WO2023046116A1 - Complexe polypeptidique d'interleukine 15 et son récepteur - Google Patents

Complexe polypeptidique d'interleukine 15 et son récepteur Download PDF

Info

Publication number
WO2023046116A1
WO2023046116A1 PCT/CN2022/121068 CN2022121068W WO2023046116A1 WO 2023046116 A1 WO2023046116 A1 WO 2023046116A1 CN 2022121068 W CN2022121068 W CN 2022121068W WO 2023046116 A1 WO2023046116 A1 WO 2023046116A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
il15rα
antibody
seq
mutation
Prior art date
Application number
PCT/CN2022/121068
Other languages
English (en)
Chinese (zh)
Inventor
芦迪
霍永庭
路力生
Original Assignee
广东菲鹏制药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202111123534.XA external-priority patent/CN113943374B/zh
Application filed by 广东菲鹏制药股份有限公司 filed Critical 广东菲鹏制药股份有限公司
Publication of WO2023046116A1 publication Critical patent/WO2023046116A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the invention relates to the field of biomedicine, in particular to a disulfide bond modified polypeptide complex comprising interleukin 15 and its receptor.
  • Cytokines play an important role in human immune regulation, and also participate in the immune regulation of tumors, which are closely related to the occurrence and development of tumors.
  • cytokines can directly act on immune effector cells in the tumor microenvironment to enhance the tumor suppressive effect.
  • many cytokines have been proven to have significant anti-tumor activity, and several cytokines have been approved by the FDA for marketing.
  • Interleukin 15 is a cytokine of about 12-14kD discovered by Grabstein et al. in 1994, which can play a role in the normal immune response of the body, such as promoting T cells, B cells, natural killing Proliferation of (NK) cells.
  • IL-15 is a member of the small four ⁇ -helix bundle family of cytokines. IL-15 exerts its biological activity through binding to its receptor.
  • the IL-15 receptor consists of three receptor subunits: IL-15 receptor ⁇ (IL-15R ⁇ ), IL-2 receptor ⁇ (IL-2R ⁇ , also known as IL-15R ⁇ or CD122) and ⁇ c (also known as CD132).
  • IL-15R ⁇ contains a Sushi domain, which can bind IL-15 and is necessary for the biological function of IL-15 after binding.
  • IL15 and IL15 receptors have been more used to construct fusion proteins.
  • this application introduces a disulfide bond between interleukin 15 and its receptor.
  • the present invention provides an IL15/IL15R ⁇ polypeptide complex, there is a non-natural interchain bond between IL15 and IL15R ⁇ , and the non-natural interchain bond is formed by the first mutation residue of IL15 and the second mutation of IL15R ⁇ Among the residues, the first mutated residue of IL15 is E at position 90 to C, and the second mutated residue of IL15R ⁇ is P at position 67 to C.
  • the amino acid residue mutation site of IL15 refers to the natural sequence numbering site corresponding to SEQ ID NO:26, and the amino acid residue mutation site of IL15R ⁇ refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
  • the D mutation at position 61 of the IL15 is N
  • the E mutation at position 64 is Q
  • the N mutation at position 65 is D
  • the N-glycosylation site of the IL15 is absent; in some embodiments, the N-glycosylation site is selected from N71, N79 and/or N112; in some embodiments, the IL15 comprises The following amino acid mutations: N71Q, N79Q and/or N112Q; and/or at least one O-glycosylation site of IL15R ⁇ does not exist; in some embodiments, the O-glycosylation site is selected from T2, T81 and/or or T86; in some embodiments, the IL15R ⁇ comprises the following amino acid mutations: T2A, T81A and/or T86A;
  • amino acid residue mutation site of IL15 refers to the natural sequence numbering site corresponding to SEQ ID NO:26
  • amino acid residue mutation site of IL15R ⁇ refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
  • the IL15/IL15R ⁇ polypeptide complex described in any one of the above is the amino acid sequence shown in SEQ ID NO.84 or its mutant sequence; in some embodiments, the mutant sequence includes Amino acid mutation selected from D61N, E64Q and/or N65D, and/or amino acid mutation selected from N71Q, N79Q and/or N112Q; the amino acid residue mutation site of IL15 refers to the natural sequence numbering position corresponding to SEQ ID NO:26 point.
  • the IL15/IL15R ⁇ polypeptide complex described in any one of the above comprises SEQ ID NO.28 or its mutant sequence, SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or Its mutant sequence, SEQ ID NO.79 or its mutant sequence, SEQ ID NO.80 or its mutant sequence, or SEQ ID NO.81 or its mutant sequence; Amino acid mutations of T81A and/or T86A; in some embodiments, the mutant sequence includes a P67C amino acid mutation; in some embodiments, the mutant sequence includes a P67C amino acid mutation, and also includes an amino acid mutation; the amino acid residue mutation site of IL15R ⁇ refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
  • the mutant sequence includes P67C amino acid mutation;
  • the mutant sequence Including P67C amino acid mutation also includes amino acid mutation selected from T2A, T81A and/or T86A; in some embodiments, the mutant sequence includes amino acid mutation selected from T2A, T81A and/or T86A; the amino acid residue of IL15R ⁇
  • the base mutation site refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
  • the IL15/IL15R ⁇ polypeptide complex described in any one of the foregoing further comprises an antibody Fc constant region; in some embodiments, the antibody Fc constant region is a heterodimer; in some embodiments In this way, the Fc constant region of the antibody is based on KiH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction and/or increased flexibility to associate into a heterodimer; in some embodiments, the IL15 Or the C-terminus of IL15R ⁇ is connected to the N-terminus of the Fc constant region.
  • the IL15/IL15R ⁇ polypeptide complex according to any one of the above, the polypeptide complex is a bispecific fusion polypeptide, and the bispecific fusion polypeptide comprises a first antigen-binding moiety, wherein,
  • said first antigen-binding portion comprises: a first polypeptide comprising, from N-terminus to C-terminus, the first heavy chain variable domain VH1 of a first antibody operably linked to to IL15; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15R ⁇ ; or
  • said first antigen-binding portion comprises: a first polypeptide comprising, from N-terminus to C-terminus, the first heavy chain variable domain VH1 of a first antibody operably linked to to IL15R ⁇ ; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15.
  • the polypeptide complex further comprises a second antigen-binding moiety, wherein the second antigen-binding moiety comprises: a third polypeptide, wherein The third polypeptide comprises from the N-terminus to the C-terminus the second heavy chain variable domain VH2 of the second antibody, which is operably linked to the antibody heavy chain constant region CH1, and a fourth polypeptide, the fourth The polypeptide comprises from N-terminus to C-terminus the second light chain variable domain VL2 of the second antibody operably linked to the antibody light chain constant region CL.
  • the first antigen-binding portion and the second antigen-binding portion bind to different antigens or bind to different epitopes of the same antigen;
  • the first antigen-binding moiety targets immune cells and the second antigen-binding moiety targets tumor cells;
  • both the first antigen binding portion and the second antigen binding portion target tumor cells
  • both the first antigen binding moiety and the second antigen binding moiety target immune cells
  • the first antigen-binding portion targets human PD-L1
  • the second antigen-binding portion targets human TIGIT
  • the first antigen-binding portion targets human TIGIT
  • the second antigen-binding portion targets Human PD-L1.
  • the present invention also relates to an isolated nucleic acid encoding the IL15/IL15R ⁇ polypeptide complex according to any one of the preceding items.
  • the present invention also relates to a vector comprising a nucleic acid as described above.
  • the present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
  • the present invention also relates to a method for preparing the IL15/IL15R ⁇ polypeptide complex, comprising the steps of: transforming a host cell with the above-mentioned vector; cultivating the transformed host cell; and collecting the IL15/IL15R ⁇ polypeptide complex expressed in the host cell .
  • the present invention also relates to a pharmaceutical composition, which comprises the above-mentioned IL15/IL15R ⁇ polypeptide complex and a pharmaceutically acceptable carrier, excipient or stabilizer.
  • the present invention also relates to the application of the aforementioned IL15/IL15R ⁇ polypeptide complex or pharmaceutical composition in the preparation of medicines for treating diseases.
  • the present invention also relates to the IL15/IL15R ⁇ polypeptide complex or pharmaceutical composition described in any one of the preceding items for use as a medicament for treating diseases or conditions.
  • the present invention also relates to a method for treating diseases, the method comprising administering a therapeutically effective amount of the IL15/IL15R ⁇ polypeptide complex or pharmaceutical composition described above to a subject in need.
  • Figure 1 shows four classic double antibody platforms: Figure 1A shows the KiH heterodimerization Fc engineering technology; Figure 1B shows the CrossMab bispecific antibody technology; Figure 1C shows Wuhan Youzhiyou YBody double antibody technology (asymmetric scFv double antibody technology) anti); Figure 1D is a symmetric scFv double antibody;
  • Fig. 2 is a novel bispecific antibody FiBody provided by the present invention, in which CH1 and CL of Fab on one side are replaced by ligand receptors with specific affinity;
  • Figure 3 is an exemplary display of the 4 feasible schemes of FiBody:
  • Figure 3-1 is a modified ligand receptor with non-naturally occurring interchain bonds between ligand receptors;
  • Figure 3-2 is CH1, Fab on both sides. CL is replaced by receptors and ligands, and both sides are selected from different ligand receptors;
  • Figure 3-3 shows that CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and the CH3 segment in the Fc dimer is also replaced by ligand receptors.
  • Figure 3-4 shows that CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and CH2 in the Fc dimer is also replaced by ligand receptors; there are many other feasible transformation methods;
  • Figure 4 is an example of when the bispecific antibody of the present invention is used to treat tumors, the targeted binding of the antigen-binding part of the bispecific antibody includes exemplary three types: Figure 4-A The first antigen-binding part target To T cells, the second antigen-binding part targets tumor cells; Figure 4-B The first antigen-binding part and the second antigen-binding part both target tumor cells; Figure 4-C The first antigen-binding part and the second antigen-binding part Both target T cells; Figure 4-D exemplarily reflects that the bispecific antibody of the present invention can optionally be a trifunctional fusion protein, which can not only play different antigen binding, but also activate the ligand receptor pathway and stimulate the biological activity of the ligand receptor ;
  • Figure 5 is a three-dimensional conformation diagram of interleukin and its receptors, which can be divided into four categories: Type A is lifting type, Type B is bow-tie type, Type C is baseball player type, and Type D is pincer type;
  • Figure 6 is an example of four types of interleukins and their receptors in three-dimensional configurations.
  • Class A holds IL2/IL2R, B bow-tie IL22/IL22R, C bow-tie IL18/IL18R, and D clamps IL21/IL21R;
  • Figure 7 is the FiBody design based on IL15 (ligand) and IL15RA (receptor) in the embodiment of the present invention, the second antigen-binding region VH is connected to IL15RA, and the second antigen-binding region VL is connected to IL15;
  • Figure 8 is a schematic diagram of the optimized structure of disulfide bond reformation
  • Figure 9 is a schematic diagram of the three-dimensional structure of the interaction between IL15/IL15RA and IL2/15R ⁇ / ⁇ C complex;
  • Figure 10 is a schematic diagram of the structure of the mismatch molecule R1042/R1124 in the embodiment of the present invention.
  • Fig. 11 is the HPLC-SEC detection result of sample R0951 in the embodiment of the present invention.
  • Fig. 12 is the HPLC-SEC detection result of sample R1042 in the embodiment of the present invention.
  • Fig. 13 is the HPLC-SEC detection result of sample R0809 in the embodiment of the present invention.
  • Fig. 14 is the HPLC-SEC detection result of sample R1110 in the embodiment of the present invention.
  • Figure 15 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody TIGIT end and CHO-Tigit cells (R0950, R0951, R0952, R0954, R0955, R0960);
  • Figure 16 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R1123/R1119/R1120/R1124);
  • Figure 17 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R1042/R1043);
  • Figure 18 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R0810);
  • Figure 19 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody PD-L1 end and CHO-PD-L1 cells (R0950, R0951, R0952, R0954, R0955, R0960);
  • Figure 20 is the detection of the binding activity of double antibody PD-L1 end to CHO-PD-L1 cells (R1072, R1115-R1120, R1123-R1124) by FCM method in the embodiment of the present invention
  • Figure 21 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody PD-L1 end and CHO-PD-L1 cells (R0950, R1042, R1043);
  • Figure 22 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end and CHO-PD-L1 cells (R1072, R1081-R1086);
  • Figure 23 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody PD-L1 end and CHO-PD-L1 cells (R1072, R1109-R1111);
  • Figure 24 is the binding activity (R0950, R0951, R0952, R0954, R0955, R0960) of the sample receptor complex (IL15/IL15R) in the example of the present invention.
  • Figure 25 is the binding activity (R1042, R1043) of the sample receptor ligand complex (IL15/IL15R) in the embodiment of the present invention.
  • Figure 26 is the results of gel electrophoresis detection of disulfide bond transformation optimization samples in the embodiment of the present invention (R1072, R1081, R1082, R0954, R1084-R1086)
  • Figure 27 is a schematic diagram of the three-dimensional structure of the disulfide bond mutation pairing between IL15 (ligand) and IL15RA (receptor) molecules;
  • Figure 28 is a schematic diagram of the structure of an exemplary IL15/IL15R ⁇ complex
  • Figure 29 is the gel electrophoresis detection result of complex 1-4
  • Figure 30 is the detection result of IL15/IL15R ⁇ complex binding to the targeting region (@TIGIT);
  • Figure 31 shows the detection results of the IL15/IL15R ⁇ complex binding to the targeting region (@PD-L1).
  • antigen-binding portion or "antigen-binding domain” means the portion of an antigen-binding molecule that confers its binding specificity for an antigenic determinant.
  • the "antigen-binding portion” is a functional fragment of an antibody.
  • wild type or WT means the amino acid sequence or nucleotide sequence found in nature, including allelic variations.
  • a WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.
  • antibody encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific (bivalent) antibody or bispecific fusion polypeptide that binds a particular antigen.
  • a natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (“HCVR” or VH) and first, second, and third constant regions (CH1, CH2, CH3, respectively), and each light chain consists of a variable region (“LCVR” " or VL) and a constant region (CL).
  • HCVR variable region
  • LCVR variable region
  • Mammalian heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇
  • mammalian light chains can be classified as ⁇ or ⁇ .
  • Antibodies have a "Y" shape, with a backbone consisting of the second (CH2), third (CH3), and optionally fourth (CH4) constant regions of the two heavy chains, joined by disulfide bonds.
  • Each arm of the "Y"-shaped structure contains the variable region (VH) and the first constant region (CH1) of one of the heavy chains, which are combined with the variable region (VL) and constant region (CL) of one of the light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR), (the CDR of the light (L) chain includes LCDR1, LCDR2, LCDR3, and the CDR of the heavy (H) chain includes HCDR1, HCDR2, HCDR3.
  • CDR complementarity determining regions
  • the three CDRs are separated by parts called framework regions (FR), which are more highly conserved than CDRs and form a scaffold to support hypervariable loops.
  • HCVR and LCVR each contain 4 FRs, and the CDRs and FRs Arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but have various effector functions.
  • Antibodies can be classified into several classes based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms based on the presence or absence of alpha, delta, epsilon, gamma, and mu heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • the hypervariable region typically comprises amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the variable region of the light chain and 31-35B (HCDR1) in the variable region of the heavy chain , 50-65 (HCDR2) and 95-102 (HCDR3) amino acid residues (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed.
  • residues that form hypervariable loops such as residue 26 in the light chain variable region -32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917).
  • the antibody is a bispecific antibody (BiAb).
  • BiAb bispecific antibody
  • the term "bispecific” herein refers to two different antigens, or when the two are the same antigen, each of them has binding specificities for different epitopes. The epitopes may be derived from different antigens or from the same antigen.
  • the terms "bispecific fusion polypeptide” and “bispecific antibody” herein refer to all products made with full-length antibodies or fragments with antigen-binding sites.
  • the antibody can be a human antibody, a non-human antibody (such as a mouse-derived antibody), a humanized antibody, or a chimeric antibody (such as a human-mouse chimeric antibody or a chimeric antibody of different subtypes).
  • antibody variants are obtained by conservative modifications or conservative substitutions or substitutions on the antibody sequences provided by the present invention.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.
  • nucleotide and amino acid sequences indicate the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
  • Fab is a Fab fragment of an immunoglobulin that contains no or a small portion of the residual Fc fragment, eg, a Fab fragment includes the variable domains of the heavy and light chains, and all or part of the first constant domain.
  • Fc or “Fc region” or “Fc domain” means comprising the constant region of an antibody, in some cases excluding all or a portion of the first constant region immunoglobulin domain (e.g. CH1), and in some cases Polypeptides that are all or part of the hinge are further excluded.
  • Fc can refer to the last two constant region immunoglobulin domains (eg CH2 and CH3) of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM (eg CH2, CH3 and CH4) , and optionally all or a portion of the flexible hinge N-terminus of these domains.
  • Fc may contain a J chain.
  • the Fc domain comprises the immunoglobulin domains CH2 and CH3 (C ⁇ 2 and C ⁇ 3) and a lower hinge region located between CH1 (C ⁇ 1) and CH2 (C ⁇ 2).
  • the human IgG heavy chain Fc region is generally defined to include residues E216, C226 or A231 at its carboxy-terminus, with numbering according to the EU index as in Kabat.
  • amino acid modifications are made to the Fc region, eg, the Fc is a heterodimer.
  • Modification herein refers to amino acid substitutions, insertions and/or deletions in the polypeptide sequence or changes in the part chemically linked to the protein.
  • Amino acid modification herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are amino acids encoded by DNA, e.g., the 20 amino acids with codons in DNA and RNA.
  • Epitope means herein a determinant of interaction with a particular antigen-binding domain, eg, the variable region of an antibody molecule, called a paratope.
  • An epitope is a grouping of molecules such as amino acids or sugar side chains, and usually has specific structural characteristics as well as specific charge characteristics. A single molecule may have more than one epitope.
  • An epitope may comprise amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides; In other words, the amino acid residues are within the coverage area of the specific antigen binding peptide.
  • Epitopes can be either conformational or linear. Conformational epitopes result from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain.
  • a linear epitope is one that arises from contiguous amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished by the loss of binding to the former but not to the latter in the presence of denaturing solvents.
  • An epitope usually includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Antigen-binding molecules that recognize the same epitope can be validated in a simple immunoassay showing the ability of one antigen-binding molecule to block the binding of another antigen-binding molecule to the target antigen.
  • the present invention includes not only the antigen-binding molecules and antigen-binding domains recited herein, but also antigen-binding molecules and antigen-binding structures that compete for binding with epitopes that bind to the recited antigen-binding molecules or antigen-binding domains. area.
  • binding refers to a certain ligand that is directed and can be competitively blocked by the corresponding substance in vitro or in vivo.
  • Biological binding processes of structural site interactions Such as the binding between antigen and antibody or receptor and ligand.
  • KD dissociation constant
  • Binding properties can be determined by methods well known in the art, such as biolayer interferometry and surface plasmon resonance based methods.
  • One such method entails measuring the rates of association and dissociation of antigen-binding site/antigen or receptor/ligand complexes, where the rates depend on the concentration of the complex partner, the affinity of the interaction, and the equivalent in both directions. Geometric parameters that affect the velocity. Therefore, the association rate (ka) and the dissociation rate (kd) can be determined, and the ratio of kd/ka is equal to the dissociation constant KD ("Nature (Nature)" 361:186-187 (1993) and Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • Immune cell includes cells of the immune system involved in protecting the body against both infectious disease and foreign substances.
  • Immune cells can include, for example, neutrophils, eosinophils, basophils, lymphocytes, such as B cells and T cells, and monocytes.
  • T cells can include, for example, CD4+, CD8+, T helper cells, cytotoxic T cells, ⁇ T cells, regulatory T cells, suppressor T cells, and natural killer cells.
  • multifunctional fusion polypeptide means a non-naturally occurring binding molecule designed to target two or more antigens.
  • a “multifunctional fusion polypeptide” as described herein is typically a genetically engineered fusion protein, eg, designed to bring two different desired biological functions into a single binding molecule.
  • a multifunctional fusion polypeptide can be a multifunctional binding molecule.
  • the term “FiBody” refers to a bispecific fusion polypeptide or a multifunctional fusion protein obtained by substituting part or all of the constant region of a bispecific antibody with specific affinity for a ligand and its receptor.
  • the "YBody” technology mentioned in this invention was developed by Wuhan Youzhiyou Company in 2012. This technology is based on the "Knob-into-Holes” technology, and one of the heterodimers formed is a normal heavy chain , and the other one is the N-terminal link scFv of the Fc functional region to form an asymmetric bispecific antibody.
  • IL15/IL15R ⁇ polypeptide complex refers to a polypeptide complex comprising IL15 and IL15R ⁇ , and the IL15 specifically binds to IL15R ⁇ to form a polypeptide complex.
  • the words “comprising”, “including” and “containing” will be understood to mean the inclusion of stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements.
  • Consisting of means including and being limited to what the phrase “consisting of” follows. Thus, the phrase “consisting of” indicates that the listed elements are required or required and that no other elements may be present.
  • Consisting essentially of means including any element listed after the phrase, and being limited to other elements that contribute to or do not interfere with the activity or effect of the listed element as specified in the present invention. Thus, the phrase “consisting essentially of” means that the listed elements are required or essential, but that other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements. exist.
  • references throughout this disclosure to "one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “another embodiment,” or “further embodiments” ” or a combination thereof means that a particular feature, structure or characteristic described in relation to said embodiment is included in at least one embodiment of the present invention. Therefore, appearances of the foregoing terms in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
  • the present invention provides a novel bispecific fusion polypeptide, which comprises a ligand (or a fragment thereof) and a receptor (or a fragment thereof), and the ligand (or a fragment thereof) and a receptor (or a fragment thereof) are respectively independently replacing CH1 and CL of Fab on one side of the antibody, specifically, the bispecific fusion polypeptide comprises a first antigen-binding portion, and the first antigen-binding portion comprises: a first polypeptide, the first polypeptide is from N-terminus to C-terminus comprising the first heavy chain variable domain VH1 of the first antibody, which is operably linked to the first conjugated fragment; and a second polypeptide from N-terminus to C-terminus The end comprises the first light chain variable domain VL1 of the first antibody, which is operably linked to a second conjugated fragment; wherein the first conjugated fragment is a receptor and the second conjugated fragment is a ligand; or the first conjugated fragment is a ligand and the second conjugated fragment is
  • the bispecific fusion polypeptide has: the first polypeptide, which is sequenced from N-terminus to C-terminus: [VH1]-[Linker 1]-[IL15]-[Linker 2]- [CH2]-[CH3], the second polypeptide, from N-terminus to C-terminus: [VL1]-[Linker 3]-[[IL15RA];
  • the bispecific fusion The polypeptide has: the first polypeptide, which is in order from N-terminal to C-terminal: [VH1]-[Linker 1]-[IL15RA]-[Linker 2]-[CH2]-[CH3], the second polypeptide , which from N-terminus to C-terminus are: [VL1]-[Linker 3]-[[IL15].
  • the CH2 and CH3 are heavy chain constant region subunits, and the linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may or may not be the same; in some embodiments,
  • the linker 1, linker 2 and linker 3 are independently selected from (GxS)y linkers, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6.
  • the bispecific fusion polypeptide further comprises a second antigen-binding moiety that is different from the first antigen-binding moiety; the second antigen-binding moiety may be selected from:
  • CH1 and CL of the Fab on the other side of the antibody are replaced by another ligand (or its fragment) and its receptor (or its fragment), that is, the second antigen binding part includes: the third polypeptide, the The third polypeptide comprises from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to the third conjugated fragment, and a fourth polypeptide which The second light chain variable domain VL2 comprising the second antibody from the N-terminus to the C-terminus is operably linked to a fourth conjugated fragment; wherein the third conjugated fragment is a receptor, and the second conjugated fragment is The four conjugated fragments are ligands; or the third conjugated fragment is a ligand, and the fourth conjugated fragment is a receptor; the third conjugated fragment/fourth conjugated fragment is the same as the first conjugated fragment a conjugate fragment/second conjugate fragment are selected from different receptors/ligands, said third conjugate fragment and said fourth conjugate fragment are capable of specific binding; or
  • the Fab on the other side of the antibody retains the original CH1 and CL, that is, the second antigen-binding part includes: a third polypeptide, and the third polypeptide includes the second heavy chain of the second antibody from the N-terminus to the C-terminus Chain variable domain VH2, which is operably linked to antibody heavy chain constant region CH1, and a fourth polypeptide comprising from N-terminus to C-terminus the second light chain variable region of the second antibody. Domain VL2, which is operably linked to the antibody light chain constant region CL.
  • the present invention utilizes the specific binding force unique to the ligand and its receptor itself, and creatively connects it to the antigen-binding region (antibody variable region VH/VL), and the connection includes binding to one of the antigens region, another antigen-binding region is still connected to CH1 and CL; or both antigen-binding regions are connected to ligands/receptors, but the two antigen-binding regions are connected to different kinds of ligands/receptors, thereby avoiding different antigens
  • the light and heavy chains in the binding region are mismatched.
  • the bispecific fusion polypeptide provided by the present invention is a multifunctional fusion polypeptide comprising two Fabs, wherein CH1 and CL of one Fab are independently replaced by a ligand and its receptor, and the other The CH1 and CL of the Fab are not substituted, and the receptor includes not only the active site for recognizing and binding the ligand, but also the functional active site for generating a response; the light chain of the first antigen-binding part will not interact with the second Heavy chain mismatch of two antigen-binding portions.
  • CH1 and CL of one Fab are independently replaced by a first ligand and its receptor
  • CH1 and CL of the other Fab are independently replaced by a second ligand and its receptor
  • said The first ligand and its receptor are different from the second ligand and its receptor.
  • the multifunctional fusion protein can not only exert dual-target specificity, but also exert ligand/receptor conduction biological activity.
  • the ligand and its receptors are IL15 and IL15R ⁇ , and the multifunctional fusion polypeptide not only has a dual-target targeting effect, IL15R ⁇ can also present IL-15 to IL-2/15R ⁇ dimer forms a ternary complex, activates JAK and STAT pathways, promotes target cell proliferation and activation, increases IFN- ⁇ , TNF- ⁇ secretion levels; JAK/STAT, Ras/MAPK—enhances proliferation signals ; Up-regulation of Bcl-2, Bcl-XL (anti-apoptotic proteins), down-regulation of Bim, Puma (pro-apoptotic proteins) -- weakening of apoptotic signals.
  • the bispecific fusion polypeptide has: the first polypeptide, which is sequenced from N-terminus to C-terminus: [VH1]-[Linker 1]-[IL15]-[Linker 2]- [Fc1], the second polypeptide, its sequence from N-terminal to C-terminal is: [VL1]-[Linker 3]-[[IL15RA], the third polypeptide, its sequence from N-terminal to C-terminal is: [ VH2]--[Fc2], and the fourth polypeptide, which are: [VL2]-[CL] from N-terminus to C-terminus; in some embodiments, the bispecific fusion polypeptide has: the first polypeptide Peptide, its order from N-terminal to C-terminal is: [VH1]-[Linker 1]-[IL15RA]-[Linker 2]-[Fc1], the second polypeptide, its order from N-terminal to C-terminal is : [VL
  • the Fc1 and Fc2 are two subunits of the heavy chain constant region Fc, which may be the same or different, and preferably the Fc constant region is a heterodimer (heterodimer Fc fusion protein); In some embodiments, the Fc constant regions are associated into heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility.
  • the linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may or may not be the same; in some embodiments, the linker 1, linker 2 and linker 3 are independently selected From (GxS)y linker, wherein, x is selected from the integer of 1-5, and y is selected from the integer of 0-6.
  • the VH1 and VL1 cooperate to form an antigen binding site that specifically binds TIGIT
  • the VH2 and VL2 cooperate to form an antigen binding site that specifically binds PD-L1.
  • the VH1 and VL1 cooperate to form an antigen binding site that specifically binds PD-L1
  • the VH2 and VL2 cooperate to form an antigen binding site that specifically binds TIGIT.
  • the antigen-binding portion that binds to TIGIT comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO.
  • the light chain variable region comprises SEQ ID NO.74 or has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99%) sequence identity
  • the light chain variable region comprises SEQ ID NO.74 or has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the heavy chain variable region of the antigen-binding portion in conjunction with TIGIT includes HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.73, respectively
  • said light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions, said LCDR1, LCDR2 and LCDR3 respectively comprising LCDR1, LCDR2 and LCDR3 in SEQ ID NO.74;
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • the antigen-binding portion that binds to PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.36 or has at least 80% sequence therewith A sequence of identity, the light chain variable region comprising SEQ ID NO.37 or a sequence having at least 80% sequence identity thereto;
  • the antigen-binding portion that binds PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO.71 or a sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO.72 or has at least 80% sequence identity thereto Sequences of sequence identity; in some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.75 or A sequence having at least 80% sequence identity with it, the light chain variable region includes
  • the heavy chain variable region of the antigen-binding portion that binds to PD-L1 includes HCDR1, HCDR2, and HCDR3 regions
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions
  • the HCDR1 , HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.36
  • said LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.37
  • said HCDR1, HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.71
  • said LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.72
  • said HCDR1, HCDR2 and HCDR3 comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO.75 respectively, and said LCDR1, LCDR
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system system definition.
  • the non-natural interchain bond is formed between the first mutated residue of IL15 and the second mutated residue of IL15R ⁇ , at
  • the first mutation residue of IL15 is the E mutation at the 90th position to C
  • the second mutation residue of IL15R ⁇ is the P mutation position C at the 67th position
  • the IL15 The first mutation residue is the E mutation at position 93 to C
  • the second mutation residue of IL15R ⁇ is the R mutation position C at position 35
  • the amino acid residue mutation site of IL15 refers to SEQ ID NO: 26
  • the corresponding natural sequence numbering site, the amino acid residue mutation site of IL15R ⁇ refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
  • the IL15 is the amino acid sequence shown in SEQ ID NO.84 or its mutation sequence
  • the mutation sequence includes, for example, an amino acid mutation selected from D61N, E64Q and/or N65D, and/or selected from N71Q, N79Q and/or N112Q amino acid mutations.
  • the IL15R ⁇ comprises SEQ ID NO.28 or its mutant sequence, SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or its mutant sequence, SEQ ID NO.79 or its mutant sequence, SEQ ID NO.80 or Its mutant sequence, or SEQ ID NO.81 or its mutant sequence;
  • the mutant sequence includes an amino acid mutation selected from T2A, T81A and/or T86A;
  • the mutant sequence includes P67C amino acid mutation; in some embodiments, the mutant sequence includes a P67C amino acid mutation, and also includes an amino acid mutation selected from T2A, T81A and/or T86A.
  • the IL15R ⁇ is SEQ ID NO.77 or a mutant sequence thereof, SEQ ID NO.78 or a mutant sequence thereof, SEQ ID NO.79 or a mutant sequence thereof, SEQ ID NO.80 or a mutant sequence thereof, Or SEQ ID NO.81 or its mutant sequence;
  • the mutation is a P67C amino acid mutation;
  • the mutation is P67C and an amino acid mutation selected from T2A, T81A and/or T86A;
  • the mutation is an amino acid mutation selected from T2A, T81A and/or T86A.
  • the aforementioned amino acid residue mutation site of IL15R ⁇ refers to the natural sequence numbering site corresponding to SEQ ID NO:27.
  • the IL15 is selected from the group that has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the sequence of SEQ ID NO.84) %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, said IL15RA is selected from the sequence with SEQ ID NO .77-81 any one of the indicated sequences has at least 80% (e.g.
  • Receptor is a substance on the cell membrane or in the cell that can recognize and bind to bioactive molecules, and the bioactive substances that can bind to receptors are collectively referred to as "ligands”.
  • the receptor itself contains at least two active sites: one is the active site that recognizes and binds to the ligand; the other is the functional active site that is responsible for generating a response. Only after that can a response reaction be generated, thereby initiating a series of biochemical reactions, which eventually lead to biological effects in target cells.
  • Receptors are generally glycoproteins, and the binding between wild-type receptors and ligands is not mediated by covalent bonds, but mainly by ionic bonds, hydrogen bonds, van der Waals forces and hydrophobic interactions. When the receptor binds to the ligand, it has the characteristics of saturation, high affinity and specificity.
  • Cooperating receptors and ligands have relatively specific binding affinities, and optionally biological effects.
  • the receptor only includes an active site that recognizes and binds a ligand, and does not include a functional active site that produces a response (such as the function of activating a biological effect of a downstream signaling pathway).
  • the receptor and/or ligand is a natural ligand structure, and the receptor not only includes an active site that recognizes and binds a ligand, but also includes a functional active site that is responsible for generating a response, and can exert According to the corresponding biological function, the bispecific fusion protein is a multifunctional fusion protein, which not only has bispecificity, but also can exert the function of ligand receptor.
  • the receptor and/or ligand has been modified on the basis of the natural sequence, and the modification includes but is not limited to: truncation, insertion and/or mutation; the purpose of these modifications includes but is not limited to : increase or decrease the binding force between ligand and receptor; enhance, decrease or eliminate the biological function of ligand receptor; increase, decrease or eliminate the glycosylation site in receptor and or ligand protein; reduce or eliminate Ligand toxicity.
  • the amino acid sequences of the receptors and/or ligands are each independently composed of 10 to 1000 amino acids; in some embodiments, the amino acid sequences of the receptors and/or ligands are each independently It consists of 20-800 amino acids; in some embodiments, the amino acid sequence of the receptor and/or ligand is independently composed of 30-600 amino acids; in some embodiments, the receptor and/or The amino acid sequences of the ligands each independently consist of 40-400 amino acids; in some embodiments, the amino acid sequences of the receptors and/or ligands each independently consist of 50-300 amino acids; in some embodiments , the amino acid sequences of the receptors and/or ligands each independently consist of 55-260 amino acids.
  • the amino acid sequence of the receptor and/or ligand can also be independently selected from 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 amino acids.
  • the molecular weights of the receptors and/or ligands are each independently selected from 1KD to 100KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 2KD to 80KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 3KD to 70KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 60KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 50KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 40KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 5KD to 30KD.
  • the molecular weight of the receptor and/or ligand can be independently selected from 1KD, 2KD, 3KD, 4KD, 4.5KD, 5KD, 6KD, 7KD, 8KD, 9KD, 10KD, 11KD, 15KD, 18KD, 20KD, 25KD, 30KD , 35KD, 40KD, 45KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD.
  • the receptor (or fragment thereof) and its corresponding ligand (or fragment thereof) can be bound by covalent binding, non-covalent interaction, or a combination thereof; examples of non-covalent bonds include, but are not limited to, hydrogen bonds , hydrophobic, ionic, and van der Waals bonds.
  • the affinity between the inserted or replaced conjugated fragments when the affinity between the inserted or replaced conjugated fragments is lower than expected (for example, the two variable regions in the antigen-binding moiety cannot be brought together to allow them to specifically recognize the antigen, or Inability to prevent heavy chain mismatches between two or more heavy chain constant regions, or inability to prevent mismatches between antigen-binding moieties to achieve specific VL-VH moiety combinations), can be achieved by targeting the ligand and/or or receptors engineered to increase affinity.
  • At least one non-natural interchain bond is included between the receptor and the ligand, and the non-natural interchain bond can enhance the specific binding force between the receptor and the ligand; in some embodiments wherein the non-native interchain bond is formed between a first mutated residue comprised by the receptor and a second mutated residue comprised by the ligand; in some embodiments, the first and the second mutated residue at least one of the radicals is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.
  • Non-natural interchain linkages refers to interchain linkages not found in wild-type polypeptide polymers.
  • a non-natural interchain bond can be formed between a mutated amino acid residue of one polypeptide and a mutated amino acid residue of another polypeptide.
  • At least one native glycosylation site is absent in said receptor and/or ligand.
  • the receptor and ligand are selected from interleukins and their receptors.
  • the ligand and its receptor are selected from class A interleukins and their receptors, such as IL15/IL15R, IL2/IL2R, IL4/IL-4R ⁇ +R ⁇ , IL-6/IL-6R , IL-11/IL-11R, IL-13/IL-13R1, IL-20/IL20R ⁇ +IL20R ⁇ , IL24/IL20R ⁇ +IL20R ⁇ .
  • class A interleukins and their receptors such as IL15/IL15R, IL2/IL2R, IL4/IL-4R ⁇ +R ⁇ , IL-6/IL-6R , IL-11/IL-11R, IL-13/IL-13R1, IL-20/IL20R ⁇ +IL20R ⁇ , IL24/IL20R ⁇ +IL20R ⁇ .
  • the ligand and its receptor are selected from class D interleukins and their receptors, such as IL7/IL7R, IL21/IL21R, IL23A/IL12B.
  • the interleukin and its receptor have the following amino acid sequences in Table 2:
  • the receptor-ligand combination is selected from IL15 and IL15R ⁇ , and “IL15R ⁇ ” and “IL15RA” are interchangeable in the present invention.
  • the IL15 and IL15R ⁇ have the following amino acid sequences:
  • IL-15R ⁇ sushi human, sushi domain amino acid sequence
  • IL-15R ⁇ means the polypeptide fragment of amino acid residues 1 to 77 from the N-terminus in the sushi domain of human IL-15R ⁇ , also marked as IL15R ⁇ sushi-77a.a., and so on.
  • the IL15 and IL15R ⁇ comprise at least one non-natural interchain bond, in some embodiments, the non-natural interchain bond is a disulfide bond, and the IL15 and/or the IL15R ⁇ comprise at least one One amino acid is mutated to cysteine.
  • the mutation is located at the contact interface of IL-15 and IL15R ⁇ .
  • the cysteine mutation of IL15 is selected from E90C, the The cysteine mutation of IL15R ⁇ is selected from P67C; in some technical solutions, the cysteine mutation of IL15 is selected from E93C, and the cysteine mutation of IL15R ⁇ is selected from R35C.
  • the IL15 is the mutant sequence of SEQ ID NO.26, and the mutation includes E90C mutation and/or E93C mutation; in some embodiments, the IL15R ⁇ is SEQ ID NO.27, 29, 30 Or the mutated sequence of 31, said mutation includes P67C mutation and/or R35C mutation.
  • the IL15 amino acid residue mutation position refers to the natural sequence numbering corresponding to the amino acid sequence (SEQ ID NO.26) of the mature form of human IL-15
  • the IL15R ⁇ amino acid residue mutation position refers to the sushi of human IL-15R ⁇ .
  • the natural order number corresponding to the structural domain (SEQ ID NO.27).
  • the D mutation at position 61 of IL15 described above is N
  • the E mutation at position 64 is Q
  • the N mutation at position 65 is D
  • the IL15 is the mutant sequence of SEQ ID NO.26
  • the mutation comprises a mutation selected from D61N, E64Q and/or N65D.
  • the IL15 is the mutant sequence of SEQ ID NO.26, and the mutation includes the E90C mutation, and also includes the D61N, E64Q and/or N65D mutation.
  • At least one N-glycosylation site of IL15 described above does not exist, and in some embodiments, the N-glycosylation site is selected from N71, N79 and/or N112; in some embodiments
  • the IL15 comprises the following amino acid mutations: N71Q, N79Q and/or N112Q.
  • the IL15 is the mutant sequence of SEQ ID NO.26, and the mutation comprises a mutation selected from N71Q, N79Q and/or N112Q.
  • the IL15 is the mutant sequence of SEQ ID NO.26, the mutation includes the E90C mutation, also includes the N71Q, N79Q and/or N112Q mutation, and/or also includes the N71Q, N79Q and/or N112Q mutation .
  • At least one O-glycosylation site of IL15R ⁇ described above does not exist; in some embodiments, the O-glycosylation site is selected from T2, T81 and/or T86; in some embodiments
  • the IL15R ⁇ comprises the following amino acid mutations: T2A, T81A and/or T86A.
  • the IL15R ⁇ is a mutant sequence of SEQ ID NO.27-31, and the mutation includes a mutation selected from T2A, T81A and/or T86A; in some embodiments, it also includes a P67C mutation.
  • amino acid sequences of IL15 and IL15R ⁇ are as follows in Table 4:
  • the IL15 mutation position is numbered according to the natural sequence corresponding to the amino acid sequence of the mature form of human IL-15 (SEQ ID NO.26), and the IL15R ⁇ mutation position is referenced to the sushi domain of human IL-15R ⁇ (SEQ ID NO.27 ) corresponds to the natural order number.
  • the insertion or replacement position of the mutually coordinated receptor (or its fragment) and ligand (or its fragment) can be located, for example: the receptor or its fragment inserts or replaces the CL region, and the ligand or its fragment inserts or replaces the CH1 region; Or the receptor or its fragment inserts or replaces the CH1 region, and the ligand or its fragment inserts or replaces the CL region.
  • the bispecific fusion polypeptide provided by the present invention comprises a first antigen-binding portion and a second antigen-binding portion, and has two antigen specificities, the first antigen-binding portion and the second antigen-binding portion are different, and may be the first antigen
  • the binding moiety and the second antigen-binding moiety may bind different antigens, or the first antigen-binding moiety and the second antigen-binding moiety may bind different epitopes of the same antigen.
  • the target of the bispecific fusion protein is a tumor.
  • both the first antigen-binding moiety and the second antigen-binding moiety bind to targets expressed on tumor cells; in some embodiments, the first antigen-binding moiety binds to tumor cells, and the second antigen-binding moiety binds to tumor cells The portion binds to an immune cell; in some embodiments, both the first antigen-binding moiety and the second antigen-binding moiety bind to an immune cell.
  • T cell redirected killing is a desirable mechanism of action in many therapeutic areas.
  • Various bispecific antibody formats are involved in T cell redirection in preclinical and clinical trials (May C et al. (2012) Biochem Pharmacol, 84(9)): 1105-1112, pp.; Frankel SR, and Baeuerle PA, (2013) CURR OPIN Chemical Biology, Vol. 17(3): 385-92, pp.).
  • All T cell retargeting bispecific antibodies or fragments thereof have been engineered to have at least two antigen binding sites, one of which binds to a surface antigen on the target cell and the other binds to a T cell surface antigen .
  • T cell surface antigens the ⁇ subunit of human CD3 derived from the TCR protein complex is most commonly targeted as a target for redirected T cell killing.
  • Tumor-associated antigens that can be targeted include, but are not limited to: ⁇ -fetoprotein (AFP), ⁇ -actinin-4, A3, antigen specific to A33 antibody, ART-4, B7, Ba 733 , BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a- e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147,
  • T-cell antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69, and CD90.
  • Immune checkpoints are inhibitory pathways in the immune system that are critical for maintaining self-tolerance and regulating the duration and magnitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage.
  • the targets of the first antigen-binding moiety and the second antigen-binding moiety are both immune checkpoints or their ligands, and the immune checkpoint molecules include but are not limited to: TIGIT, PD-1, TIM- 3.
  • the target to which the first antigen-binding moiety binds is PD-1, and the target to which the second antigen-binding moiety binds is PD-L1; in some embodiments, the target to which the first antigen-binding moiety binds is PD-1, the target of the second antigen-binding part is TIGIT; in some embodiments, the target of the first antigen-binding part is PD-1, and the target of the second antigen-binding part is GTLA4; in some In some embodiments, the target to which the first antigen-binding moiety binds is PD-1, and the target to which the second antigen-binding moiety binds is LAG3; in some embodiments, the target to which the first antigen-binding moiety binds is PD-1, The target that the second antigen-binding portion binds to is TIM-3; in some embodiments, the target that the first antigen-binding portion binds to is PD-1, and the target that
  • the target to which the antigen-binding moiety binds is 4-1-BB; in some embodiments, the target to which the first antigen-binding moiety binds is PD-L1, and the target to which the second antigen-binding moiety binds is 4-1-BB; In some embodiments, the target to which the first antigen-binding moiety binds is PD-L1, and the target to which the second antigen-binding moiety binds is TIGIT.
  • the first antigen binding moiety targets a tumor-associated antigen and the second antigen binding moiety targets an immune checkpoint.
  • the first antigen-binding portion targets HER2, and the second antigen-binding portion targets PD-1; in some embodiments, the first antigen-binding portion targets VEGF, and the second antigen-binding portion targets PD-1 L1; in some embodiments, the first antigen-binding moiety targets Claudin18.2 and the second antigen-binding moiety targets PD-L1; in some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety targets Targets CTLA-4; in some embodiments, the first antigen-binding moiety targets CD20 and the second antigen-binding moiety targets CD47; in some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety Targets CD47.
  • the first antigen binding moiety and the second antigen binding moiety target tumor heterogeneity simultaneously.
  • exemplary co-targets for tumors include, but are not limited to, HGF and VEGF, IGF-IR and VEGF, Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 and Fc ⁇ RIIIa, CD20 and Fc ⁇ RIIIa, Her2 and Fc ⁇ RIIIa.
  • the bispecific fusion polypeptide of the present invention can bind VEGF and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 and
  • exemplary co-targets for autoimmune and inflammatory disorders include, but are not limited to, IL-1 and TNF ⁇ , IL-6 and TNF ⁇ , IL-6 and IL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13, IL-9 and IL-13, CD19 and Fc ⁇ RIIb, and CD79 and Fc ⁇ RIIb.
  • Exemplary targets for the treatment of inflammatory diseases include, but are not limited to: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF and IL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE and IL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R and RANKL; IL-17A and IL-1 ⁇ ; IL-1 ⁇ and RANKL;
  • Targets involved in rheumatoid arthritis include, but are not limited to: TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1 ⁇ ; TNF and MIF; TNF and IL-17 ; and TNF and IL-15.
  • Targets for the treatment of systemic lupus erythematosus include but are not limited to: CD20, CD22, CD19, CD28, CD4, CD24, CD37, CD38, CD40, CD69, CD72, CD74, CD79A, CD79B, CD80, CD81, CD83, CD86, IL-4, IL-6, IL10, IL2, IL4, IL11, TNFRSF5, TNFRSF6, TNFRSF8, C5, TNFRSF7, TNFSF5, TNFSF6, TNFSF7, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, IFNB1, AICDA, BLNK, GALNAC4S-6ST, INHA, INHBA, KLF6, DPP4, FCER2, R2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC, CHSTIO, HLA-A, HLA-DRA
  • MS multiple sclerosis
  • MS including but not limited to: IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200 , IFN ⁇ , GM-CSF, FGF, C5, CD52 and CCR2.
  • Targets for treating sepsis include but not limited to: TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-10, IL-23, FasL, LPS, Toll-like receptor, TLR-4, tissue factor, MIP-2, ADORA2A, IL-1B, CASP1, CASP4, NF ⁇ B1, PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NF ⁇ B1, PTAFR, TLR2, TLR4, GPR44 , HMOX1, Midkine, IRAK1, NF ⁇ B2, SERPINA1, SERPINE1, and TREM1.
  • antibodies may be prepared against any combination of these antigens; that is, each of these antigens may optionally and independently be included or excluded by the multispecific antibody according to the invention .
  • the first antigen binding portion and the second antigen binding portion target different epitopes of the same antigen.
  • At least one of the two antigen-binding fragments may also include a secretion signal sequence.
  • the secretory signal sequence refers to a sequence that induces the secretion of expressed protein or peptide by linking to the N-terminus of the coding sequence outside the cell membrane or outside the cell, and the signal sequence may be a peptide sequence consisting of about 18-30 amino acids . All proteins that can be transported to the outside of the cell membrane have different signal sequences that are cleaved by signal peptidases on the cell membrane. Typically, for a foreign protein that is not natively expressed by the host cell, a secretion signal sequence that enables secretion of the protein into the periplasm or culture medium, or a modified sequence, may be used.
  • the VH1 and VL1 cooperate to form an antigen binding site that specifically binds TIGIT
  • the VH2 and VL2 cooperate to form an antigen binding site that specifically binds PD-L1.
  • the VH1 and VL1 cooperate to form an antigen binding site that specifically binds PD-L1
  • the VH2 and VL2 cooperate to form an antigen binding site that specifically binds TIGIT.
  • the antigen-binding portion that binds to TIGIT comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO.
  • the light chain variable region comprises SEQ ID NO.74 or has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99%) sequence identity
  • the light chain variable region comprises SEQ ID NO.74 or has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the heavy chain variable region of the antigen-binding portion in conjunction with TIGIT includes HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.73, respectively
  • said light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions, said LCDR1, LCDR2 and LCDR3 respectively comprising LCDR1, LCDR2 and LCDR3 in SEQ ID NO.74;
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • the antigen-binding portion that binds to PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.36 or has at least 80% sequence therewith A sequence of identity, the light chain variable region comprising SEQ ID NO.37 or a sequence having at least 80% sequence identity thereto;
  • the antigen-binding portion that binds PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO.71 or a sequence having at least 80% sequence identity thereto, and the light chain variable region comprises SEQ ID NO.72 or has at least 80% sequence identity thereto Sequences of sequence identity; in some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO.75 or A sequence having at least 80% sequence identity with it, the light chain variable region includes
  • the heavy chain variable region of the antigen-binding portion that binds PD-L1 includes HCDR1, HCDR2, and HCDR3 regions
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions
  • the HCDR1 , HCDR2 and HCDR3 respectively comprise HCDR1, HCDR2 and HCDR3 in SEQ ID NO.36
  • said LCDR1, LCDR2 and LCDR3 respectively comprise LCDR1, LCDR2 and LCDR3 in SEQ ID NO.37
  • said HCDR1, HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.71
  • said LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.72
  • said HCDR1, HCDR2 and HCDR3 comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO.75 respectively, and said LCDR1, LCDR2
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system system definition.
  • it comprises a heavy chain constant region, Fc, which is a heterodimer (heterodimer Fc fusion protein).
  • the Fc includes but is not limited to the following combinations:
  • the Fc constant region was mutated to avoid heavy chain mismatches.
  • the introduction of mutations into the Fc constant region is based on KiH technology (Knob-into-Holes), that is, amino acid mutations are introduced into a heavy chain of the Fc constant region, and the volume of the introduced amino acids is greater than the volume of the original amino acid residues, A protruding "knob"-like structure (Knob) is formed, and another mutation is introduced in the other chain region of the Fc constant region.
  • the volume of the introduced amino acid is smaller than the volume of the original amino acid residue, forming a depression, similar to the "hole” structure ( Hole), so that the convex heavy chain is more inclined to pair with the concave heavy chain, so as to avoid heavy chain mispairing.
  • KiH technology KiH technology
  • the introduction of mutations in the Fc constant region is based on electrostatic interactions, such as ART-lg technology, which is developed by Roche subsidiary Chugai, which specifically changes the charge of the Fc constant region domain and promotes heterologous recombination.
  • the pairing of chains is equivalent to the KiH technology of the charge version, which is described in the patent application WO2006106905, which is incorporated in the present invention in its entirety.
  • the introduction of mutations in the Fc constant region is based on SEED technology.
  • SEED heterodimerization is another design strategy based on spatial mutation, which utilizes the CH3 domain of IgG and IgA (also known as AG SEED). Complementarity of alternate sequences derived from CH3 and GA SEED (CH3). IgG and IgA CH3 derivatives generate complementary sequences, thus excluding the assembly of homodimers lacking complementarity while assembling two complementary heavy chain heterodimers. This technology is described in patent application WO2007110205, which is incorporated in the present invention in its entirety.
  • the introduction of mutations into the Fc constant region is based on the change of the isoelectric point, which facilitates the modification of improving the formation rate of heterodimers and maintaining the stability of the Fc region.
  • This technology is described in WO2014145806, which is incorporated in this patent in its entirety. .
  • the Fc constant regions associate as heterodimers based on hydrophilic interactions or increased flexibility.
  • the Fc constant regions are associated into heterodimers based on any combination of the above techniques, for example, in some embodiments, the Fc constant regions are mutated based on a combination of KIH and electrostatic interactions .
  • the XmAb bispecific platform approach can improve the thermal stability of bispecific antibodies by combining electrostatic interactions, CH3 domain conformation, and hydrogen bonding. Specifically, this strategy swaps the Fc side chain mutations of native IgG1 for S364K and K370S heterodimers to form hydrogen bonds between the two, followed by L368D/K370S substitutions to drive salt bridge interactions to promote heterodimers.
  • all or part of the CH2, CH3 or CH4 region is inserted or replaced with a receptor and its ligand.
  • the region to be inserted or replaced is independently located in the CH2, CH3 or CH4 region, or any position between adjacent regions (such as CH1-CH2 junction, CH2-CH3 junction, CH3- CH4 junction);
  • the two matching suffixes of the replacement region Affinity between combined fragments, KD ⁇ 1 ⁇ 10 -3 (M), such as x ⁇ 10 -4 (M), x ⁇ 10 -5 (M), x ⁇ 10 -6 (M), x ⁇ 10 -7 (M), x ⁇ 10 -8 (M), x ⁇ 10- 9 (M), x ⁇ 10 -10 (M), x ⁇ 10 -11 (M); the value of x can be selected from 1 ⁇ 9, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • the N-terminal and/or C-terminal of the conjugated fragment is linked to the antigen-binding fragment via a linker peptide.
  • operably linked means that parts (eg, two polypeptides) are linked by a covalent bond, either directly or via one or more linkers (connecting peptides).
  • the number of amino acids of the connecting peptide is 1-30; it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5-20.
  • the amino acids of the linking peptide are nonsense polypeptides that do not have additional functions (such as protein localization, enzyme cleavage sites, etc.) other than linking.
  • the connecting peptide is a flexible connecting peptide
  • the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.
  • the amino acid sequence of the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.
  • the connecting peptide is usually flexible, which can reduce the steric hindrance between the fusion protein and the target protein, which is more conducive to the correct folding of the protein.
  • the connecting peptide is a rigid linker peptide; ie a relatively inflexible peptide linker.
  • a rigid linker peptide does not require complete lack of flexibility, but is less flexible than a flexible linker peptide such as a glycine-rich peptide linker. Due to their relative lack of flexibility, the rigid linker peptide reduces the movement of two protein domains (in the present case the stabilizer protein and the thermostable reverse transcriptase) linked together by the rigid linker peptide.
  • Linker peptides that provide ordered chains eg, alpha helical structures
  • arginine, leucine, glutamic acid, glutamine, and methionine all show a relatively high propensity for helical linker structures.
  • non-helical linkers containing many proline residues can also exhibit significant rigidity.
  • rigid linking peptides include polylysine and poly-DL-alanine polylysine.
  • rigid linker peptides are described in the linker database described by George et al., Protein Engineering, 15, pp. 871-79 (2003).
  • the rigid linker peptide is also a non-cleavable linker peptide, ie a non-cleavable rigid linker peptide.
  • the invention also relates to an isolated nucleic acid encoding a bispecific fusion polypeptide or a multifunctional fusion protein as described above.
  • isolated nucleic acid refers herein to a polymer of deoxyribonucleic acid or ribonucleic acid in single- or double-stranded form.
  • Isolated nucleic acids include RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, polypeptides include native polynucleotides, sugar, or base altered analogs.
  • said polynucleotide is a light chain polynucleotide.
  • the isolated nucleic acid includes the nucleotide sequence encoding the amino acid sequence of the protein complex, as well as the nucleotide sequence complementary thereto.
  • the complementary sequence includes a fully complementary sequence and a substantially complementary sequence, which refers to a sequence that can hybridize to a nucleotide sequence encoding an amino acid sequence of a protein complex under stringent conditions known in the art.
  • nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such changes include additions, deletions, or non-conservative or conservative substitutions.
  • a polynucleotide encoding an amino acid sequence of a protein complex may be construed to include nucleotide sequences having substantial identity relative to the isolated nucleic acid. The substantial identity is when the nucleotide sequence is aligned with another random sequence in such a way that they correspond maximally, which may show greater than 80% identity when the aligned sequences are analyzed using algorithms common in the art. % homology, greater than 90% homology, or greater than 95% homology.
  • the present invention also relates to a vector comprising a nucleic acid as described above.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses adeno-associated viruses
  • herpesviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • the vector may contain a selection marker (such as a tag that is convenient for enrichment, such as his tag; or a tag that is convenient for detection, such as GFP), and an origin of replication that matches the cell type specified by the cloning vector, and the expression vector then contain regulatory elements such as enhancers, promoters, internal ribosomal entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly U sequence, etc.).
  • the vector can be a cloning vector and an expression vector. When expressing or preparing antibodies or fragments, prokaryotic expression vectors and eukaryotic expression vectors are often involved.
  • Prokaryotic expression vectors are commonly used in PET series and pGEX series, and eukaryotic expression vectors are commonly used in pcDNA3.1, pcDNA3.4, pcDNA4, pEGFP-N1, pEGFP-N1, pSV2, etc.
  • the carrier can be a composition, such as a mixture of various plasmids, and different plasmids carry a part of the antibody or its fragment.
  • the present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
  • a variety of cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as Escherichia coli or Bacilis stearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), insect Cells (such as Lepidoptera cells including Spodoptera cells) or mammalian cells (such as Chinese hamster ovary (CHO) cells, NSO cells, baby hamster kidney (BHK) cells, monkey kidney cells, Hela cells, human Hepatocellular carcinoma cells or 293 cells, etc.).
  • prokaryotic cells such as Escherichia coli or Bacilis stearothermophilus
  • fungal cells such as Saccharomyces cerevisiae or Pichia pastoris
  • insect Cells such as Lepidoptera cells including Spodoptera cells
  • mammalian cells such as Chinese hamster ovary (CHO) cells, NSO cells, baby hamster kidney (B
  • the bispecific fusion polypeptide or multifunctional fusion protein of the present invention can be prepared by any method known in the art.
  • the bispecific fusion polypeptide or multifunctional fusion protein expressed in the host cells is collected.
  • bispecific antibodies Early methods for constructing bispecific antibodies include chemical cross-linking or hybrid hybridoma or tetravalent tumor method (for example, Staerz UD et al., Nature, 314:628-31, 1985; Milstein C et al., Nature, 305:537 -540, 1983; Karpovsky B et al., J. Exp. Med., 160:1686-1701, 1984).
  • the chemical coupling method is to link two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies. For example the chemical combination of two different monoclonal antibodies, or for example the chemical combination of two antibody fragments such as two Fab fragments.
  • the heterozygous-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or ternary hybridomas. These cell hybridomas or ternary hybridomas are fused by established hybridomas, or established hybridomas and hybridomas from childhood obtained from the fusion of mouse lymphocytes. Although these techniques are used to make BiAbs, various production issues make the use of such complexes difficult, such as generation of mixed populations containing different combinations of antigen-binding sites, difficulties in protein expression, need to purify the BiAb of interest, low yields, production The cost is high.
  • More recent approaches utilize genetically engineered constructs capable of producing a homogeneous product of a single BiAb without extensive purification to remove unwanted by-products.
  • Such constructs include tandem scFv, diabodies, tandem diabodies, dual variable domain antibodies, and heterodimerization using motifs such as Ch1/Ck domains or DNLTM (Chames & Baty, Curr. Opin. Drug. Discov. Devel., 12:276-83, 2009; Chames & Baty, mAbs, 1:539-47).
  • the relevant purification techniques are well known.
  • Antibodies can also be produced using the monolymphocyte antibody approach by cloning and expressing immunoglobulin variable region cDNA produced by a single lymphocyte selected for production of specific antibodies, e.g. by Babcook J et al., Proc. Natl. Acad. Sci.USA.93:7843-7848,1996; WO 92/02551; methods described in WO 2004/051268 and WO 2004/106377.
  • Antigenic polypeptides for the production of antibodies can be obtained from genetically engineered hosts comprising expression systems by methods well known in the art. Cells are prepared, or they may be recovered from natural biological sources.
  • nucleic acids encoding one or both polypeptide chains of bispecific antibodies can be introduced into cultured host cells by various known methods (eg, transformation, transfection, electroporation, bombardment with nucleic acid-coated particles, etc.).
  • the nucleic acid encoding the bispecific antibody can be inserted into a vector suitable for expression in the host cell before being introduced into the host cell.
  • vectors will contain sequence elements that enable the expression of the inserted nucleic acid at the RNA and protein levels.
  • the bispecific antibodies of the invention can be used to detect any or all of these antigens by conventional immunological assay methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry (eg in biological samples such as serum or plasma).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • tissue immunohistochemistry eg in biological samples such as serum or plasma.
  • the present invention provides a method for detecting an antigen in a biological sample, the method comprising: contacting the biological sample with the bispecific antibody of the present invention that can specifically recognize the antigen, or part of the antigen of the antibody, and detecting the antigen-binding An antibody (or antibody portion), or a non-binding antibody (or antibody portion), thereby detecting said antigen in said biological sample.
  • the antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • suitable enzymes include, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase;
  • suitable repair group complexes include streptavidin/biotin and Avidin/biotin;
  • suitable fluorescent substances include 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, basalin B, dichlorotriazinylamine fluorescein, dan Sulfonyl chloride or phycoerythrin;
  • luminescent substances include 3-aminophthaloyl cyclic hydrazine;
  • suitable radioactive substances include I125, I131, 35S or 3H.
  • the bispecific fusion polypeptide or multifunctional fusion protein of the present invention or nucleic acid encoding it can be applied to the preparation of pharmaceutical compositions or sterile compositions, for example, combining bispecific fusion polypeptide or multifunctional fusion protein with pharmaceutically acceptable Carriers, excipients or stabilizers are mixed.
  • a pharmaceutical composition may comprise one or a combination (eg, two or more different) functional fragments of the antibodies of the present invention.
  • a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on a target antigen.
  • Formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions.
  • the bispecific fusion polypeptide or multifunctional fusion protein in the pharmaceutical composition can be in the form of combining with the second activator (functional molecule).
  • the second activator may be a random functional molecule capable of preventing or treating a target disease, and may include compounds, peptides, polypeptides, nucleic acids, carbohydrates, lipids, or inorganic particles.
  • the bispecific fusion polypeptide or multifunctional fusion protein may itself have therapeutic activity; but it may function to target the second activator to a specific disease area.
  • the diseased areas may be those organs, tissues, or cells where the bispecific antibody specifically binding to the antigen is accumulated and distributed.
  • the drug targeted to the diseased area is present in a high concentration such that the effect of the drug is increased compared to the amount injected. Therefore, the pharmaceutical composition can be used to treat drug-resistant tumors, and can reduce side effects and adverse drug reactions caused by non-specific drug distribution.
  • the activator comprising bispecific fusion polypeptide or multifunctional fusion protein in the pharmaceutical composition can be accommodated in microcapsules, or contained in colloidal drug delivery systems (such as liposomes, albumin spheres, microemulsions, nanoparticles and nanocapsules), or contained in macroemulsions (macroemulsions), the microcapsules can be prepared by techniques such as coacervation (coacervation) or interfacial polymerization, examples are hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules.
  • colloidal drug delivery systems such as liposomes, albumin spheres, microemulsions, nanoparticles and nanocapsules
  • macroemulsions macroemulsions
  • the microcapsules can be prepared by techniques such as coacervation (coacervation) or interfacial polymerization, examples are hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) micro
  • the present invention also relates to the application of the above-mentioned bispecific fusion polypeptide or multifunctional fusion protein in the preparation of medicines for treating diseases.
  • the present invention also relates to a bispecific fusion polypeptide or a multifunctional fusion protein as described above for use as a medicine; said medicine is used for the treatment of diseases.
  • the disease may be, for example, cancer, immune disorders, metabolic diseases, and microbial infections.
  • cancer refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body.
  • Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases.
  • the microorganism in a microbial infection can be an exogenous pathogen or a population of cells harboring an exogenous pathogen such as a virus.
  • the invention is applicable to exogenous pathogens such as bacteria, fungi, viruses, mycoplasma and parasites.
  • Pathogens that may be treated by the present invention may be any infectious organisms known in the art that cause disease in animals, including organisms such as: Gram-negative or Gram-positive cocci or bacilli, bacteria, DNA viruses, and RNA viruses , including but not limited to DNA viruses such as papillomaviruses, parvoviruses, adenoviruses, herpesviruses, and vaccinia viruses, and DNA viruses such as arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial virus, influenza viruses, picornaviruses , paramyxoviruses, reoviruses, retroviruses and rhabdoviruses are RNA viruses.
  • DNA viruses such as papillomaviruses, parvoviruses, adenoviruses, herpesviruses, and vaccinia viruses
  • DNA viruses such as arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial virus,
  • antibiotic-resistant bacteria such as antibiotic-resistant Streptococcus species and Staphlococcus species, or those that are susceptible to antibiotics but cause recurrent infections treated with antibiotics so that resistant organisms eventually develop bacteria.
  • antibiotics can be treated with the ligand-immunogen conjugates of the invention in combination with lower doses of antibiotics than are normally administered to patients to avoid the development of these antibiotic resistant bacterial strains.
  • the invention is also applicable to any fungi, mycoplasma species, parasites or other infectious organisms that cause disease in animals.
  • fungi examples include fungi that grow as molds or yeasts, including, for example, fungi that cause diseases such as: ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichia mycosis, coccidioidomycosis, coccidioidomycosis and candidiasis.
  • the present invention may be used to treat parasitic infections including, but not limited to, infections caused by the following parasites: Taenia spp. Leishmania and Toxoplasma species.
  • Parasites of particular interest are those that express folate receptors and bind folate; however, there are numerous references in the literature to ligands that exhibit high affinity for infectious organisms.
  • penicillins and cephalosporins whose antibiotic activity is known and which bind specifically to bacterial cell wall precursors, can likewise be used as ligands for the preparation of ligand-immunogen conjugates used in accordance with the present invention.
  • the ligand-immunogen conjugates of the invention can also be directed against cell populations bearing endogenous pathogens, wherein the pathogen-specific antigens are preferentially expressed on the surface of cells bearing the pathogens and used as specific agents for the antigens.
  • Receptors for sex-binding ligands are those that express folate receptors and bind folate; however, there are numerous references in the literature to ligands that exhibit high affinity for infectious organisms.
  • penicillins and cephalosporins whose antibiotic activity is known and which bind specifically to bacterial
  • the present invention also relates to a method of prophylaxis and/or treatment and administration of a therapeutically effective amount of the pharmaceutical composition to prevent and/or treat diseases as described above.
  • the methods of the invention can be used in human clinical and veterinary applications.
  • the host animal carrying the pathogenic population and being treated with the ligand-immunogen conjugate may be a human, or in the case of veterinary applications, a laboratory animal, an agricultural animal, a domesticated animal or a wild animal.
  • the present invention may be applicable to host animals including, but not limited to: humans; laboratory animals such as rodents (e.g.
  • mice rats, hamsters, etc.
  • rabbits monkeys, chimpanzees
  • domesticated animals such as dogs, cats and rabbits
  • farm animals such as cattle, horses, pigs, sheep, goats
  • captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.
  • compositions can be injected into entities, including rats, mice, domestic animals, and/or humans, by a variety of routes. All injection methods are contemplated, for example, oral, rectal, intravenous, nasal, abdominal, subcutaneous, or local injections are all possible.
  • the compositions can be injected by other methods known in the art.
  • a “therapeutically effective amount” herein refers to an amount sufficient to treat a disease with a reasonable benefit to loss ratio.
  • a therapeutically effective amount may vary depending on the patient for a variety of reasons, for example, type of disease, severity, onset, age of the entity, body weight, rate of excretion, susceptibility to reactions, health status, and/or complications; And/or drug activity, injection route, injection cycle and injection times, and/or drug combination; it can also be appropriately selected by those of ordinary skill in the art according to the purpose of treatment.
  • the injection amount can be randomly divided into multiples so that the amount is about 0.001-100 mg/kg body weight of an adult.
  • a bispecific fusion polypeptide or multifunctional fusion protein of the invention, or a nucleic acid or polynucleotide encoding an antibody of the invention can also be administered in combination with, for example, standard cancer treatments (eg, surgery, radiation, and chemotherapy).
  • standard cancer treatments eg, surgery, radiation, and chemotherapy
  • antitumor therapy using the compositions of the invention and/or effector cells equipped with these compositions is used in combination with chemotherapy.
  • Non-limiting examples of antibody combination therapy of the invention include surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and combinations thereof.
  • Embodiment 1 FiBody design
  • FiBody is a recombinantly obtained bispecific antibody that uses the specific affinity between the ligand and its receptor to replace CL and CH1 on one side of the bispecific antibody, which can avoid or reduce the Mismatches between light and heavy chains.
  • interleukin and its receptor As an example to construct FiBody, it is divided into four categories according to the three-dimensional conformation of interleukin and its receptor, as shown in Table 5 and Figure 5 below:
  • Bispecific antibodies were constructed based on the above four types of interleukins and their receptors.
  • Example 2 construction based on interleukin and its receptor FiBody
  • the VH targeting the first antibody is selected to be connected to the receptor protein through a Linker, and then connected to the Fc of the antibody through a Hinge; the VL targeting the first antibody is connected to the ligand protein through a Linker to reduce or avoid light chains and heavy chains.
  • the chain is mismatched; the other end is the complete Fab structure of the targeting second antibody (anti-TIGIT antibody), and the Fc of the first antibody and the Fc of the second antibody have a conventional KiH modification to reduce or avoid the heavy chain. match. or
  • the VH targeting the first antibody is selected to be connected to the ligand protein through a Linker, and then connected to the Fc of the antibody through a Hinge; the VL targeting the second antibody is connected to the receptor protein (IL15) through a Linker to reduce or avoid light There is a mismatch between the chain and the heavy chain; the other end is the complete Fab structure targeting the first antibody, and the Fc that makes up the first antibody and the Fc that make up the second antibody have conventional KiH modifications to reduce or avoid heavy chain mismatches.
  • the bispecific antibody is modified with a disulfide bond, as shown in Figure 8.
  • Ligand receptor disulfide bond modification select the VH targeting the second antibody (anti-TIGIT) to connect to the receptor protein (IL15RA) through the Linker, and then connect to the Fc of the antibody through the Hinge; the VL targeting the second antibody is connected to the receptor protein (IL15RA) through the Linker Linked to the ligand protein (IL15); the other end is the complete Fab structure targeting the first antibody.
  • the Fc of the first antibody and the Fc of the second antibody have a conventional KiH modification to avoid heavy chain mismatch.
  • the receptor and ligand proteins are mutated to form an intermolecular disulfide bond to further improve the stability of the molecule, as shown in Table 8 below:
  • Disulfide bond modification positions include but are not limited to the following mutation sites:
  • Embodiment 4 the mutation transformation that eliminates glycosylation
  • the construction method of Fab-IL15/IL15RA_Fc specifically selects the VH targeting the second antibody (anti-TIGIT) to connect to the receptor protein (IL15RA) through a Linker, and then connects to the Fc of the antibody through a Hinge; the VL targeting the second antibody Linked to the ligand protein (IL15) through Linker; the other end is the complete Fab structure targeting the first antibody, the Fc of the first antibody and the Fc of the second antibody have conventional KiH modification to avoid heavy chain mismatch .
  • the receptor and ligand protein are mutated to form intermolecular disulfide bonds to further improve the stability of the molecule; further, the glycosylation sites on the receptor and ligand protein are modified to eliminate Molecular heterogeneity, for example:
  • Example 5 IL15/IL15RA modified to reduce the affinity of IL15/IL15RA and IL2/15R ⁇ / ⁇ C complex and bispecific antibody composed of it
  • our bispecific antibody against IL15/IL15RA and its component Antibodies engineered to reduce or completely lose affinity for IL15/IL15RA and IL2/15R ⁇ / ⁇ C complexes.
  • MOE Chemical Computing Group, Montreal, Quebec, Canada
  • Fab-IL15/IL15RA_Fc specifically select the VH of the targeting second antibody (anti-TIGIT) to be connected to the receptor protein (IL15RA) through Linker, and then connect to the Fc of the antibody through Hinge; VL is connected to the ligand protein (IL15) through a Linker; the other end is the complete Fab structure targeting the first antibody, and the Fc of the first antibody and the Fc of the second antibody have a conventional KiH modification to avoid heavy chain misalignment match.
  • the purpose of mutating the ligand protein is to reduce or inactivate the biological function of the receptor-ligand complex, for example:
  • the scFv structure double antibody Based on the construction method of the scFv structure double antibody, specifically select the VH of the targeting second antibody (anti-TIGIT) to connect to the VL of the second antibody through a Linker to form a scFv structure, and then connect to the Fc of the antibody through a Hinge;
  • the complete Fab structure of the first antibody (this double antibody platform was developed by Wuhan Youzhiyou and named YBody), the Fc of the first antibody and the Fc of the second antibody have conventional KiH modification to avoid heavy chain mismatch .
  • this double antibody platform was developed by Wuhan Youzhiyou and named YBody
  • the Fc of the first antibody and the Fc of the second antibody have conventional KiH modification to avoid heavy chain mismatch .
  • the VH of the targeting second antibody is selected to connect to the VL of the second antibody through a Linker to form a scFv structure, and then through the Linker and the complete Fc targeting the first antibody
  • the C-terminal connection form a symmetrical structure.
  • the VH of the targeting second antibody is specifically selected to be connected to the CL domain, and then connected to the Fc of the antibody through Hinge, and the VL of the targeting second antibody (anti-TIGIT) is connected to
  • the CH1 domain forms the light chain; the other end is the complete Fab structure targeting the first antibody, and the Fc that makes up the first antibody and the Fc that make up the second antibody have conventional KiH modifications to avoid heavy chain mismatches.
  • R1042 specifically select the VH targeting the first antibody (anti-PD-L1 antibody) to be connected to the receptor protein (IL15RA) through a Linker, and then connect to the Fc of the antibody through a Hinge; targeting the second antibody (anti-TIGIT antibody)
  • the VL is connected to the ligand protein (IL15) through a Linker; the other end is connected to the VH targeting the second antibody to CH1 through a conventional sequence, and then connected to the Fc of the antibody through a Hinge, and the VL targeting the primary antibody is connected through a conventional sequence
  • the two Fcs have conventional KiH remodeling to avoid heavy chain mismatches.
  • IL15RA receptor protein
  • IL15RA receptor protein
  • Fc of the antibody anti-TIGIT antibody
  • R1042 PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL ( Figure 10 left) first polypeptide @PD-L1_VH_IL15RA_Fc-Knob (SEQ ID NO.1) second polypeptide @TIGIT_VL_IL15(SEQ ID NO.6) third polypeptide @TIGIT_VH_CH1_Fc-Hole (SEQ ID NO.3) The fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.8)
  • R1043 Specifically select the VH targeting the second antibody (anti-TIGIT antibody) to be connected to the receptor protein (IL15RA) through a Linker, and then connect to the Fc of the antibody through a Hinge; targeting the first antibody (anti-PD-L1 antibody)
  • the VL is connected to the ligand protein (IL15) through a Linker; the other end is connected to the VH targeting the first antibody to CH1 through a conventional sequence, and then connected to the Fc of the antibody through a Hinge, and the VL targeting the second antibody is connected through a conventional sequence
  • the two Fcs have conventional KiH remodeling to avoid heavy chain mismatches.
  • molecular number R1043 PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL first polypeptide @TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO:5) second polypeptide @PD-L1_VL_IL15(SEQ ID NO:2) third polypeptide @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) The fourth polypeptide @TIGIT_VL_CL(SEQ ID NO.4)
  • the VH targeting the second antibody is selected to connect to the receptor protein (IL21R) through Linker, and then connected to the Fc of the antibody through Hinge;
  • the VL targeting the first antibody is connected through the Linker Linked to the ligand protein (IL21); the other end is linked to the VL of the targeting second antibody (anti-PD-L1 antibody) on the CL, and the VH of the targeting structure first antibody (anti-TIGIT antibody) is linked to CH1, It is then connected to the Fc of the antibody through Hinge, and the Fc at both ends has a conventional KiH modification.
  • Embodiment 8 the preparation of FiBody sample
  • the plasmid containing the target gene is formed into a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293.
  • the exogenous gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
  • Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm, and the cells were counted before transfection.
  • the 2E6 cells were inoculated into a 1L shake flask, and the culture system was about 300ml.
  • the transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22 ⁇ m filter membrane.
  • Purification using ProA affinity chromatography The process is as follows, use AKTA york 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, make the target protein adsorb on the chromatography column and Other impurities are separated by breakthrough.
  • HPLC-SEC detection results of sample R0951 are shown in Figure 11
  • HPLC-SEC detection results of sample R1042 are shown in Figure 12
  • HPLC-SEC detection results of sample R0809 are shown in Figure 13
  • HPLC-SEC detection results of sample R1110 are shown in Figure 13.
  • the results of SEC detection are shown in Figure 14.
  • the FiBody platform prepares bispecific antibodies (including various modified and optimized antibodies) Higher expression and/or higher purity.
  • the binding activity of the double antibody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay.
  • Disulfide bond transformation optimization samples R1081, R1085 and glycosylation samples have similar tigit end affinity results compared with the molecules before transformation.
  • R1042, R1043 and R1124 are mismatch test molecules, and their TIGIT binding activity is significantly reduced; R0810 is a ScFv molecule, and its binding activity is also weaker than that of the control molecule R0226.
  • the binding activity of the double antibody molecule (PD-L1 end) to CHO-PD-L1 cells was detected by FCM assay.
  • R0802 is (PD-L1 monoclonal antibody, 176F9, the VH sequence is shown in SEQ ID NO:36, and the VL sequence is shown in SEQ ID NO:37),
  • R0514 is (PD-L1 monoclonal antibody, Avelumab), and
  • R0919 is ( PD-L1 monoclonal antibody, VH sequence is shown in SEQ ID NO:75, VL sequence is shown in SEQ ID NO:76),
  • R0968 is (PD-L1 monoclonal antibody, VH sequence is shown in SEQ ID NO:71 , the VL sequence is shown in SEQ ID NO:72)
  • Example 11 the binding activity of FiBody receptor ligand complex (IL15/IL15R)
  • Antibody dilution use FACS buffer to dilute all molecules to an initial concentration of 400nM, volume 180 ⁇ l, 3-fold serial dilution (60+120), 10 concentrations; cell counting and plating: R0255-2(CHO-mTigit)/293T-IL15R -28 cells were centrifuged at 250g for 5min, then the supernatant was discarded, the cell density was adjusted to 2E+06 with FACS buffer, and 100 ⁇ L/tube was evenly divided into 96-well V-shaped plates; the above-mentioned diluted antibody was added to the cells, 100 ⁇ L/ Well, incubate at 2-8 degrees for 0.5h; take out the 96-well plate, centrifuge at 250g for 5min, carefully remove the supernatant, add 200 ⁇ L/well of FACS buffer, centrifuge again at 250g for 5min, carefully remove the supernatant; use FACS buffer to prepare PE fluorescent secondary antibody (1:500 dilution), add 100 ⁇ L/well to the corresponding 96-
  • R1042, R1043 have the same activity as R0951, indicating that there is no mismatch, and even the wrong Fv can be expressed; among them, R0655 is (IL15/IL15RFc fusion protein, see SEQ ID NO:38)
  • the modified disulfide bonds of light and heavy chains were R1081, R1082, and R1084. Electrophoresis results showed that there was no non-covalent light chain (no band between 25KD and 35KD), indicating that the disulfide bonds of R1081, R1082, and R1084 were successfully modified.
  • variable region of the heavy chain variable region (VH) targeting the antigen is connected to IL15R ⁇ through a Linker, and then connected to the Fc of the antibody through a Hinge; targeting the same antigen
  • VL variable light chain region
  • IL15/IL15R ⁇ complex See Figure 28 for a schematic structural view of the IL15/IL15R ⁇ complex.
  • amino acid sequence of the IL15/IL15R ⁇ complex is shown in Table 21 below:
  • the plasmid containing the target gene is formed into a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293.
  • the exogenous gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
  • Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm, and the cells were counted before transfection.
  • the 2E6 cells were inoculated into a 1L shake flask, and the culture system was about 300ml.
  • the transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22 ⁇ m filter membrane.
  • Purification using ProA affinity chromatography The process is as follows, use AKTA york 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, make the target protein adsorb on the chromatography column and Other impurities are separated by breakthrough.
  • the binding activity of the double antibody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay.
  • the binding activity of the double antibody molecule (PD-L1 end) to CHO-PD-L1 cells was detected by FCM assay.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne le domaine de la biomédecine, et en particulier un complexe polypeptidique d'interleukine 15 et son récepteur, ainsi que son utilisation. Une liaison interchaîne non naturelle est présente entre l'IL15 et l'IL15Rα dans le complexe polypeptidique de la présente invention.
PCT/CN2022/121068 2021-09-24 2022-09-23 Complexe polypeptidique d'interleukine 15 et son récepteur WO2023046116A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202111123534.XA CN113943374B (zh) 2021-09-24 2021-09-24 一种白介素15及其受体的多肽复合物
CN202111121937 2021-09-24
CN202111121937.0 2021-09-24
CN202111123534.X 2021-09-24

Publications (1)

Publication Number Publication Date
WO2023046116A1 true WO2023046116A1 (fr) 2023-03-30

Family

ID=85720127

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/121068 WO2023046116A1 (fr) 2021-09-24 2022-09-23 Complexe polypeptidique d'interleukine 15 et son récepteur

Country Status (2)

Country Link
TW (1) TW202321315A (fr)
WO (1) WO2023046116A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110461871A (zh) * 2017-02-16 2019-11-15 索纳特生物疗法公司 白蛋白结合结构域融合蛋白
CN113135996A (zh) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 一种双特异抗体及其应用
CN113307879A (zh) * 2020-02-27 2021-08-27 启愈生物技术(上海)有限公司 一种taa/ctla-4/il15三功能融合蛋白及其应用
CN113943374A (zh) * 2021-09-24 2022-01-18 广东菲鹏制药股份有限公司 一种白介素15及其受体的多肽复合物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110461871A (zh) * 2017-02-16 2019-11-15 索纳特生物疗法公司 白蛋白结合结构域融合蛋白
CN113135996A (zh) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 一种双特异抗体及其应用
CN113307879A (zh) * 2020-02-27 2021-08-27 启愈生物技术(上海)有限公司 一种taa/ctla-4/il15三功能融合蛋白及其应用
CN113943374A (zh) * 2021-09-24 2022-01-18 广东菲鹏制药股份有限公司 一种白介素15及其受体的多肽复合物

Also Published As

Publication number Publication date
TW202321315A (zh) 2023-06-01

Similar Documents

Publication Publication Date Title
US11524991B2 (en) PD-1 targeted heterodimeric fusion proteins containing IL-15/IL-15Ra Fc-fusion proteins and PD-1 antigen binding domains and uses thereof
CN111138547B (zh) 针对cd19和cd3的同源二聚体型双特异性抗体及其制备方法和用途
US11084863B2 (en) Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains
TWI788286B (zh) 三特異性和/或三價結合蛋白
CN112105645A (zh) Il-15/il-15ra异二聚体fc融合蛋白及其用途
TWI672317B (zh) 單價不對稱串聯Fab雙特異性抗體
JP2019533444A (ja) ＩＬ−１５／ＩＬ−１５ＲアルファＦｃ融合タンパク質およびＰＤ−１抗体の断片を含む二重特異性ヘテロ二量体融合タンパク質
US11618776B2 (en) Targeted heterodimeric Fc fusion proteins containing IL-15/IL-15RA and NKG2D antigen binding domains
US20130129723A1 (en) Heterodimer Binding Proteins and Uses Thereof
JP2022521937A (ja) ＮＫｐ３０に結合する抗体分子およびその使用
CN114127113A (zh) 与钙网蛋白结合的多功能分子及其用途
WO2022174813A1 (fr) Anticorps trispécifique anti-gprc5d × bcma × cd3 et son utilisation
WO2022171080A1 (fr) Anticorps anti-cd112r et son utilisation
WO2023046093A1 (fr) Complexe polypeptidique bispécifique
CN114026122A (zh) 结合t细胞相关癌细胞的多功能分子及其用途
WO2022223001A1 (fr) Polypeptide de fusion multifonctionnel bispécifique
JP2022522662A (ja) Ｔ細胞に結合する多機能性分子および自己免疫障害を処置するためのその使用
WO2023046116A1 (fr) Complexe polypeptidique d'interleukine 15 et son récepteur
KR20240019786A (ko) Cd20, nkp46, cd16에 결합하고 il-2에 접합된 다중특이적 항체
KR20230074144A (ko) NKp30에 결합하는 항체 분자 및 이의 용도
TW202104261A (zh) 三特異性結合蛋白、方法及其用途
JP2022517809A (ja) Ｌｉｌｒｂ３結合分子とその使用
WO2023273914A1 (fr) Anticorps anti-cd3 et son utilisation
WO2024041435A1 (fr) Anticorps bispécifique anti-her2/cd3 activé de manière spécifique dans un micro-environnement tumoral
TW202313699A (zh) 新型抗ｓｉｒｐａ抗體

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22872160

Country of ref document: EP

Kind code of ref document: A1