WO2023036215A1 - Molécule de liaison à un antigène bispécifique et son utilisation - Google Patents

Molécule de liaison à un antigène bispécifique et son utilisation Download PDF

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WO2023036215A1
WO2023036215A1 PCT/CN2022/117704 CN2022117704W WO2023036215A1 WO 2023036215 A1 WO2023036215 A1 WO 2023036215A1 CN 2022117704 W CN2022117704 W CN 2022117704W WO 2023036215 A1 WO2023036215 A1 WO 2023036215A1
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seq
sequence
vhh
chain
antigen
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PCT/CN2022/117704
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English (en)
Chinese (zh)
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孙中翠
罗娟
武云
王玉霞
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上海齐鲁制药研究中心有限公司
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Priority to CN202280054801.9A priority Critical patent/CN117940460A/zh
Publication of WO2023036215A1 publication Critical patent/WO2023036215A1/fr

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present disclosure is in the field of immunology and generally relates to bispecific antigen binding molecules.
  • the present disclosure also relates to polynucleotides, vectors and host cells encoding such bispecific antigen binding molecules.
  • the present disclosure also relates to pharmaceutical compositions comprising bispecific antigen-binding molecules and their related applications in the treatment and prevention of cancer.
  • Bispecific antigen binding molecules capable of binding two antigens are known in the art. Such bispecific binding proteins can be produced by fusion of hybridoma cells, chemical conjugation or genetic engineering.
  • Bispecific antigen-binding molecules are of great interest for therapeutic applications because they allow simultaneous binding of two target antigens, thereby reducing the need for combination therapy.
  • Another application of bispecific antigen-binding molecules is as a recruiter of immune effector cells for tumor immunotherapy.
  • bispecific antigen binding molecules are designed that bind to surface antigens on target cells and to activating components on the surface of immune T cells. Simultaneous binding of this antibody to both of its targets will force a transient interaction between target cells and T cells, resulting in activation of any cytotoxic T lymphocytes and subsequent target cell lysis.
  • CD3 is a T cell co-receptor composed of four distinct chains. In mammals, a complex formed by CD3 multisubunits associates with molecules of the T cell receptor to generate an activation signal in T lymphocytes. In the absence of CD3, the T cell receptor (TCR) does not assemble properly and degrades. The study found that CD3 binds the membranes of all mature T cells and hardly any other cell types.
  • CD3 of the TCR complex on T cells has been used as a target to prompt the formation of an immunological synapse between T cells and tumor cells.
  • Co-binding of CD3 and tumor antigens activates T cells, thereby lysing tumor cells expressing tumor antigens.
  • IL-13 is mainly a 12KD pleiotropic cytokine produced by activated T cells, which can pass through IL-4R ⁇ , IL-13R ⁇ 1 and IL-13R ⁇ 2 in many cells such as B lymphocytes, endothelial cells, and monocytes.
  • a complex receptor system exerts the effect.
  • IL-13R ⁇ 1 has a weak ability to bind IL-13 alone, but it can form a high-affinity IL-13 receptor complex after forming a heterodimer with IL-4R ⁇ , and activate Janus protein kinase/signal transducer and The activator of transcription (JAK/STAT) signaling pathway functions.
  • IL-13 is closely related to many diseases such as asthma, eczema, fibrosis and tumors.
  • IL-13R ⁇ 2 binds to IL-13 more easily than IL-13R ⁇ 1, so it can compete with IL-13R ⁇ 1 for IL-13 and block the function mediated by JAK/STAT signaling pathway.
  • IL-13R ⁇ 2 is a glycosylated protein with a relative molecular weight of 56,000 Da, including 380 amino acids, and the homology of human and mouse protein sequences is 59%.
  • Human IL-13R ⁇ 1 has 33% homology with the extracellular region of IL-13R ⁇ 2, and the intracellular region of IL-13R ⁇ 2 has 17 amino acid residues, lacking the region interacting with JAK.
  • IL-13R ⁇ 2 is not expressed or expressed in a small amount in normal tissues (such as human testis). It is highly expressed in various cancer cells, such as human glioblastoma, melanoma, pancreatic cancer, ovarian cancer, etc.
  • glioma Human glioma is the most common intracranial malignant tumor, accounting for 48.3% of intracranial malignant tumors. The annual incidence rate is 3-10/100,000, the average survival rate is less than 15 months, and the 5-year survival rate is less than 3%.
  • IL-13R ⁇ 2 is not expressed in normal brain tissue, but expressed in glioma, and the higher the pathological grade, the stronger the expression.
  • IL-13R ⁇ 2 Patients with high expression of IL-13R ⁇ 2 have a poor prognosis and can be used as a specific target for the treatment of glioma.
  • the organization has successively prepared the drug IL-13-PE38 targeting human IL-13R ⁇ 2 and the drug targeting human IL-13R ⁇ 2.
  • IL-13-PE38 has achieved curative effect in the treatment of malignant tumors such as glioma, head and neck cancer, ovarian cancer and kidney cancer, and has been approved by the FDA to enter clinical treatment, but during the treatment, IL-13 -PE38 not only binds to IL-13R ⁇ 2 specifically expressed on the surface of tumor cells, but also binds to IL-13R ⁇ 1 expressed on the surface of normal tissue cells, damaging normal tissues and cells. Due to the lack of strict targeting, the further application of IL-13-PE38 is limited. Cell therapy and vaccine therapy targeting IL-13R ⁇ 2 are currently in development.
  • the present disclosure relates to a bispecific antigen binding molecule or fragment thereof comprising:
  • VHH antibody specifically binding to a first antigen selected from the group consisting of:
  • VHH(1) and VHH(2) Two VHH antibodies VHH(1) and VHH(2) that specifically bind to the first antigen, wherein VHH(1) and VHH(2) are the same or different;
  • scFv specifically binding to a second antigen, said scFv comprising VH and VL selected from:
  • an Fc domain comprising a first Fc domain subunit Fc(1) and a second Fc domain subunit Fc(2), the Fc(1) and Fc(2) forming a heterodimer;
  • the bispecific antigen-binding molecule is composed of two peptide chains, wherein 1 or 2 VHH antibodies and/or scFv antibodies are fused to the N-terminal or C-terminal of Fc(1) in the first peptide chain, and the second peptide chain 1 or 0 VHH antibodies and/or scFv antibodies in the chain fused to the N- or C-terminus of Fc(2); the bispecific antigen binding molecule provides monovalent or bivalent/bivalent binding to the first antigen , while the bispecific antigen-binding molecule provides monovalent binding to the second antigen; optionally, at least one peptide linker is further included.
  • connection sequence of the peptide chains or fragments of the bispecific antigen-binding molecule is selected from one of various modes: (i) the connection sequence of the first peptide chain is: the first peptide chain that binds the first antigen
  • the VHH antibody VHH(1) is fused at its C-terminus to the N-terminus of the scFv antibody, and the scFv antibody is fused at its C-terminus to the N-terminus of the first Fc domain subunit Fc(1);
  • the connection sequence of the peptide chains is: the second VHH antibody VHH(2) that binds to the first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit Fc(2); or
  • the connection sequence of a peptide chain is: the VHH(1) is fused at its C-terminus to the N-terminus of the Fc(1), and the Fc(1) is fused to the N-terminus of the scFv antibody at its C-termin
  • the bispecific antigen binding molecule provides monovalent or bivalent/bivalent binding to said first antigen. In other embodiments, the bispecific antigen binding molecule provides monovalent or bivalent/bivalent binding to said second antigen.
  • the VHH antibody that specifically binds the first antigen is a Nanobody; preferably, a humanized Camelidae Nanobody.
  • the scFv antibody specifically binding to the second antigen comprises VH and VL; preferably, the connection sequence is that the C-terminus of VH is fused to the N-terminus of VL.
  • connection sequence of the first peptide chain of the bispecific antigen-binding molecule or fragment thereof, from the N-terminal to the C-terminal is: VHH(1)-VH-VL-Fc(1), VHH( 1)-Fc(1)-VH-VL, VH-VL-Fc(1) or VHH(1)-VHH(2)-Fc(1); the connection sequence of the second peptide chain, starting from the N-terminal To the C-terminus: VHH(2)-Fc(2), VH-VL-Fc(2) or Fc(2).
  • the bispecific antigen-binding molecules or fragments thereof are connected using one or more peptide linkers, and the connection method of the first peptide chain, from the N-terminal to the C-terminal, is: VHH(1)- Peptide Linker-VH-Peptide Linker-VL-Peptide Linker-Fc(1), VHH(1)-Peptide Linker-Fc(1)-Peptide Linker-VH-Peptide Linker-VL, VH-Peptide Linker-VL-Peptide Linker -Fc(1) or VHH(1)-peptide linker-VHH(2)-Fc(1);
  • the connection method of the second peptide chain, from N-terminal to C-terminal is: VHH(2)-peptide linker - Fc(2), VH-peptide linker-VL-peptide linker-Fc(2) or peptide linker-Fc(2).
  • the peptide linker of the bispecific antigen-binding molecule or fragment thereof is selected from L1, L2, L3 or L4, wherein the sequence of L1 is shown in SEQ ID NO: 26, and the sequence of L2 is shown in SEQ ID As shown in NO:27, the sequence of L3 is shown in SEQ ID NO:28, and the sequence of L4 is shown in SEQ ID NO:29.
  • the L2 peptide linker is (G 4 S)xA, and the x is 3 or 4.
  • the bispecific antigen binding molecules or fragments thereof are connected using one or more peptide linkers, from N-terminus to C-terminus: VHH(1)-L1-VH-L2-VL-L3 -Fc(1), VHH(1)-L3-Fc(1)-L1-VH-L2-VL, VH-L1-VL-L3-Fc(1) or VHH(1)-L1-VHH(2) -L3-Fc(1); the connection method of the second peptide chain, from N-terminal to C-terminal: L4-Fc(2), VHH(2)-L3-Fc(2) or VH-L2- VL-L3-Fc (2).
  • the VHH antibody specifically binding to the first antigen targets a tumor cell surface antigen; preferably, the VHH antibody targets IL-13R ⁇ 2.
  • the VHH antibody targeting IL-13R ⁇ 2 comprises 3 HCDRs selected from the sequences shown in SEQ ID NO: 13-24. More preferably, comprising HCDR1 selected from SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19 or SEQ ID NO:22; and, selected from SEQ ID NO:14, SEQ ID NO:17 , HCDR2 shown in SEQ ID NO:20 or SEQ ID NO:23; and, selected from HCDR3 shown in SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21 or SEQ ID NO:24.
  • the bispecific antigen binding molecule or fragment thereof is capable of specifically binding IL-13R ⁇ 2, and its heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 13, ID NO:14 and SEQ ID NO:15; or SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18; or SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21; or SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • the bispecific antigen-binding molecule or its fragment can specifically bind IL-13R ⁇ 2, which comprises HCDR1, HCDR2 and HCDR3, and HCDR1, HCDR2 and HCDR3 are from SEQ ID NO: 9-12 heavy chain variable region.
  • said bispecific antigen binding molecule comprises variable region domains having at least 95% identity to the complementarity determining regions (CDRs) of SEQ ID NO: 9-12, said bispecific antigen binding molecule The specific antigen binding molecule specifically binds IL-13R ⁇ 2.
  • the bispecific antigen-binding molecules or fragments thereof are capable of specifically binding IL-13R ⁇ 2, comprising a heavy chain variable region (VHH), and the heavy chain variable region has the same expression as SEQ ID NO: 9-12. at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or 100% identity.
  • VHH heavy chain variable region
  • the bispecific antigen binding molecule or fragment thereof comprises a scFv antibody targeting a T cell surface antigen (second antigen); preferably, the scFv antibody is targeting CD3.
  • the CDR sequence of the scFv antibody targeting CD3 is shown in SEQ ID NO: 1-6; preferably, the sequence of HCDR1 is shown in SEQ ID NO: 1; the sequence of HCDR2 is shown in SEQ ID NO: 2; The sequence of HCDR3 is shown in SEQ ID NO: 3; the sequence of LCDR1 is shown in SEQ ID NO: 4; the sequence of LCDR2 is shown in SEQ ID NO: 5; the sequence of LCDR3 is shown in SEQ ID NO: 6.
  • the bispecific antigen-binding molecule or fragment thereof is capable of specifically binding to CD3, which comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are derived from the heavy chain variable region shown in SEQ ID NO:7; And it also comprises LCDR1, LCDR2 and LCDR3, and described LCDR1, LCDR2 and LCDR3 are from the light chain variable region shown in SEQ ID NO:8.
  • the scFv antibody targeting CD3 comprises a heavy chain variable region (VH) having a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and comprising a light chain variable region (VL) having at least 80%, 81%, 82%, 83%, 84% of the sequence shown in SEQ ID NO:8 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of sequence identity.
  • VH heavy chain variable region
  • VL light chain variable region
  • VH and VL may or may not use a peptide linker, and the connection sequence may be VH-VL or VL-VH.
  • the sequence of the scFv antibody is shown in SEQ ID NO:25.
  • the bispecific antigen binding molecule or fragment thereof comprises light chain and heavy chain variable region domains at least 95% identical to the complementarity determining regions (CDRs) in SEQ ID NO: 25, said The bispecific antigen binding molecule or fragment thereof specifically binds CD3.
  • the bispecific antigen binding molecule or fragment thereof comprises a first polypeptide chain and a second polypeptide chain comprising an amino acid sequence that specifically binds IL-13R ⁇ 2 as disclosed herein, and, the present disclosure Amino acid sequences that specifically bind CD3 are shown.
  • the bispecific antigen-binding molecule or fragment thereof further comprises heavy chain constant regions CH2 and CH3, and does not contain light chain constant region and/or heavy chain constant region CH1; preferably, the heavy chain constant region Including Fc domain or variant Fc; more preferably, Fc is of murine or human origin.
  • the Fc domain of the bispecific antigen-binding molecule or fragment thereof is an IgG Fc domain; preferably, the Fc domain is an IgG1 domain or an IgG4 domain.
  • the Fc domain comprises modifications that facilitate binding of said Fc(1) domain to said Fc(2) domain.
  • the two polypeptide sequences constituting the Fc domain have different sequences from each other, and the amino acid residues at position 366 of the Knob chains constituting the two polypeptides constituting the Fc domain are mutated to tryptophan according to the EU numbering.
  • the amino acid at position 366 is mutated to serine, the amino acid at position 368 is mutated to alanine, and the amino acid at position 407 is mutated to valine; more preferably, the Knob chain includes S354C and T366W amino acid substitutions
  • the Hole chain includes Y349C, T366S, L368A and Y407V amino acid substitutions; further preferably, the Hole chain also includes H435R substitutions; still more preferably, the Fc segment also includes L234A and L235A substitutions.
  • the Fc(1) chain may be a Knob chain (Knob-Fc) or a Hole chain (Hole-Fc); the Fc(2) chain may be a Knob chain or a Hole chain.
  • the sequence of the Fc domain is as shown in SEQ ID NO:30 and/or SEQ ID NO:31, for example, Fc(1) is as shown in SEQ ID NO:30, and the Fc(2) As shown in SEQ ID NO:31.
  • the bispecific protein molecule further comprises a first polypeptide chain and a second polypeptide chain, the sequence of the first polypeptide chain is as SEQ ID NO: 32-35, 40, 42 or 43 As shown in any one, and/or the sequence of the second polypeptide chain is shown in any one of SEQ ID NO: 36-39, 41 or 44; preferably, the first polypeptide of the bispecific protein molecule
  • the peptide chain and the second polypeptide chain are selected from one of the combinations: a.
  • the sequence of the first polypeptide chain is as shown in SEQ ID NO:32, and the sequence of the second polypeptide chain is as shown in SEQ ID NO:36 shown; or b.
  • the sequence of the first polypeptide chain is as shown in SEQ ID NO: 33, and the sequence of the second polypeptide chain is as shown in SEQ ID NO: 37; or c. the sequence of the first polypeptide chain The sequence is as shown in SEQ ID NO:34, and the sequence of the second polypeptide chain is as shown in SEQ ID NO:38; or d. the sequence of the first polypeptide chain is as shown in SEQ ID NO:35, and the sequence The sequence of the second polypeptide chain is as shown in SEQ ID NO:39; or e. the sequence of the first polypeptide chain is as shown in SEQ ID NO:33, and the sequence of the second polypeptide chain is as shown in SEQ ID NO :39; or f.
  • the sequence of the first polypeptide chain is as shown in SEQ ID NO:40, and the sequence of the second polypeptide chain is as shown in SEQ ID NO:36; or g. the first polypeptide
  • the sequence of the chain is as shown in SEQ ID NO:32, and the sequence of the second polypeptide chain is as shown in SEQ ID NO:41; or h.
  • the sequence of the first polypeptide chain is as shown in SEQ ID NO:42, And the sequence of the second polypeptide chain is as shown in SEQ ID NO:36; or i. the sequence of the first polypeptide chain is as shown in SEQ ID NO:43, and the sequence of the second polypeptide chain is as shown in SEQ ID NO:43 ID NO:44 shows.
  • the bispecific protein molecule or bispecific antigen binding molecule comprises at least 95% identity to a complementarity determining region (CDR) in SEQ ID NO: 32-35, 40, 42 or 43 A heavy chain variable (VH) domain and at least 80%, 81%, 82%, 83%, 84%, 85% with a complementarity determining region (CDR) in SEQ ID NO: 36-39, 41 or 44 , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain A variable (VL) domain, the bispecific protein molecule or bispecific antigen binding molecule specifically binds IL-13R ⁇ 2 and CD3.
  • CDR complementarity determining region
  • the present disclosure also protects a polynucleotide encoding the bispecific antigen-binding molecule or fragment thereof or the bispecific protein molecule of the present disclosure.
  • the polynucleotide is an isolated polynucleotide.
  • the present disclosure also protects a recombinant vector comprising the polynucleotide of the present disclosure.
  • the present disclosure also protects a host cell comprising the polynucleotide of the present disclosure or the recombinant vector of the present disclosure.
  • the present disclosure also protects methods of producing the disclosed bispecific antigen binding molecules or fragments thereof or bispecific protein molecules.
  • the present disclosure also protects a pharmaceutical composition, comprising a pharmaceutically acceptable carrier, and comprising the bispecific antigen binding molecule of the present disclosure or its fragment, the bispecific protein molecule of the present disclosure, the polynucleotide of the present disclosure, the recombinant vector of the present disclosure and/or One or more of the group consisting of host cells of the present disclosure.
  • the present disclosure also protects the use of bispecific antigen binding molecules or fragments thereof, bispecific protein molecules, polynucleotides, recombinant vectors, host cells or pharmaceutical compositions in the preparation of drugs for treating and/or preventing diseases in individuals in need thereof
  • the disease is cancer; more preferably, the cancer is an IL-13R ⁇ 2 positive tumor.
  • the cancer may be selected from glioma, melanoma, pancreatic cancer, ovarian cancer, kidney cancer, head and neck cancer, breast cancer, leukemia, aggressive lymphoma, non-Hodgkin's lymphoma, cervical cancer, rectal cancer, liver cancer, Lung cancer or stomach cancer etc.
  • the present disclosure also protects the use of bispecific antigen binding molecules or fragments thereof, bispecific protein molecules, polynucleotides, recombinant vectors, host cells or pharmaceutical compositions to treat and/or prevent diseases in individuals in need; preferably , the disease is cancer; more preferably, the cancer is an IL-13R ⁇ 2 positive tumor.
  • the cancer may be selected from glioma, melanoma, pancreatic cancer, ovarian cancer, kidney cancer, head and neck cancer, breast cancer, leukemia, aggressive lymphoma, non-Hodgkin's lymphoma, cervical cancer, rectal cancer, liver cancer, Lung cancer or stomach cancer etc.
  • the present disclosure also protects a method for treating and/or preventing a disease related to the expression of IL-13R ⁇ 2 in a subject, comprising administering to a patient in need a therapeutically and/or preventively effective amount of the bispecific antigen-binding molecule or Its fragment, bispecific protein molecule, polynucleotide, recombinant vector, host cell or pharmaceutical composition; preferably, the disease related to the expression of IL-13R ⁇ 2 is cancer; more preferably, the cancer is selected from glioma , melanoma, pancreatic cancer, ovarian cancer, kidney cancer, head and neck cancer, breast cancer, leukemia, aggressive lymphoma, non-Hodgkin's lymphoma, cervical cancer, rectal cancer, liver cancer, lung cancer or gastric cancer, etc.
  • Figure 1 shows a schematic diagram of a bispecific antigen-binding molecule, wherein A in Figure 1 is Format-1, B is Format-2, C is Format-3, D is Format-4, and E is Format-5; There may be one or two VHH antibodies that sexually bind to the first antigen, and when there are two, they may be the same or different.
  • Figure 2 shows the binding of the bispecific antigen-binding molecule to the tumor cell line A375 cells highly expressing IL-13R ⁇ 2 detected by flow cytometry.
  • Figure 2A shows the binding experiments of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules in different formats
  • Figure 2B shows the binding experiments of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules and IL-13R ⁇ 2 monovalent bispecific antigen-binding molecules
  • Fig. 2C is the binding experiment of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules with different anti-IL-13R ⁇ 2 antibody monoclonal antibody sequences in the same format.
  • Figure 3 shows the binding of the bispecific antigen-binding molecule to the tumor cell line U87 cells highly expressing IL-13R ⁇ 2 detected by flow cytometry.
  • Figure 4 shows the binding of the bispecific antigen-binding molecule to the tumor cell line U251 cells highly expressing IL-13R ⁇ 2 detected by flow cytometry.
  • Figure 5 shows the binding of the bispecific antigen-binding molecule to the T cell line Jurkat cells with high expression of CD3 detected by flow cytometry.
  • Figure 5A shows the binding experiments of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules in different formats
  • Figure 5B shows the binding experiments of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules and IL-13R ⁇ 2 monovalent bispecific antigen-binding molecules
  • FIG. 5C is the binding experiment of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules with different anti-IL-13R ⁇ 2 antibody monoclonal antibody sequences in the same format.
  • Figure 6 shows the binding of the bispecific antigen-binding molecule to the stable cell line (cynomolgus IL-13R ⁇ 2-CHOK1 cells) highly expressing cynomolgus IL-13R ⁇ 2 detected by flow cytometry.
  • Figure 7 shows the killing effect of bispecific antigen-binding molecules detected by LDH method on tumor cells when T cells were co-incubated with A375 tumor cell line cells highly expressing human IL-13R ⁇ 2.
  • Figure 7A is the killing experiment of IL-13R ⁇ 2 monovalent or bivalent bispecific antigen-binding molecules on A375 cells
  • Figure 7B is the killing experiment of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules with different fomats on A375 cells
  • Figure 7C is the same IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules with different anti-IL-13R ⁇ 2 antibody monoclonal antibody sequences in different formats kill A375 cells.
  • Figure 8 shows the killing effect of the bispecific antigen-binding molecules detected by the LDH method on tumor cells when T cells were co-incubated with the MCF-7 tumor cell line that does not express human IL-13R ⁇ 2.
  • Figure 9 shows the killing effect of the bispecific antigen-binding molecules detected by the LDH method on tumor cells when T cells were co-incubated with the U87 tumor cell line that highly expresses human IL-13R ⁇ 2.
  • Figure 10 shows the killing effect of bispecific antigen-binding molecules detected by LDH method on tumor cells when T cells were co-incubated with U251 tumor cell line cells highly expressing human IL-13R ⁇ 2.
  • FIG. 11 shows the release of cytokines IFN ⁇ and IL-2 when T cells were co-incubated with A375 tumor cell line cells highly expressing human IL-13R ⁇ 2, as detected by flow cytometry.
  • 11A shows the release of the cytokine IFN ⁇
  • FIG. 11B shows the release of the cytokine IL-2.
  • Figure 12 shows the release of cytokines IFN ⁇ and IL-2 when T cells were co-incubated with MCF-7 tumor cell line cells not expressing human IL-13R ⁇ 2, as detected by flow cytometry.
  • Figure 12A shows the release of the cytokine IFN ⁇
  • Figure 12B shows the release of the cytokine IL-2.
  • Figure 13 shows the activation of T cells detected by flow cytometry when the bispecific antigen-binding molecules were co-incubated with the A375 tumor cell line that highly expresses human IL-13R ⁇ 2.
  • Figure 14A shows the activation of Jurkat cells when the bispecific antigen-binding molecules detected by the chemiluminescence method were co-incubated with the A375 tumor cell line that highly expresses human IL-13R ⁇ 2
  • Figure 14B shows the activation of the Jurkat cells by the chemiluminescence method When the detected bispecific antigen-binding molecules were co-incubated with Jurkat-NFAT cells and U251 tumor cell line cells that highly express human IL-13R ⁇ 2, the activation of Jurkat cells
  • Figure 14C shows the binding of bispecific antigens detected by chemiluminescence Molecular activation of Jurkat cells when Jurkat-NFAT cells were co-incubated without other tumor cells.
  • Figure 15 shows the killing effect of bispecific antigen-binding molecules detected by LDH method on tumor cells when NK cells were co-incubated with A375 tumor cell line cells highly expressing cynomolgus monkey human IL-13R ⁇ 2.
  • Figure 16 shows the detection of anti-tumor efficacy of bispecific antigen-binding molecules in the mouse A375 model reconstituted from human PBMCs.
  • Figure 16A shows the change of tumor volume after administration
  • Figure 16B shows the change of mouse body weight after administration.
  • Figure 17 shows the detection of anti-tumor efficacy of bispecific antigen-binding molecules in the mouse U251 model reconstituted from human PBMCs.
  • Figure 17A shows the change of tumor fluorescence signal value (Radiance) after administration
  • Figure 17B shows the change of mouse body weight after administration.
  • antigen binding molecule refers to a molecule that specifically binds an antigenic determinant.
  • antigen binding molecules are immunoglobulins and derivatives, eg fragments thereof.
  • bispecific means that the antigen binding molecule is capable of specifically binding two different antigenic determinants.
  • a bispecific antigen binding molecule comprises two antigen binding sites, each of the two antigen binding sites is specific for a different antigenic determinant.
  • a bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, especially two antigenic determinants expressed on two different cells.
  • an antigen refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment.
  • an antigen can include any immunogenic fragment or determinant of a selected target, including single-epitope, multi-epitope, single-structure domains, multiple domains, or complete extracellular domains (ECDs) or proteins.
  • ECDs extracellular domains
  • Peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof can constitute antigens.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens, among others.
  • Antigen can also refer to a molecule that elicits an immune response.
  • antigen or cells or preparations containing the antigen can be used to generate antibodies specific for an antigenic determinant.
  • the antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein) or protein Constructs (eg, Fc antigens).
  • the antigen can be produced in genetically modified cells. Any of the foregoing antigens may be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art.
  • the DNA encoding the antigen may be genomic or non-genomic (eg, cDNA), and may encode at least a portion of the ECD sufficient to elicit an immunogenic response.
  • Any vector may be used to transform the cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids that are juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents.
  • Epitopes generally exist in a unique spatial conformation and comprise at least 3-15 amino acids. Methods for determining the epitope bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, among others. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as x-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • polypeptide peptide
  • protein protein
  • the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length.
  • the polymer may be linear, cyclic or branched, it may comprise modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • amino acid polymers that have been modified, for example, by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, elimination Amino acid polymers modified by spinylation, selenoylation, transfer-RNA-mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with labeling components.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide.
  • the term also includes polypeptides expressed from a specified nucleic acid sequence.
  • amino acid modification includes amino acid substitutions, insertions and/or deletions in a polypeptide sequence.
  • amino acid substitution or “substitution” or “substitution” herein means the replacement of an amino acid at a specified position in a parent polypeptide sequence with another amino acid.
  • substitution S32A refers to the replacement of serine at position 32 with alanine.
  • Specifically binds means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind a specific epitope can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument ) (Liljeblad et al., Glyco J, 17, 323-329 (2000)) and traditional binding assays (Heeley Endocr, Res, 28, 217-229 (2002)).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • Binding affinity refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, an antigen-binding portion and an antigen, or a receptor and its ligand).
  • KD dissociation constant
  • equivalent affinities can contain different rate constants as long as the ratio of rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A specific method used to measure affinity is Surface Plasmon Resonance (SPR).
  • bivalent/bivalent bispecific antigen-binding molecule refers to a bispecific antigen-binding molecule that has two antigen-binding regions for a certain target, such as IL-13R ⁇ 2 bivalent/bivalent bispecific antigen The binding molecule means that the bispecific antigen-binding molecule contains two antigen-binding regions for IL-13R ⁇ 2.
  • bispecific antigen-binding molecule refers to a bispecific antigen-binding molecule that has only one antigen-binding region for a certain target, for example, IL-13R ⁇ 2. Contains an antigen-binding domain for IL-13R ⁇ 2.
  • Antigen binding site refers to the site of an antigen binding molecule, ie, one or more amino acid residues, which provide for the interaction with the antigen.
  • the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs).
  • antibody herein is used in the broadest sense to encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.
  • the term "antigen-binding molecule that specifically binds IL-13R ⁇ 2”, “anti-IL-13R ⁇ 2 antibody”, “antibody targeting IL-13R ⁇ 2” refers to an antibody that is capable of binding to IL-13R ⁇ 2 with sufficient affinity IL-13R ⁇ 2 protein or a fragment thereof, and does not significantly bind IL-13R ⁇ 1, so that the antibody can be used as a diagnostic and/or therapeutic agent targeting IL-13R ⁇ 2.
  • anti-CD3 antibody or “antibody targeting CD3” refers to a CD3 chain that can specifically bind to a single CD3 chain (such as CD3 ( ⁇ ) chain, CD3 ( ⁇ ) chain or CD3 ( ⁇ ) chain) or is composed of two CD3 chains. Complexes of one or more individual CD3 chains (e.g. complexes of more than one CD3( ⁇ ) chain, complexes of CD3( ⁇ ) and CD3( ⁇ ) chains, CD3( ⁇ ) chains CD3( ⁇ ) chain complex) antibodies.
  • the anti-CD3 antibody is capable of specifically binding CD3( ⁇ ), CD3( ⁇ ) or CD3( ⁇ ) or any combination thereof, more preferably, is capable of specifically binding CD3( ⁇ ).
  • Anti-human CD3 antibody and “anti-hCD3 antibody” refer to an antibody that can specifically bind to human-derived CD3.
  • sequence identity or “sequence similarity” or “sequence homology” means that the sequences are aligned (and introducing gaps where necessary) for maximum percent sequence identity, without any The percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after conservative substitutions are considered part of the sequence identity. Alignment of sequences to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • VHH variable domain of heavy chain of heavy-chain antibody
  • the VHH may have an additional disulfide bond between CDR1 and CDR3 in dromedaries, and between CDR2 and CDR3 in llamas, in addition to the regular disulfide bonds (Harmsen and De Haard, 2007, Appl Microbiol Biotechnol., 77, 13-22; Muyldermans, 2001, J Biotechnol., 74, 277-302).
  • the enlarged CDR3 loop can adopt a convex conformation, whereas the conventional paratope is restricted to a concave or planar structure (Muyldermans, 2001, J Biotechnol., 74, 277-302). These features allow VHHs to recognize unique epitopes that are less immunogenic for conventional antibodies (Lafaye, 2009, Mol Immuno., 46, 695-704; Wernery, 2001, J Vet Med B Infect Dis Vet Public Health., 48 , 561-568).
  • VHH is defined as a monovalent antibody, any avidity effect is excluded by default, the biological activity measured as in vitro IC50 can be similar to that of conventional bivalent antibody molecules (Thys et al., 2010, Antiviral Res., 87, 257-264) .
  • VHH antibody “heavy chain variable region (VHH)” and “Nanobody VHH” are used interchangeably.
  • the present disclosure may relate to chimeric camelid/human antibodies, particularly wherein the VH and/or VL domains are entirely camelid sequences (e.g. llama or alpaca), The rest of the antibody is a chimeric antibody that is completely human sequence.
  • "humanized” or “germlined” camelid antibodies and camelid/human chimeric antibodies are also included, wherein the VH and/or VL domains are relative to those obtained by active immunization Camelidae VH and/or VL domains comprising one or more amino acid substitutions in the framework regions.
  • the present disclosure includes natural, recombinant VHH or VH.
  • VHHs according to the present disclosure can be in the form of monomers or homomultimers, such as homodimers or homotrimers.
  • Antibodies of the present disclosure include camelid antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • chimeric antibody is a construct in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, and this or these The remainder of the chain is identical or homologous to the corresponding sequence in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies.
  • chimeric antibodies comprise all or a majority of selected murine heavy and light chain variable regions operably linked to human light and heavy chain constant regions. Constant region sequences or variants or derivatives thereof can be operably associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide anti-IL- Full-length antibody to 13R ⁇ 2.
  • humanized antibody is a hybrid immunoglobulin containing minimal sequence derived from non-human immunoglobulin, immunoglobulin chains or fragments thereof.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the CDRs of the recipient are replaced by a non-human species with the desired specificity, affinity, and properties (donor antibody). ), such as mouse, rat, rabbit or primate.
  • donor antibody such as mouse, rat, rabbit or primate.
  • framework region residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • back mutations can be introduced into humanized antibodies in which residues in one or more FRs of the variable region of the recipient human antibody are replaced by corresponding residues from the donor antibody of a non-human species.
  • Such back mutations can help maintain the proper three-dimensional configuration of the grafted CDR(s) and thus improve affinity and antibody stability.
  • Antibodies from various donor species including, but not limited to, mouse, rat, rabbit or non-human primate can be used.
  • humanized antibodies may contain novel residues not found in the recipient antibody or in the donor antibody in order to further improve antibody properties.
  • Single domain antibody also known as Nanobody
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 refer to framework regions (Frame) 1-4 respectively
  • CDR1-CDR3 refer to complementarity determining regions 1-3 respectively.
  • VHH refers to the variable antigen-binding domain of heavy chain antibodies from Camelidae (camelidae, dromedary, llama, alpaca, etc.) (see Nguyen, 2000 EMBO J., 19, 921-930; Muyldermans, 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot, 2011, Antiviral Res., 92, 389-407).
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (for example via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv may have the VL and VH variable regions described in any order (e.g. relative to the N- and C-terminus of the polypeptide), the scFv may include VL-peptide linker-VH or may include VH-peptide linker-VL .
  • IgA Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chain constant domains are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • IgG and IgA can be further divided into different For example, IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • this constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain, CH4).
  • CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length.
  • Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.
  • Fc is used herein to define the C-terminal region of an immunoglobulin heavy chain, ie, the dimer-forming two polypeptide chains comprising a C-terminal constant region of an immunoglobulin heavy chain capable of stabilizing association with itself.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxy-terminus of the heavy chain, e.g., an IgG Fc domain comprising IgG CH2 and IgG CH3 constant domains.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Modifications that promote association of the first and second subunits of an Fc domain are manipulations of the peptide backbone or post-translational modifications of an Fc domain subunit that reduce or prevent a polypeptide comprising that Fc domain subunit from being identical to The peptides combine to form homodimers.
  • Binding-promoting modifications as used herein include, inter alia, separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) to which binding is desired, wherein the modifications are complementary to each other so that Promotes association of two Fc domain subunits.
  • modifications that promote binding may alter the structure or charge of one or both of the Fc domain subunits in order to render their binding sterically or electrostatically favorable, respectively.
  • (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may occur in other components fused to each subunit (e.g. a Fab fragment).
  • the binding-promoting modification comprises amino acid mutations, specifically amino acid substitutions, in the Fc domain.
  • the binding-promoting modification comprises separate amino acid mutations, in particular amino acid substitutions, in each of the two subunits of the Fc domain.
  • both the CH3 domain of the first polypeptide chain of the Fc domain and the CH3 domain of the second polypeptide chain of the Fc domain are engineered in a complementary manner such that each CH3 domain (or comprises Its heavy chain) can no longer homodimerize with itself but is forced to heterodimerize with complementary engineered other CH3 domains (so that the first and second CH3 domains heterodimerize and the two first CH3 domains or no homodimers are formed between the two second CH3 domains).
  • the modification that promotes the association of the first polypeptide chain and the second polypeptide chain of the Fc domain is a so-called “knob-into-hole” (Knob-into-Hole) modification, which is contained in A "knob” modification in one of the two polypeptide chains of the Fc domain and a "hole” modification in the other of the two polypeptide chains of the Fc domain.
  • the method involves introducing a bump ("knob") at the interface of a first polypeptide chain and a corresponding cavity ("cavity") in the interface of a second polypeptide chain, so that the bump can be placed in the cavity This promotes heterodimer formation and hinders homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide chain with larger side chains such as tyrosine or tryptophan.
  • a complementary cavity of the same or similar size as the bump is created in the interface of the second polypeptide chain by replacing large amino acid side chains with smaller amino acid side chains (eg alanine or threonine).
  • one amino acid residue is replaced with an amino acid residue with a larger side chain volume , thereby creating a bulge in the CH3 domain of the first polypeptide chain, which can be placed in the cavity in the CH3 domain of the second polypeptide chain, and in the CH3 domain of the second polypeptide chain of the Fc domain, an amino acid residue
  • the base is replaced with an amino acid residue with a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second polypeptide chain, in which the bulge within the CH3 domain of the first polypeptide chain can be accommodated.
  • said amino acid residues with larger side chain volumes are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • said amino acid residues with smaller side chain volumes are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
  • Protuberances and cavities can be generated by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.
  • Knob-Fc in the present disclosure refers to a point mutation containing T366W in the Fc region of an antibody to form a Knob-like spatial structure.
  • “Hole-Fc” refers to mutations in the Fc region of the antibody including T366S, L368A, and Y407V to form a hole-like spatial structure. Knob-Fc and Hole-Fc are more likely to form heterodimers due to steric hindrance. In order to reduce the generation of hole-hole homodimers, the H435R mutation of the hole can reduce the binding of protein A during purification.
  • the S354C and Y349C site mutations can also be introduced into Knob-Fc and Hole-Fc, respectively, to further promote the formation of heterodimers through disulfide bonds.
  • substitution mutations of 234A and 235A can also be introduced into Fc.
  • the preferred Knob-Fc and Hole-Fc of the present disclosure are shown in SEQ ID NO: 30 and 31, respectively.
  • Knob-Fc and Hole-Fc can serve as both the Fc region of the first polypeptide chain and the Fc region of the second polypeptide chain.
  • the Fc regions of the first polypeptide chain and the second polypeptide chain are not both Knob-Fc and Hole-Fc.
  • Homologous and “heterologous” used in this article are a set of relative concepts, which can refer to the same or different sources of different elements in the construct, or refer to the original source of the same or “homologous” element after the construction of the construct is completed. Among the multiple elements of ", some elements have been transformed and have changed compared with other original elements that have not been transformed, thus becoming “heterogeneous”.
  • Fusion means that the components (eg scFv antibody and Fc domain subunit) are joined by peptide bonds, either directly or via one or more peptide linkers.
  • linker refers to any means for joining two different functional units (eg antigen-binding fragments). Types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of the polypeptide linker ("peptide linker") is not limited. Polypeptide linkers are preferably non-immunogenic and flexible, such as those comprising serine and glycine sequences. Linkers can be long or short depending on the particular construct.
  • linkers linking different functional units preferably comprise flexible peptide linkers, such as glycine-serine peptide linkers.
  • the peptide linker comprises the amino acid sequence (G4S)x or (G4S)xA, wherein x is any integer selected from 1-6, preferably comprises the amino acid sequence (G4S) 3 or (G4S) 3A .
  • the peptide linker linking the VH and VL domains to form a VH-VL or a VL-VH scFv domain preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker.
  • the peptide linker comprises the amino acid sequence (G4S)x or (G4S)xA, wherein x is any integer selected from 1-6, preferably comprising the amino acid sequence G4S (SEQ ID NO: 45), (G 4 S) 3 (SEQ ID NO:46), (G 4 S) 4 (SEQ ID NO:49), (G 4 S) 3 A (SEQ ID NO:47) or (G 4 S) 4 A (SEQ ID NO:48).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • engineing includes any manipulation of the backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Modifications include modification of amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these methods.
  • composition refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered. .
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient or vehicle with which the therapeutic agent is administered.
  • adjuvant eg, Freund's adjuvant (complete and incomplete)
  • an effective amount refers to the dose of a pharmaceutical formulation comprising an active ingredient of the present disclosure which, when administered to a patient in single or multiple doses, produces the desired effect in a treated patient.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as ethnic differences; body weight, age and health; the particular disease involved; the severity of the disease; the response of the individual patient; The specific antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom, regardless of passage number. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.
  • a host cell is any type of cellular system that can be used to produce the bispecific antigen binding molecules of the disclosure.
  • Host cells include cultured cells, such as mammalian cultured cells, such as Jurkat cells, PBMC cells, A375 cells, U251 cells and U87 cells or hybridoma cells, yeast cells, insect cells and plant cells, etc., as well as those contained in transgenic animals, transgenic A cell in a plant or cultured plant or animal tissue.
  • mammalian cultured cells such as Jurkat cells, PBMC cells, A375 cells, U251 cells and U87 cells or hybridoma cells, yeast cells, insect cells and plant cells, etc.
  • transfection refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • stable transfection or “stable transfection” refers to the introduction and integration of exogenous nucleic acid, DNA or RNA into the genome of a transfected cell.
  • stable transfectant refers to a cell that has stably integrated foreign DNA into genomic DNA.
  • isolated polynucleotide refers to a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment.
  • a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present disclosure.
  • Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • An isolated polynucleotide includes a polynucleotide molecule contained in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the disclosure, as well as positive and negative strand and double-stranded forms.
  • An isolated polynucleotide or nucleic acid of the present disclosure also includes such molecules produced synthetically.
  • a polynucleotide or nucleic acid may or may not include regulatory elements, such as promoters, ribosomal binding sites, or transcription terminators.
  • the antibody or its antigen-binding fragment described in the invention uses genetic engineering methods to add one or more human FR regions to the non-human CDR region.
  • Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website (http://imgt.cines.fr), or from The Immunoglobulin Facts Book (2001) ISBN: 012441351 .
  • Antibodies or antigen-binding fragments thereof engineered in the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid that secretes the antibody can be purified and collected using conventional techniques.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange.
  • subject refers to any animal, such as a mammal or a marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (such as cynomolgus or rhesus or other types of rhesus monkeys), mice, pigs, horses, donkeys, cows, sheep, rats, and any species of poultry.
  • the term “disease” or “condition” or “disorder” and the like refers to any change or disorder that damages or interferes with the normal function of a cell, tissue or organ.
  • the “disease” includes, but is not limited to: tumor, pathogenic infection, autoimmune disease, T cell dysfunction disease, or immune tolerance deficiency (such as transplant rejection).
  • treatment refers to clinical intervention in an attempt to alter the course of a disease caused by an individual or a cell, either for prevention or for intervention in the course of clinical pathology.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing down the progression of the disease, improving or mitigating the condition, mitigating or improving the prognosis, etc.
  • the DNA sequence was determined by double-strand sequencing.
  • VL light chain variable region
  • VH heavy chain variable region
  • Table 1 Exemplary anti-CD3 CDR, VH, VL or scFv sequences are shown in Table 1:
  • the molecular structure of the bispecific antigen-binding molecule in some embodiments disclosed herein is shown in Figure 1 A-E, which consists of two peptide chains, and the bispecific antigen-binding molecule comprises: Nanobody VHH that specifically binds to the first antigen , an scFv antibody that specifically binds a second antigen, an Fc domain capable of forming first and second subunits of a stable heterodimer, and one or more peptide linkers ( Figure 1, C and D); or comprising: A first and a second Nanobody VHH specifically binding to a first antigen (wherein the first and second Nanobody VHH may be the same or different), an scFv antibody specifically binding to a second antigen, capable of forming a stable heterodimer The Fc domains of the first and second subunits and one or more peptide linkers (A, B, and E of FIG.
  • Bispecific antigen binding molecules provide monovalent binding to the second antigen, and, monovalent or bivalent binding to the first antigen.
  • the nanobody VHH that specifically binds to the first antigen targets tumor cell surface antigens
  • the scFv antibody that specifically binds to the second antigen targets T cell surface antigens.
  • the molecular structure (Format) of the specific bispecific antigen-binding molecule is shown in Table 3 below, and Format 1-5 in Table 3 correspond to A-E in Figure 1.
  • L1, L2, L3, and L4 represent peptide linkers for connecting the respective antigen-binding domains as well as the Fc region.
  • the peptide linker used to connect the antigen-binding domain and the Fc region can be selected from any other peptide linker that can be used to connect the antibody functional domain, and is not limited to the peptide linker limited by the following sequence.
  • the peptide linker is shown in Table 4, including amino acid sequences (G 4 S)x(SEQ ID NO:26), (G 4 S)xA(SEQ ID NO:27), EPKSSDKTHTCPPCP(SEQ ID NO:28 ) or DKTHTCPPCP (SEQ ID NO:29), wherein x is any integer from 1 to 6, preferably, x is 3, and the amino acid sequence of the peptide linker comprises (G 4 S) 3 or (G 4 S) 3 A.
  • the repeating unit G 4 S sequence number is SEQ ID NO:45.
  • the bispecific antigen-binding molecule can also include an Fc region (ie, an Fc domain), as shown in Table 3, which can be Knob-Fc and Hole-Fc.
  • the Fc region can maintain the normal half-life and good stability of the antibody.
  • the bispecific antigen-binding molecule can use an Fc domain that contains a modification that promotes the binding of the first and second Fc domain subunits. Through the design of two chains, the mismatching is greatly reduced. For the purpose of probability, the uniformity of the sample and the yield of the target antibody are improved.
  • the bispecific antigen-binding molecule is a bispecific heterodimer, consisting of a Knob-Fc chain and a Hole-Fc chain, and the structure of the Knob-Fc chain includes amino acid substitutions at two positions of S354C and T366W , the structure of the Hole-Fc chain, including the amino acid substitutions at four positions of Y349C, T366S, L368A and Y407V.
  • the Hole-Fc chain can also be substituted with H435R.
  • amino acid substitutions of L234A and L235A can also be carried out in the Fc segment.
  • the modified Knob-Fc chain (Fc(1)) sequence is shown in SEQ ID NO:30
  • the Hole-Fc chain (Fc(2)) sequence is shown in SEQ ID NO:31 .
  • the molecular structures of the bispecific antigen-binding molecules 002-1, 039, 040, 042, 044, 005-1, 025, 027 and 003 in Format-1, Format-2, Format-3, Format-4 or Format-5 are as follows: As shown in A-E of Figure 1 and Table 3, the specific sequences of bispecific antigen-binding molecules 002-1, 039, 040, 042, 044, 005-1, 025, 027 and 003 are shown in Table 6.
  • the target gene fragments encoding the first polypeptide chain and the second polypeptide chain of the aforementioned bispecific antigen-binding molecule were respectively cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid, the first polypeptide chain (chain 1) :
  • the second polypeptide chain (chain 2) was 1:1, transfected into ExpiCHO cells (Thermo Fisher Scientific), seeded the cells in shake flasks (Corning Company), and placed in an environment of 37°C and 8% CO 2 Grow on a shaker. After 8-10 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and protein A column was used for affinity purification.
  • A375 (ATCC, Cat. No.: CRL-1619), U87 (ATCC, Cat. No.: HTB-14) and U251 (Chinese Academy of Sciences, Cat. No.: TcHu 58 ) tumor cell lines that highly express human IL-13R ⁇ 2, and stable transfection of cynomolgus monkey IL -13R ⁇ 2 cynomolgus monkey IL-13R ⁇ 2-CHOK1 cell line (namely, Cyno IL-13R ⁇ 2-CHO-K1), after digesting with 0.25% trypsin (containing EDTA), complete medium was added to terminate the digestion reaction. Centrifuge at 1500 rpm for 5 minutes to discard the supernatant, resuspend in PBS containing 1% BSA and count.
  • bispecific antigen-binding molecules obtained in Example 1 were taken, and bispecific antigen-binding molecule solutions were prepared in PBS containing 1% BSA, and diluted in multiples to obtain samples at multiple concentration points.
  • IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules could bind to cell lines that highly express human or cynomolgus IL-13R ⁇ 2, and there was a concentration-dependent effect.
  • IL-13R ⁇ 2 monovalent bispecific antigen-binding molecules Binding to cell lines that highly express human IL-13R ⁇ 2 also has a concentration-dependent effect, but its binding is weaker than that of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules. It indicates that IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules, compared with IL-13R ⁇ 2 monovalent bispecific antigen-binding molecules, have more obvious binding advantages on cells with high expression of IL-13R ⁇ 2, and will have a better safety window .
  • bispecific antigen-binding molecules bind to the Jurkat cell line in a concentration-dependent manner.
  • the binding EC 50 values of bispecific antigen-binding molecules with different design methods are close to those of Jurkat.
  • This example studies the T cell killing ability of bispecific antigen binding molecules in vitro.
  • the killing effect of T cells on tumor cells mediated by bispecific antigen binding molecules is realized by detecting the level of lactate dehydrogenase (LDH). Lactate dehydrogenase exists in the cell, and when the cell is damaged, it will be quickly released into the cell culture medium. By detecting the activity of LDH in the cell culture supernatant, the degree of cell damage can be judged, which is suitable for many kinds of cytotoxicity analysis. Killing experiments can also be achieved by quantitatively detecting cell proliferation.
  • Use cell titer-glo to detect the content of ATP in the cells. ATP is an indicator of the metabolism of living cells and is directly proportional to the number of cells in the culture.
  • melanoma cell line A375 ATCC, catalog number: CRL-1619
  • glioma cell line U251 Choinese Academy of Sciences, catalog number: TcHu 58
  • U87 ATCC, catalog number: HTB-14
  • Tumor cell lines and an MCF-7 cell line ATCC, HTB-22
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules with the same antigen-binding domain but different formats, such as Format-1 (002-1), Format-2 (005-1), Format-5 (003)
  • Format-1 002-1
  • Format-2 (005-1)
  • Format-5 003
  • the tumor cell killing activity was tested in parallel, and the results showed that IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules with different structural sequences all had significant killing effects on A375 cells.
  • the killing activity of bispecific antigen-binding molecule 003 and 005-1 was slightly lower than that of bispecific antigen-binding molecule 002-1.
  • the experimental results are shown in Figure 7B and Table 9.
  • bispecific antigen-binding molecules 002-1, 039, 040 and 044 were examined.
  • the results of parallel experiments showed that the bispecific antigen-binding molecules 002-1, 039, 040 and 044 all had obvious killing activity, and the activity of 044 was slightly stronger than that of 002-1, 039 and 040.
  • the experimental results are shown in Figure 7C and Table 10.
  • This example investigates the in vitro T cell activation of bispecific antigen binding molecules.
  • the bispecific antigen binding molecule not only kills tumor cells, but also activates T cells.
  • ELISA is used to detect the levels of cytokines IFN ⁇ and IL-2 in the cell culture supernatant, and in addition, it is realized by detecting the levels of CD25 and CD69, the activated surface markers on the surface of T cells.
  • Two different target cells were shared, including the melanoma cell line A375 and a MCF-7 cell line that does not express IL-13R ⁇ 2 but expresses IL-13R ⁇ 1, and the effector cells were T cells isolated from PBMCs of healthy volunteers. The target cells were seeded in a 96-well plate and cultured overnight. On the next day, an equal amount of freshly extracted PBMC or T cells and serially diluted bispecific antigen-binding molecules to be tested were added to each well.
  • cytokine release results of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules 002-1, 005-1 and 003 in the co-incubation of A375 or MCF-7 tumor cell lines with T cells were tested.
  • 002-1, 005-1 and 003 can significantly increase the levels of cytokines IFN ⁇ and IL-2 in a dose-dependent manner, and the release level of cytokines can be combined with bispecific antigens
  • the killing activity of the molecules is positively correlated, and the experimental results are shown in Figure 11 and Table 12.
  • CD25 and CD69 uses A375 cells, and other systems are the same as cytokine release experiments. After 24 hours of culture, PBMC or T cells were collected, CD25 and CD69 fluorescently labeled antibodies were added, after incubation, free antibodies were washed away, and the expression levels of CD25 and CD69 on the surface of T cells were analyzed by flow cytometry. According to the proportion of CD25 and CD69 double-positive cells on the surface of T cells, the data were processed and analyzed with Graphpad Prism5.
  • the ratio of CD25 and CD69 double-positive cells of IL-13R ⁇ 2 bivalent bispecific antigen-binding molecule 002-1 on the system in which A375 was co-incubated with T cells was tested, in which NC-hG1AA was (Shanghai Baiying Bio, Cat. No.: B109802 ) negative control.
  • NC-hG1AA was (Shanghai Baiying Bio, Cat. No.: B109802 ) negative control.
  • the experimental results are shown in Figure 13 and Table 13, 002-1 can dose-dependently increase the levels of CD25 and CD69 on the surface of T cells.
  • the NFAT-driven luciferase reporter gene system was constructed on Jurkat cells to obtain Jurkat-NFAT recombinant cell lines.
  • Experiment 2 utilized the Jurkat-NFAT recombinant cell line to detect the expression of NFAT-driven luciferase reporter gene after Jurkat activation in the presence or absence of the A375/U251 tumor cell line.
  • a 96-well cell culture plate 4 ⁇ 10 5 /mL, 100 ⁇ L/well
  • 50 ⁇ L of Jurkat cell suspension (1 ⁇ 10 6 /mL)
  • 50 ⁇ L of serially diluted bispecific antigen-binding molecule to each well, place at 37°C, and incubate 6 hours.
  • the activation of non-tumor cell-specific Jurkat recombinant cells is to directly add Jurkat recombinant cells and the antibody to be tested to a blank 96-well culture plate.
  • IL-13R ⁇ 2 bivalent bispecific antigen-binding molecules 002-1, 039, 040, 042 and 044 were tested for activation of Jurkat recombinant cells in the presence or absence of A375/U251 to verify bispecific antigen binding
  • the experimental results are shown in Figures 14A, 14B and 14C, and Table 14, all bispecific antigen-binding molecules can effectively activate Jurkat recombination in the presence of A375 ( Figure 14A) or U251 ( Figure 14B) tumor cell lines cell line, significantly induced the expression of luciferase, the negative control antibody could not induce the expression of luciferase.
  • the activation of Jurkat recombinant cells requires the co-recruitment of Jurkat recombinant cells expressing CD3 and tumor cells expressing IL-13R ⁇ 2 through bispecific antigen-binding molecules.
  • luciferase The expression of is relatively low, and only some weak signals can be detected at high concentration points (Fig. 14C).
  • Detecting whether the Fc-mutated bispecific antigen-binding molecule 002-1 has an ADCC effect can be achieved by detecting the level of lactate dehydrogenase (LDH). Lactate dehydrogenase exists in the cell, and when the cell is damaged, it will be quickly released into the cell culture medium. By detecting the activity of LDH in the cell culture supernatant, the degree of cell damage can be judged, which is suitable for many kinds of cytotoxicity analysis.
  • LDH lactate dehydrogenase
  • A375 cells on a 96-well cell culture plate (1 ⁇ 10 5 /mL, 100 ⁇ L/well), and culture overnight at 37° C. in an environment of 5% CO 2 .
  • the next day isolate NK cells from fresh PBMCs, remove the cell culture supernatant in the 96-well plate, add 50 ⁇ L of NK cell suspension (1 ⁇ 10 6 /mL) to each well, and 50 ⁇ L of serially diluted
  • NC-IgG1AA was used as a negative control.
  • LDH or CTG was used to detect, the signal value was read by a microplate reader, and finally converted into the percentage of cell killing, and the data was processed and analyzed with Graphpad Prism5.
  • IL-13R ⁇ 2 monoclonal antibody hI-8 with wild-type Fc has a dose-dependent ADCC effect, and the corresponding bispecific antigen-binding molecule 002-1 and negative None of the control antibodies had significant ADCC effect.
  • the NOG mouse (Beijing Weitong Lihua Experimental Animal Co., Ltd.) A375 model reconstituted with human PBMC was used to evaluate the anti-tumor efficacy of bispecific antigen-binding molecules in mice.
  • A375 cells were inoculated subcutaneously in the right axilla of NOG mice, with an inoculation volume of 0.1 mL/mouse, and PBMCs were inoculated into NOG mice by tail vein injection on the same day.
  • G1 NC-hIgG1AA (1000 ⁇ g/kg) group
  • G2 002-1 (1000 ⁇ g /kg) group
  • G3 002-1 (200 ⁇ g/kg) group
  • G4 002-1 (40 ⁇ g/kg) group
  • G5 002-1 (8 ⁇ g/kg) group.
  • the way of administration is intraperitoneal injection (ip), once a week, for 3 consecutive administrations. Tumor volume and animal weights were monitored and data recorded twice a week.
  • the bispecific antigen-binding molecule 002-1 1000 ⁇ g/kg had a certain tumor-inhibiting effect after 7 days of administration, and the tumors in all mice completely regressed after 14 days of administration, and maintained until The experiment is over.
  • the tumor inhibition rate (TGI) reached 99.6%, 60% of the mice's tumors regressed, and 87% of the mice's tumors regressed at the end of the experiment.
  • Example 7 Drug efficacy experiment of the mouse U251 model rebuilt by human PBMC
  • the U251-luc orthotopic model of NCG mice reconstituted with human PBMCs was used to evaluate the anti-tumor efficacy of bispecific antigen-binding molecules in mice.
  • U251-luc cells Karl, Cat. No. CBP30207L
  • human PBMCs were inoculated into the tail veins of mice to establish human-derived immune cells to reconstitute small mouse model. The mice were randomly grouped according to the tumor volume, and the administration began on the day of grouping (D0).
  • G1 NC1-hG1AA (1000 ⁇ g/kg) group
  • G2 002-1 group (300 ⁇ g/kg)
  • G3 002-1 (1000 ⁇ g/kg) group.
  • the administration method is intravenous administration (iv), 2 times a week, 4 consecutive administrations. Tumor volume and animal weights were monitored and data recorded twice a week.
  • the experimental results are shown in Figure 17.
  • the bispecific antigen-binding molecule 002-1 at 1000 ⁇ g/kg and 300 ⁇ g/kg already had a certain tumor-inhibiting effect 5 days after administration, and the tumor inhibition rates were 92% at 10 days after administration. % and 90%, and there are significant differences compared with the control group. At the end of the experiment, the tumor inhibition rate was 99.6%.
  • the antibody has three transition regions, Tm1 corresponds to the unfolding temperature of the non-Fc part (higher Tm determines higher stability), Tm2 corresponds to the unfolding temperature of the CH2 domain of the Fc part, and T agg is the initiation Gather temperature.
  • Tm1 corresponds to the unfolding temperature of the non-Fc part (higher Tm determines higher stability)
  • Tm2 corresponds to the unfolding temperature of the CH2 domain of the Fc part
  • T agg is the initiation Gather temperature.
  • the thermal stability of different antibodies in pH7.4PBS buffer was detected by NanoDSF (micro differential fluorescence scanning technology).
  • the sample concentration is about 1mg/mL, and it is detected by Prometheus NT.Plex instrument.
  • Each sample was centrifuged at 10,000g for 10 minutes before detection. Add 40 ⁇ L of sample to each well of the sample plate (the instrument loading volume is 10 ⁇ L, and each sample has a duplicate well).
  • the scanning temperature starts at 30°

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Abstract

L'invention concerne une molécule de liaison à un antigène bispécifique ou un fragment de liaison à l'antigène de celle-ci, un dérivé contenant la molécule de liaison à un antigène bispécifique ou un fragment de liaison à l'antigène de celle-ci, et une composition pharmaceutique. L'invention concerne en outre l'utilisation associée de la molécule de liaison à un antigène bispécifique ou du fragment de liaison à l'antigène de celle-ci dans le traitement de cancers et dans le dépistage et le diagnostic.
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