, antibodies lacking the light chains, can be produced by methods well known in the art. See Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al., (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and WO94/04678, WO94/25591 and WO01/44301.
  • antibodies that immunospecifically bind to an antigen can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” an antigen using techniques well known to those skilled in the art. (See, e.g., Greenspan N S & Bona C A (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438).
  • a multispecific antibody described herein which binds to the same epitope of an antigen of interest (e.g., human serum albumin) as an antibody described herein, is a human antibody.
  • an antibody described herein which competitively blocks (e.g., in a dose-dependent manner) 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, transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes, can be used.
  • the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination.
  • homozygous deletion of the J H region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of an antigen.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • IgG, IgA, IgM and IgE antibodies For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93.
  • mice capable of producing human antibodies include the XenomouseTM (Abgenix, Inc.; U.S. Pat. Nos. 6,075,181 and 6,150,184), the HuAb-MouseTM (Mederex, Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), the Trans Chromo MouseTM (Kirin) and the KM MouseTM (Medarex/Kirin).
  • Human antibodies which specifically bind to an antigen can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; and WO98/46645, WO98/50433, WO98/24893, WO98/16654, WO96/34096, WO96/33735, and WO91/10741.
  • human antibodies can be produced using mouse-human hybridomas.
  • human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas secreting human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine ones which secrete human monoclonal antibodies that immunospecifically bind to a target antigen.
  • EBV Epstein-Barr virus
  • Such methods are known and are described in the art, see, e.g., Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31.
  • polynucleotides comprising a nucleotide sequence encoding an antibody described herein or a fragment thereof (e.g., a variable light chain region and/or variable heavy chain region) that immunospecifically binds to an antigen
  • vectors e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).
  • polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.
  • an “isolated” polynucleotide or nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source (e.g., in a mouse or a human) of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free” includes preparations of polynucleotide or nucleic acid molecule having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular less than about 10%) of other material, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals.
  • a nucleic acid molecule(s) encoding an antibody described herein is isolated or purified.
  • polynucleotides comprising nucleotide sequences encoding antibodies, which immunospecifically bind to an antigen polypeptide (e.g., human serum albumin) and comprises an amino acid sequence as described herein, as well as antibodies that compete with such antibodies for binding to an antigen polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope as that of such antibodies.
  • an antigen polypeptide e.g., human serum albumin
  • antibodies that compete with such antibodies for binding to an antigen polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope as that of such antibodies.
  • polynucleotides comprising a nucleotide sequence encoding the light chain or heavy chain of an antibody described herein.
  • the polynucleotides can comprise nucleotide sequences encoding a light chain comprising the VL FRs and CDRs of antibodies described herein.
  • the polynucleotides can comprise nucleotide sequences encoding a heavy chain comprising the VH FRs and CDRs of antibodies described herein.
  • polynucleotides comprising a nucleotide sequence encoding a multispecific antibody comprising a Fab comprising three VH chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of an antibody to human serum albumin described herein and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of an antibody to human serum albumin described herein.
  • polynucleotides comprising a nucleotide sequence encoding a multispecific antibody or a fragment thereof comprising a VL domain.
  • a polynucleotide described herein comprises a nucleotide sequence encoding a multispecific antibody provided herein comprising a light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:46), wherein the antibody immunospecifically binds to serum albumin (e.g., human serum albumin).
  • serum albumin e.g., human serum albumin
  • a polynucleotide described herein comprises a nucleotide sequence encoding an antibody provided herein comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:45), wherein the antibody immunospecifically binds to serum albumin (e.g., human serum albumin).
  • serum albumin e.g., human serum albumin
  • a polynucleotide comprising a nucleotide sequence encoding an antibody comprising a light chain and a heavy chain, e.g., a separate light chain and heavy chain.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding a kappa light chain.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding a lambda light chain.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein comprising a human kappa light chain or a human lambda light chain.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody, which immunospecifically binds to serum albumin (e.g., human serum albumin), wherein the antibody comprises a light chain, and wherein the amino acid sequence of the VL domain can comprise the amino acid sequence set forth in SEQ ID NO:46 and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region.
  • serum albumin e.g., human serum albumin
  • the amino acid sequence of the VL domain can comprise the amino acid sequence set forth in SEQ ID NO:46 and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region.
  • human constant region sequences can be those described in U.S. Pat. No
  • polynucleotides encoding a multispecific antibody or a fragment thereof that are optimized, e.g., by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements.
  • Methods to generate optimized nucleic acids encoding a multispecific antibody or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.
  • potential splice sites and instability elements within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression.
  • the alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid.
  • an optimized polynucleotide sequence encoding a multispecific antibody described herein or a fragment thereof can hybridize to an antisense (e.g., complementary) polynucleotide of an unoptimized polynucleotide sequence encoding a multispecific antibody described herein or a fragment thereof (e.g., VL domain or VH domain).
  • an optimized nucleotide sequence encoding a multispecific antibody described herein or a fragment hybridizes under high stringency conditions to antisense polynucleotide of an unoptimized polynucleotide sequence encoding a multispecific antibody described herein or a fragment thereof.
  • an optimized nucleotide sequence encoding a multispecific antibody described herein or a fragment thereof hybridizes under high stringency, intermediate or lower stringency hybridization conditions to an antisense polynucleotide of an unoptimized nucleotide sequence encoding a multispecific antibody described herein or a fragment thereof.
  • Information regarding hybridization conditions has been described, see, e.g., US 2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.
  • the polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Nucleotide sequences encoding antibodies described herein and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody.
  • Such a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-246), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • chemically synthesized oligonucleotides e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-246
  • a polynucleotide encoding an antibody or fragment thereof described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and/or heavy chain of an antibody.
  • Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and/or the variable heavy chain region of an antibody.
  • the amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies.
  • a nucleic acid encoding the immunoglobulin or fragment can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, such as poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
  • DNA encoding multispecific antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the multispecific antibodies).
  • Hybridoma cells can serve as a source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS SystemTM (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of multispecific antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS SystemTM (Lonza)), or myeloma cells that do not otherwise produce immuno
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • the PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, e.g., human kappa or lambda constant regions.
  • the vectors for expressing the VH or VL domains comprise an EF-1 ⁇ promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
  • the VH and VL domains can also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the murine sequences, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • polynucleotides that hybridize under high stringency, intermediate or lower stringency hybridization conditions to polynucleotides that encode an antibody described herein.
  • polynucleotides described herein hybridize under high stringency, intermediate or lower stringency hybridization conditions to polynucleotides encoding a VH domain and/or VL domain provided herein.
  • Hybridization conditions have been described in the art and are known to one of skill in the art.
  • hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2 ⁇ SSC/0.1% SDS at about 50-65° C.
  • hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6 ⁇ SSC at about 45° C. followed by one or more washes in 0.1 ⁇ SSC/0.2% SDS at about 68° C.
  • Hybridization under other stringent hybridization conditions are known to those of skill in the art and have been described, see, for example, Ausubel F M et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3.
  • expression vectors comprising:
  • the second nucleic acid molecule can encode 2, 3, 4, 5, 6, or more bioactive effector moieties and linkers.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising
  • the first nucleic acid molecule can comprise a nucleic acid sequence encoding a Fab comprising: a heavy chain variable domain comprising (a) above and a light chain variable domain comprising (g) above; a heavy chain variable domain comprising (b) above and a light chain variable domain comprising (h) above; a heavy chain variable domain comprising (c) above and a light chain variable domain comprising (i) above; a heavy chain variable domain comprising (d) above and a light chain variable domain comprising (j) above; a heavy chain variable domain comprising (e) above and a light chain variable domain comprising (k) above; a heavy chain variable domain comprising (f) above and a light chain variable domain comprising (1) above; or any combination of a heavy chain variable domain above and a light chain variable domain above.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab (SL335) comprising
  • CDR1 a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76), and
  • CDR1 a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising 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 to SEQ ID NO:94, 95, 96, 97, 98, or 99.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising 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 to SEQ ID NO:100, 101, 102, 103, 104, or 105.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising 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 to SEQ ID NO:94, 95, 96, 97, 98, or 99, 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 to SEQ ID NO:100, 101, 102, 103, 104, or 105, respectively.
  • the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab (SL335) comprising a heavy chain domain comprising an amino acid sequence of SEQ ID NO:45 (V H -C H1 domain) and a light chain domain comprising an amino acid sequence of SEQ ID NO:46 (V L -C L domain).
  • the bioactive effector moieties are anti-TNF- ⁇ Fv, anti-TNF- ⁇ dsFv, anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1 Fv, and/or anti-IFNAR1 dsFv.
  • the second nucleic acid molecule can comprise a nucleotide sequence encoding the 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 to one or more of SEQ ID NOs: 49-60.
  • the second nucleic acid molecule can comprise a nucleotide 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 to one or more of SEQ ID NOs:6-15, 39, and 40.
  • cells e.g., host cells
  • cells expressing (e.g., recombinantly) multispecific antibodies described herein which specifically bind to serum albumin (e.g., human serum albumin) and related polynucleotides and expression vectors.
  • vectors e.g., expression vectors
  • host cells comprising such vectors for recombinantly expressing multispecific antibodies described herein (e.g., human or humanized antibody).
  • Recombinant expression of an antibody or fragment thereof described herein that specifically binds to involves construction of an expression vector containing a polynucleotide that encodes the antibody or fragment.
  • an expression vector containing a polynucleotide that encodes the antibody or fragment Once a polynucleotide encoding an antibody or fragment thereof (e.g., heavy or light chain variable domains) described herein has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody or antibody fragment (e.g., light chain or heavy chain) encoding nucleotide sequence are described herein.
  • Such vectors can, for example, include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., WO86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464) and variable domains of the antibody can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein.
  • host-expression vector systems can be utilized to express antibody molecules described.
  • Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule described herein in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B.
  • 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 systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii ) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.
  • cells for expressing antibodies described herein are CHO cells, for example CHO cells from the CHO GS SystemTM (Lonza).
  • cells for expressing antibodies described herein are human cells, e.g., human cell lines.
  • a mammalian expression vector is pOptiVECTM or pcDNA3.3.
  • bacterial cells such as Escherichia coli , or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary (CHO) cells in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking M K & Hofstetter H (1986) Gene 45: 101-105; and Cockett M I et al., (1990) Biotechnology 8: 662-667).
  • antibodies described herein are produced by CHO cells or NS0 cells.
  • the expression of nucleotide sequences encoding antibodies described herein is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited to, the E.
  • coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M (1989) J Biol Chem 24: 5503-5509); and the like.
  • pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems can be utilized.
  • the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan J & Shenk T (1984) PNAS 81: 3655-3659).
  • Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol 153: 516-544).
  • a host cell strain can be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product 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 murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells.
  • multispecific antibodies described herein e.g., an antibody comprising the CDRs are produced in mammalian cells, such as CHO cells.
  • the antibodies described herein have reduced fucose content or no fucose content.
  • Such antibodies can be produced using techniques known one skilled in the art.
  • the antibodies can be expressed in cells deficient or lacking the ability of to fucosylate.
  • cell lines with a knockout of both alleles of ⁇ 1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content.
  • the Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.
  • stable expression cells For long-term, high-yield production of recombinant proteins, stable expression cells can be generated.
  • cell lines which stably express multispecific antibodies can be engineered.
  • a cell provided herein stably expresses a light chain/light chain variable domain and a heavy chain/heavy chain variable domain which associate to form an antibody described herein (e.g., an antibody comprising the CDRs).
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method can advantageously be used to engineer cell lines which express a multispecific antibody described herein or a fragment thereof.
  • Such engineered cell lines can be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.
  • a number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-232), hypoxanthineguanine phosphoribosyltransferase (Szybalska E H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-823) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: 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 mycophenolic acid (Mulligan R C & Berg P (1981) PNAS 78(4): 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C (1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) Ann Rev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) Trends Biotechnol 11(5)
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington C R & Hentschel C C G, 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)).
  • vector amplification for a review, see Bebbington C R & Hentschel C C G, 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)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse G F et al., (1983) Mol Cell Biol 3: 257-
  • the host cell can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • the host cells can be co-transfected with different amounts of the two or more expression vectors. For example, host cells can be transfected with any one of the following ratios of a first expression vector and a second expression vector: 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.
  • a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides.
  • the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot N J (1986) Nature 322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199).
  • the coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.
  • the expression vector can be monocistronic or multicistronic.
  • a multicistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotide sequences.
  • a bicistronic nucleic acid construct can comprise in the following order a promoter, a first gene (e.g., heavy chain of an antibody described herein), and a second gene and (e.g., light chain of an antibody described herein).
  • a promoter e.g., a promoter
  • a first gene e.g., heavy chain of an antibody described herein
  • a second gene and e.g., light chain of an antibody described herein.
  • the transcription of both genes can be driven by the promoter, whereas the translation of the mRNA from the first gene can be by a cap-dependent scanning mechanism and the translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.
  • the vector can comprise a first nucleic acid molecule encoding an antigen binding fragment (Fab) that bind to serum albumin, and a second nucleic acid molecule encoding a bioactive effector moiety and a linker.
  • Fab antigen binding fragment
  • an antibody molecule described herein can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • an antibody described herein is isolated or purified.
  • an isolated antibody is one that is substantially free of other antibodies with different antigenic specificities than the isolated antibody.
  • a preparation of an antibody described herein is substantially free of cellular material and/or chemical precursors.
  • the language “substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”) and/or variants of an antibody, for example, different post-translational modified forms of an antibody.
  • heterologous protein also referred to herein as a “contaminating protein”
  • variants of an antibody for example, different post-translational modified forms of an antibody.
  • culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation.
  • the antibody or fragment When the antibody or fragment is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody or fragment have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody or fragment of interest.
  • antibodies described herein are isolated or purified.
  • compositions comprising a multispecific antibody described herein having the desired degree of purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.). Also disclosed herein are pharmaceutical compositions comprising a multispecific antibody described herein and a pharmaceutically acceptable excipient. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition of the present disclosure can provide rapid, sustained or delayed release of an active ingredient after being administered to a subject and can be formulated using a method well known to those skilled in the art.
  • the formulations can be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, sterile powder, or the like.
  • Suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the formulations can additionally include a filler, an anti-agglutinating agent, a lubricating agent, a wetting agent, a favoring agent, an emulsifier, a preservative, and the like.
  • compositions described herein can be useful in enhancing, inducing, or activating the activities of multispecific antibodies and treating a disease or condition, such as autoimmune conditions or diseases.
  • compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.
  • Autoimmune disease or conditions that can be treated include but are not limited to neuromyelitis optica spectrum disorders, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, systemic lupus erythematosus, ANCA-associated vasculitis, ulcerative colitis and Crohn's disease.
  • presented herein are methods for modulating one or more immune functions or responses in a subject, comprising to a subject in need thereof administering a multispecific antibody described herein, or a composition thereof.
  • methods for activating, enhancing or inducing one or more immune functions or responses in a subject comprising to a subject in need thereof administering a multispecific antibody or a composition thereof.
  • methods for preventing and/or treating diseases in which it is desirable to activate or enhance one or more immune functions or responses comprising administering to a subject in need thereof a multispecific antibody described herein or a composition thereof.
  • presented herein are methods of treating an autoimmune disease or condition comprising administering to a subject in need thereof a multispecific antibody or a composition thereof.
  • a multispecific antibody described herein activates or enhances or induces 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 in the range of between 10% to 25%, 25% to 50%, 50% to 75%, or 75% to 95% relative to the immune function in a subject not administered the multispecific antibody described herein using assays well known in the art, e.g., ELISPOT, ELISA, and cell proliferation assays.
  • assays well known in the art, e.g., ELISPOT, ELISA, and cell proliferation assays.
  • compositions of the present disclosure can be administered to a subject through a variety of administration routes including oral, transcutaneous, subcutaneous, intravenous, and intramuscular administration routes.
  • an antibody or composition which will be effective in the treatment and/or prevention of a condition will depend on the nature of the disease and can be determined by standard clinical techniques.
  • the amount of the multispecific antibody actually administered is determined in light of various relevant factors including the disease to be treated, a selected route of administration, the age, sex and body weight of a patient, and severity of the disease, and the type of a bioactive polypeptide as an active ingredient. Since the multispecific antibody of the present disclosure has a very excellent sustainability in blood, the number and frequency of administration of the peptide preparations comprising the fusion protein of the present disclosure can be noticeably reduced.
  • dose to be employed in a composition will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each subject's circumstances.
  • effective doses can also vary depending upon means of administration, target site, physiological state of the patient (including age, body weight and health), whether the patient is human or an animal, other medications administered, or whether treatment is prophylactic or therapeutic.
  • the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • an in vitro assay is employed to help identify optimal dosage ranges.
  • Effective doses can be extrapolated from dose response curves derived from in vitro or animal model test systems.
  • human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible
  • kits comprising one or more antibodies described herein or conjugates thereof.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more antibodies provided herein.
  • the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • kits that can be used in the above methods.
  • kits described herein comprise an antibody described herein, e.g., a purified antibody, in one or more containers.
  • kits described herein contain a substantially isolated antigen(s) (e.g., human serum albumin) that can be used as a control.
  • the kits described herein further comprise a control antibody which does not react with a serum albumin antigen.
  • kits described herein contain one or more elements for detecting the binding of an antibody to a serum albumin antigen (e.g., the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody can be conjugated to a detectable substrate).
  • a kit provided herein can include a recombinantly produced or chemically synthesized serum albumin antigen.
  • the serum albumin antigen provided in the kit can also be attached to a solid support.
  • the detecting means of the above described kit includes a solid support to which a serum albumin antigen is attached.
  • Such a kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse/rat antibody. In binding of the antibody to the serum albumin antigen can be detected by binding of the said reporter-labeled antibody.
  • hu5c8 scFv (SEQ ID NO:5) was synthesized through codon optimization suitable for mammalian cells (Cosmo Genetech, Korea). Primers commercially available from Macrogen (Seoul, Korea) were used for cloning, and initial cloning was conducted on pcDNA3.3 and pOptiVEC vector (Thermo Fisher Scientific) through a polymerase chain reaction (PCR) by linking the synthesized hu5c8 scFv to the N-terminals of SL335 heavy and light chains by means of a flexible linker, respectively.
  • PCR polymerase chain reaction
  • cloning was identified by protein expression on the ExpiCHO-STM cell line (Thermo Fisher Scientific, Waltham, Mass.).
  • cloning was performed using animal cell expressing vectors, that is, pd2535nt (Horizon Discovery) for a heavy chain and pd2539 (Horizon Discovery) for a light chain.
  • the PCR was performed under conditions in which a total of 25 cycles are performed at 95° C. for 30 seconds, at 61° C. for 30 seconds, and at 72° C. for 1 minute, and finally for 5 minutes for extension, followed by lowering the temperature to 4° C.
  • Table 1 indicates primer sets for producing recombinant vectors by cloning the APB-A1 heavy-chain and light-chain genes inserted into pcDNA3.3 and pOptiVEC vectors to pd2535nt and pd2539 vectors, respectively.
  • pGL3c(1b) (Satorius) plasmid vectors were used as additional vectors.
  • the PCR was performed in such a manner as described above, and a PCR product of APB-A1 heavy and light chains of about 1,600 base pairs (bp) in length were acquired. Ends of the heavy chain of the PCR product and pd2535nt vector were treated with BbsI (Thermo Fisher Scientific); and 5′ ends of the light chain of the PCR product and the pd2539 vector were treated with BsrGI (Thermo Fisher Scientific), 3′ ends of the light chain of the PCR product and pd2539 vector were treated with a restriction enzyme BbsI, and then treated with T4 DNA ligase (Takara, Japan). Subsequently, E.
  • BbsI Thermo Fisher Scientific
  • BsrGI Thermo Fisher Scientific
  • APB-A1 plasmid DNA sequences of SEQ ID NO:41 and SEQ ID NO:42, respectively, wherein the 104 th amino acid from the N-terminal of those amino acid sequences corresponds to glycine (G) or glutamine (Q).
  • the bioactive-effector can be an anti-TNF- ⁇ Fv (certolizumab having a heavy chain of SEQ ID NO:6 and a light chain of SEQ ID NO:7) or anti-TNF- ⁇ dsFv (certolizumab having a heavy chain of SEQ ID NO:8 and a light chain of SEQ ID NO:9), anti-IL-23 Fv (ustekinumab having a heavy chain of SEQ ID NO:10 and a light chain of SEQ ID NO:11) or anti-IL-23 dsFv (ustekinumab having a heavy chain of SEQ ID NO:12 and a light chain of SEQ ID NO:13), or anti-IFNAR1 Fv (anifrolumab having a heavy chain of SEQ ID NO:14
  • PCR was performed for 30 cycles with a T100TM thermal cycler instrument (Bio-Rad, Hercules, Calif.) using a Taq DNA polymerase (Takara, Japan) under conditions of each cycle being performed at 94° C. for 1 minute, at 60° C. for 1 minute, and at 72° C. for 1 minute.
  • a T100TM thermal cycler instrument Bio-Rad, Hercules, Calif.
  • Taq DNA polymerase Takara, Japan
  • an assembly PCR was performed under conditions of cycling at 94° C. for 1 minute, at 60° C. for 1 minute, and at 72° C.
  • the heavy chain assembled product obtained by the PCR and a pD2535NT vector were treated with a Bbs I restriction enzyme (Thermo Fisher Scientific, Waltham, Mass.); the light chain assembled product and a pd2539 vector (Horizon Discovery) were treated with Bbs I and Bsr GI restriction enzymes (New England Biolabs, Ipswich, Mass.).
  • the chain reaction assembled products treated with the respective restriction enzymes and the plasmid vectors were assembled with each other using a T4 DNA ligase (Takara), and the assembled products were put into soluble competent cells treated with CaCl 2 , followed by applying heat-shock for transformation. Next, the transformed clones were screened using a culture medium containing a Kanamycin antibiotic.
  • cloning assays were conducted in the same manner as described above.
  • the recombinant human CD40L gene (SEQ ID NO:38) synthesized by Cosmo Genetech was used and cloned to pcDNA3.3TM® vector (Thermo Fisher Scientific) using restriction enzymes Xba I (Takara) and Not I (Takara).
  • Proteins were produced by transient expression using a CHO cell.
  • an ExpiCHO cell (Thermo Fisher Scientific) was incubated in a shaking incubator under conditions of 37° C., 140 rpm, 5% CO 2 , and 80% humidity with ExpiCHO expression media (Thermo Fisher Scientific).
  • the cells were seeded to a 125 ml-culture flask under the condition of a concentration of 6.0 ⁇ 10 6 cells/ml, and plasmid vectors pD2535NT and pD2539, having 3 sequence-identified genes (certolizumab, ustekinumab and anifrolumab) heavy and light chains inserted thereto, and a recombinant human CD40L gene inserted vector pcDNA3.3-TOPO were transfected to the seeded cells using an ExpiFectamine CHO transfection kit (Thermo Fisher Scientific).
  • the cells incubated in the shaking incubator for 16 hours were treated with ExpiCHO feed and an enhancer, followed by incubating for 3 days in the incubator being under the same conditions.
  • ExpiCHO feed was additionally treated and incubated under conditions of 32° C., 140 rpm, 5% CO 2 , and not less than 80% humidity.
  • the culture medium was collected and then centrifuged under conditions of 4,000 rpm, 15 minutes, and 4° C., thereby isolating cells from the culture medium.
  • the isolated culture medium was filtered through a 0.2 ⁇ m-filter sheet, thereby removing impurities.
  • a glutamine synthesis (GS)-null CHO K1 cell line (Horizon Discovery) was used.
  • Transfection was performed using a FreestyleTMMax reagent (Invitrogen, Thermo Fisher Scientific) according to the procedure modified from the standard protocol provided by Horizon Discovery.
  • the transfection was performed by co-transfecting light chains and heavy chains in a ratio of 1:1 to 1:3 (pd2539: pd2535nt) using a total of 37.6 ⁇ g plasmid vectors. 2 days after transfection, the incubated cells were taken out, transferred to a 50 ml conical tube (Nunc, Denmark) for centrifugation, and then dissolved in a CDfortiCHO culture medium not containing L-glutamine, followed by identifying the cell concentration and viability using a COUNTESS II automated cell counter (Invitrogen, Thermo Fisher Scientific). 50 ⁇ M methionine sulfoximine (MSX) (Sigma-Aldrich, St.
  • HD-BIOP3 GS-null CHO-K1 cell (Horizon Discovery) seeded to a CD FortiCHO (Thermo Fisher Scientific) culture medium supplemented with 4 mM L-glutamine was prepared under the condition of a concentration 3.0 ⁇ 10 5 cells/ml, and seed culture was performed in a shaking incubator being under conditions of 37° C., 5% CO 2 , and not less than 80% humidity, for 1 day.
  • cells were seeded at a concentration of 4.8 ⁇ 10 5 cells/ml, additionally incubated for 1 day, and finally prepared at a concentration of 1.0 ⁇ 10 6 cells/ml.
  • Plasmid vectors (pD2535NT and pD2539) containing heavy and light chain genes of a sequence-identified, certolizumab-related SAFA-based bispecific antibody were transfected to the seeded cells using OptiPRO SFM culture media and a Freestyle max reagent (Invitrogen, Carlsbad, Calif.), and then incubated for 2 days under conditions of 37° C., 5% CO 2 , and not less than 80% humidity. The incubated cells were all transferred to a CD FortiCHO culture medium not containing L-glutamine and then treated with 50 ⁇ m of methionine sulfoximine (MSX) (Sigma-Aldrich, St.
  • MSX methionine sulfoximine
  • the pre-existing culture medium was removed using a centrifuge and then replaced by a CD FortiCHO culture medium containing both MSX and puromycin at an interval of 7 to 10 days, and incubation was performed for 21 days so as to maintain the number of cells to be 5.0 ⁇ 10 5 cells/ml.
  • rhCD40L a recombinant hCD40L antigen prepared according to the present disclosure (AprilBio, Chuncheon, South Korea) was coated onto a 96-well MaxiSorp ELISA plate (Nunc) at a concentration of 100 ng/well overnight at 4° C. using a carbonate coating buffer (pH of 9.6). The plate was blocked by treating with a blocking buffer (Starting BlockTM (PBS) (Thermo Fisher Scientific) at room temperature for 3 hours.
  • PBS Starting BlockTM
  • APB-A the supernatant of the recombinant antibody having a structure of anti-CD40L scFv) 2 -anti-HSA Fab produced from GS null CHOK1 cell, termed APB-A 1, was continuously diluted with a dilute buffer (0.1% PBST+0.3% BSA; 0.3% PBA), and was allowed to react at room temperature for 1 hour.
  • HRP horseradish peroxidase conjugated goat-anti-human Fd antibody
  • TMB tetramethylbenzidine
  • BD science Franklin Lakes, N.J.
  • PK ELISA was performed such that the rhCD40L antigen was diluted in PBS (Roman Industries, Japan) at a concentration of 1 ⁇ g/ml and then coated on an ELISA plate at a volume of 100 ⁇ l overnight at 4° C.
  • a blocking buffer (0.3% BSA in PBS, 300 ⁇ l) was added to each well to perform blocking at 25° C. for 3 hours, and standard and QC samples were transferred to each well at a volume of 100 ⁇ l and were allowed to react at 25° C. for 1.5 hours.
  • a wash buffer 300 ⁇ l/well
  • the anti-human light chain goat IgG-biotin monkey absorbed; Immuno-Biological Laboratories, Japan was seeded to each well at a 100 ⁇ l/well concentration and were allowed to react at 25° C. for 1 hour.
  • the GS null CHO K1 cell line expressing the produced APB-A1 was incubated in a CDfortiCHO culture medium using a WAVE bioreactor (GE Healthcare) for 11 days, the resulting supernatant and cell pellets were centrifuged at 4° C. at 4,000 rpm for 20 minutes, and the culture supernatant was filtered with a 0.2 ⁇ m filter.
  • APB-A1 protein was purified through a 3-step chromatography process. First, an affinity chromatography step was performed using a CaptureSelect IgG-C H1 affinity matrix resin (Life Technologies). After washing a matrix with PBS of 5 column volumes (CVs), sample binding was performed at a flow rate of 20 ml/min.
  • a washing step was performed using 4 CVs of a high-salt wash buffer (PBS, 500 mM NaCl, pH 7.4) and 2 CVs of a low-salt wash buffer (25 mM sodium phosphate, pH 7.6) at a flow rate of 25 mf/min.
  • An elution buffer (20 mM citric acid pH 3.0, 150 mM sodium chloride) was passed through the matrix at a flow rate of 20 mf/min, and a protein solution having peaks of UV 50 mAU or greater was collected and then transferred to a 250 me container for cold storage for 1 hour.
  • the collected protein solution was neutralized by adding a 1 M tris-HCl (pH 8.0) solution, and impurities were removed using a 0.2 ⁇ m all filter, thereby eluting the APB-A1.
  • cation exchange purification was performed using a CaptoTM SP ImpRes resin equilibriated by 25 mM of a sodium phosphate (pH 7.6) solution.
  • An affinity chromatography elution sample which was 4 ⁇ diluted with sterilized distilled water was coupled to a column with a flow rate set to 5 ml/min, and 30%-50%-100% elution steps were performed using an elution buffer (25 mM sodium phosphate, pH 7.6, 1 M sodium chloride).
  • a wash buffer (2 M sodium chloride) was passed through at a flow rate of 3 ml/min to wash a resin column, 5 CVs of 20 mM sodium phosphate (pH 6.5) solution was passed through the resin column for equilibration.
  • a sample was dialyzed with 20 mM of a sodium phosphate pH 6.5 buffer, and the pH level and salt concentration were adjusted. The sample was passed through the resin column at a flow rate of 5 ml/min to collect a protein solution, followed by removing impurities using a 0.2 ⁇ m filter, and the obtained protein was quantified and analyzed.
  • the protein sample was loaded onto a 4 to 15% gradient gel (Bio-Rad, Hercules, Calif.) at a concentration of 1 ⁇ g/well, and then electrophoresed at 150 V for 50 minutes, thereby performing SDS-PAGE analysis.
  • the gel separated after the electrophoresis was stained with an Ez-Gel stain solution (DoGenBio, South Korea) for 1 hour, and then decolorized with water.
  • SE-HPLC size exclusion high-performance liquid chromatography
  • HPLC Shimadzu, Japan
  • TSK gel Ultra SW aggregate column Tosoh Bioscience, Japan
  • the sample was diluted with 100 mM Na 2 HPO 4 , 100 mM Na 2 SO 4 , and 0.05% (w/v) NaN 3 (pH 6.7), and 50 ⁇ g of the diluted sample was injected by an automatic sample injector at 15° C., and then eluted using a mobile phase (200 mM phosphate, pH 6.7, 0.05% (w/v) NaN 3 (flow rate: 0.5 mf/min).
  • the UV absorbance was measured at a wavelength of 280 nm.
  • Molecular weights of reduced and nonreduced APB-A1 were measured using LC-ESI MS spectrometry, and then analyzed in combination with Dionex UHPLC (Thermo Fisher Scientific) and Q-TOF 5600+MS/MS system (AB SCIEX, CA, USA).
  • Dionex UHPLC Thermo Fisher Scientific
  • Q-TOF 5600+MS/MS system AB SCIEX, CA, USA.
  • the pl value of the isolated protein was measured using an isoelectric focusing gel (pH 3 to 10). After loading 1 ⁇ g, 3 ⁇ g, and 5 ⁇ g of the sample onto the gel at a density of 1 mg/ml, isoelectric focusing was performed at 100 V for 1 hour, at 200 V for 1 hour, and at 500 V for 2 hours, and 12% trichloroacetic acid (TCA) staining and coomassie brilliant blue (CBB) staining were performed, followed by analyzing by ImageMasterTM 2D Platinum (GE healthcare, ver 5.0).
  • TCA trichloroacetic acid
  • CBB coomassie brilliant blue
  • ion change chromatography was performed using Protein-Pak HiRes CM. 20 ⁇ g of the protein sample was injected by an automatic sample injector at 30° C., and then eluted using a mobile phase [25 mM 2-(Nmorpholino) ethanesulfonic (MES), 500 nM NaCl, pH 6.5] with a gradient of 0 to 40% for 30 minutes. The flow rate was 0.3 ml/min, and the UV absorbance was measured at a wavelength of 280 nm.
  • MES 2-(Nmorpholino) ethanesulfonic
  • affinity chromatography For purification of a bispecific antibody protein sample present in a CHO cell culture medium, affinity chromatography (AC) was performed using a CaptureSelect IgG-C H1 affinity matrix resin (Life Technologies, Carlsbad, Calif.) and an AKTA pure 150 L instrument (GE Healthcare, Chicago, Ill.) in the following manner.
  • a phosphate-buffered saline (PBS) (pH 7.4) buffer was passed through a resin-packed column for equilibration, a culture medium containing a protein expressed from a transient expression cell was isolated and then passed through the resin column at a flow rate of 1.5 ml/min.
  • PBS phosphate-buffered saline
  • the PBS pH 7.4 buffer containing 500 mM NaCl was passed through the column to wash materials non-specifically binding to the resin.
  • the equilibration and washing steps for each material were performed with 10 column volumes (CVs).
  • a 20 mM citric acid, pH 3.0 buffer containing 150 mM NaCl was used.
  • the eluted buffer was treated with 1 M Tris-HCl (pH 8.0) to be neutralized to have a neutral pH level, and the concentration of the purified protein was measured using a microplate spectrophotometer (BMG LABTECH, Germany) at a wavelength of A280 nm.
  • anion exchange chromatography was performed using a Q sepharose HP resin (GE Healthcare) in the following manner. After equilibrating about 10 CVs of a 20 mM citrate, pH 6.0 buffer without NaCl added thereto was passed through a Q sepharose HP resin, the protein primarily purified through affinity chromatography was passed through the resin, and then the protein not binding to the resin was recovered. The concentration of the recovered protein was measured using a microplate spectrophotometer instrument at a wavelength of A280 nm.
  • cation exchange chromatography was performed by packing a CM sepharose FF resin (GE Healthcare) into a column. Prior to purification, a pre-treatment step was performed by dialyzing the protein purified by affinity chromatography with a 20 mM citric acid, pH 6.0 binding buffer without NaCl added thereto.
  • the binding buffer was passed through the resin-packed column at a flow rate of 1.0 ml/min for equilibration, and a pre-treated protein was then treated at the same flow rate and was allowed to react with the resin.
  • 5 CVs of the binding buffer was passed through the column at the same flow rate, and 3 CVs of 100 mM NaCl added binding buffer was further treated, thereby washing and removing nonspecifically binding materials.
  • the bispecific antibody protein existing in the form of monomer was eluted from the resin by adding 120 mM of a NaCl added binding buffer. The concentration of the purified protein was measured at A280 nm.
  • the protein sample was purified using 15 mM of a sodium phosphate, pH 7.4 buffer containing 1 M NaCl in anion chromatography and cation chromatography.
  • the concentration of the purified protein was measured using a microplate spectrophotometer at A280 nm.
  • the purified bispecific antibody protein sample was diluted with a nonreducing 4 ⁇ SDS sample buffer (Thermo Fisher Scientific) and a reducing sample buffer containing 2-mercaptoethanol.
  • a sample heated at 100° C. for 5 minutes and an unheated sample were prepared, and for size comparison, a protein size marker (SMOBio, Taiwan) and a (scFv) 2 -Fab protein sample were treated together.
  • the prepared protein samples were loaded onto a 4-15% 15-well Miniprotein TGX precast gel (Bio-Rad) at a density of 2 ⁇ g/well, and electrophoresis was performed in a tris-glycine SDS running buffer at 150 V for 1 hour. After completion of the electrophoresis, the SDS-PAGE gel was stained with an EZ-Gel staining solution (DoGenBio, South Korea) for 1 hour, and decolorized in distilled water for one day.
  • EZ-Gel staining solution DoGenBio, South Korea
  • the protein melting temperature was analyzed using a hydrophobic dye (5,000 ⁇ , SYPRO Orange) and Light Cycler 480 II (Roche, Switzerland) as a real time PCR instrument.
  • the protein sample was diluted in a sodium phosphate pH 7.0 buffer at a 300 ⁇ g/ml concentration, and then placed in an ultraAmp PCR plate (Sorenson Bioscience, Salt Lake City, Utah) with a 5 ⁇ reagent and a SYPRO Orange dye until the final concentration of each well reached 5.4 ⁇ g/well.
  • an excitation filter and an emission filter were set to 465 nm and 580 nm, respectively, and the denaturation of protein was assessed according to the temperature increasing at a rate of 1° C./min within a range from 20° C. to 85° C.
  • the purity of the purified bispecific antibody was analyzed using a column T SKgel UltraSW Aggregate 7.8 ⁇ 300 mm (Tosoh Bioscience, Japan) and a 1260 infinity II LC system (Agilent Technologies, Santa Clara, Calif.) as a HPLC instrument. Prior to sample analysis, the column and the HPLC instrument were equilibrated with 100 mM of a 20 mM citric acid (pH 5.5) buffer. The sample to be analyzed was diluted with 20 mM citric acid pH 5.5 buffer, and the sample was loaded onto the column at a density of up to 25 ⁇ g. SE-HPLC analysis was performed under conditions of 0.7 mf/min in the flow rate and 120 bar in the maximum pressure limit for 30 minutes, and the absorbance was measured at A280 nm.
  • Human serum albumin Sigma-Aldrich
  • CD40L protein a recombinant human CD40L protein
  • TNF- ⁇ protein BioLegend, San Diego, Calif.
  • Human serum albumin, CD40L and TNF- ⁇ protein were diluted in sodium carbonate pH 9.6 buffer at a 1 ⁇ g/ml concentration, and each 100 ⁇ l was then seeded to each well of a 96-well maxisorp plate (Nunc, Denmark), followed by coating at 4° C. for one day.
  • the non-coated protein and the buffer were completely removed, and blocking was then performed by adding each 300 ⁇ l of a PBS pH 7.4 buffer containing 3% bovine serum albumin (BSA) (Sigma-Aldrich) and 0.1% tween-20 to each well. After blocking for 2 hours, washing was performed by repeatedly performing a process of completely removing the added buffer, adding each 300 ⁇ l of a buffer (PBS-T) containing 0.1% tween-20 PBS (pH 7.4), and then removing the buffer 3 times in total.
  • BSA bovine serum albumin
  • the respective antibodies were serially diluted 10 folds at concentrations decreasing from 100 nM to 1.0 ⁇ 10 ⁇ 4 nM in a PBS pH 7.4 buffer (0.3% PBA) containing 0.3% bovine serum albumin and 0.1% tween-20, and each 100 ⁇ l of the diluted antibody samples were added to each well to be allowed to react at room temperature for 2 hours.
  • HRP-conjugated goat anti-human Fd antibodies (Southern Biotechnology, Birmingham, Ala.) were diluted with a 0.3% PBA buffer in 1:4,000, and each 100 ⁇ l of the diluted antibody samples were added to each well to then be allowed to react at room temperature for 1 hour.
  • TMB substrate (BD Bioscience, Franklin Lakes, N.J.) was added and reacted, and the absorbance was measured using a microplate spectrophotometer at A450 nm.
  • TNF- ⁇ protein (30 ⁇ g/ml), CD40L protein (10 ⁇ g/ml) and human serum albumin (20 ⁇ g/ml) were immobilized to an amine reactive second generation (AR2G) biosensor (Forte Bio) using a pH 5.0 sodium acetate buffer, materials that are not immobilized were removed with 1 M ethanolamine (pH 8.5), and the bispecific antibody was allowed to react at serially diluted concentrations, followed by measuring binding and dissociation constants for the respective antigens.
  • AR2G amine reactive second generation
  • CD40L protein (10 ⁇ g/ml) was immobilized to the AR2G biosensor using the pH 5.0 sodium acetate buffer, and binding capacities were measured in the order of the bispecific antibody (3.2 ⁇ g/d), human serum albumin (13.2 ⁇ g/ml), human serum albumin (13.2 ⁇ g/ml) and TNF- ⁇ (2 ⁇ g/ml). The assessment results were analyzed using DataAnalysis8 software.
  • HSA human serum albumin
  • SL335 Real-time binding assays between human serum albumin (HSA) (Sigma-Aldrich) and SL335 and between rhCD40L antigen and APB-A1 were performed using biolayer interferometry equipped with an Octet RED system.
  • HSA binding affinity 20 ⁇ g/mf of HSA and 10 ⁇ g/ml of rhCD40L were immobilized to the AR2G biosensor (pH 5.0), non-binding molecules were removed from the surface of the biosensor using a kinetics buffer (1 M ethanol amine, pH 8.5).
  • D1.1 cells were centrifuged to remove a supernatant and then resuspended in a MACS buffer (0.5% BSA, 2 mM EDTA in 1 ⁇ PBS, 0.22 ⁇ m filtered) at a concentration of 1.0 ⁇ 10 6 cells/mf. After the cells were seeded at each concentration of 100 ⁇ l (1.0 10 5 cells/test) to a 1.5 me tube, centrifugation was performed for 5 minutes under 4° C., and 500 ⁇ g conditions, thereby removing the supernatant.
  • APB-A1, hu5c8 IgG1 and SL335 were continuously diluted by one tenth ( 1/10) at 5 time points each starting from at an amount of 1 ⁇ g/ml, and each 100 ⁇ l of the first diluted antibodies were transferred to cell pellets of 1.5 ml tubes using a pipette, followed by incubating at 4° C. for 30 minutes.
  • the MACS buffer was added by each 500 ⁇ l to the respective tubes, and then washed by centrifuging under conditions of 4° C., 500 ⁇ g, and 5 minutes.
  • cell pellets were dissolved by adding each 50 ⁇ l of the goat-anti-human kappa-FITC samples diluted in 1:1000 to the MACS buffer (Lifespan Biosciences, Washington, Seattle), followed by incubating at 4° C. for 30 minutes, and the washing step was repeated once again. After removing the supernatant, the cell pellets were dissolved by adding 200 ⁇ l of 0.4% paraformaldehyde (PFA in PBS) buffer, followed by storage at 4° C. for immobilization, and the cells were analyzed by a BD FACS verse instrument.
  • PFA in PBS paraformaldehyde
  • HEKBlueTM CD40L reporter cells (InvivoGen, San Diego, Calif.) were used, and D1.1 cells expressing mCD40L, and rhCD40L, were used as CD40L donors. Buffers with and without 20 M HSA added to a Dulbecco's PBS buffer (Corning) containing 0.2% bovine serum albumin were used, and APB-A 1, hu5c8 IgG1 and SL335 were diluted by one third (1 ⁇ 3) starting from a concentration of 200 nM.
  • the D1.1 cells were added by an equal volume of 1 ⁇ 10 4 cells/well, and incubated in an incubator being under conditions of 37° C. and 5% CO 2 , for 3 hours.
  • HEKBlueTMCD40L cells were added at a density of 5 ⁇ 10 4 cells per well, and then incubated in an incubator being under the same condition for 21 hours.
  • the supernatant was transferred by each 40 ⁇ l from the plate to other 96-well EIA/RIA plates (Corning) using a multi-pipette.
  • Each 160 ⁇ l of a QUANTI-BlueTM solution (InvivoGen) was added to each well, which was wrapped with a foil to block light, and was kept in a 37° C. CO 2 incubator for 1 hour, followed by measuring the absorbance at 655 nm using a spectrophotometer.
  • Each 70 ⁇ l of 300 ng/ml rhCD40L antigen was added to a tube containing 70 ⁇ l of a diluted sample obtained by diluting the three samples by one third (1 ⁇ 3) at the same concentration starting from 200 nM, and was then allowed to react at 37° C. in a CO 2 incubator for 30 minutes.
  • PRP Human platelet-rich-plasma
  • KRBC Korean Red Cross Blood Centers
  • PRP anticoagulated in an acid-citrate dextrose solution (0.8% citric acid, 2.2% sodium citrate, 2.45% glucose) was centrifuged at 120 ⁇ g for 10 minutes to eliminate red blood cells, and then centrifuged at 360 ⁇ g for 15 minutes to obtain plate pellets. Platelets were dissolved in platelet poor plasma (PPP) at a final concentration of 5.0 ⁇ 10 8 /ml, and all procedures were performed at room temperature (23 ⁇ 2° C.).
  • APB-A1 proteins were administered to each 3 cynomolgus monkeys (males) of each group at a dose of 5 mg/kg (group 1) or 20 mg/kg (group 2) through a single intravenous injection. After the administration, blood samples were collected from a total of 17 points in time: 1 point prior to administration; and 16 points; 0.25, 1, 2, 6 and 24 hours and 4, 7, 10, 13, 16, 19, 22, 25, 28, 34 and 40 days after administration. The concentration of APB-A1 present in the platelet of each cynomolgus monkey was measured by ELISA.
  • TT anti-tetanus-toxoid
  • DXT dexamethasone
  • DXT was injected a total of 4 times at each dose of 1 mg/kg, that is, 2 days before the first TT injection and on days 1, 5 and 8 after the first TT injection, and APB-A1 was injected once at the time of the first TT injection (on day 1).
  • the blood samples to be analyzed were collected prior to injection, on days 10, 12, 14, 16, 20, 27, 30 and 40, and anti-TT IgG antibody values were measured by ELISA.
  • the blood samples were collected a total of 5 times about 2 hours after TT injection, and on days 20, 27, 30 and 40 after TT injection, and the collected blood samples were stored in K2-EDTA containing tubes to prevent blood coagulation from non-injected portions.
  • Immunophenotyping was performed using antibody panels for markers such as CD45, CD20, CD27, Ki67 and IgD, and 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.
  • L929 mouse cell expressing a TNF receptor on a cell surface and a recombinant soluble TNF- ⁇ protein were used.
  • L929 cells Korean Cell Line Bank
  • an RPMI1640 culture medium containing 10% fetal bovine serum in an incubator being under conditions of 37° C., 5% CO 2 , and not less than 80% humidity.
  • L929 cells were plated to a 96-well cell culture plate (Corning Inc., New York City, N.Y.) under the condition of a concentration of 5.0 ⁇ 10 4 cells/well, and then incubated in an incubator being under the same condition for 24 hours. After 24 hours of incubation, the existing culture medium was removed, actinomycin D (Sigma-Aldrich) diluted with an RPMI1640 culture medium containing 10% fetal bovine serum at a 1 ⁇ g/ml concentration was treated on each well, and then reacted for 30 minutes in an incubator being under conditions of 37° C., 5% CO 2 , and not less than 80% humidity.
  • actinomycin D Sigma-Aldrich
  • the antibodies serially diluted according to varying concentrations were treated on each well, and recombinant soluble TNF- ⁇ proteins were then treated at a 10 ng/ml concentration to then be allowed to react for 24 hours in an incubator being under conditions of 37° C., 5% CO 2 , and not less than 80% humidity.
  • 10 ⁇ l of CCK-8 (Dojindo, Japan) was treated by transferring the same using a multi-channel pipette to the wells containing the respective reactants.
  • the supernatant was transferred to another plate using a pipette, and the absorbance was measured at A450 nm wavelength.
  • the analysis was performed using a CD40L HEK-BlueTM reporter cell (InvivoGen, San Diego, Calif.) expressing secreted embryonic alkaline phosphatase (SEAP) by reacting with CD40L and TNF- ⁇ , D1.1 cells expressing cellular membrane CD40L on cell surfaces, and recombinant soluble TNF- ⁇ proteins.
  • the CD40L HEK-BlueTM cells were incubated with a DMEM (Thermo Fisher Scientific) culture medium containing 10% fetal bovine serum and an antibiotic (Normocin, Blasticidin and Zeocin) in an incubator being under conditions of 37° C., 5% CO 2 , and not less than 80% humidity.
  • the D1.1 cells were incubated in an incubator being under the same conditions using an RPMI1640 culture medium containing 10% fetal bovine serum.
  • the D1.1 cells were plated to a 96-well cell culture plate under the condition of a 5.0 ⁇ 10 4 cells/well concentration, or the recombinant soluble TNF- ⁇ proteins were plated to the 96-well cell culture plate at a concentration of 10 ng/ml.
  • D1.1 cells and recombinant soluble TNF- ⁇ proteins were plated together to a 96-well cell culture plate.
  • serially diluted samples pre-treated with human serum albumin
  • the serially diluted samples were treated on the 96-well cell culture plate to which either or both of the cells and the recombinant proteins were plated, and then allowed to react for 3 hours in an incubator being under conditions of 37° C., 5% CO 2 , and not less than 80% humidity.
  • the CD40L HEK-BlueTM cells were treated on all wells of the plate at a concentration of 5 ⁇ 10 4 cells/well, and then reacted in an incubator being under the same conditions for 21 hours.
  • each 160 ⁇ l of a QUANTI-Blue reagent SEAP detection reagents, InvivoGen
  • APB-A1 a hu5c8 scFv (V L +V H )-flexible linker (SEQ ID NO:3-GGGGSGGGGSGGGGS; linker 1)-SL335 Fd (V H +C H 1) (termed APB-A1 heavy chain) gene, and a hu5c8 scFv (V L +V H )-flexible linker (SEQ ID NO:4-GSTSGSGKPGSGEGSTKG; linker 2)-SL335 kappa (V L +C L ) (termed APB-A1 L light chain) gene were linked by a linking PCR.
  • the genes were transfected to ExpiCHO-STM cell line for transient expression, and it was identified by Western blotting whether to normally express 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).
  • APB-A1 H and APB-A1 L genes were cloned to pd2535nt and pd2539vector, respectively, to produce recombinant vectors ( FIG. 1A ), and it was confirmed by amino acid sequencing that no abnormality was found ( FIG. 1B ).
  • FIG. 1A represents APB-A1 heavy and light chains inserted into pd2535nt and pd2539 vectors.
  • FIG. 1B represents amino acid sequences of APB-A1 H (total of 483 amino acids) and APB-A1 L (total of 478 amino acids), which are SL335 H and SL335 L having hu5c8 scFv linked by a linker 1 and a linker 2.
  • the recombinant vectors were transfected to GS null CHO K1 cells prior to use, and screened using MSX and puromycin, thereby establishing a stable CHO cell line.
  • APB-A1 protein For production of APB-A1 protein to be used for evaluation of in vitro and in vivo effects, the stable CHO cell line was cultured in a bioreactor, a supernatant was acquired, and purification was performed through a total of three steps including affinity chromatography, cation exchange chromatography and anion exchange chromatography.
  • the APB-A1 protein was acquired with yield of 95% greater through the affinity chromatography as the first step, and the APB-A1 sample was obtained with yield of 82% using the second and third steps of cation and anion exchange chromatography steps for removing impurity and endotoxin.
  • FIG. 2A represents one of the repeated experiments.
  • the obtained sample was analyzed by SDS-PAGE under reducing, nonreducing (boiled), and nonreducing (not boiled) conditions ( FIG. 2B ).
  • FIG. 2A represents the analysis result of APB-A1 characteristics identified by HPLC
  • FIG. 2B represents the analysis result of APB-A1 characteristics identified by SDS-PAGE.
  • Characteristics of the APB-A1 purified from the supernatant of the purified GS null CHO K1 cell culture medium were identified on a 4 to 15% gradient gel under reducing (R), nonreducing (boiled) (NR (B)) and nonreducing (not boiled) (NR (NB)) conditions by (A) HPLC and (B) SDS-PAGE
  • R reducing
  • B nonreducing
  • NR (NB) nonreducing
  • SDS-PAGE SDS-PAGE
  • APB-A1 protein To accurately measure the mass of APB-A1 protein, Q-TOF analysis was performed under reducing and nonreducing conditions. The measured masses of the heavy (H) and light (L) chains of APB-A1 were 50.77 kDa and 50.98 kDa ( FIG. 3 ). In FIG. 3 , characteristics of the purified APB-A1 protein were identified using a mass spectrometry instrument (ProteomeTech, South Korea). In mass spectrometry, the protein sample was analyzed under reducing and nonreducing conditions. The theoretical molecular weights of the APB-A1 heavy and light chains were 50,777 Da and 50,994 Da, which are substantially identical with the values measured by the mass spectrometry analysis of this example.
  • the APB-A1 had no N-linked glycosylation site, as confirmed using glycosylation prediction software, and that any peaks other than APB-A1 H and L peaks were not actually observed through Q-TOF analysis, it was predicted that the APB-A1 would not comprise N-linked glycosylation.
  • the APB-A1 had a theoretical pI value of 8.65, and the actual measurements of isoelectric focusing (IEF) at pH 3-10 and capillary isoelectric focusing (cIEF) were 9.16 and 9.2, respectively ( FIGS. 4A and 4B ).
  • IEF isoelectric focusing
  • cIEF capillary isoelectric focusing
  • the pl analysis for the purified APB-A1 protein was performed by ProteomeTech.
  • the pI values identified by isoelectric focusing (IEF) gel at pH 3-10, shown in FIG. 4A , and capillary isoelectric focusing (cIEF), shown in FIG. 4B were 9.16 and 9.2, respectively.
  • charge variant experiments were repeatedly conducted using ultra performance liquid chromatography (UPLC), and the UPLC assay resulted in a peak profile showing that 76.3% of the samples had main peaks with constant charges, 4.6% had acid peaks and 19.1% had basic peaks ( FIG. 5 ).
  • APB-A1 simultaneously bind to HSA and rhCD40L antigens
  • biolayer interferometry was performed such that the rhCD40L antigen was immobilized to an AR2G biosensor and APB-A1 and HSA were allowed to sequentially react therewith.
  • APB-A1 was capable of simultaneously binding to HSA and rhCD40L antigens ( FIG. 6 ).
  • a D1.1 cell expressing mCD40L was used, and as a preliminary experiment, it was identified by flow cytometry analysis whether APBA1 and hu5c8 IgG1 as a control group bind to the mCD40L expressed by the D1.1 cell.
  • HEKBlueTM CD40L reporter cell was combined with the D1.1 cell or the rhCD40L antigen with or without HSA and then reacted by adding thereto APB-A1, hu5c8 IgG1 and SL335 (at concentrations ranging from 0.01 to 22.2 nM), followed by measuring alkaline phosphatase (AP) responses of the reporter cell ( FIGS. 8A to 8D ).
  • APB-A1, hu5c8 IgG1 and SL335 at concentrations ranging from 0.01 to 22.2 nM
  • AP alkaline phosphatase
  • the capacities of APB-A1 and hu5c8 IgG1 inhibiting the CD40L-CD40 interaction in the absence of HSA were 0.9907 nM and 0.289 nM ( FIG. 8B ) and 1.031 nM and 0.4729 nM in the presence of HSA ( FIG. 8A ).
  • the IC 50 values for the inhibiting capacities of APB-A1 and hu5c8 IgG1 on the interaction between soluble CD40L and CD40 in the absence of HSA were 1.031 nM and 0.4729 nM ( FIG. 8D ) and 0.6371 nM and 0.501 nM in the presence of HSA ( FIG. 8C ).
  • APB-A1 demonstrated low suppressive potency that is about 3 times lower than that of hu5c8 IgG1 in the absence of HSA, while APB-A1 and hu5c8 IgG1 demonstrated substantially the same suppressive efficacy in the presence of HSA ( FIGS. 8A and 8B ).
  • the suppressive potency of APB-A1 was about 2.1 times lower than that of hu5c8 IgG1 in the absence of HSA, while APB-A1 and hu5c8 IgG1 demonstrated substantially the same suppressive efficacy in the presence of HSA ( FIGS. 8C and 8D ).
  • SL335 used as a negative control demonstrated no suppressive efficacy under any condition ( FIGS. 8A-8D ).
  • the IC 50 values representing the CD40L-CD40 interaction inhibiting capacity which were derived from the results shown in FIGS.
  • hu5c8 IgG1 as a positive control was identified to be about 0.2 to 0.3 nM, indicating that the presence or absence of HSA did not affect the IC 50 value of hu5c8 IgG1 ( FIGS. 8A and 8B ).
  • the IC 50 values were 1.031 nM in the absence of HSA and 0.6371 nM in the presence of HSA, and the IC 50 value of hu5c8 IgG1 was identified to be about 0.47 to 0.5 nM, which was substantially the same result as in the experiment stated above ( FIGS. 8C and 8D ).
  • PRP was pre-cultured with hCD40L (30 ⁇ g/ml) and different concentrations (6 ng/ml, 60 ng/ml and 600 ng/ml) of hu5c8 IgG1 or different concentrations (4 ng/ml, 40 ng/ml and 400 ng/ml) of APB-A1, in the presence of 5 to 10 mM CaCl 2 ) at 37° C. for 2 minutes.
  • the platelet was further stimulated by ADP at a concentration less than the optimum concentration, while continuously stirring.
  • the platelet aggregation occurred ( FIGS.
  • FIGS. 9A and 9B show that the rate of platelet aggregation was low in the case of the sample rhCD40L+APB-A1 ( FIGS. 9C and 9D ).
  • the data represents the standard deviation (SD) of the experiments with at least 6 different donors.
  • platelet aggregation % was calculated, and the calculation result showed that the sample rhCD40L+hu5c8 IgG1 IC demonstrated a response of about 80% or greater at concentrations of 60 ng/ml and 600 ng/ml, while the sample rhCD40L+APB-A1 demonstrated less than about 10% of platelet aggregation at a concentration of 400 ng/ml ( FIG.
  • APB-A1 was administered in two dosages of 5 mg/kg and 20 mg/kg through a single intravenous injection.
  • the concentrations of APB-A1 in blood plasma were measured using PK ELISA ( FIG. 11 ).
  • the APB-A1 concentrations of samples at the respective points were measured by ELISA, and the data represents the average of the experiments conducted.
  • Half-lives were calculated using the data based on the ELISA result using Phoenix WinNonlin software (ver 6.4; Certara LP, Princeton, N.J., USA), and the half-lives of 5 mg/kg APB-A1 and 20 mg/kg APB-A1 were identified to be about 7 and 9.6 days, respectively. Therefore, it was understood that the APB-A1 half-life was increased at a dose of 20 mg/kg to be about 1.4 times longer than that at a dose of 5 mg/kg.
  • C max values were 143 ⁇ g/ml and 509 ⁇ g/ml at doses of 5 mg/kg and 20 mg/kg, respectively, and renal clearance (CL) rates were 4.44 ml/day/kg and 4.72 me/day/kg, which were similar levels regardless of dose (Table 6).
  • TT was intramuscularly injected twice to animals to induce first and memory anti-TT antibody immune responses, and a vehicle (negative control, one single-dose injection), a positive control (DXT; 1 mg/kg, 4-dose injections), and APBA1 (5 mg/kg or 20 mg/kg, one single-dose injection) were intravenously administered to each animal, and concentrations of anti-TT IgG antibodies produced in serum were measured by ELISA.
  • A Anti-TT antibody levels were measured by ELISA (*p ⁇ 0.02 versus vehicle control by t-test).
  • Memory B cell percentages were significantly reduced in both groups of 5 mg/kg APB-A1 and 20 mg/kg APB-A1 (* p ⁇ 0.03 versus vehicle control by t-test). 20 days after the primary TT injection, to induce a memory anti-TT IgG immune response, second TT injection was performed, and then concentrations of anti-TT IgG antibodies produced in serum were measured.
  • APB-A1 injected groups demonstrated statistically significant effects in inhibiting second anti-TT IgG immune responses on day 27 in a dose-dependent manner. It was confirmed from initial CD40-CD40L responses that APB-A1 possessed suppressive potency, and population percentages of the respective groups were compared through immunophenotyping. Memory B cells (CD45+20+/27+IgD ⁇ /Ki67+) and dividing populations (CD45+/20+/Ki67+) of B cells demonstrated statistically significant suppressive potencies of APB-A1 up until day 27 after the second TT injection ( FIG. 12B ).
  • cysteine forming an inter-chain disulfide bond between C H 1 (hinge region, EPKSC-) of a heavy chain and CL (NRGEC-) of a light chain was substituted with serine (Ser), and resulting mutant forms of SL335 Fab, EPKSS- and NRGES-having two inter-chain disulfide bonds removed therefrom, were used.
  • An scFv antibody fragment [with V H and V L gene sequences derived from ruplizumab (or hu5c8)] binding to human CD40L were fused to N-terminals of heavy and light chains of the SL335 Fab using a (GGGGS)3 or GSTSGSGKPGSGEGSTKG peptide linker, and an Fv antibody fragment (with V H and V L gene sequences derived certolizumab pegol) binding to TNF- ⁇ , an Fv antibody fragment (with V H and V L gene sequences derived from ustekinumab) binding to IL-23, and an antibody fragment (with V H and V L gene sequences derived from anifrolumab) binding to INFAR1, were fused to the C-terminal of SL335 Fab using peptide linkers, respectively.
  • APB-B1a (a), (scFv) 2 -Fab-Fv construct and APB-B1b (b), (scFv) 2 -Fab-dsFv construct, having anti-CD40L scFv, anti-HSA Fab and anti-TNF- ⁇ Fv (with or without a disulfide bond) are linked by (GGGGS)3 (SEQ ID NO:3) and GSTSGSGKPGSGEGSTKG peptide linkers.
  • the (scFv) 2 -Fab-dsFv comprises an inter-chain disulfide bond (ss) between variable light chain (V L ) and variable heavy chain (V H ) of anti-TNF- ⁇ dsFv fragment, while the (scFv) 2 -Fab-Fv does not comprises an inter-chain disulfide bond.
  • FIG. 13C is a diagram representing recombinant pD2539 and recombinant pD2535NT after DNA cloning.
  • bispecific antibodies fused with Fv and dsFv derived from certolizumab were termed APB-B1a and APB-B1b, respectively ( FIGS. 13A and 13B ).
  • the thus produced SAFA-based bispecific antibody protein has a theoretical size of up to 128 kDa, and in order to utilize a CHO cell expression system, two polypeptide coding genes (N′-anti-CD40L scFv-SL335 H chain-anti-TNF- ⁇ V H -C′ and N′-anti-CD40L scFv-SL335 L chain-anti-TNF- ⁇ V L -C′) constructing the APB-B1a or APB-B1b were cloned to pD2535NT and pD2539 vectors, which are mammalian expression vectors, respectively, thereby producing recombinant pD2535NT and recombinant pD2539 vector ( FIG.
  • the produced recombinant pD2535NT and recombinant pD2539 vectors were used in transient expression and stable pool production.
  • the recombinant CHO cells were cultured in a flask for 7 to 9 days and then centrifuged, thereby acquiring culture media in 90% cell viability.
  • the expression quantity of (scFv) 2 -Fab-Fv constructs without a disulfide bond was 1.5 to 3 times higher than that of (scFv) 2 -Fab-dsFv constructs with a disulfide bond in all of three SAFA-based bispecific antibodies derived from certolizumab, ustekinumab and anifrolumab genes.
  • APB-B1a fragments produced with a stable pool were cultured in a flask for 7 days, yielding about 150 mg/L.
  • FIGS. 15A, 15B and 15C represent the results of SDS-PAGE analysis performed under the reducing, nonreducing and nonreducing (not boiled) conditions, respectively.
  • FIG. 15D represents size exclusion HPLC analysis for purified (scFv) 2 -Fab-Fv (up to 25 ⁇ g) constructs.
  • a dimer of the (scFv) 2-Fab-dsFv is indicated by an arrow.
  • FIGS. 15A to 15D Prior to purification of APB-B1a and APB-B1b proteins isolated through affinity chromatography, the protein samples were analyzed using SDS-PAGE and SE-HPLC ( FIGS. 15A to 15D ). To remove proteins identified at dimer positions of APB-B1b (up to 245 kDa) ( FIGS. 15C and 15D ), cation exchange chromatography was performed using a CM sepharose FF resin. For the APB-B1a protein sample identified only by affinity chromatography with high purity, anion exchange chromatography was performed using a Q sepharose HP. The respective purification steps were performed using an AKTA pure 150 L system, and a final purification product was determined by SE-HPLC.
  • APB-B1b proteins of dimer positions were further removed through cation exchange chromatography ( FIG. 16B ), and a peak was identified at a point in retention time, which is similar to that of APB-B1a ( FIG. 16A ).
  • the SAFA-based construct was analyzed on a TSKgel UltraSW aggregation column (in 20 mM citric acid, pH 5.5 buffer) under a native condition at 280 nm.
  • APB-B1a (a) was purified by CaptureSelect IgG-C H 1 affinity and Q sepharose HP anion exchange chromatography.
  • APB-B1b (b) was purified by CaptureSelect IgG-C H1 affinity and CM sepharose FF cation exchange chromatography.
  • three target proteins that is, human serum albumin ( FIG. 18A ), CD40L ( FIG. 18B ) and TNF- ⁇ ( FIG. 18C ) were coated on a 96-well MaxiSorp plate at a density of 1 ⁇ g/ml.
  • APB-B1a, APB-B1b and parental antibody were allowed to bind to the targets at pH 7.4.
  • HRP-conjugated goat anti-human Fd antibodies were used as secondary antibodies. Data was analyzed using an ELISA reader at 450 nm. * parental antibodies: anti-HSA Fab (SL335), anti-CD40L IgG (ruplizumab), anti-TNF- ⁇ IgG (adalimumab), and anti-TNF- ⁇ Fab (certolizumab).
  • the ELISA result showed that the binding strength of APB-B1a or APB-B1b to human serum albumin was reduced to about 2 to 3 times compared to SL335 Fab used as a control ( FIG.
  • BLI was performed (Table 8). 500 nM CD40L was reacted in an AR2G biosensor to be immobilized on the sensor, and 25 nM APB-B1a was reacted to be associated with the immobilized CD40L. Next, human serum albumin was allowed to react with APB-1a at a concentration of up to 10 folds higher than APB-1a to allow the albumin binding site of APB-B1a to be saturated, and the identical concentration of human serum albumin was allowed to react with 50 nM TNF- ⁇ . The results are represented in FIG. 19 . In FIG.
  • binding affinities of purified APB-B1a and APB-B1b were analyzed using an Octet RED instrument.
  • the respective antigens, HSA, CD40L and TNF- ⁇ were immobilized onto the amine reactive second-generation (AR2G) biosensor at concentrations of 20 ⁇ g/ml, 10 ⁇ g/ml and 30 ⁇ g/ml in a pH 5.0 sodium acetate buffer.
  • the purified antibodies were continuously treated in a 1 ⁇ kinetic buffer at pH 7.4 for two-fold dilution. Data was analyzed using Octet Data Analysis8 software.
  • D1.1 cells were prepared at concentration of 3.0 ⁇ 10 5 cells/reaction, an FITC-conjugated goat anti-human kappa antibody was used as a secondary antibody for detecting SL335-based construct, SL335 Fab (negative control group) and anti-CD40L IgG (positive control group). Binding signals indicated by the cell counts were measured using a FASCVerse flow cytometer.
  • APB-B1a and APB-B1b are capable of inhibiting biological functions of TNF- ⁇
  • in vitro inhibiting capacities were assessed using L929 mouse cells expressing cell membrane TNF receptors and exhibiting cell cytotoxicity in a TNF- ⁇ dependent manner in the presence of actinomycin D ( FIG. 21 ).
  • a TNF- ⁇ sample was continuously diluted 3 folds at concentrations decreasing from 20 nM to 0.24 nM and then allowed to react with L929 mouse cells expressing the TNF receptors and TNF- ⁇ (10 ng/ml) on the cell membranes in the presence of actinomycin D.
  • An anti-TNF- ⁇ -Fab′ (certolizumab) was used as a positive control group.
  • APB-B1a and APB-B1b maintained their capacities of inhibiting the TNF ⁇ -TNFR interaction.
  • APB-B1a and APB-B1b were capable of simultaneously binding to the cell membrane CD40L expressed on the cell surface and the soluble TNF- ⁇ in the presence of human serum albumin to thus simultaneously inhibit CD40L-CD40 and TNF ⁇ -TNFR interaction pathways.
  • the simultaneous inhibiting capacities were observed using HEK-BlueTM CD40L reporter cells that express both of the cell membrane CD40 and the cell membrane TNF receptor.
  • FIGS. 22A to 22C antibody constructs for CD40L or TNF- ⁇ were continuously diluted 4 folds at concentrations from 50 nM to 0.0122 nM.
  • Anti-CD40L IgG1 and anti-TNF- ⁇ were both used as control groups for the respective target molecules.
  • the secreted embryonic alkaline phosphatase (SEAP) expressed by the reaction of HEK-BlueTM reporter cell with CD40L or TNF- ⁇ was measured using a QUANTI-Blue reagent, and signals were measured at A655 nm.
  • the inhibiting capacities of APB-B1a and APB-B1 were identified by measuring HEK-BlueTM reporter cell responses to various interactions including (a) an interaction between D1.1 cell expressing CD40L and HEK-BlueTM cell expressing CD40 ( FIG. 22A ) and (b) an interaction between HEK-BlueTM cell expressing TNF receptor and soluble TNF- ⁇ ( FIG.
  • FIG. 22B (c) and both interactions between D1.1 cell and HEK-BlueTM cell and between HEK-BlueTM cell and soluble TNF- ⁇ ( FIG. 22C ). It was identified that the IC 50 values of APB-B1a and APB-B1b for the cell membrane CD40L were 0.15 to 0.18 nM, and the IC 50 value of the parental antibody (ruplizumab, IgG1) was 0.30 nM ( FIG.
  • IC 50 values of APB-B1a and APB-B1b were 1.98 nM and 3.79 nM, which means that both of the antigens were totally (100%) inhibited by APB-B1a and APB-B1b.
  • the parental antibody anti-TNF- ⁇ Fab′ could inhibit only the TNF- ⁇ antigen, which means that only about 60% inhibition of total responses was achieved.
  • the anti-CD40L IgG1 could not inhibit any of the responses due to extremely high SEAP activity of the reporter cell for the uninhibited TNF- ⁇ , which is similar to a case of the SL335 Fab (negative control).
  • the two parental antibodies, anti-TNF- ⁇ Fab′ and anti-CD40L IgG1 were simultaneously treated (combined treatment), the two species of targets were totally (100%) inhibited by the two parental antibodies, like in the case of the bispecific antibody, and the measured IC 50 value was 0.47 nM, which is 3 to 8 times higher than APB-B1. Consequently, notwithstanding similar inhibiting capacity levels for CD40L, it is considered that the two antibodies had lower inhibiting capacities than in the case of combined treatment due to a difference in the inhibiting capacity for TNF- ⁇ between the two antibodies ( FIG. 22C ).
  • the multispecific antibody which is capable of binding to CD40L, TNF- ⁇ or other bioactive effectors, can be usefully applied as therapeutic agents for various autoimmune diseases.
  • the multispecific antibody of the present disclosure can be used in development of an autoimmune disease therapeutic agent having extended in vivo retention time, while reducing a side effect, such as thromboembolism.
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