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WO2021071319A1 - Protéine de fusion multispécifique et utilisation associée - Google Patents

Protéine de fusion multispécifique et utilisation associée Download PDF

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WO2021071319A1
WO2021071319A1 PCT/KR2020/013805 KR2020013805W WO2021071319A1 WO 2021071319 A1 WO2021071319 A1 WO 2021071319A1 KR 2020013805 W KR2020013805 W KR 2020013805W WO 2021071319 A1 WO2021071319 A1 WO 2021071319A1
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region
amino acid
acid sequence
cdr3
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Korean (ko)
Inventor
박범찬
송재호
김나영
장세일
백정현
목진걸
송영자
김혜난
이재민
장창호
김영철
이은진
백기선
이현미
양소영
유재님
윤재봉
박영우
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Y Biologics Inc
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Y Biologics Inc
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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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2827—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 immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2809—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 immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2818—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 immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K16/2863—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 growth factors, growth regulators
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07K2317/50—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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a multispecific fusion protein that binds to cells, a method for preparing a multispecific fusion protein, a composition comprising a multispecific fusion protein, and a use thereof.
  • Antibodies and developed materials based on antibodies are used in the treatment of various diseases and disorders today.
  • the present inventors studied to develop a new structure of a multispecific antibody that enhances immune activity, as a result of the heterodimer form in which the Fc region of the antibody is substituted with the antibody variable region consisting of the heavy and light chain variable regions of the antibody.
  • the multispecific fusion protein of was developed.
  • the present invention was completed by confirming that the fusion protein is effective as an anticancer agent.
  • one aspect of the present invention provides a multispecific fusion protein comprising the following structural formulas (I) and (II):
  • the N' is the N-terminus of the fusion protein
  • the C' is the C-terminus of the fusion protein
  • a and B are each specific for a first antigen.
  • L1 is a peptide linker containing at least one Cys
  • X is an antibody variable heavy chain (VH) region or an antibody variable light chain (VL) region
  • Y is an antibody variable light chain (VL) region or an antibody variable heavy chain ( VH) region, wherein X and Y form an antibody variable region that specifically binds to a second antigen.
  • Another aspect of the present invention provides a pharmaceutical composition for treating or preventing cancer comprising the fusion protein as an active ingredient.
  • the multispecific fusion protein according to the present invention can specifically bind to two or more antigens.
  • a multispecific fusion protein constructed to include an antibody variable region that specifically binds to immune cells and an antibody variable region that specifically binds to a cancer antigen can be used as an anticancer agent.
  • FIG. 1 is a diagram showing the "Knob-in-Hole" structure of the variable domains of anti-CD3 UCHT1, anti-PD-L1 dervalumab, and anti-CTLA-4 ipilimumab.
  • the CDR loops are denoted as "VH CDR3", “VL CDR1”, “VL CDR2”, and "VL CDR3".
  • variable domain fragments of UCHT1 are shown on the right, and the variable formed by electrostatic interaction (top), hydrophobicity and knob-into-hole interaction (bottom). Domain fragments are shown.
  • Figure 3a is a schematic diagram of the structure of the ALiCE.
  • 3b, 3c, 3d, 3e, and 3f are schematic structures of ACE-05, ACE-31, BiTE-05, YBL-007 and UCHT1 representing variable domains for PD-L1 and CD3 binding. .
  • Figure 3g is a schematic diagram of the structures of ACE-11 and ACE-19.
  • Figure 3h is a schematic diagram of the structure of ACE-28.
  • YBL-007 an anti-PD-L1 antibody (YBL-007) having an activity similar to that of avelumab in PD-L1/PD-1 signal blocking ability.
  • Figure 5a shows whether the expression of ACE-HC-VH, ACE-HC-VL and ACE-LC was confirmed by SDS-PAGE and Western blotting.
  • Figure 5b is a view confirming ACE-18 by SDS-PAGE under reducing and non-reducing conditions.
  • 5C and 5D are diagrams confirming ACE-11 and ACE-19 by SDS-PAGE under reducing and non-reducing conditions.
  • 5E is a view confirming ACE-11 and ACE-19 by capillary electrophoresis (CE) under reducing and non-reducing conditions.
  • 5F shows the results of analysis of ACE-11 and ACE-19 by SEC HPLC.
  • 5G is a result of analyzing ACE-19 by SEC-HPLC.
  • 5h shows the ACE-19 fraction confirmed by Native-PAGE.
  • 5i is a view confirming the ACE-19 fraction obtained in FIG. 5g by CE under reducing and non-reducing conditions.
  • FIG. 6 is a schematic diagram of a multispecific fusion protein capable of specifically binding to PD-L1 and CD3, ACE-LC, ACE-HC-VH and ACE-HC-VL through capillary electrophoresis analysis and size-exclusion chromatography. I confirmed it.
  • Figure 7a is through capillary electrophoresis analysis, confirming the ACE-LC, ACE-HC-VH and ACE-HC-VL of ACE-05. As a result of the analysis, it was confirmed that ACE-LC, ACE-HC-VH, and ACE-HC-VL were present in a 2:1:1 ratio.
  • 7B is a result of analyzing ACE-10 and ACE-18 by SEC-HPLC.
  • 7c is a result of confirming ACE-11 and ACE-19 with CE under reducing and non-reducing conditions.
  • 7D is a result of confirming ACE-11 with CE in reducing and non-reducing conditions.
  • 7G is a result of analysis by SEC-HPL after dividing ACE-19 into complex and ACE-19 using SEC-FPLC and purifying.
  • Figure 7h is a view confirming the presence and absence of SDS, ACE-19 and complex according to the heating conditions in Native-PAGE.
  • Figure 8 is the result of mass spectrometry (MS) using size-exclusion chromatography and LC-ESI/TOF. As a result, it was confirmed that ACE-05 is a homogeneous heterotetramer.
  • FIG. 9 is a schematic diagram of ACE-00, capillary electrophoresis analysis, and size-exclusion chromatography to confirm ACE-LC, ACE-HC-VH and ACE-HC-VL.
  • Figure 11a confirms the thermal stability of ACE-05.
  • Figure 11b confirms the thermal stability of ACE-11 and ACE-19.
  • FIG. 13 is a schematic diagram of the immunological synaptic bridge of ACE-05.
  • Figure 14a confirms the binding strength of ACE-05 and ACE-31.
  • Figure 14b confirms the binding force of ACE-19.
  • Figure 14c confirms the binding strength of ACE-11 and ACE-19.
  • Figure 15a confirms the binding strength of ACE-05 and ACE-31.
  • Figure 15b confirms the binding ability of ACE-19 to EGFR.
  • Figure 15c confirms the binding ability of ACE-19 to CD3.
  • Figure 16a confirms the simultaneous binding of ACE-05 to PD-L1 and CD3.
  • 16B and 16C confirm the simultaneous binding of ACE-19 to EGFR and CD3.
  • Figure 17 shows the binding power of ACE-05 and ACE-31 to Jrukat T cells.
  • 18A shows the binding power of ACE-05 and ACE-31 to Karpas-299 cells, Jrukat T cells, and Raji cells.
  • Figure 18b shows the binding power of ACE-18 to Raji cells.
  • Figure 18c shows the binding ability of ACE-18 to CD20 + Raji cells or CD20R - aji cells.
  • 18D shows the amount of CD20 expression in cells by using the cell avidity of the anti-CD20 antibody in each cell.
  • Fig. 18e shows the ability of ACE-18 to kill Karpass-299 cells.
  • Figure 18f confirms the killing ability of ACE-18 on Raji cells.
  • Fig. 18g is a confirmation of the killing ability of ACE-18 on Toledo cells and Jeko-1 cells.
  • 18h is a view confirming the expression level of EGFR present in the tumor through flow cytometry and the killing ability of ACE-19 on SW48 cells, HCT116 cells, and HT29 cells.
  • FIG. 19 shows HEK cells into which the PD-L1 expression vector has been introduced.
  • FIG. 21 shows the NFAT-luciferase reporter activity was measured, and the activities of ACE-05 and ACE-31 were compared with other substances.
  • Figure 22 is a comparison of the cytolytic ability of ACE-05, ACE-31 and BiTE-05 against PD-L1 + tumor cells (HCC827).
  • 24 is a flow cytometer confirming the expression of PD-L1 in HCC827 and MDA-MB-231.
  • Figure 25a is a measure of the anti-cancer activity of ACE-05.
  • Figure 25b is a measure of the killing ability against tumor cells after simultaneous administration of ACE-11 and PBMC.
  • Figure 26a confirms whether ACE-05 has the ability to specifically activate T cells.
  • Figure 26b confirms whether or not ACE-18 has the ability to specifically activate T cells.
  • Figure 27 confirms whether ACE-05, ACE-31 and BiTE-05 have the ability to activate T cells.
  • Fig. 28 shows the effect of ACE-05, ACE-31 and BiTE-05 on the release of Granzyme B in CD8 + T cells.
  • FIG. 30 confirms whether ACE-05, ACE-31, and BiTE-05 have the ability to specifically activate T cells.
  • Fig. 32 shows the effects of ACE-05, ACE-31 and BiTE-05 on T cell stimulation.
  • Figure 34 shows the binding affinity of ACE-05 and its variants, ACE-47 (K55Q variant of ACE-05), ACE-49 (D104N variant of ACE-05), and ACE-56 (K55Q, D104N variant of ACE-05). Is measured.
  • Figure 35 confirms the non-target (Off Target) Jurkat T cell activation of ACE-05 and its variants.
  • Fig. 36 shows the ability of ACE-05 and its variants to kill PD-L1 HCC827 cancer cells.
  • Figure 37 shows that ACE-05, ACE-31, and BiTE-05 confirm the ability to secrete non-target cytokines in CD4 + and CD8 + T cells.
  • 39 shows the degree of activation of non-target T cells of the fusion protein according to the present example when cancer cells are not present.
  • Figure 42 shows the change in body weight of mice after administration of ACE-05, YBL-007 and BiTE-05.
  • 49 shows changes in tumor size after administration of ACE-05, YBL-007 and UCHT1.
  • FIG. 50 shows CD45 + lymphocytes present in tumors obtained after administration of ACE-05, YBL-007 and UCHT1.
  • FIG. 51 shows the confirmation of CD45 + lymphocyte count and CD3 + T cell ratio after administration of ACE-05, YBL-007 and UCHT1.
  • 54 shows the amount of expression of EGFR and PD-L1 present in the tumor through flow cytometry.
  • ACE-05-HC-VH and ACE-05 show ACE-05-HC-VH and ACE-05 that can be presented on MHC class I molecules to confirm the immunogenicity of ACE-05-HC-VH and ACE-05-HC-VL. This is an analysis of the peptide in -HC-VL.
  • 58 and 59 are views confirming ACE-02 and ACE-03 by SDS-PAGE under reducing and non-reducing conditions.
  • FIG. 60 is a diagram showing the results of confirming ACE-05 and ACE-16 by SDS-PAGE (left) and CE (right) under reducing and non-reducing conditions.
  • 61 to 63 are views confirming ACE-06, ACE-10, and ACE-11 by SDS-PAGE under reducing and non-reducing conditions.
  • Figure 64 is a view confirmed by Western blot (top and middle panels) and SDS-PAGE (bottom panels) of ACE-15 under reducing and non-reducing conditions.
  • Lane 1 is the total supernatant of the ACE-05 culture
  • Lane 2 is the total supernatant of the ACE-15 culture
  • Lane 3 is loaded with the purified protein of ACE-05.
  • the upper panel shows an anti-CH1 antibody
  • the middle panel shows a Western blot image using an anti-Kappa antibody.
  • 65 and 66 are diagrams confirming ACD-20 and ACE21 by SDS-PAGE and Western blot under reducing and non-reducing conditions.
  • 67 is a view confirming ACE-23 by SDS-PAGE under reducing and non-reducing conditions.
  • FIG. 68 is a diagram showing the results of analyzing ACE-25 by SDS-PAGE and CE under reducing and non-reducing conditions and by SEC-HPLC.
  • 69 is a diagram showing ACE-VH-LC, ACE-VL-LC and ACE-VH-VL-LC of ACE-26 confirmed by SDS-PAGE and Western blot under reducing and non-reducing conditions.
  • FIG. 70 is a diagram showing the results of analyzing ACE-26 by SDS-PAGE and CE under reducing and non-reducing conditions.
  • 71 is a diagram showing the results of analysis of ACE-28 by SDS-PAGE, Western blot and CE under reducing and non-reducing conditions.
  • 72 is a view showing the results of SDS-PAGE and Western blot analysis of ACE-28 containing a CH3 region under reduced and non-reducing conditions.
  • 73 is a diagram showing the results of CE analysis of ACE-28 containing a CH3 region under reduced and non-reducing conditions.
  • 74 and 75 are diagrams showing the results of analysis of ACE-30 and ACE-32 by SDS-PAGE, Western blot, and CE under reducing and non-reducing conditions.
  • 76 is a diagram showing the results of analysis of ACE-33 by SDS-PAGE and CE under reducing and non-reducing conditions.
  • the term "multispecific fusion protein” refers to a substance that binds to at least one target or antigen. Specifically, the fusion protein may specifically bind or selectively bind to an antigen, for example, when the dissociation constant (KD) is ⁇ 10 -7 M. In one embodiment, the fusion protein may specifically bind to an antigen with high affinity when the KD is ⁇ 10 -8 M or the KD is ⁇ 10 -9 M.
  • the multispecific fusion protein may include an antibody and a molecule derived from the antibody.
  • the multispecific fusion protein or antigen binding domain thereof may be a "humanized" form of a non-human antibody that is a chimeric antibody comprising a human immunoglobulin comprising a native CDR.
  • a multispecific fusion protein or antigen binding domain may comprise a portion of a “fully human antibody” or “human antibody”.
  • the multispecific fusion protein or antigen binding domain may be a “monoclonal antibody” or a portion thereof.
  • antibody refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction.
  • antibodies are also referred to as immunoglobulins.
  • the antibody may mean any one selected from IgG, IgE, IgM, IgD and IgA, and may be IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2, which are subclasses of IgG. Further, the antibody may be an agonistic antibody or an antagonistic antibody.
  • Fab refers to an antibody region that binds to an antigen.
  • Conventional IgG generally contains two Fab regions.
  • Each Fab region typically consists of one variable region and one constant region of each heavy and light chain.
  • the variable and constant regions of the heavy chain are VH and CH1 regions
  • the variable and constant regions of the light chain are VL and CL regions.
  • the VH, CH1, VL and CL of the Fab region can be arranged in a variety of ways to confer antigen binding capacity according to the present disclosure.
  • the term “heavy chain” refers to a polypeptide chain of about 50-70 kDa.
  • the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids
  • the carboxy terminal portion includes a constant region.
  • the constant region may be one of five types: alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ), and mu ( ⁇ ).
  • ⁇ , ⁇ , and ⁇ include about 450 amino acids
  • ⁇ and ⁇ include about 550 amino acids.
  • the term “light chain” refers to a polypeptide chain of about 25 kDa. Wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and the carboxy-terminal portion includes a constant region. There are two types of light chain constant domains: kappa ( ⁇ ) or lambda ( ⁇ ). In addition, the constant region of the light chain is referred to as "CL".
  • the heavy chain C domain (CH domain) is numbered from amino-terminus to carboxy-terminus (eg, CH1, CH2, CH3, etc.). Any of the CL and CH1 regions of these antibody classes can be used in the present disclosure.
  • the CL and CH1 regions provided herein are of the IgG type (eg, IgG1).
  • a representative CL region of the Fab region provided herein has the following amino acid sequence: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 59).
  • the representative CH1 region of the Fab region provided herein has the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 60).
  • the term "Fc region” refers to the C-terminal region of an immunoglobulin heavy chain including a native Fc region, a recombinant Fc region and a variant Fc region.
  • the variant Fc region may have at least one amino acid substitution, for example about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acids substitutions compared to the native sequence Fc region.
  • the variant Fc region may have at least about 80% homology, at least about 90% homology, at least about 95% homology with the native sequence Fc region.
  • the term "antigen" is a structure capable of selectively binding to an antibody.
  • the target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring compound or a synthetic compound.
  • the antigen is a polypeptide, and may be a protein present on or within a cell.
  • vector refers to a material for carrying or expressing a nucleic acid sequence comprising a nucleic acid sequence encoding a multispecific fusion protein (eg, an antibody) described herein.
  • vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes.
  • polynucleotide is also referred to as “nucleic acid” and refers to a polymer of nucleotides of any length. Specifically, the polynucleotide may be DNA or RNA.
  • One aspect of the present invention provides a multispecific fusion protein in which the Fc region of an antibody is substituted with a variable region of an antibody.
  • the present invention provides a novel multispecific fusion protein having multiple binding domains that bind to cells. These fusion proteins may be referred to herein as "antibody like cell engagers (ALICE)".
  • ALICE antibody like cell engagers
  • the ALiCE molecule provided in the present invention has two antigen binding domains. The first antigen binding domain has two Fab regions and the second antigen binding domain has an Fv region. Examples of ALiCE molecules are shown in FIGS. 3A, 3B, 3C, 3G, and 3H.
  • the Fv and Fab regions can be linked through the hinge region of the heavy chain.
  • the first antigen binding domain comprises a Fab region
  • the second antigen binding domain is usually at a position where the CH2 and CH3 domains will be located on the native antibody structure. Attached (directly or indirectly).
  • the C-terminus of the first heavy chain comprises a VH domain other than the CH2 domain
  • the C-terminus of the second heavy chain comprises a VL domain other than the CH2 domain.
  • the multispecific fusion proteins disclosed herein provide many advantages over conventional antibodies and conventional multispecific antibodies (eg, bispecific antibodies). Due to the multiple antigen binding domains and overall configuration design, the multispecific fusion proteins provided in the present invention can be used as cell linkers providing multiple cells. For example, a first antigen binding domain can bind an antigen expressed on a first cell, and a second antigen binding domain can bind an antigen expressed on a second cell. Therefore, when the multispecific fusion protein is used, the two cells can be effectively linked.
  • the multispecific fusion proteins provided herein may be useful for binding and activating immune cells.
  • the divalent Fab portion of the ALiCE molecule retains the function of a conventional antibody, while the second Fc-deficient monovalent antigen-binding region (i.e., the Fv region) is an effector cell of the immune system,
  • T cells can be recognized, bound, redirected and/or activated.
  • the Fv portion of the ALiCE molecule retains the function of a conventional antibody, and the Fab region is capable of recognizing, binding, redirecting and/or activating effector cells, such as T cells, of the immune system.
  • ACE-05 and ACE-31 are both ALiCE molecules composed of anti-PD-L1 and anti-CD3 domains, but ACE-05 binds to PD-L1 through the Fab region and to CD3 through the Fv region. Combine.
  • ACE-31 binds to CD3 through the Fab region and to PD-L1 through the Fv region.
  • the absence of a properly functioning Fc region or the absence of a complete CH2 and/or CH3 region eliminates or reduces Fc-mediated effector cytotoxicity.
  • the natural interaction between the VH and VL chains of the Fv moiety can promote heterodimerization of ALiCE molecules without conferring undesirable immunogenicity through artificial manipulation.
  • the multispecific fusion protein comprises two antigen binding domains, wherein the first antigen binding domain comprises two antibody Fab regions and the second antigen binding domain comprises an antibody Fv region.
  • each of the two Fab regions includes two portions.
  • the first portion comprises an antibody variable heavy chain (VH) region and an antibody CH1 region.
  • the second portion comprises an antibody variable light chain (VL) region and an antibody light chain constant region (CL).
  • Each of the two Fab regions is capable of binding to an antigen.
  • the Fv region of the second antigen binding domain comprises a VH region and a VL region.
  • an embodiment of the multispecific fusion protein may include the following four polypeptides:
  • a fourth polypeptide comprising a third VH region, a second CH1 region and a VL region.
  • the first VH region and the first CH1 region of the first polypeptide and the third polypeptide form a first antigen-binding Fab region
  • the third VH region and the second CH1 region of the second and fourth polypeptides are second.
  • the antigen binding Fab region is formed, and the second VH region of the third polypeptide and the VL region of the fourth polypeptide form the antigen binding Fv region.
  • the C-terminal Fv also plays an important role in the heterodimerization of two different heavy chain-like chains (third and fourth polypeptides). Because the interaction between the VH and VL regions is much stronger than that of the VL-VL interaction, VH-VL binding occurs. Thus, it can be heterodimerized to produce the multispecific fusion protein of the present application.
  • the efficiency of heterodimerization was very high, and most of the multispecific fusion proteins expressed and purified in mammalian cells are in heterodimerized form. It was confirmed that the heterodimerization efficiency was more than 99%.
  • this structure provides an optimal synaptic distance between the target cell and the effector cell.
  • the distance between the two N-terminal Fab regions and the C-terminal Fv region was estimated to be 40 ⁇ .
  • the multispecific fusion proteins provided herein have more folding complexity (molecular size) than other known bispecific antibodies such as BiTE, DART and other ScFv based bispecific antibody formats, and thus improved thermodynamic stability. Is expected to have.
  • the VH region and the VL region of the Fv region are on separate polypeptides.
  • the distance between the two N-terminal Fab regions and the C-terminal Fv regions in the multispecific fusion proteins provided herein is in the range of about 40 ⁇ to about 70 ⁇ . In one embodiment, the distance between the two N-terminal Fab regions and the C-terminal Fv regions in the multispecific fusion proteins provided herein was estimated to be about 42 ⁇ . In some other embodiments, the distance between the N-terminal two Fab regions and the C-terminal Fv regions in the multispecific fusion proteins provided herein was estimated to be about 60 ⁇ .
  • the binding affinity of the first antigen-binding domain for the first antigen may be higher than that of the second antigen-binding domain for the second antigen.
  • the binding kinetics of ACE-05 to human PD-L1 was similar to that of the parental anti-PD-L1 antibody (ie, YBL-007 from Y-Biologics Inc.).
  • the binding affinity of ACE-05 for CD3 was much lower than that of the parental anti-CD3 antibody (UCHT1, BioLegend, USA).
  • the multispecific fusion proteins provided herein are about 1 ⁇ M or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 It is possible to bind one or more targets, antigens or epitopes with a dissociation constant (K D ) of pM or less, 10 pM or less, or 1 pM or less.
  • K D dissociation constant
  • a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with a K D of about 20 nM or less.
  • the multispecific fusion protein is capable of binding to a target, antigen or epitope with a K D of about 10 nM or less. In one embodiment, the multispecific fusion protein is capable of binding to a target, antigen or epitope with a K D of about 1 nM or less. In one embodiment, the multispecific fusion protein is capable of binding to a target, antigen or epitope with a K D of about 0.5 nM or less. In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with a K D of about 0.1 nM or less.
  • a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with a K D of about 50 pM or less. In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with a K D of about 25 pM or less. In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with a K D of about 10 pM or less. In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with a K D of about 1 pM or less.
  • the K D of the multispecific fusion protein for the first antigen is about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times that of the multispecific fusion protein K D for the second antigen. , May be 8 times, 9 times, 10 times, 15 times, 20 times, 50 times or more. In some embodiments, the K D of the multispecific fusion protein for the first antigen may be about 10, 10 2 , 10 3 or 10 4 times that of the multispecific fusion protein K D for the second antigen.
  • the multispecific fusion proteins herein may be in chemically modified form.
  • the fusion protein is chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting groups/blockers, proteolytic cleavage and/or binding of cellular ligands or other proteins. Can be. Many of these chemical modifications can be performed by known techniques.
  • One embodiment of the fusion protein may include the following structural formulas (I) and (II):
  • N' is the N-terminus of the fusion protein
  • Each of A and B is specific for a first antigen
  • the L1 is a peptide linker containing at least one Cys
  • X is an antibody variable heavy chain (VH) region or an antibody variable light chain (VL) region,
  • Y is an antibody variable light chain (VL) region or an antibody variable heavy chain (VH) region
  • the X and Y form an antibody variable region (Fv) that specifically binds to the second antigen.
  • Fv antibody variable region
  • the A and B are each antibody Fab region, (i) a first portion including an antibody variable heavy chain (VH) region and an antibody CH1 region; And (ii) an antibody variable light chain (VL) region and an antibody light chain constant region (CL).
  • a and B may bind to the same antigen by two Fab regions. In addition, A and B may bind to the same epitope of the same antigen. In addition, A and B bind to different epitopes of the same antigen. In another embodiment, A and B bind different antigens.
  • the first antigen binding domain and the second antigen binding domain bind different antigens, wherein the first antigen binding domain binds a first antigen and a second antigen binding domain is capable of binding a second antigen.
  • 3B and 3C show examples of such ALiCE molecules.
  • the first antigen-binding domain two Fab regions
  • the second antigen-binding domain binds to immune cells such as T cells through antigens such as CD3.
  • the first antigen binding domain (two Fab regions) binds to immune cells such as T cells through antigens such as CD3.
  • the second antigen binding domain binds to the cancer antigen (PD-L1).
  • These ALiCE molecules can bind immune cells (eg, T cells) to cancer cells, and thus can be used as a therapeutic agent for cancer treatment.
  • structural formula (I) may include the following structural formulas (I') and (I''):
  • A' is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;
  • A'' is a light chain region of an antibody, or as a heavy chain region of an antibody, includes a variable region and a CH1 region,
  • variable region of the antibody, wherein the variable region specifically binds to the first antigen.
  • the light chain region may include a light chain variable region and a light chain constant region.
  • N', L1, X and C' are as defined above.
  • structural formula (II) may include the following structural formulas (II') and (II''):
  • B' is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;
  • B'' is the light chain region of the antibody, or as the heavy chain region of the antibody comprises a variable region and a CH1 region;
  • variable region of the antibody, wherein the variable region specifically binds to the first antigen.
  • the light chain region may include a light chain variable region and a light chain constant region.
  • N', L1, Y and C' are as defined above.
  • Another embodiment of the fusion protein may be one comprising the following structural formulas (III) and (IV):
  • N' is the N-terminus of the fusion protein
  • Each of A and B specifically binds to a first antigen
  • the L2 is a peptide linker containing at least one Cys
  • X is an antibody variable heavy chain (VH) region or an antibody variable light chain (VL) region,
  • Y is an antibody variable light chain (VL) region or an antibody variable heavy chain (VH) region
  • the X and Y form an antibody variable region (Fv) that specifically binds to the second antigen.
  • Fv antibody variable region
  • the A and B are each antibody Fab region, (i) a first portion including an antibody variable heavy chain (VH) region and an antibody CH1 region; And (ii) an antibody variable light chain (VL) region and an antibody light chain constant region (CL).
  • structural formula (III) may include the following structural formulas (III') and (III"):
  • A' is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;
  • A'' is the light chain region of the antibody, or as the heavy chain region of the antibody comprises a variable region and a CH1 region;
  • variable region of the antibody, wherein the variable region is specific for the first antigen.
  • structural formula (IV) may include the following structural formulas (IV') and (IV''):
  • B' is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;
  • B'' is the light chain region of the antibody, or as the heavy chain region of the antibody comprises a variable region and a CH1 region;
  • variable region is specific for the first antigen.
  • the L1 and L2 may include a hinge region derived from an immunoglobulin.
  • the L1 and L2 may include 1, 2 or 3 Cys.
  • each of L1 and L2 may have the following structural formula (V):
  • the L1' and L1'' are each a linker consisting of 1 to 15 amino acids
  • n and m are integers of 0 or 1
  • the hinge is an immunoglobulin-derived hinge region.
  • the antibody hinge region is an IgG hinge region.
  • the IgG hinge regions provided herein can be selected from, for example, antibody hinge regions of various IgG subtypes.
  • IgG subtype Core hinge sequence Sequence number IgG1 EPKSCDKTHTCPPCP 55 IgG2 ERKCCVECPPCP 56 IgG3 ELKTPLDTTHTCPRCP(EPKSCDTPPPCPRCP) 3 57 IgG4 ESKYGPPCPSCP 58
  • the hinge can be modified to introduce additional disulfide bonds.
  • the hinge region may contain more than two disulfide bonds.
  • the hinge region may contain an even number of disulfide bonds such as, for example, 2, 4, 6, 8 or 10 disulfide bonds, 4, 6, 8, 10 disulfide bonds, and 4 disulfide bonds.
  • the hinge region may comprise an odd number of disulfide bonds such as 1, 3, 5, 7 or 9 disulfide bonds, 3, 5, 7 or 9 disulfide bonds, 3 disulfide bonds.
  • 3G shows a representative example of a multispecific fusion protein, wherein the hinge region was prepared such that two and three disulfide bonds were formed between the heavy chains of the multispecific fusion protein.
  • the hinge region comprises an amino acid sequence (PPC) n , where n is an integer.
  • n can be an even number, such as 2, 4, 6, 8 or 10, for example 4, 6, 8 or 10, for example 4.
  • n can be an odd number, for example 1, 3, 5, 7, or 9, for example 3, 5, 7, or 9, for example 3.
  • an inter-hinge disulfide bond is formed between a cysteine residue in one hinge region and a cysteine residue in another hinge region.
  • L1' and L1'' may be a peptide consisting of 1 to 20 amino acids.
  • the L1′ and L1′′ may be amino acids such as (G4S)p (p is an integer of 1 to 10).
  • L1' and L1'' may be (G4S) 1 , (G4S) 2 , (G4S) 3 , or (G4S) 4 .
  • L1′ and/or L1′′ may vary. In some embodiments, LI' and/or LI'' is 5 amino acids long. In another embodiment, L1' and/or L1'' is 9 amino acids long. In another embodiment, L1' and/or L1'' is 10 amino acids long.
  • the table below shows representative examples of L1' and/or L1''.
  • the first antigen may be a cancer antigen (Tumor-Specific Antigens), or a protein present on the surface of an immune cell, or may be a cytokine.
  • the first antigen is PD-L1, PD-1, EGFR, TNFR, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, cMET, DLL3, GD2, Nectin-4, RANKL, SLAMF7, TROP2, Claudin 18.2, TNFR, TNF, CD3, HER2, CD20, CD19, CTLA-4, VEGFR, VEGF, NCAM1, ICAM-1, ICAM-2, CEACAM6, Carcinoembryonic antigen (CEA), CA-125, Alphafetoprotein (AFP ), MUC-1, Epithelial tumor antigen (ETA), Melanoma-associated antigen (MAGE), Immature laminin receptor, TAG-72, HPV E6/E7, BING-4
  • the first antigen may be a cancer antigen.
  • the cancer antigens are PD-L1, EGFR, BCMA, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, HER2, cMET, DLL3, GD2, Nectin-4, RANKL, It may be any one selected from the group consisting of SLAMF7, TROP2, Claudin 18.2, MUC-1, Mesothelin, EpCAM and CEA.
  • the first antigen may be a cell surface molecule that regulates T cell function.
  • the first antigen may be an immune checkpoint inhibitor.
  • the first antigen may be a cancer antigen.
  • the first antigen may be a protein expressed on the surface of immune cells such as lymphocytes and monocytes. Specifically, it may be a protein expressed in cells such as T cells, B cells, dendritic cells, granulocytes, megakaryocytes, monocytes, and NK cells. In addition, the first antigen may be a protein expressed on CD8 + T cells or CD4 + T cells.
  • a and B can recognize the same antigen. In addition, A and B can recognize different antigens.
  • a and/or B may specifically bind to any one antigen selected from the group consisting of PD-L1, HER2, CD19, CD20, EGFR, CD3, TNF, and CTLA-4.
  • a and/or B may include any one variable region selected from:
  • VH region comprising the amino acid sequence of SEQ ID NO: 5 (VH-CDR1), SEQ ID NO: 6 (VH-CDR2) and SEQ ID NO: 7 (VH-CDR3), and SEQ ID NO: 9 (VL-CDR1), SEQ ID NO: 10 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 11 (VL-CDR3);
  • VH-CDR1 VH-CDR1
  • SEQ ID NO: 160 VH-CDR2
  • SEQ ID NO: 161 VH-CDR3
  • SEQ ID NO: 171 VL-CDR1
  • SEQ ID NO: 172 VL-CDR2
  • VL-CDR3 VL-CDR3
  • VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 314 (VL-CDR1), SEQ ID NO: 315 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 316 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 118 (VH-CDR1), SEQ ID NO: 119 (VH-CDR2) and SEQ ID NO: 120 (VH-CDR3), and SEQ ID NO: 121 (VL-CDR1), SEQ ID NO: 122 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 123 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 140 (VH-CDR1), SEQ ID NO: 141 (VH-CDR2) and SEQ ID NO: 142 (VH-CDR3), and SEQ ID NO: 152 (VL-CDR1), SEQ ID NO: 153 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 154 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 62 (VH-CDR1), SEQ ID NO: 63 (VH-CDR2) and SEQ ID NO: 64 (VH-CDR3), and SEQ ID NO: 66 (VL-CDR1), SEQ ID NO: 67 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 68 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 223 (VH-CDR1), SEQ ID NO: 224 (VH-CDR2) and SEQ ID NO: 225 (VH-CDR3), and SEQ ID NO: 227 (VL-CDR1), SEQ ID NO: 228 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 229 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 436 (VH-CDR1), SEQ ID NO: 437 (VH-CDR2) and SEQ ID NO: 438 (VH-CDR3), and SEQ ID NO: 441 (VL-CDR1), SEQ ID NO: 442 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 443 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 245 (VH-CDR1), SEQ ID NO: 246 (VH-CDR2) and SEQ ID NO: 247 (VH-CDR3), and SEQ ID NO: 249 (VL-CDR1), SEQ ID NO: 250 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 251 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 256 (VH-CDR1), SEQ ID NO: 257 (VH-CDR2) and SEQ ID NO: 258 (VH-CDR3), and SEQ ID NO: 260 (VL-CDR1), SEQ ID NO: 261 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 262 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 326 (VH-CDR1), SEQ ID NO: 327 (VH-CDR2) and SEQ ID NO: 328 (VH-CDR3), and SEQ ID NO: 338 (VL-CDR1), SEQ ID NO: 339 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 340 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 396 (VH-CDR1), SEQ ID NO: 397 (VH-CDR2) and SEQ ID NO: 398 (VH-CDR3), and SEQ ID NO: 402 (VL-CDR1), SEQ ID NO: 403 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 404 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 410 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 150 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 70 (VH-CDR1), SEQ ID NO: 71 (VH-CDR2) and SEQ ID NO: 72 (VH-CDR3), and SEQ ID NO: 74 (VL-CDR1), SEQ ID NO: 75 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 76 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 84 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 295 (VH-CDR1), SEQ ID NO: 296 (VH-CDR2) and SEQ ID NO: 297 (VH-CDR3), and SEQ ID NO: 299 (VL-CDR1), SEQ ID NO: 300 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 301 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 87 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 183 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 187 (VH-CDR1), SEQ ID NO: 188 (VH-CDR2) and SEQ ID NO: 189 (VH-CDR3), and SEQ ID NO: 191 (VL-CDR1), SEQ ID NO: 192 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 193 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 211 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 306 (VH-CDR1), SEQ ID NO: 307 (VH-CDR2) and SEQ ID NO: 308 (VH-CDR3), and SEQ ID NO: 310 (VL-CDR1), SEQ ID NO: 311 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 312 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 349 (VH-CDR1), SEQ ID NO: 350 (VH-CDR2) and SEQ ID NO: 351 (VH-CDR3), and SEQ ID NO: 353 (VL-CDR1), SEQ ID NO: 354 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 355 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 359 (VH-CDR1), SEQ ID NO: 360 (VH-CDR2) and SEQ ID NO: 361 (VH-CDR3), and SEQ ID NO: 363 (VL-CDR1), SEQ ID NO: 364 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 365 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 197 (VH-CDR1), SEQ ID NO: 198 (VH-CDR2) and SEQ ID NO: 199 (VH-CDR3), and SEQ ID NO: 201 (VL-CDR1), SEQ ID NO: 202 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 203 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 124 (VH-CDR1), SEQ ID NO: 125 (VH-CDR2) and SEQ ID NO: 126 (VH-CDR3), and SEQ ID NO: 127 (VL-CDR1), SEQ ID NO: 128 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 129 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 280 (VH-CDR1), SEQ ID NO: 281 (VH-CDR2) and SEQ ID NO: 282 (VH-CDR3), and SEQ ID NO: 284 (VL-CDR1), SEQ ID NO: 285 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 286 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 369 (VH-CDR1), SEQ ID NO: 370 (VH-CDR2) and SEQ ID NO: 371 (VH-CDR3), and SEQ ID NO: 373 (VL-CDR1), SEQ ID NO: 374 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 375 (VL-CDR3).
  • a or B may include any one variable region selected from the following group:
  • the second antigen may be cancer antigens (Tumor-Specific Antigens), proteins present on the surface of immune cells, or cytokines.
  • Second antigens are PD-L1, PD-1, EGFR, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, cMET, DLL3, GD2, Nectin-4, RANKL, SLAMF7, TROP2, Claudin 18.2, TNFR, TNF, CD3, HER2, CD20, CD19, CTLA-4, VEGFR, VEGF, NCAM1, ICAM-1, ICAM-2, CEACAM6, Carcinoembryonic antigen (CEA), CA-125, Alphafetoprotein (AFP), MUC-1, Epithelial tumor antigen (ETA), Melanoma-associated antigen (MAGE), Immature laminin receptor, TAG-72, HPV E6/E7, BING-4, Calcium-activated chloride channel 2, Cyclin-B1,
  • X and Y combine to form the Fv of the antibody.
  • the Fv may specifically bind to a predetermined antigen.
  • X and Y may be a light chain variable region or a heavy chain variable region specific for an antigen.
  • X and Y may include light chain and heavy chain CDRs.
  • the X and Y combine to form Fv, and the Fv may specifically bind to the above-described second antigen.
  • the Fv formed by binding of X and Y specifically binds to any one antigen selected from the group consisting of PD-L1, HER2, CD19, CD20, EGFR, CD3, TNF, and CTLA-4. can do.
  • X and Y may each include a variable heavy chain (VH) region or a variable light chain (VL) region of any one variable region selected from the following group:
  • VH region comprising the amino acid sequence of SEQ ID NO: 5 (VH-CDR1), SEQ ID NO: 6 (VH-CDR2) and SEQ ID NO: 7 (VH-CDR3), and SEQ ID NO: 9 (VL-CDR1), SEQ ID NO: 10 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 11 (VL-CDR3);
  • VH-CDR1 VH-CDR1
  • SEQ ID NO: 160 VH-CDR2
  • SEQ ID NO: 161 VH-CDR3
  • SEQ ID NO: 171 VL-CDR1
  • SEQ ID NO: 172 VL-CDR2
  • VL-CDR3 VL-CDR3
  • VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 314 (VL-CDR1), SEQ ID NO: 315 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 316 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 118 (VH-CDR1), SEQ ID NO: 119 (VH-CDR2) and SEQ ID NO: 120 (VH-CDR3), and SEQ ID NO: 121 (VL-CDR1), SEQ ID NO: 122 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 123 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 140 (VH-CDR1), SEQ ID NO: 141 (VH-CDR2) and SEQ ID NO: 142 (VH-CDR3), and SEQ ID NO: 152 (VL-CDR1), SEQ ID NO: 153 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 154 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 62 (VH-CDR1), SEQ ID NO: 63 (VH-CDR2) and SEQ ID NO: 64 (VH-CDR3), and SEQ ID NO: 66 (VL-CDR1), SEQ ID NO: 67 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 68 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 223 (VH-CDR1), SEQ ID NO: 224 (VH-CDR2) and SEQ ID NO: 225 (VH-CDR3), and SEQ ID NO: 227 (VL-CDR1), SEQ ID NO: 228 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 229 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 436 (VH-CDR1), SEQ ID NO: 437 (VH-CDR2) and SEQ ID NO: 438 (VH-CDR3), and SEQ ID NO: 441 (VL-CDR1), SEQ ID NO: 442 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 443 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 245 (VH-CDR1), SEQ ID NO: 246 (VH-CDR2) and SEQ ID NO: 247 (VH-CDR3), and SEQ ID NO: 249 (VL-CDR1), SEQ ID NO: 250 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 251 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 256 (VH-CDR1), SEQ ID NO: 257 (VH-CDR2) and SEQ ID NO: 258 (VH-CDR3), and SEQ ID NO: 260 (VL-CDR1), SEQ ID NO: 261 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 262 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 326 (VH-CDR1), SEQ ID NO: 327 (VH-CDR2) and SEQ ID NO: 328 (VH-CDR3), and SEQ ID NO: 338 (VL-CDR1), SEQ ID NO: 339 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 340 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 396 (VH-CDR1), SEQ ID NO: 397 (VH-CDR2) and SEQ ID NO: 398 (VH-CDR3), and SEQ ID NO: 402 (VL-CDR1), SEQ ID NO: 403 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 404 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 410 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 150 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 70 (VH-CDR1), SEQ ID NO: 71 (VH-CDR2) and SEQ ID NO: 72 (VH-CDR3), and SEQ ID NO: 74 (VL-CDR1), SEQ ID NO: 75 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 76 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 84 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 295 (VH-CDR1), SEQ ID NO: 296 (VH-CDR2) and SEQ ID NO: 297 (VH-CDR3), and SEQ ID NO: 299 (VL-CDR1), SEQ ID NO: 300 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 301 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 87 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 183 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 187 (VH-CDR1), SEQ ID NO: 188 (VH-CDR2) and SEQ ID NO: 189 (VH-CDR3), and SEQ ID NO: 191 (VL-CDR1), SEQ ID NO: 192 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 193 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 211 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 306 (VH-CDR1), SEQ ID NO: 307 (VH-CDR2) and SEQ ID NO: 308 (VH-CDR3), and SEQ ID NO: 310 (VL-CDR1), SEQ ID NO: 311 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 312 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 349 (VH-CDR1), SEQ ID NO: 350 (VH-CDR2) and SEQ ID NO: 351 (VH-CDR3), and SEQ ID NO: 353 (VL-CDR1), SEQ ID NO: 354 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 355 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 359 (VH-CDR1), SEQ ID NO: 360 (VH-CDR2) and SEQ ID NO: 361 (VH-CDR3), and SEQ ID NO: 363 (VL-CDR1), SEQ ID NO: 364 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 365 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 197 (VH-CDR1), SEQ ID NO: 198 (VH-CDR2) and SEQ ID NO: 199 (VH-CDR3), and SEQ ID NO: 201 (VL-CDR1), SEQ ID NO: 202 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 203 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 124 (VH-CDR1), SEQ ID NO: 125 (VH-CDR2) and SEQ ID NO: 126 (VH-CDR3), and SEQ ID NO: 127 (VL-CDR1), SEQ ID NO: 128 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 129 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 280 (VH-CDR1), SEQ ID NO: 281 (VH-CDR2) and SEQ ID NO: 282 (VH-CDR3), and SEQ ID NO: 284 (VL-CDR1), SEQ ID NO: 285 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 286 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3);
  • VH region comprising the amino acid sequence of SEQ ID NO: 369 (VH-CDR1), SEQ ID NO: 370 (VH-CDR2) and SEQ ID NO: 371 (VH-CDR3), and SEQ ID NO: 373 (VL-CDR1), SEQ ID NO: 374 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 375 (VL-CDR3).
  • X or Y may be a variable heavy (VH) region or a variable light (VL) region of any one variable region selected from the following group:
  • the X and/or Y may further include CH3.
  • CH3 may be an immunoglobulin-derived CH3 region.
  • CH3 may be bonded to the N-terminus or C-terminus of X and/or Y.
  • CH3 may be included in both the C-terminus of X and Y.
  • CH3 may be included only at the C-terminus of X or may be included only at the C-terminus of Y.
  • CH3 region can provide the ability of the multispecific fusion protein to bind to the Fc receptor.
  • a kiH (knobs-into-holes) structure may be introduced.
  • 1A shows the "Knob-in-Hole" structure of the variable domains of anti-CD3 UCHT1, anti-PD-L1 dervalumab, and anti-CTLA-4 ipilimumab.
  • the A and B may be designed to specifically bind to any one antigen selected from the above-described first antigen.
  • X/Y may be designed to specifically bind to any one antigen selected from the above-described second antigen.
  • the A and B specifically bind to any one first antigen selected from the group consisting of PD-L1, EGFR, CD20, HER2, TNF, CD19, CD3 and CTLA4, and X and Y are Fv formed by binding specifically binds to any one second antigen selected from the group consisting of PD-L1, EGFR, CD20, HER2, TNF, CD19, CD3 and CTLA4, but the A and B and X and Y The formed Fv can be characterized as not binding to the same antigen.
  • the multispecific fusion protein may specifically bind to HER2 as a first antigen and specifically bind to TNF as a second antigen.
  • the multispecific fusion protein may specifically bind to PD-L1 as a first antigen and specifically bind to CD3 as a second antigen.
  • the multispecific fusion protein may specifically bind to EGFR as a first antigen and specifically bind to CD3 as a second antigen.
  • the multispecific fusion protein may specifically bind to CD20 as a first antigen and specifically bind to CD3 as a second antigen.
  • the multispecific fusion protein may specifically bind to CD3 as a first antigen and specifically to PD-L1 as a second antigen.
  • the multispecific fusion protein may specifically bind to HER2 as a first antigen and specifically bind to CD3 as a second antigen.
  • the multispecific fusion protein may specifically bind to CD19 as a first antigen and specifically bind to CD3 as a second antigen.
  • the multispecific fusion protein may specifically bind to PD-L1 as a first antigen and specifically bind to CTLA-4 as a second antigen.
  • the multispecific fusion protein may specifically bind to PD-L1 with a first antigen and specifically bind to PD-1 with a second antigen.
  • the multispecific fusion protein may specifically bind to PD-1 with a first antigen and specifically bind to PD-L1 with a second antigen.
  • the multispecific fusion protein may specifically bind to CD20 as a first antigen and to CTLA-4 as a second antigen.
  • ACE-HC-VH and ACE-HC-VL were generated by replacing the Fc domains of the two HCs of the parent IgG with the VH and VL domains of the IgG specific for the second antigen (Fig. 3a). .
  • the present inventors used anti-CD3 antibody UCHT1 and anti-PD-L1 antibody YBL-007, and the YBL-007 has PD-L1/PD-1 signal blocking ability of avelumab. It is an anti-PD-L1 antibody produced by the present inventors similar to (Fig. 4).
  • the LC of parent YBL-007 was used for ACE-LC, and the hinge regions of VH-CH1 and YBL-007 HC were fused to VH or VL of UCHT1 to generate two HCs of ALiCE.
  • Expression vectors of various combinations encoding ACE-HC-VH, ACE-HC-VL and ACE-LC were transfected into FreeStyle 293-F cells, and the expression of these molecules in culture medium was SDS-PAGE and Western blotting. It was analyzed as (Fig. 5).
  • each chain could not be expressed due to folding and secretion problems (FIG. 5, lanes 1 to 3).
  • the ACE-HC-VH chain having a knob structure in the CDR 3 loop of the UCHT1 VH domain cannot assemble into a homodimer due to inappropriate knob-knob interaction (Fig. 5, lane 4).
  • Homodimer formation between the same ACE-HC-VL chains was hardly detected (Fig. 5, lane 5).
  • the suitably assembled complex was highly expressed and secreted only when ACE-HC-VL, ACE-HC-VH and ACE-LC were present together (FIG. 5, lane 6).
  • the resulting ALiCE anti-PD-L1 Fab ⁇ anti-CD3 Fv; hereinafter referred to as ACE-05
  • ACE-05 was transiently expressed in FreeStyle 293-F cells and purified by CH1 affinity chromatography, and the yield was about 20-30 mg. /L.
  • ACE-31 anti-CD3 Fab x anti-PD-L1 Fv using UCHT1 and YBL-007
  • ACE-00 anti-HER2 mAb [Herceptin] and anti-TNF- ⁇ mAb [Humira]
  • Anti-HER2 Fab x anti-TNF- ⁇ Fv was generated to investigate whether the ALiCE platform could be applied to other antibody pairs in general. Similar to ACE-05, ACE-31 and ACE-00 were assembled into the corresponding heterotetrameric complex, ACE-HC-VL, ACE-HC-VH and two ACE-LCs, and secreted in a homogeneous form ( 6 and 9).
  • the distance between the outer and inner binding domains of ALiCE was found to be about 60 ⁇ , which is the likely distance for the formation of an immunological synaptic bridge between the tumor and effector cells (Arnett, KL et.al. , Proc Natl Acad. Sci USA , 101:16268-16273, 2004) ( Figures 3 and 13).
  • ALiCE acting as a tumor-specific T cell linker, can potentially improve anti-tumor efficacy while reducing off-target T-cell cytotoxicity.
  • ACE-05 anti-PD-L1 Fab ⁇ anti-CD3 Fv
  • ACE-31 anti-CD3 Fab ⁇ anti-PD-L1 Fv
  • the binding affinity (K D ) of ACE-05 for PD-L1 was similar to that of YBL-007 (6.46 ⁇ 10 -10 M), but BiTE-05 It was confirmed to be higher than the binding affinity of (1.39 ⁇ 10 -9 M). This is probably due to the two PD-L1 binding sites in ACE-05 and YBL-007.
  • the binding affinity for CD3 of both ACE-31 (2.39 ⁇ 10 -10 M) and UCHT1 (2.65 ⁇ 10 -10 M), including bivalent anti-CD3 Fab cancer is BiTE-05 (1.01 ⁇ It was higher than the binding affinity of 10 -9 M).
  • the binding affinity of the monovalent stem Fv of ACE-05 for CD3 (2.15 ⁇ 10 -8 M) and ACE-31 (2.72 ⁇ 10 -8 M) for PD-L1 is the parent antibody for CD3, respectively.
  • the binding affinity of UCHT1 (2.65 ⁇ 10 -10 M) and that of the parent antibody YBL-007 for PD-L1 (6.46 ⁇ 10 -10 M) were 40 to 80 fold lower.
  • the binding affinity of the stem Fv of ACE-05 to CD3 and of ACE-31 to PD-L1 is the binding affinity of BiTE-05 to CD3 (1.01 ⁇ 10 -9 M) and BiTE to PD-L1. It was much lower than the binding affinity of -05 (1.39 ⁇ 10 -9 M), which may be due to steric hindrance between the Fab cancer and the second antigen-binding region of stem Fv.
  • the present inventors investigated the effect of the valence of ALiCE paratope on tumor and T-cell binding.
  • the apparent binding affinity of ACE-05 or ACE-31 for PD-L1 of Karpas-299 tumor cells and CD3 of Jurkat T cells was consistent with the in vitro binding affinity for PD-L1 and CD3.
  • the present inventors described wild-type (WT) PD-L1 - HEK cells or maintain a fully operating the PD-L1 + HEK cells NFAT- luciferase reporter gene PD-1 expressing - Jurkat T cells were incubated with the ball (19). Then, after treatment with the T-cell binding agents ACE-05, ACE-31 or BiTE-05, NFAT-luciferase reporter activity was measured to evaluate Jurkat T cell activation.
  • WT wild-type
  • ACE-05 showed the highest on-target NFAT activation when co-cultured with PD-L1 + HEK cells and PD-1 -Jurkat T cells.
  • BiTE-05 and ACE-31 showed higher off-target T-cell activation than ACE-05 when co-cultured with WT PD-L1- HEK cells and PD-1 -Jurkat T cells.
  • It is mediated by direct binding to CD3 on Jurkat T cells in the absence of PD-L1 targeting (FIG. 20 ).
  • the binding affinity is different, as described above, since ACE-05, ACE-31, BiTE-05 and YBL-007 bound to PD-L1, the present inventors stably expressed PD-1 and NFAT-luciferase reporters.
  • the ability to inhibit PD-L1/PD-1 activity was evaluated using Jurkat T cells and CHO-K1 cells expressing human PD-L1 and engineered cell surface proteins.
  • the engineered cell surface protein was designed to activate cognate T-cell receptor (TCR) in an antigen-independent manner (Cheng, ZJJ et al., Cancer Res ., 75 (2015)).
  • T-cell activation was higher after treatment with ACE-05 or BiTE-05 during cell culture than after treatment with YBL-007, which can only interfere with PD-L1/PD-1 interaction (FIG. 21).
  • ACE-31 is much less effective than ACE-05, BiTE-05 and YBL-007 in re-activating T cells, and the affinity of ACE-31 for PD-L1 is low compared to the other three molecules. Reflects (Fig. 21).
  • PBMC peripheral blood mononuclear cells
  • HCC827 or MDA-MB-2331 tumor cells
  • PBMC peripheral blood mononuclear cells
  • ACE-31 tumor cells
  • CD8 + cytotoxic T cells are often regarded as major effector cells in solid tumors
  • CD8 + T cells isolated from PBMCs were cytotoxic upon ACE-05 treatment. Consistently, incubation of CD8 + T cells and PD-L1 + MDA-MB-231 and ACE-05 showed the most potent cytolytic activity, and direct proteolysis of target tumor cells in cooperation with perforin.
  • caspase-mediated apoptosis inducing granzyme B (Granzyme B) showed the highest level of secretion (FIGS. 27 and 28).
  • the present inventors investigated whether ACE-05 can stimulate the activation and expansion of human effector cells in the presence of PD-L1 + tumor cells.
  • the present inventors cultured human CD3 + T cells with PD-L1 + MDA-MB-231 cells and 1 nM ACE-05 or IgG for 24 hours, and then monitored the surface expression of the activation markers CD69 and CD25.
  • Initial activation marker CD69 was upregulated in both CD4 + and CD8 + T cells in the presence of ACE-05 and PD-L1 + tumor cells, but not by IgG (FIG. 29 ).
  • CD25 a late activation marker
  • ACE-05 in the presence of PD-L1 + tumor cells
  • Activation of T cells and subsequent differentiation into effector cells are also associated with T-cell clustering and aggregation (Zhou, J. et al. , PLoS One , 13:e0191634, 2018). Therefore, the present inventors cultured CytoLight-stained human CD3 + T cells with PD-L1 + MDA-MB-231 cells and 1 nM ACE-05, ACE-31, BiTE-05 or IgG for 90 hours and then cluster The area was measured.
  • ACE-05 stimulates clustering of CD3 + T cells with respect to PD-L1 + tumor cells, and can induce T-cell activation more effectively than ACE-31 or BiTE-05 (FIG. 31 ). Moreover, ACE-05 strongly induced CD3 + T cell proliferation/expansion (Fig. 32).
  • the inventors of the present invention have different binding affinity for CD3 (ACE-05> ACE-49> ACE-47> ACE-56), but ACE with the same binding affinity for PD-L1.
  • the -05 variant was prepared ( Figure 34).
  • ACE-05 which has the highest affinity for CD3, showed the highest off-target T-cell activation in the NFAT reporter assay, and non-target T-cell activation was found in CD3 for the ACE-05 variant. The binding affinity for this decreased continuously with decreasing.
  • mice were implanted into female NCG mice 3 days before subcutaneous inoculation of PD-L1 + HCC827 tumor cells in the right posterior flank.
  • mice are given 3 doses of ACE-05 or BiTE-05 (0.5 mg/kg body weight), or 3 doses of YBL-007 or IgG (5.0 mg/kg body weight). It was injected intravenously.
  • mice treated with ACE-05 In 9 of 10 mice treated with ACE-05, the pre-established tumors completely regressed on day 12 (FIG. 41 ). In addition, the present inventors monitored body weight changes as signs of side effects. Mice treated with BiTE-05 showed a significant decrease in body weight ( ⁇ 20%), whereas mice treated with ACE-05 or YBL-007 did not show significant weight loss (FIG. 42 ).
  • the present inventors further investigated the in vivo T cytotoxicity and anti-tumor efficacy of ACE-05 and BiTE-05 in hCD3 ⁇ transgenic (TG) mice, where the T cells were genetically engineered to express both hCD3 ⁇ and mCD3 ⁇ . Became. Both IL-2 and IFN- ⁇ are pleiotropic cytokines, important effector molecules for anti-tumor immunity through various mechanisms.
  • IL-2 immune-related side effects of IL-2 and immune-avoidance function of IFN- ⁇ have also been reported (Berraondo, P. et. al. , Br J Cancer , 120:6-15, 2019).
  • pro-inflammatory cytokines IL-6 and TNF- ⁇ which are released from activated antigen presenting cells (APCs) such as macrophages, dendritic cells or B cells, are central mediators of cytokine-releasing syndrome (CRS) toxicity. It has been proposed (Shimabukuro-Vornhagen, A. et. al. , J Immunother Cancer , 6, 56 (2016)).
  • non-tumor-bearing hCD3 ⁇ mice received higher levels of cytokines than ACE-05 when administered BiTE-05, particularly The release of IL-2, IFN- ⁇ and IL-6 was induced, and the highest cytokine release level was shown 6 hours after administration (FIG. 45).
  • the non-tumor bearing hCD3 ⁇ mice treated with BiTE-05 showed severe weight loss, whereas the mice treated with ACE-05 or IgG showed much less weight loss (Figure 46), and the low of ACE-05.
  • Non-target cytotoxicity was demonstrated.
  • hCD3 ⁇ TG mice transplanted with hPD-L1 + CT26 tumors treated with ACE-05 It was confirmed that the tumor size was effectively reduced so as not to cause a significant change in body weight, and complete regression was caused in 1 out of 6 mice by the 15th day (FIGS. 47 and 48). Thereafter, the present inventors collected tumors from each mouse at the end of the study, and analyzed tumor-infiltrating lymphocytes (TIL).
  • TIL tumor-infiltrating lymphocytes
  • ACE-05 and YBL-007 is a CD4 + T cells are not induced the proliferation and expansion of CD8 + T cells (Fig. 52 and Fig. 53), which possibly PD-1 and PD-L1 be due to inhibition of the interaction (Beyrend, G. et. al. , J Immunother Cancer , 7 , 217 (2019)).
  • the present inventors also investigated the T-cell class I MHC immunogenicity of the ACE-05-HC-VH and ACE-05-HC-VL chains using an in silico immunogenicity prediction tool.
  • the immunogenicity of ACE-05-LC was not analyzed because it was identical to that of the native antibody LC.
  • the present inventors investigated peptides treated with ACE-05-HC-VH and ACE-05-HC-VL that can be presented on MHC class I molecules using a percentile ranking (based on cut-off value of 0.3). (Fig. 54 and Fig. 55, left).
  • Immunogenic peptides (score> 0) possible within the ACE-05-HC-VH and ACE-05-HC-VL chains are listed and shown in FIGS. 56 and 57 (right). Potential immunogenic peptides of the ACE-05-HC-VH and ACE-05-HC-VL chains were mainly found in the CDRs, FRs and CH1s within the variable domains.
  • ACE-05 has improved anti-tumor efficacy with less promiscuous cytotoxicity through tumor-specific on-target T-cell activation and prolonged pharmacokinetic profile, indicating that PD on tumor cells
  • ACE-05 has improved anti-tumor efficacy with less promiscuous cytotoxicity through tumor-specific on-target T-cell activation and prolonged pharmacokinetic profile, indicating that PD on tumor cells
  • Another aspect of the present invention is to provide a polynucleotide encoding the structural formula (I), (I'), (I''), (II), (II') or (II'').
  • the polynucleotide may be in the form of RNA or DNA. It may be double-stranded or single-stranded, and if single-stranded, it may be a coding strand or a non-coding (antisense) strand.
  • the polynucleotide may include a sequence encoding a marker or tag sequence.
  • One embodiment of the tag sequence is a hexa-histidine tag.
  • the polypeptide is at least about 70% with a polynucleotide encoding the formula (I), (I'), (I''), (II), (II') or (II''), At least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, At least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity. have.
  • the polynucleotide may additionally include a nucleic acid encoding a signal sequence or a leader sequence.
  • signal sequence refers to a signal peptide that directs the secretion of a protein of interest.
  • the signal peptide is cleaved after translation in the host cell.
  • the signal sequence is an amino acid sequence that initiates the movement of a protein through the ER (endoplasmic reticulum) membrane.
  • the signal sequence is well known in the art, and usually includes 16 to 30 amino acid residues, but may include more or less amino acid residues.
  • a typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.
  • the central hydrophobic region contains 4 to 12 hydrophobic residues that fix the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide.
  • the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases.
  • the signal sequence may be a tissue plasma activation (tPa), a signal sequence of Herpes simplex virus glycoprotein D (HSV gDs), or a secretion signal sequence of growth hormone.
  • tPa tissue plasma activation
  • HSV gDs Herpes simplex virus glycoprotein D
  • a secretion signal sequence of growth hormone e.gDs
  • a secretion signal sequence used in higher eukaryotic cells including mammals and the like can be used.
  • it can be used by substituting codons with high expression frequency in host cells.
  • Another aspect of the present invention is a vector comprising a polynucleotide encoding the above structural formula (I), (I'), (I''), (II), (II') or (II''). to provide.
  • the vector can be introduced into a host cell and recombined and inserted into the host cell genome.
  • the vector is understood as a nucleic acid means comprising a polynucleotide sequence capable of spontaneously replicating as an episome.
  • Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof.
  • examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.
  • the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis S-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.), animal virus vectors (retrovirus (retrovirus) ), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the vector has different expression levels and modifications of proteins depending on the host cell, it is preferable to select and use the vectors
  • the term "gene expression” or “expression” of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof.
  • Useful expression vectors may be RcCMV (Invitrogen, Carlsbad) or a variant thereof.
  • the expression vector includes a human CMV (cytomegalovirus) promoter for promoting the continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence for increasing the stable state level of RNA after transcription. can do.
  • Another aspect of the present invention provides a transformed cell into which the vector has been introduced.
  • Host cells of the transformed cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, mammals, plants, insects, fungi, or cells of cellular origin.
  • prokaryotic cell E. coli may be used.
  • yeast may be used as an example of eukaryotic cells.
  • CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells, HEK293T cells, etc. can be used as the mammalian cells. , It is not limited thereto, and all cells that can be used as mammalian host cells known to those skilled in the art may be used.
  • the Hanahan method electroporation, calcium phosphate precipitation method, which improved efficiency by using a reducing material called dimethyl sulfoxide (DMSO) in CaCl 2 precipitation method and CaCl 2 precipitation method.
  • DMSO dimethyl sulfoxide
  • Protoplasm fusion method agitation method using silicon carbide fibers, Agrobacteria mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/inhibition mediated transformation method, and the like may be used.
  • the glycosylation-related gene of the host cell for optimizing the properties of the fusion protein as a therapeutic agent or for other purposes is manipulated through a method known to those skilled in the art, and the sugar chain pattern of the fusion protein (e.g., Sialic acid, fucosylation, saccharification) can be adjusted.
  • the sugar chain pattern of the fusion protein e.g., Sialic acid, fucosylation, saccharification
  • composition comprising a fusion protein
  • Another aspect of the present invention is to provide a pharmaceutical composition for the treatment or prevention of cancer comprising the above-described multi-specific fusion protein as an active ingredient.
  • the cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and It may be any one selected from the group consisting of lymphoma.
  • the cancer may be overexpression of any one protein selected from the group consisting of PD-L1, EGFR, and HER2.
  • the active ingredient is an arbitrary amount (effective amount) according to the use, formulation, purpose of combination, etc., as long as it exhibits anti-cancer activity or can exhibit a therapeutic effect on infectious diseases.
  • a typical effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition.
  • effective amount refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such effective amounts can be determined empirically within the range of ordinary skill in the art.
  • the term “treatment” may be used to include both therapeutic treatment and prophylactic treatment. In this case, prevention may be used in the sense of alleviating or reducing a pathological condition or disease of an individual. In one embodiment, the term “treatment” encompasses any form of dosage or application to treat a disease in mammals, including humans. In addition, the term includes inhibiting or slowing the progression of a disease or disease; Restore or repair impaired or impaired function to partially or completely alleviate the disease; Or stimulating an inefficient process; Includes the meaning of alleviating the severity of the disease.
  • the term “efficacy” refers to one or more parameters such as survival over a period of time, such as 1, 5, or 10 years, or disease-free survival. Can be determined.
  • the parameter may include suppressing the size of at least one tumor in the individual.
  • “enhanced efficacy” eg, improved efficacy
  • improved efficacy can be attributed to improved pharmacokinetic parameters and improved efficacy, comparing clearance rates and tumor growth in test animals or human subjects, or survival, recurrence rates or disease. It can be measured by comparing parameters such as survival in the absence.
  • terapéuticaally effective amount or “pharmaceutically effective amount” is an amount of a compound or composition effective to prevent or treat the subject disease, which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and It means the amount that does not cause side effects.
  • the level of the effective amount is the health condition of the patient, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including the drugs used in combination or concurrently, and Other factors well known in the medical field can be determined.
  • a therapeutically effective amount refers to an amount of a drug effective to treat cancer.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohols, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.
  • the pharmaceutical composition may be prepared in a parenteral formulation according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.
  • pharmaceutically acceptable means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.
  • the pharmaceutical composition When the pharmaceutical composition is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation, and a suppository according to a method known in the art together with a suitable carrier.
  • a suitable carrier When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterilization for injection. Water, isotonic solutions such as 5% dextrose, etc. can be used.
  • a pharmaceutical composition it is known in the art, and specifically, Remington's Pharmaceutical Sciences (19th ed., 1995), etc. may be referred to. This document is considered part of this specification.
  • the preferred dosage of the pharmaceutical composition may be in the range of 0.01 ⁇ g to 10 g per 1 kg of body weight per day, or in the range of 0.01 mg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.
  • compositions of the present application are mammals and humans, particularly preferably humans.
  • the pharmaceutical composition of the present application may further include any compound or natural extract, which has already been verified for safety and has a therapeutic effect on anti-cancer activity or infectious disease, in order to increase and reinforce anti-cancer activity.
  • Another aspect of the present invention provides a pharmaceutical composition further comprising the above-described multispecific fusion protein and an anticancer agent.
  • the fusion protein includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be formulated to be suitable for the route of administration to the subject.
  • Another aspect of the present invention provides the use of the multispecific fusion protein for treating or preventing cancer.
  • the cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. Can be selected from the group.
  • Another aspect of the present invention provides the use of the multispecific fusion protein for use as a cell engager.
  • Another aspect of the present invention provides a method for treating or preventing cancer comprising administering the multispecific fusion protein to an individual.
  • the subject may be a subject suffering from cancer or an infectious disease.
  • the individual may be a mammal, preferably a human.
  • the multispecific fusion protein is as described above.
  • the multispecific fusion protein may be administered to a subject in various methods and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal administration route, dosage, and frequency of administration may be selected within an appropriate range by a person skilled in the art.
  • the fusion protein may be administered in combination with other drugs or physiologically active substances known to have a therapeutic effect on the disease to be treated, or may be formulated in the form of a combination formulation with other drugs.
  • Another aspect of the present invention provides a method for producing a multispecific fusion protein comprising culturing the transformed cells described above. Specifically, the production method comprises the steps of: i) culturing the transformed cells to obtain a culture; And ii) recovering the fusion protein from the culture.
  • the method of culturing the transformed cells may be performed using a method well known in the art. Specifically, the culture may be continuously cultured in a batch process or in a fed batch or repeated fed batch process.
  • any method known in the art for purification of immunoglobulins to recover the fusion protein for example chromatography (ion exchange, affinity, especially Protein A, sizing column chromatography and kappa-select affinity chromatography Thereafter, in the case of specific antigens, purification by affinity), centrifugation, differential solubility or other standard techniques for protein purification can be used.
  • kappa-select eg, kappa select developed by GE Healthcare Life Science
  • Fab kappa select developed by GE Healthcare Life Science
  • the multispecific fusion proteins provided herein can be fused to heterologous polypeptide sequences described herein or other sequences known in the art to facilitate purification.
  • ALiCE variants ACE-05 (anti-PD-L1 Fab ⁇ anti-CD3 Fv), ACE-31 (anti-CD3 Fab ⁇ anti-PD-L1 Fv), ACE-18 (anti-CD20 Fab ⁇ CD3 Fv) and HC pair of ACE-00 (anti-HER2 Fab ⁇ anti-TNF- ⁇ Fv), ACE-HC-VH and ACE-HC-VL are parent antibodies YBL-007 (anti-PD-L1), UCHT1 (Anti-CD3), Rituximab (anti-CD20), Herceptin (anti-HER2) and Humira (anti-TNF- ⁇ ) were used as templates, and VHA-CH1-hinge, VHB and VLB were encoded The sequence was amplified by PCR.
  • PCR primers for constructing ALiCE HC were designed to contain a short linker (G4S) between the VHB or VLB at the hinge and 5'end, and the Sap I restriction site required for the Electra TM cloning system at the 3'end.
  • G4S short linker
  • ACE-LC the LC used to construct ALiCE
  • the sequence encoding the LC containing the leader peptide at the N-terminus was PCR-amplified and subcloned into the p293 expression vector containing the Nhe I/ Xho I restriction site.
  • the resulting constructs were transformed with DH5 ⁇ competent cells (# CP010, Enzynomics) and confirmed through sequencing.
  • ALiCE molecule and BiTE-05 are purified by centrifuging the culture at 4°C for 30 minutes at 4,800 rpm, and then filtering the supernatant using a 0.22 ⁇ m TOP-filter to remove debris. I did.
  • the supernatant containing ALiCE molecules was loaded on a CaptureSelect CH1-XL pre-packed column (#494346201, ThermoFisher), and the supernatant containing BiTE-05 was Ni-NTA agar. It was loaded on OS resin (Ni-NTA agarose resin, # R90101, ThermoFisher).
  • the ALiCE molecule was eluted from the column using 0.1 M glycine (pH 3.0), and BiTE-05 was eluted using 3 M imidazole/20 mM sodium phosphate (pH 6.0).
  • the eluted ALiCE molecules and BiTE-05 were dialyzed with phosphate buffered saline (PBS; pH 7.4) using Slide-A-Lyzer Dialysis Cassette Kits (#66372, ThermoFisher).
  • SPR binding kinetic assay Surface plasmon resonance binding kinetic assay
  • a 6 ⁇ -His-tag was removed from BiTE-05 using a Thrombin Cleavage Capture kit (#69022, Merck).
  • anti-CD3 antibody UCHT (PBD ID: 1XIW), anti-PD-L1 antibody dervalumab, and anti-CTLA-1 antibody ipilimumab (PBD ID: 5TRU) are described in Protein Data Bank (PBD, www.rcsb. org) and visualized using PyMOL software (Schrodinger, LLC The PyMOL Molecular Graphics System, Version 1.8. (2015)).
  • the ALiCE molecule was SDS-PAGE; Fully automated capillary electrophoresis (CE) using an Agilent 2100 Bioanalyzer (Agilent Technology); Analytical Size-Exclusion Chromatography (SEC) using a Superdex 200A column (GE Healthcare Life Science); And analytical cation exchange chromatography (CEX) using a MabPac SCX-10 column (ThermoFisher). Heterodimer formation by two different ALiCE HCs was evaluated by analyzing ALiCE molecules by SDS-PAGE and CE under reducing and non-reducing conditions.
  • the protein-analytical solution mixture was loaded onto a microfluidic protein chip and separated by molecular weight using the Bioanalyzer Protein 230 assay kit (Agilent) under reducing and non-reducing conditions according to the manufacturer's protocol. I did.
  • the stoichiometric ratio of various chains in ACE-05 was determined under reducing conditions using a Bioanalyzer Protein 80 assay kit (Agilent).
  • VH and VL including anti-CD3 antibody UCHT1 (PDB ID: 1XIW), anti-PD-L1 antibody dervalumab (PDB ID: 5X8M) and anti-CTLA4 antibody ipilimumab (PDB ID: 5TRU)
  • the crystal structure of the complex is clear that the key determinant of VH and VL interaction is the CDR3 region of VH, which forms a "knob-into-Hole" structure and binds to the CDR1, CDR2 and CDR3 regions of VL. Proved to be (Fig. 1).
  • the hydrophobic interaction surrounded by electrostatic interactions in the VH-VL interface contributes to the autonomous assembly of the VH and VL domains and the stabilization of the Fv complex (FIG. 2).
  • the molecular weight of purified ACE-05 was determined by liquid chromatography-electrospray ionization (LC-ESI/TOF) through time-of-flight (TOF) analysis using a ZORBAX 300SB-C8 (2.1 ⁇ 50 nm; Agilent) column. Confirmed.
  • the formic acid concentration was constant over the entire run at 0.2%, while the mobile phase consisted of a gradient of water and acetonitrile over 35 minutes, starting with 5% acetonitrile (initial conditions) to 100% acetonitrile.
  • MS spectrometry detection was performed using a micro A-TOF III mass spectrometer (Bruker Daltonics, Germany) using electrospray ionization (ESI) as a negative mode.
  • MS parameters were used: capillary voltage, 4500 V; Nebulizer pressure, 0.8 psi; Dry gas flow, 5.5/min; Dry gas temperature, 190°C.
  • the thermal stability of ACE-05, BiTE-05, YBL-007 and UCHT1 was determined by Thermofluor assay (Lavinder, JJ et.al. , J Am Chem Soc , 131:3794-3795, 2009) using SYPRO orange dye (dye). Analyzed using. Specifically, 3 ⁇ M solution of each purified antibody was mixed with 10 ⁇ l of SYPRO orange dye (#S6650, ThermoFisher) diluted 1:25, and 50 ⁇ l of each mixture was incubated at 25° C. for 30 minutes.
  • Binding kinetics of ALiCE molecules to various antigens was measured by surface plasmon resonance using a Biacore 8K system equipped with a certified-grade CM5 series S sensor chip (# BR100399, GE Healthcare).
  • HEPES-buffered saline (0.01 M HEPES, 0.15 M NaCl) containing 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.05% (v/v) P20 surfactant (HBS-EP+) was added to the reaction and running buffer. (#BR100669, GE Healthcare).
  • Antigens PD-L1-his (0.1 ⁇ g/ml; self-synthesizing in-house) and CD3 ⁇ -flag-his (0.2 ⁇ g/ml; #CT038-H2508H, Sino Biological) were assigned to the CM5 sensor chip according to the manufacturer's instructions. It was fixed on the surface of (#BR100399, GE Healthcare). Thereafter, bispecific T-cell linkers (ACE-05, BiTE-05, and ACE-31) and parent mAb (YBL-007 and UCHT1) diluted in HBS-EP + buffer were added.
  • biolayer light interferometry was performed with the Octet QKe system (Pall Forte Bio) (Abdiche, Y. et.al. , Anal Biochem , 377:209-217, 2008).
  • the first ligand, PD-L1-Fc (2 ⁇ g/ml; synthesized in-house) and CD3 ⁇ -flag-his (3 ⁇ g/ml; #CT038-H2508H, Sino Biological) have a binding of 0.5 to 1.0
  • Each hydrated AHC (#18-5064, Pall Forte Bio) or Ni-NTA (#18-5013, Pall Forte Bio) biosensor was loaded until nM was reached.
  • the biosensor was washed with kinetics buffer (0.1% bovine serum albumin [BSA] and 0.02% Tween-20-containing PBS) for 2 minutes (ACE-05) or 1 minute (ACE-31) to obtain unbound protein.
  • kinetics buffer (0.1% bovine serum albumin [BSA] and 0.02% Tween-20-containing PBS) for 2 minutes (ACE-05) or 1 minute (ACE-31) to obtain unbound protein.
  • BSA bovine serum albumin
  • ACE-31 1 minute
  • a second ligand that is, 200 nM CD3 ⁇ -flag-his (ACE-05) or 120 nM PD-L1-Fc (ACE-31) was added to the biosensor loaded with the first ligand and ACE-05 or ACE-31.
  • the binding was measured by immersion in the containing solution, followed by washing with PBS for 3 minutes (ACE-05) or 2 minutes (ACE-31) to measure dissociation.
  • Sensorgram data was plotted using GraphPad Prism 8 software.
  • PD-L1 + cancer cells HCC827, MDA-MB-231, and Karpas-299
  • PD-L1 - Raji cells at 0.5 ⁇ 10 6 cells/ It was 100 ⁇ l and incubated with an anti-PD-L1 antibody (#55817, BD Bioscience) conjugated with phycoerythrin (PE)-Cy7 diluted 1:50 (v/v).
  • PE phycoerythrin
  • FITC fluorescein isothiocyanate conjugated anti-CD3 antibody (#130-113-138, Miltenyi Biotech) was used to evaluate CD3 levels on Jurkat T cells (0.5 ⁇ 10 6 cells/100 ⁇ l).
  • FACS buffer PBS containing 1% fetal bovine serum [FBS]
  • FBS fetal bovine serum
  • the double binding capacity of ACE-05 and ACE-31 to PD-L1 and CD3 on the cell surface was determined by CD3 + Jurkat T cells and 20 nM ACE-05, ACE-31 or ACE-18 (CD20 Fab ⁇ CD3 Fv, Ctrl-ACE). ) was investigated by incubating for 1 hour. After washing twice with 1 ml of FACS buffer, cells were incubated with PD-L1-Fc (75 ⁇ g/100 ⁇ l) and washed twice with 1 ml of FACS buffer.
  • PD-L1-Fc was detected using an anti-human Fc antibody conjugated with Alexa 647 (#109-605-098, Jackson ImmunoResearch).
  • the apparent binding affinity of ALiCE (ACE-05 and ACE-31) to PD-L1 + Karpas-299 and PD-L1 - Raji cancer cells and CD3 + Jurkat T cells was determined by the above cells (0.5 ⁇ 10 6 cells/100).
  • ACE-05 0.000932, 0.003729, 0.014915, 0.059662, 0.59459, 3.81, and 15.27 nM for Raji cells; 1.56, 15.625, 156.25, and 1562 nM for Raji cells; 1.1, 3.3 for Jurkat cells) , 9.9, 29.6, 88.9, 266.7, 800, and 2400 nM) or ACE-31 (0.594, 3.81, 15.27, 61.03, 244.37, 977.5, and 3910 nM for Karpas-299 cells; 1.56, 15.625, for Raji cells; 156.25, and 1562 nM; for Jurkat cells 0.011, 0.033, 0.101, 0.304, 0.914, 2.743, 8.320, 24.691, and 74.074 nM) were measured by treatment at the indicated concentrations.
  • ACE-05 or ACE-31 bound to the cell surface was detected with a flow cytometer (CytoPLEX-LX) after incubation with an antibody conjugated with Alexa 647 to a human Fab fragment (#109-606-097, Jackson Immunoresearch). .
  • Flow cytometric data was analyzed using FlowJo 10 software (FlowJo, LLC), and geometric mean plotted using GraphPad Prism 8 software.
  • PD-L1 - WT HEK cells or genetically engineered PD-L1 + HEK Cells (7 ⁇ 10 4 cells/well) were seeded on a white-bottom plate coated with poly-L-lysine (#P4707, Sigma).
  • Luciferase accumulation induced by T cell activation was measured by performing a Bio-Glo Luciferase assay (#G7940, Promega) according to the manufacturer's protocol. The resulting data expressed in relative light units (RLU) was plotted and analyzed using GraphPad Prism 8 software.
  • CD3 + T cells (1 ⁇ 10 5 cells/well) and human PBMC (#SER- PBMC-200-F, Zenbio) isolated CD3 + T cells (1 ⁇ 10 6 cells/well) were co-cultured, but 1 nM ACE-05, BiTE-05, ACE-31 or IgG was added. CD3 + T cells were stained with Trace Far Red (#C34564, ThermoFisher) according to the manufacturer's instructions.
  • T cells were harvested, and APC conjugated anti-CD4 antibody (#130-113-210, MiltenyBiotec), FITC conjugated anti-CD8 antibody (#130-110- 677, MiltenyBiotec), PE-Vio 770 conjugated anti-CD69 antibody (#130-122-5-4, MiltenyBiotec) and PE conjugated anti-CD25 antibody (#341009, BD Bioscience).
  • APC conjugated anti-CD4 antibody #130-113-210, MiltenyBiotec
  • FITC conjugated anti-CD8 antibody #130-110- 677, MiltenyBiotec
  • PE-Vio 770 conjugated anti-CD69 antibody #130-122-5-4, MiltenyBiotec
  • PE conjugated anti-CD25 antibody #341009, BD Bioscience.
  • T cell subsets and activated T cells were identified by flow cytometry (BD FACSCanto II) using FlowJo 10 software (FlowJo, LLC).
  • the PD-1/PD-L1 blockade bioassay was performed according to the manufacturer's protocol (#J1250, Promega). Briefly, one vial of PD-L1/aAPC + CHO-K1 cells included in the bioassay kit was suspended in recovery medium from a frozen stock (90% Ham's F-12 containing 10% FBS) and , This was seeded on a white bottom plate and incubated overnight at 37°C. Assay buffer (1% FBS) with ACE-05, BiTE-05, ACE-31, YBL-007 or IgG at concentrations of 0, 0.006, 0.032, 0.16, 0.8, 4 or 20 nM in each well.
  • RPM 1640 containing Jurkat T cells stably expressing human PD-1 and NFAT-luciferase reporters were added. After 6 hours, NFAT-mediated luciferase activity was measured using a Bio-Glo Luciferase assay system (#G7940, Promega). RLU (relative light units) data were plotted and analyzed using GraphPad Prism 8 software.
  • HCC827 (ATCC CRL2868), MDA-MB-231 (ATCC HTB-26), Karpas-299 (# 06072604, Sigma) and Raji (ATCC CCL86) cancer cells were added with 10% FBS under conditions of 37°C and 5% CO 2 It was cultured using the RPMI-1640 medium.
  • PBMC and CD8 + T cells of all healthy donors used in the present invention were purchased from AllCells (#PB004F and #PB009-3F), Zenbio (#SER-PBMC-200-F), and Lonza (#3W-270). .
  • CD3 + T cells and CD8 + T cells were human using Pan T-cell isolation kit (#130-096-535, Miltenyi Biotec) and CD8 + T-cell isolation kit (#130-096-495, Miltenyi Biotec). It was isolated from PBMC preparation.
  • the cytotoxicity of PBMCs or T-cells against cancer cells expressing PD-L1 was evaluated by measuring lactate dehydrogenase (LDH) released from killed cancer cells.
  • LDH lactate dehydrogenase
  • the on-target tumor cell-killing ability of the bispecific T cell linker is 10:1 (CD3 + T cells) or 5:1 (CD8 + T cells) of E:T (effector: target ( target)) PD-L1 + MDA-MB-231 cancer cells (1 ⁇ 10 4 cells/well) and T cells with the indicated protein (ACE-05, ACE-31, BiTE-05, or IgG) It was investigated by culturing.
  • PBMC peripheral blood mononuclear cells
  • E:T ratio 25:1
  • PD-L1 - HEK293 or Raji cancer cells were co-cultured with CD3 + T cells and 1 nM ACE-05, BiTE-05, ACE-31, IgG or ACE-18. . After 48-72 hours incubation, LDH released from PD-L1- cells was measured and calculated as described above.
  • T-cell activation and differentiation was evaluated by accurately quantifying T cells and PD-L1 + tumor cell clustering using the IncuCyte live-cell analysis system (Sartorius, USA).
  • CD3 + T cells were isolated from human PBMCs and labeled with CytoLight reagent (#4706, Sartorius) according to the manufacturer's protocol.
  • PD-L1 + MDA-MB-231 cells (4 ⁇ 10 3 cells/well) and CD3 + T cells with 1 nM ACE-05, ACE-31, BiTE-05 or IgG at an E:T ratio of 10:1. Co-cultured together.
  • Live-cell images were obtained every 6 hours during the 90-hour culture period, and the average area (mm 2) of red fluorescent clusters representing T-cell activation was measured using IncuCyte software. Data obtained in quadruplicate were plotted using GraphPad Prism 8 software.
  • T-cell proliferation assay Primary human T-cell expansion induced with ACE-05 in the presence of PD-L1 + tumor cells was investigated using a T-cell proliferation assay.
  • CD3 + T cells isolated from PBMC were stained with Cell Trace Far Red (#C34564, ThermoFisher) according to the manufacturer's instructions.
  • PD-L1 + MDA-MB-231 cancer cells were seeded in a 24-well plate at a concentration of 1 ⁇ 10 5 cells/well. The next day, the pre-cultured MDA-MB-231 cells were washed once with Dulbecco's PBS, which had been pre-warmed, and then replaced with an assay medium (RPMI-1640 containing 1% FBS).
  • Example 14 Non-target T-cell activation assay by multimeric T-cell combiner in the absence of tumor cells
  • CD3 + T cells isolated from human PBMC (#4W-270C, Lonza) (1 ⁇ 10 6 cells/well) were cultured in a medium containing 5% FBS, but 1 nM ACE-05, BiTE-05 or multimer ACE-05 was added directly.
  • Clustered ACE-05 was prepared by mixing 5 ⁇ l CH1 beads (#1943462250, ThermoFisher) with 1 nM ACE-05. After incubation for 48 hours, T cells were harvested, and FITC conjugated with CD4 antibody (#130-114-531, MiltenyBiotec) and CD69-PE-Vio 770 antibody (#130-122-5-4, MiltenyBiotec). Labeled.
  • T cell subsets and activated T cells were identified by flow cytometry (BD FACSCanto II) using FlowJo 10 software (FlowJo, LLC).
  • Example 15 Analysis of cytokines secreted by immune cells
  • cytokines, IL-2, IFN- ⁇ , IL-6 and TNF- ⁇ released by activated immune cells (PBMC and CD4 + T cells) in the presence or absence of PD-L1 + tumor cells Investigated.
  • PBMC and CD4 + T cells activated immune cells
  • human PBMCs with HCC827 cancer cells 25:1 E:T Co-cultured for 72 hours at a rate. Samples were collected at 0, 6, 12, 18, 24, 48 and 72 hours after drug administration. Thereafter, the concentration of cytokines was analyzed using an ELISA kit for IL-2 (#431004, BioLegend) and IFN- ⁇ (#431004, BioLegend).
  • ACE-05, BiTE-05, ACE-31 or IgG was directly added to CD4 + T cells isolated from human PBMC using a CD4 + T cell separation kit (#130-096 -533, Miltenyi Biotec). After incubation for 72 hours, the released cytokines were analyzed by ELISA as described above.
  • Granzyme B analysis in the presence of various concentrations of ACE-05, BiTE-05, ACE-31 or IgG (0, 6.4, 32, 160, 800, 4000 pM), PBMC-derived CD8 + T cells and MDA-MB -231 cells were co-cultured at an E:T ratio of 5:1. After 48 hours, Granzyme B accumulated in the assay medium was analyzed using an ELISA kit (#DGZB00, R & D Systems). The optical density (OD) of each supernatant was measured using a microplate reader, and the concentration of cytokines was analyzed using GraphPad Prism 8 software.
  • biotinylated anti-human IgG CH1 nanobodies were immobilized on the surface of streptavidin-coated Gyrolab Bioaffy CD200 (#P0004180, Gyros Protein Technologies), and serum samples were loaded.
  • biotinylated PD-L1-Fc was used to capture BiTE-05.
  • Captured ACE-05 and human IgG were detected using an Alexa 647-conjugated anti-kappa antibody (#316514, Novus).
  • BiTE-05 was detected using an Alexa 647-conjugated anti-His antibody (#362611, Novus).
  • the anti-tumor efficacy of ALiCE against PD-L1 + HCC827 tumors was evaluated in a PBMC-reconstituted humanized NCG (NOD/scid IL-2R ⁇ null) mouse model. Specifically, 7-8 week old female NCG mice (Crown Bioscience) were randomly divided into 4 groups of 10 mice each. PBMCs of 5 ⁇ 10 6 cells/100 ⁇ l obtained from two healthy donors were implanted into the veins of each mouse. After 3 days, 5 ⁇ 10 6 PD-L1 + HCC827 tumor cells were inoculated subcutaneously on the right flank of the mouse.
  • mice When the tumor volume reached ⁇ 50 mm 3 (after 4 days), mice were injected with ACE-05 or BiTE-05 every other day (Q2d, total 3 doses), or YBL-007 or IgG every 3 days. (Q3d, total 3 doses). Tumor diameter and body weight were measured every 2 days and analyzed using GraphPad Prism 8 software.
  • Example 18 In vivo cytokine analysis
  • 6-7 week old female non-tumor-bearing hCD3 ⁇ TG mice were divided into 4 groups of 6 mice each. Thereafter, 0.5 mg/kg of ACE-05 and BiTE-05 were administered to tail venous blood to each group, and 5 mg/kg of IgG was administered.
  • Blood samples for cytokine analysis were collected from each animal at 0, 6, 12, 24 and 48 hours and stored at -80°C.
  • Various cytokines present in each collected plasma sample were analyzed according to the protocols of BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (# 552364, BD Bioscience) and Mouse Th1 / Th2 Cytokine Kit (# 551287, BD Bioscience). Cytokine levels were analyzed using GraphPad Prism 8 software.
  • Tumor-infiltrating lymphocytes were analyzed in hCD3 ⁇ TG mice bearing CT26 tumors expressing hPD-L1. Specifically, 5 ⁇ 10 5 CT26-hPD-L1 cells were inoculated subcutaneously on the right flank of the mouse. When the subcutaneous tumor volume reaches ⁇ 90 mm 3 , start the intraperitoneal (ip) administration of the test formulation (ACE-05, 1 mg/kg; YBL-007, 3 mg/kg; UCHT1, 2 mg/kg) and , It was administered twice a week for 2 weeks (BIW, a total of 4 administrations). Tumors were obtained from mice one week after the last injection. Live lymphocytes and T cells in tumor tissue were analyzed by flow cytometry using lymphocyte markers (mCD45, mCD4, mCD8 and hCD3). The results were analyzed using GraphPad Prism 8 software.
  • Example 20 In silico immunogenicity prediction
  • Example 21 Examples of fusion proteins according to targets
  • This example shows exemplary multispecific fusion proteins as provided herein, in particular multispecific fusion proteins ACE-00, ACE-02, ACE-03, ACE-04, ACE-05, ACE-09, ACE- 10, ACE-11, ACE-12, ACE-18, ACE-19, ACE-31 and many other multispecific fusion proteins have been disclosed.
  • ACE-00r, ACE-02r, ACE-03r, ACE-04r, ACE-05r, ACE-09r, ACE-10r, ACE- 11r, ACE-12r, ACE-18r, ACE-19r, ACE-31r, and many other multispecific fusion proteins are illustrated.
  • Components targeting the first and second antigens in each exemplary multispecific fusion protein are summarized in Table 4 below.
  • Multispecific fusion proteins Targeting component First antigen binding domain Second antigen binding domain ACE-00 Trastuzumab (Anti-Her2 Ab)
  • Adalimumab ACE-01 Anti-CD19 Ab Anti-CD3 mouse OKT3 ACE-02 Anti-CD19 Ab Anti-CD3 humanized 12F6 ACE-03 Anti-CD19 Ab Anti-CD3 humanized OKT3 ACE-04 Anti-PD-L1 Ab Anti-CD3 chimeric OKT3 Fab ACE-05 Anti-PD-L1 Ab Anti-CD3 Ab ACE-06 Anti-PD-L1 Ab
  • Foralumab ACE-07 Anti-PD-L1 Ab
  • ITALICIZED antibody hinge region
  • ACE-00 contains two different heavy chain-like chains (ACE-00-VH and ACE-00-VL) and two identical light chains (ACE-00-LC).
  • the parent antibody used to construct the anti-Her2 domain of ACE-00 is trastuzumab
  • the parent antibody used to construct the anti-TNF alpha domain of ACE-00 is adalimumab.
  • the amino acid sequence of these three types of polypeptide is as follows:
  • ACE-00-LC amino acid sequence (anti-CD19 antibody light chain) :
  • VH and VL amino acid sequences for the bivalent Fab region targeting Her2 and the monovalent Fv region targeting TNF alpha are listed in Table 5:
  • VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 51)
  • VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR
  • CDR H1 GFNIKDTY
  • CDR L1 QDVNTA
  • CDR H2 IYPTNGYT
  • CDR L2 SAS (SEQ ID NO: 122)
  • CDR H3: SRWGGDGFYAMDY SEQ ID NO:
  • ACE-01 contains two different heavy chain-like chains (ACE-01-VH and ACE-01-VL) and two identical light chains (ACE-01-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain (divalent Fab region targeting CD19) and the second antigen-binding domain (monovalent Fv region targeting CD3) are shown in Table 6 below. Are listed:
  • VH QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 139)
  • VL DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 151)
  • CDR H3 ARRETTTVGRYYYY
  • ACE-02 contains two different heavy chain-like chains (ACE-02-VH and ACE-02-VL) and two identical light chains (ACE-02-LC).
  • the amino acid sequence of these three types of polypeptide is as follows:
  • ACE-02-LC amino acid sequence (anti-CD19 antibody light chain) :
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD19 and the second antigen binding domain monovalent Fv region of humanized 12F6 are listed in Table 7 below:
  • VH QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 61)
  • VL DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 65)
  • CDR H1 SYWMN
  • CDR H1 SYWMN
  • DNA sequences encoding ACE-02-VH, ACE-02-VL and ACE-02-LC have nucleic acid sequences of SEQ ID NOs: 100 to 102, respectively.
  • ACE-03 consists of anti-CD19 and humanized anti-CD3 OKT3 domains.
  • ACE-03 contains two different heavy chain-like chains (ACE-03-VH and ACE-03-VL) and two identical light chains (ACE-03-LC).
  • the amino acid sequence of these three types of polypeptide is as follows:
  • ACE-03-LC amino acid sequence (anti-CD19 antibody light chain) :
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD19 and the second antigen binding domain monovalent Fv region of humanized OKT3 are listed in Table 8 below:
  • VH QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 61)
  • VL DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 65)
  • CDR H1 SYWMN
  • CDR H1 SYWMN
  • DNA sequences encoding ACE-03-VH, ACE-03-VL and ACE-03-LC have nucleic acid sequences of SEQ ID NOs: 103, 104 and 102, respectively.
  • ACE-04 has two different heavy chains such as ACE-04-VH (VL-CL-VH-CH1) and ACE-04-VL (VH-CH1-VL-CL) and two identical light chains (ACE-04-LC ).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • ACE-04-LC amino acid sequence (anti-PD-L1 antibody light chain) :
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region of the chimeric OKT3 Fab region are listed in Table 9 below. have:
  • VH QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 4)
  • VL QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR
  • CDR H1 GGTFSSYA
  • CDR L1 SSNIGAGYD
  • CDR H2 IIPILGIA
  • CDR L2 GDI (SEQ ID NO: 10)
  • CDR H3 AKPRDGYNLVAFD
  • DNA sequences encoding ACE-04-VH, ACE-04-VL and ACE-04-LC have nucleic acid sequences of SEQ ID NOs: 105, 106 and 107, respectively.
  • ACE-05 contains two different heavy chain-like chains (ACE-05-VH and ACE-05-VL) and two identical light chains (ACE-05-LC).
  • ACE-05 contains a G4S linker (amino acid sequence of GGGS, SEQ ID NO: 112) in the flexible peptide region.
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 10 below. have:
  • DNA sequences encoding ACE-05-VH, ACE-05-VL and ACE-05-LC have nucleic acid sequences of SEQ ID NOs: 20, 21 and 22, respectively.
  • ACE-06 contains two different heavy chain-like chains (ACE-06-VH and ACE-06-VL) and two identical light chains (ACE-06-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • ACE-06-LC amino acid sequence (SEQ ID NO: 157)
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 11 below. have:
  • ACE-07 contains two different heavy chain-like chains (ACE-07-VH and ACE-07-VL) and two identical light chains (ACE-07-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 12 below. have:
  • ACE-08 contains two different heavy chain-like chains (ACE-08-VH and ACE-08-VL) and two identical light chains (ACE-08-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 13 below. have:
  • ACE-09 contains two different heavy chain-like chains (ACE-09-VH and ACE-09-VL) and two identical light chains (ACE-09-LC). Compared to ACE-05, ACE-09 does not contain a G4S linker (amino acid sequence of GGGS) in the flexible peptide region.
  • G4S linker amino acid sequence of GGGS
  • ACE-09-LC amino acid sequence (anti-PD-L1 antibody light chain): QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL [RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] ( SEQ ID NO: 95)
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 14 below. have:
  • VH QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 4)
  • VL QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR
  • CDR H1 GGTFSSYA
  • CDR L1 SSNIGAGYD
  • CDR H2 IIPILGIA
  • CDR L2 GDI (SEQ ID NO: 10)
  • CDR H3 AKPRDGYNLVAFD
  • the DNA sequences encoding ACE-09-VH, ACE-09-VL and ACE-09-LC have the nucleic acid sequences of SEQ ID NOs: 108, 109 and 107, respectively.
  • ACE-10 contains two different heavy chain-like chains (ACE-10-VH and ACE-10-VL) and two identical light chains (ACE-10-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the bivalent Fab region targeting CD20 and the monovalent Fv region targeting CD3 are listed in Table 15 below:
  • VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 26)
  • VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKR (SEQ ID NO: 30)
  • CDR H3 ARSTYYGGDWYFNV
  • DNA sequences encoding ACE-10-VH, ACE-10-VL and ACE-10-LC have nucleic acid sequences of SEQ ID NOs: 34, 35 and 36, respectively.
  • ACE-11 has the same overall structure as ACE-05 and ACE-10, and has two different heavy chain-like chains (ACE-11-VH and ACE-11-VL) and two identical light chains (ACE-11-LC). Includes.
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 16 below:
  • VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40)
  • VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR (SEQ ID NO: 44)
  • CDR H3 ARALTYYDYEFAY (SEQ ID NO: 43)
  • DNA sequences encoding ACE-11-VH, ACE-11-VL and ACE-11-LC have nucleic acid sequences of SEQ ID NOs: 48, 49 and 50, respectively.
  • ACE-12 comprises a G4S linker with the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 113) and GGSGGGGSG (SEQ ID NO: 114), while ACE-05 comprises a G4S linker with the GGGGS amino acid sequence of the flexible peptide region.
  • the amino acid sequences of these three types of polypeptides are as follows:
  • ACE-12-VH amino acid sequence (with 10 residues GGGGSGGGGS) :
  • ACE-12-VL amino acid sequence (with 9 residues GGSGGGGSG) :
  • ACE-12-LC amino acid sequence (anti-PD-L1 antibody light chain) :
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region of UCHT1 are listed in Table 17 below:
  • VH QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 4)
  • VL QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR
  • CDR H1 GGTFSSYA
  • CDR L1 SSNIGAGYD
  • CDR H2 IIPILGIA
  • CDR L2 GDI (SEQ ID NO: 10)
  • CDR H3 AKPRDGYNLVAFD
  • DNA sequences encoding ACE-12-VH, ACE-12-VL and ACE-12-LC have nucleic acid sequences of SEQ ID NOs: 110, 111 and 107, respectively.
  • ACE-13 contains two different heavy chain-like chains (ACE-13-VH and ACE-13-VL) and two identical light chains (ACE-13-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting PD-L1 and the second antigen-binding domain monovalent Fv region targeting PD-1 are shown in Table 18 below. It is listed:
  • ACE-14 contains two different heavy chain-like chains (ACE-14-VH and ACE-14-VL) and two identical light chains (ACE-14-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 19 below. Has :
  • ACE-15 contains two different heavy chain-like chains (ACE-15-VH and ACE-15-VL) and two identical light chains (ACE-15-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting PD-L1 and the second antigen-binding domain monovalent Fv region targeting CD3 are listed in Table 20 below. Has been:
  • ACE-16 contains two different heavy chain-like chains (ACE-16-VH and ACE-16-VL) and two identical light chains (ACE-16-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 21 below. Has been:
  • VH QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 4)
  • VL QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR
  • CDR H1 GGTFSSYA
  • CDR L1 SSNIGAGYD
  • CDR H2 IIPILGIA
  • CDR L2 GDI (SEQ ID NO: 10)
  • CDR H3 AKPRDGYNLVAFD
  • ACE-17 contains two different heavy chain-like chains (ACE-17-VH and ACE-17-VL) and two identical light chains (ACE-17-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 22 below. have:
  • ACE-18 contains two different heavy chain-like chains (ACE-18-VH and ACE-18-VL) and two identical light chains (ACE-18-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD20 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 23 below:
  • VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)
  • VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK
  • CDR H1 GYTFTSYN (SEQ ID NO: 223)
  • ACE-19 contains two different heavy chain-like chains (ACE-19-VH and ACE-19-VL) and two identical light chains (ACE-19-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 24 below:
  • VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)
  • VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
  • ACE-20 contains two different heavy chain-like chains (ACE-20-VH and ACE-20-VL) and two identical light chains (ACE-20-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 25 below:
  • ACE-21 contains two different heavy chain-like chains (ACE-21-VH and ACE-21-VL) and two identical light chains (ACE-21-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 26 below:
  • VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS (SEQ ID NO: 255)
  • VL AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKR (SEQ ID NO: 259)
  • CDR H3 ARDGITMVRGVMKDYFDY
  • ACE-22 contains two different heavy chain-like chains (ACE-22-VH and ACE-22-VL) and two identical light chains (ACE-22-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD20 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 27 below:
  • VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)
  • VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK
  • CDR H1 GYTFTSYN (SEQ ID NO: 223)
  • ACE-23 contains two different heavy chain-like chains (ACE-23-VH and ACE-23-VL) and two identical light chains (ACE-23-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 28 below. have:
  • ACE-24 contains two different heavy chain-like chains (ACE-24-VH and ACE-24-VL) and two identical light chains (ACE-24-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting HER2 and the second antigen binding domain monovalent Fv region targeting TNF are listed in Table 29 below:
  • VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 275)
  • VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 287)
  • CDR H1: GFNIKDTY SEQ ID NO: 276)
  • CDR L1: QDVNTA SEQ ID NO: 288)
  • CDR H2: IYPTNGYT SEQ ID NO: 277)
  • CDR H3: SRWGGDGFYAMDY SEQ ID NO: 27
  • ACE-25 contains two different heavy chain-like chains (ACE-25-VH and ACE-25-VL) and two identical light chains (ACE-25-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting HER2 and the second antigen binding domain monovalent Fv region targeting TNF are listed in Table 30 below:
  • VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 275)
  • VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 287)
  • CDR H1: GFNIKDTY SEQ ID NO: 276)
  • CDR L1: QDVNTA SEQ ID NO: 288)
  • CDR H2: IYPTNGYT SEQ ID NO: 277)
  • CDR H3: SRWGGDGFYAMDY SEQ ID NO: 27
  • ACE-26 contains two different heavy chain-like chains (ACE-26-VH and ACE-26-VL) and two identical light chains (ACE-26-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 31 below:
  • VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)
  • VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
  • ACE-27 contains two different heavy chain-like chains (ACE-27-VH and ACE-27-VL) and two identical light chains (ACE-27-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 32 below. have:
  • ACE-28 contains two different heavy chain-like chains (ACE-28-VH and ACE-28-VL) and two identical light chains (ACE-28-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 33 below:
  • VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)
  • VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
  • ACE-29 contains two different heavy chain-like chains (ACE-29-VH and ACE-29-VL) and two identical light chains (ACE-29-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 34 below:
  • VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)
  • VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
  • ACE-30 contains two different heavy chain-like chains (ACE-30-VH and ACE-30-VL) and two identical light chains (ACE-30-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 35 below. have:
  • VH EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)
  • VL DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
  • CDR H1 GYSFTGYT
  • CDR L1: QDIRNY CDR H2: INPYKGVS (SEQ ID NO: 327)
  • ACE-31 contains two different heavy chain-like chains (ACE-31-VH and ACE-31-VL) and two identical light chains (ACE-31-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 36 below. have:
  • VH EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)
  • VL DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
  • CDR H1 GYSFTGYT
  • CDR L1: QDIRNY CDR H2: INPYKGVS (SEQ ID NO: 327)
  • ACE-32 contains two different heavy chain-like chains (ACE-32-VH and ACE-32-VL) and two identical light chains (ACE-32-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 37 below:
  • ACE-33 contains two different heavy chain-like chains (ACE-33-VH and ACE-33-VL) and two identical light chains (ACE-33-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 38 below. have:
  • VH EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)
  • VL DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
  • CDR H1 GYSFTGYT
  • CDR L1: QDIRNY CDR H2: INPYKGVS (SEQ ID NO: 327)
  • ACE-34 contains two different heavy chain-like chains (ACE-34-VH and ACE-34-VL) and two identical light chains (ACE-34-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 39 below. have:
  • ACE-35 contains two different heavy chain-like chains (ACE-35-VH and ACE-35-VL) and two identical light chains (ACE-35-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 40 below. have:
  • ACE-36 contains two different heavy chain-like chains (ACE-36-VH and ACE-36-VL) and two identical light chains (ACE-36-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting PD-L1 and the second antigen-binding domain monovalent Fv region targeting CTLA-4 are shown in Table 41 below. It is listed:
  • ACE-37 contains two different heavy chain-like chains (ACE-37-VH and ACE-37-VL) and two identical light chains (ACE-37-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:
  • VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD20 and the second antigen binding domain monovalent Fv region targeting CTLA-4 are listed in Table 42 below. have:
  • VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)
  • VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK
  • CDR H1 GYTFTSYN (SEQ ID NO: 223)
  • ACE-38 contains two different heavy chain-like chains (ACE-38-VH and ACE-38-VL) and two identical light chains (ACE-38-LC).
  • the amino acid sequences of these three types of polypeptides are as follows:

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Abstract

La présente invention concerne une protéine de fusion multispécifique qui ne contient pas de région Fc et qui peut se lier particulièrement à deux antigènes ou plus. Dans un mode de réalisation, l'invention porte sur une protéine de fusion multispécifique comprenant les formules développées (I) et (II) ci-après : N'-A-L1-X-C' (I) ; et N'-B-L1-Y-C' (II). La protéine de fusion peut se lier à des cellules cancéreuses et à des cellules immuniitaires, et peut amplifier l'activité des cellules immunitaires. La protéine de fusion peut donc être utilisée comme agent anticancéreux.
PCT/KR2020/013805 2019-10-10 2020-10-08 Protéine de fusion multispécifique et utilisation associée Ceased WO2021071319A1 (fr)

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US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
WO2025050405A1 (fr) * 2023-09-08 2025-03-13 成都维瑾柏鳌生物医药科技有限公司 Utilisation d'une composition pharmaceutique pour la préparation d'un médicament contre une infection par le vih
EP4337794A4 (fr) * 2021-05-11 2025-07-02 Janux Therapeutics Inc Anticorps ciblant egfr et cd3 et leurs utilisations
US12433953B2 (en) 2020-04-04 2025-10-07 Janux Therapeutics, Inc. Compositions and methods related to tumor activated antibodies targeting EGFR and effector cell antigens

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12433953B2 (en) 2020-04-04 2025-10-07 Janux Therapeutics, Inc. Compositions and methods related to tumor activated antibodies targeting EGFR and effector cell antigens
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
EP4337794A4 (fr) * 2021-05-11 2025-07-02 Janux Therapeutics Inc Anticorps ciblant egfr et cd3 et leurs utilisations
WO2025050405A1 (fr) * 2023-09-08 2025-03-13 成都维瑾柏鳌生物医药科技有限公司 Utilisation d'une composition pharmaceutique pour la préparation d'un médicament contre une infection par le vih
CN117986367A (zh) * 2024-04-02 2024-05-07 上海美迪西生物医药股份有限公司 靶点为Nectin-4的抗体及其应用

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