WO2022042576A1 - Multifunctional fusion protein and use thereof - Google Patents

Multifunctional fusion protein and use thereof Download PDF

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WO2022042576A1
WO2022042576A1 PCT/CN2021/114408 CN2021114408W WO2022042576A1 WO 2022042576 A1 WO2022042576 A1 WO 2022042576A1 CN 2021114408 W CN2021114408 W CN 2021114408W WO 2022042576 A1 WO2022042576 A1 WO 2022042576A1
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heavy chain
fusion protein
multifunctional fusion
seq
cells
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PCT/CN2021/114408
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French (fr)
Chinese (zh)
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周冲
吴崇兵
王艺臻
姜晓玲
殷刘松
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盛禾(中国)生物制药有限公司
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Priority claimed from CN202110971791.2A external-priority patent/CN114106195A/en
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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

Definitions

  • the invention belongs to the field of biotechnology, in particular to a multifunctional fusion protein, in particular to a multifunctional fusion protein targeting TAA and CD3 and having the biological effect of IL-15/IL-15R ⁇ complex.
  • Cytokines are immunomodulatory molecules, which have a certain activation or inhibition effect on the immune system according to their properties, administration concentrations and active sites. Therefore, this way of giving immunostimulatory cytokine therapy can improve the immune function of cancer patients.
  • cytokines As an immunotherapy, cytokines have certain benefits, but their clinical use has the disadvantage of poor targeting of single-drug administration. Only high-concentration administration can achieve anti-tumor effect, and high-concentration administration will produce immunosuppression. effects and high toxicity. Moreover, the activation of the immune system by non-targeted cytokines is systemic, and the immune system is widely activated with lethal side effects. In addition, because cytokines are small molecular weight proteins and do not have the protective mechanism of antibodies in vivo, simple cytokines often have a short half-life and require repeated high-dose administration in a short period of time. At present, most clinical research drugs use PEGylation or Fc fusion to improve the half-life of cytokines.
  • cytokines Although the half-life is prolonged, it still cannot solve the problem of poor targeting of cytokines.
  • the role of NK cells and T cells, especially cytotoxic T cells, in tumor immunity has been validated in a variety of mouse tumor models.
  • Several clinical trials are evaluating the anticancer efficacy of certain cytokines alone or in combination with multiple chemotherapeutic agents and tumor-targeted monoclonal antibodies and other cytokines.
  • cytokines such as IL-15 under high-dose administration and the short-term repeated administration caused by the short half-life still exist, which have become a major constraint to the combination strategy.
  • CD3 is a complex molecule composed of peptide chains with non-covalent bonds. It is expressed on the surface of mature T cells and plays a major role in blocking acute allograft rejection. It also stabilizes the TCR structure and transmits activation signals. Monoclonal antibodies against CD3 molecules can activate T cell proliferation and activation. Under the combined action of other cytokines IL-15, IL-15R ⁇ , etc., it produces fast proliferation, high tumoricidal activity, broad tumoricidal spectrum and non-MHC restriction. The CIK cells with tumor-killing characteristics have remarkable curative effect on the treatment of various diseases such as cancer, chronic leukemia, liver disease and neurological disease. Therefore, by combining CD3 antibody and tumor-targeting antibody, the constructed bispecific antibody can recruit T cells to approach tumor cells and play a role in mediating T cells to kill tumor cells.
  • Bispecific antibodies are clinically expected to be the next generation of biotherapeutics for cancer, autoimmunity and infectious diseases, with functions that cannot be achieved by single-target antibodies. Bispecific antibodies provide a good way to refine the mechanism of action of antibody drugs and explore the synergistic effect of multiple mechanisms.
  • Patent CN110023336A discloses a binding agent comprising at least three binding domains, wherein the first binding domain binds to T cell-specific antigen, and the second binding domain and the third binding domain bind to claudin (Claudin6 or Claudin18.2 ) combined. It is reported for the first time that a binding agent comprising two binding domains of claudin and another binding domain targeting T cell-specific antigens such as CD3 induces potent T cell-mediated lysis and is useful in the treatment of oncological diseases. effective in. However, this antibody structure does not have the addition of cytokines, so the activation of T cells and NK cells cannot achieve a good effect.
  • multispecific antibodies are also emerging in large numbers.
  • These multifunctional antibodies often use targets such as cytokines or CD3 as immune stimulators or inhibitors against tumor antigens, achieving mechanisms that are not available in monoclonal antibodies or combined drugs or exceeding their efficacy.
  • Patent CN109496217A discloses a soluble fusion protein complex comprising at least two soluble fusion proteins, for example, the first fusion protein is an anti-CD3 antibody covalently linked to an interleukin-15 (IL-15) polypeptide or a functional fragment thereof .
  • the second fusion protein comprises a binding domain that recognizes a disease antigen, wherein this domain is covalently linked to a soluble interleukin-15 receptor alpha (IL-15R ⁇ ) polypeptide or a functional fragment thereof.
  • IL-15R ⁇ soluble interleukin-15 receptor alpha
  • target design combination to achieve good multi-mechanism synergy is a preferred strategy.
  • multi-target design also brings difficulties in structural design.
  • different architecture designs have a great relationship with the druggability and even mechanism of antibodies.
  • the formation of immune synapses, the relative distance between antibody functional sites and antigens, etc., are closely related to architectural design.
  • the present invention designs a structure mainly composed of anti-TAA/CD3 and IL-15/IL-15 ⁇ complexes.
  • This article describes the multifunctional fusion proteins CCI (anti-Claudin18.2 and CD3, plus IL-15/IL-15 ⁇ ) and BCI (anti-B7H3 and CD3, plus IL-15/IL-15 ⁇ multifunctional fusion protein) protein) as an example, using this kind of framework design and target combination, discloses the biological effect of targeting Claudin18.2 (or B7H3) and CD3 obtained by genetic engineering technology, while having IL-15/IL-15R ⁇ complex
  • the multifunctional fusion protein is disclosed, and the amino acid sequence encoding the multifunctional fusion protein, the structural design, the recombinant cell comprising the recombinant vector, the preparation method of the multifunctional fusion protein, and the medical use thereof are disclosed.
  • the technical scheme adopted in the present invention is as follows:
  • the present invention relates to a multifunctional fusion protein comprising a first heavy chain, a second heavy chain and a first light chain, a second light chain, the first heavy chain comprising VH and CH1 that specifically bind to a target, cytokines and its receptor, and the immunoglobulin Fc part;
  • the second heavy chain comprises VH and CH1 that specifically bind to the target, scFv or Fab that specifically binds the target, and the immunoglobulin Fc part;
  • the first light chain and the A heavy chain, a second light chain and a second heavy chain are specifically paired, respectively.
  • the VH and CH1 contained in the first heavy chain and the second heavy chain can specifically bind to TAA antigens, and the TAA antigens are Claudin18.2, CA125, AFP, CEA, EGFR, HER2, B7H3, B7H6, MUC1 One or more of , MUC16, GPC3, and CD24, preferably, the TAA antigen is Claudin18.2 or B7H3.
  • cytokines and their receptors contained in the first heavy chain are IL-15 and IL-15 receptors, respectively.
  • the scFv or Fab contained in the second heavy chain can specifically activate T cells, NK cells and macrophages.
  • the scFv or Fab contained in the second heavy chain specifically targets CD3.
  • immunoglobulin Fc portion of the first heavy chain and the second heavy chain is selected from the constant region amino acid sequences of IgG1, IgG2, IgG3, and IgG4, preferably selected from the constant region amino acid sequences of IgG1 or IgG4.
  • the Fc portion of the first and second heavy chains further comprises one or more amino acid substitutions selected from the group consisting of: S228P, L234F, L235E, P331S, D356K, T366W, K392D, D399K, Y407A , and K409D, preferably S228P, T366W and/or Y407A.
  • the IL-15 in the first heavy chain and its receptor and the scFv or Fab in the second heavy chain can be chimeric inside the Fc portion of the first heavy chain and the second heavy chain, respectively, It may also be present outside the Fc portion, preferably between the CH1 and CH2 domains of the corresponding heavy chain.
  • the IL-15 in the first heavy chain is covalently bound to the chain with its receptor, scFv or Fab in the second heavy chain alone or together with an additional linking peptide;
  • the linking peptide comprises glycine (G) and serine (S) residues, preferably comprising GGGGS repeats, more preferably 1-2 GGGGS repeats.
  • the IL-15 is selected from native IL-15 or a variant thereof comprising one or more selected from the group of N1D, N4D, D30N, E64Q, N65D, N72D, N79A, Q108E and N112A amino acid mutations, preferably comprising one or more amino acid mutations selected from the group of N4D, N65D, N72D, N79A and N112A;
  • the IL-15 receptor fragment is selected from IL-15R ⁇ or a variant thereof, preferably IL-15R ⁇ mutation body, more preferably the IL-15R ⁇ Sushi domain.
  • amino acid sequence of the first heavy chain is selected from SEQ ID NO:1; the amino acid sequence of the second heavy chain of the multifunctional fusion protein is selected from SEQ ID NO:2; the amino acid sequence of the first heavy chain of the multifunctional fusion protein is selected from SEQ ID NO:2; The amino acid sequences of the chain and the second light chain are selected from SEQ ID NO:3.
  • amino acid sequence of the first heavy chain is selected from SEQ ID NO: 14; the amino acid sequence of the second heavy chain of the multifunctional fusion protein is selected from SEQ ID NO: 15; the amino acid sequence of the first heavy chain of the multifunctional fusion protein is selected from SEQ ID NO: 15; The amino acid sequences of the chain and the second light chain are selected from SEQ ID NO:16.
  • the present invention also relates to a nucleic acid molecule encoding the multifunctional fusion protein, comprising a nucleotide sequence encoding a first light chain and a second light chain, or a nucleotide sequence encoding a first heavy chain, or a nucleotide sequence encoding a first light chain. Nucleotide sequence of the double chain.
  • nucleotide sequence encoding the first heavy chain is selected from SEQ ID NO: 4; the nucleotide sequence encoding the second heavy chain is selected from SEQ ID NO: 5; the encoding first light chain and the nucleotide sequence of the second light chain is selected from SEQ ID NO:6.
  • nucleotide sequence encoding the first heavy chain is selected from SEQ ID NO: 17; the nucleotide sequence encoding the second heavy chain is selected from SEQ ID NO: 18; the encoding first light chain and the nucleotide sequence of the second light chain is selected from SEQ ID NO:19.
  • nucleotide sequences can be fused to a polynucleotide encoding a signal peptide native to the original antibody or a heterologous signal peptide.
  • the nucleic acid molecule may further comprise a nucleotide sequence encoding a signal peptide at the 5' ends of the nucleotide sequence encoding its light chain and the nucleotide sequence encoding its heavy chain, respectively, and the signal peptide may It is a natural signal peptide, and can also be a heterologous signal peptide; it further comprises a stop codon at the 3' end of the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain, respectively.
  • the signal peptide is selected from the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:9, and the nucleotide sequence encoding the signal peptide is selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:10.
  • the present invention also relates to a recombinant vector, such as an expression vector, comprising a first heavy chain, and/or a second heavy chain, and/or a first light chain, and/or a second light chain encoding the multifunctional fusion protein
  • a recombinant vector such as an expression vector, comprising a first heavy chain, and/or a second heavy chain, and/or a first light chain, and/or a second light chain encoding the multifunctional fusion protein
  • the nucleotide sequence of the chain may be operably linked to one or more regulatory elements.
  • the regulatory elements are selected from expression control sequences, such as promoters, enhancers and the like.
  • the vectors of the present invention include a regulatory element (eg, a promoter) operably linked to the nucleic acid sequence encoding the first heavy chain, second heavy chain, first light chain and/or second light chain of the multifunctional fusion protein or enhancer).
  • a regulatory element eg, a promoter
  • operably linked refers to an arrangement of nucleic acid sequences constructed such that their normal function is performed. Accordingly, a regulatory element operably linked to the nucleotide sequence encoding said first heavy chain, second heavy chain, first light chain or second light chain is capable of directing transcription, replication and/or translation to obtain said Multifunctional fusion protein.
  • the vector encodes the amino acid sequence of the first heavy chain, second heavy chain, first light chain or second light chain of the multifunctional fusion protein.
  • the expression vector is, for example, a prokaryotic expression vector, a eukaryotic expression vector, a phage vector or a viral vector. Further, the vector is selected from eukaryotic vectors. The heavy and light chains of the fusion protein can be expressed separately in the vector.
  • the 5' end of the nucleotide sequence encoding the first light chain and/or the second light chain is sequentially added with a HindIII restriction site, a kozak cleavage site Sequence and signal peptide sequence, add a stop codon and XhoI restriction site at the 3' end, and insert into pcDNA3.4-G418 by restriction enzyme ligation; in the nucleotide sequence encoding the first heavy chain (SEQ ID NO: 4) and the 5' end of the nucleotide sequence (SEQ ID NO: 5) of the second heavy chain are respectively added with HindIII restriction site, kozak sequence and signal peptide sequence sequence, and a stop codon is added at the 3' end and XhoI restriction site, and inserted into the vector by restriction restriction.
  • the present invention also relates to a recombinant cell comprising the recombinant vector of any one of the third aspect of the present invention.
  • the cells include human embryonic kidney cells HEK293 or HEK293T, HEK293E, HEK293F modified by HEK293, Chinese hamster ovary cells (CHO), CHO-S, CHO-DHFR-, CHO/DG44, ExpiCHO, and CHO-modified ExpiCHO, and its combination.
  • the present invention also relates to a method for preparing the multifunctional fusion protein, which specifically includes: culturing the recombinant cell described in the fourth aspect of the present invention under conditions sufficient to express the multifunctional fusion protein described in the first aspect of the present invention; expressing and The multifunctional fusion protein was purified.
  • the present invention also relates to a medicament containing the multifunctional fusion protein as an active ingredient, the medicament optionally containing a pharmaceutically acceptable carrier or excipient.
  • the present invention also relates to the use of the multifunctional fusion protein in the preparation of a medicament for preventing or treating TAA-related diseases or disorders such as tumors.
  • the tumor is a tumor that is ineffective against Claudin18.2 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against Claudin18.2 antibody monotherapy; further preferably, gastric cancer, esophageal cancer, pancreatic cancer, and the like.
  • the tumor is a tumor that is ineffective against B7H3 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against anti-B7H3 antibody monotherapy; further preferably, gastric cancer, esophageal cancer, pancreatic cancer, and the like.
  • the present invention also provides a method of treating tumors, comprising administering to a cancer patient a therapeutically effective amount of the multifunctional fusion protein.
  • the tumor is a tumor related to pathogenesis, preferably a tumor that is ineffective against Claudin18.2 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against Claudin18.2 antibody monotherapy; more preferably gastric cancer, esophageal cancer , pancreatic cancer.
  • the present invention also provides a method of treating tumors, comprising administering to a cancer patient a therapeutically effective amount of the multifunctional fusion protein.
  • the tumor is a tumor related to the pathogenesis, preferably a tumor that is ineffective against B7H3 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against anti-B7H3 antibody monotherapy; further preferably gastric cancer, esophageal cancer, and pancreatic cancer.
  • the present invention also relates to a pharmaceutical preparation, pharmaceutical composition or kit containing the multifunctional fusion protein as described above as an active ingredient.
  • the present invention designs and obtains a tumor-related antigen and CD3 targeting tumor-related antigens and has IL-15/IL through gene recombination, codon optimization, molecular biology and other technologies.
  • a multifunctional fusion protein for biological effects of the -15R ⁇ complex On the basis of targeting tumor antigens, the multifunctional fusion protein can effectively expand and activate T cells and NK cells in PMBC by using IL-15/IL-15R ⁇ complex, and increase the number of immune cells and killer cytokines.
  • IL-15 or IL-15/IL-15 receptor complex is more effective than IL-15 or IL-15/IL-15 receptor complex.
  • the serum half-life is prolonged, the tumor targeting ability is improved, and its toxic and side effects are reduced.
  • the combination of CD3 antibody and tumor-targeting antibody can target tumors and recruit T cells to approach tumor cells, which can mediate T cells to kill tumor cells.
  • IL-15 and IL-15R ⁇ are easily degraded by enzymes in vivo and difficult to express in vitro
  • the inventors innovatively designed a fusion protein of IL-15 and IL-15R ⁇ using the stability of natural antibodies in vivo. Inside the multifunctional fusion protein structure, it is protected inside the multifunctional fusion protein from being exposed and degraded by enzymes in the body, and at the same time, the difficulty of expression is reduced.
  • the designed CCI multifunctional fusion protein has been proved by experiments to have extremely high expression ability and stability.
  • the present invention rationally designs the relative distance of TAA, CD3 and IL-15 fusion protein, which is theoretically the optimal contact distance, which not only retains the recognition site of the antibody that recognizes the TAA end, but also ensures the recognition of the specific antibody end and IL-15.
  • the fusion protein functions, and the multifunctional fusion protein CCI can contact the antigen in all directions and maintain other synergistic functional activities.
  • the multifunctional fusion protein obtained by the present invention has efficient Claudin18.2 antigen affinity and IL-15 affinity, as well as good binding effect to CD3, and also has good purity, stability and Biological activity, in the commonly used antibody expression host cell CHO, can also get a better expression level.
  • using the target combination or structure design does not affect the function of the functional domain, increases the stability of the multifunctional fusion protein structure, and has a good synergistic mechanism, which can be applied to immune or tumor therapy.
  • antibody refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis.
  • Antibodies can be isolated from natural resources such as plasma or serum in which the antibodies are naturally present, or from culture supernatants of antibody-producing hybridoma cells, from animal immune sera, or from phage library screening. Alternatively, it may be partially or completely synthesized by techniques using genetic recombination or the like.
  • Preferred antibodies include, for example, antibodies of immunoglobulin isotypes or subclasses of these isotypes.
  • Human immunoglobulins are known to include 9 classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM.
  • the antibodies of the invention may include IgGl, IgG2, IgG3, IgG4.
  • multifunctional fusion protein refers to a protein comprising two or more antigen binding domains capable of binding two or more different epitopes (eg, two, three or more different epitopes) , the epitopes can be fusion proteins on the same or different antigens, and the multifunctional fusion proteins can also contain cytokines (such as IL-15, IL-15R ⁇ , etc.) and the like.
  • cytokines such as IL-15, IL-15R ⁇ , etc.
  • Claudin18.2 and “CLDN18.2” have the same meaning and can be used interchangeably.
  • variable region or “variable domain” of an antibody refers to the amino-terminal domain of an antibody heavy or light chain.
  • the variable domains of heavy and light chains may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding site.
  • single chain antibody both refer to antibody fragments of a single polypeptide chain that contain variable regions derived from heavy and light chains, but no constant regions.
  • single chain antibodies also contain a polypeptide linker between the VH and VL domains, which enables the formation of the desired structure thought to allow antigen binding.
  • Single chain antibodies are discussed in detail in "The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994)". See also International Patent WO1988/001649, US Patents US4946778 and US5260203.
  • single chain antibodies may be bispecific and/or humanized.
  • polypeptide refers to an amino acid chain of any length, regardless of modification (eg, phosphorylation or glycosylation).
  • polypeptide includes proteins and fragments thereof.
  • Polypeptides may be "foreign”, meaning that they are “heterologous”, ie foreign to the host cell being utilized, eg, human polypeptides produced by bacterial cells.
  • Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written left to right in amino-terminal to carboxy-terminal direction.
  • amino acid residue sequences are named with three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), Partic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F) , proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • variant refers to a polypeptide or polynucleotide that differs from a participating polypeptide or polynucleotide but retains essential properties.
  • a typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Often, the differences are limited such that the sequences involved in polypeptides and variants are generally very similar and identical in many regions.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (eg, substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code.
  • Variants of polypeptides may be naturally occurring, such as allelic variants, or may be variants not known to occur in nature.
  • the term "specificity" means that one of the molecules involved in specific binding does not show any significant binding to molecules other than one or more of the binding molecules.
  • the term is also used when the antibody variable region-containing domain is specific for a particular epitope of multiple epitopes in an antigen.
  • an antigen-binding molecule comprising the antibody variable region-containing domain can bind to various antigens having the epitope.
  • tumor-associated antigen preferably relates to specific expression in a limited number of tissues and/or organs under normal conditions or in a specific developmental stage as well as expression or abnormal expression in one or more tumor or cancer tissues of protein.
  • tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not or only rarely expressed in normal tissues.
  • the three-letter and one-letter codes for amino acids used in the present invention are as described in J. Boil. Chem., 243, p3558 (1968).
  • the "interaction" between the Fc of the first heavy chain or its variant and the Fc of the second heavy chain or its variant in the present invention refers to an inter-Fc interaction or an inter-Fc variant interaction.
  • An "Fc variant” refers to a change in Fc structure or function by the presence of one or more amino acid substitution, insertion or deletion mutations at appropriate sites in the Fc.
  • “Interaction between Fc variants” refers to the formation of space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions and the like between Fc variants designed by mutation.
  • Fc variants contributes to the formation of stable heterodimers.
  • Preferred mutagenesis designs are those in the "Knob-in-hole" format.
  • the Fc of the present invention may also have other mutations that lead to changes in its function, such as glycosylation mutations, Fc ⁇ R binding region mutations (to adjust ADCC activity), and amino acid mutations to improve antibody stability.
  • IL-15 or "IL-15 fragment” can be any IL-15 or a mutant thereof, such as human IL-15 or non-human mammalian or non-mammalian IL-15.
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, etc.; preferably human interleukin 15 mature molecules (see database UniProtKB, accession number P40933, 49-162aa).
  • IL-15 variant refers to a mutation that increases or decreases the affinity between IL-15 and its receptor, or that stimulates T cells or NK cells, by one or more amino acid substitutions, additions, or deletions mutations that increase or decrease the activity of body molecules.
  • the "IL-15 fragment” of the present invention is preferably a variant form thereof, more preferably IL-15N72D (SEQ ID NO: 11).
  • IL-15 and “IL-15 fragment” of the present invention can be used interchangeably and are not contradictory.
  • IL-15R ⁇ can be IL-15R ⁇ of any species or a functional fragment thereof, such as human IL-15R ⁇ or non-human mammalian IL-15R ⁇ or non-mammalian IL-15R ⁇ .
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, and the like.
  • IL-15R ⁇ variant refers to a functional mutant, preferably human IL-
  • the 15R ⁇ molecule is more preferably a shortened form of the human IL-15R ⁇ extracellular domain fragment, that is, a molecule with human IL-15 receptor ⁇ activity obtained by one or more amino acid deletion mutations from the C-terminal of the extracellular domain fragment, preferably retaining 65- 120 amino acid deletion mutant forms, more preferably 65-102 amino acid deletion mutant shortened forms, such as IL-15R ⁇ Sushi(65) (SEQ ID NO:12) or IL-15R ⁇ Sushi(77) (SEQ ID NO:13) .
  • covalently bound together with an additional linker peptide means that the coding regions of two or more genes can be covalently bound at one or several positions by the sequence encoding the linker peptide.
  • immunoglobulin refers to a globulin with antibody activity or chemical structure similar to that of an antibody molecule.
  • immunoglobulins There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into Subclasses (isotypes) such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • immunoglobulin Fc part refers to the C-terminal region of immunoglobulins, which has no antigen-binding activity, is the site of interaction between antibody molecules and effector molecules and cells, and is an antibody heavy chain Fc region polypeptide comprising two disulfide linkages. of dimer molecules. Fc regions can be produced by papain digestion or IdeS digestion into trypsinization of intact (full length) antibodies or can be produced recombinantly.
  • the "Fc portion” preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 regions of IgG.
  • Fc variants have been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion proteins.
  • Representative is the "Knob-in-Hole" form proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp617-621, 1996); Amgen company technicians use electrostatic steering (Electronic Steering) to form Fc-containing heterologous Dimeric form (US2010286374A1); heterodimeric form (SEEDbodies) formed by IgG/IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.
  • the Knob-in-Hole structure on the Fc variant fragment of the present invention refers to the mutation of the two Fc fragments, which can be combined in the form of "Knob-in-Hole" after the mutation.
  • the "Knob-in-Hole" model of Cater et al. is preferably used for site mutation engineering in the Fc region, so that the resulting first and second Fc variants can be in the form of "Knob-in-Hole" bind together to form heterodimers.
  • the selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is within the purview of those skilled in the art.
  • the Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are preferred, and the Fc regions of human antibodies IgG1 and IgG4 are more preferred.
  • One of the first Fc variant or the second Fc variant is randomly selected to mutate the knob and the other to mutate the hole.
  • the first Fc variant is mutated with knob; the second Fc variant is mutated with hole.
  • linker peptide is used in the present invention to link IL-15 and IL-15R ⁇ , VH and VL of CD3 into the corresponding heavy chains to ensure proper protein folding and peptide stability.
  • the "linking peptide” of the present invention is preferably (GGGGS)n, wherein n can be 0, 1, 2, 3, 4, 5 or more, preferably n is 1-2. If the connecting peptide sequence is too short, it may affect the folding of the higher-order structures of the two proteins, thereby interfering with each other; if the connecting peptide sequence is too long, it will involve the problem of immunogenicity, because the connecting peptide sequence itself is a new antigen.
  • heterodimer is preferably the product of gene co-expression. Such as co-expression in prokaryotic cells in E. coli; or co-expression in eukaryotic cells, such as 293, CHO.
  • co-expression refers to the co-expression of multiple genes in a cell and the simultaneous appearance of their products. These genes can be co-existing and individually or jointly controlled expression. In the present invention, three genes are preferably co-expressed in one eukaryotic cell.
  • the gene expression product obtained by co-expression is favorable for the efficient and simple formation of complexes; in the present invention, it is favorable for the formation of heterodimers.
  • nucleic acid is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded.
  • RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.
  • the nucleic acid can be contained in a vector.
  • vector herein includes any vector known to the skilled artisan, including plasmid vectors, cosmid vectors, bacteriophage vectors (eg, lambda phage), viral vectors (eg, adenovirus or baculovirus vectors), or artificial chromosomal vectors ( For example bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or P1 artificial chromosome (PAC)).
  • the vectors include expression vectors and cloning vectors.
  • Expression vectors include plasmids and viral vectors and generally contain the desired expectations for expression of operably linked coding sequences in a particular host organism (eg, bacteria, yeast, plants, insects, or mammals) or in in vitro expression systems Coding sequences and suitable DNA sequences.
  • Cloning vectors are typically used to engineer and amplify a desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment.
  • a “therapeutically effective amount” as used herein refers to the multifunctional fusion protein of the present invention, or the drug, required to achieve the desired disease or condition (eg, a tumor, for example, to regress or reduce the size of a tumor).
  • the effective amount can be determined for a particular purpose by practice, in a conventional manner.
  • the therapeutically effective amount may be that amount required to: reduce the number of cancer cells; reduce tumor size; inhibit (ie, slow or stop) infiltration of cancer cells into peripheral organs; inhibit (ie, slow or stop) ) tumor metastasis; inhibit tumor growth; and/or alleviate one or more symptoms associated with cancer.
  • the "tumor” of the present invention can be selected from B cell lymphoma, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nerve cancer Cancer of the system, esophagus, cervix, melanoma, uterine or endometrial, oral or laryngeal, liver, kidney, bile duct, small bowel or appendix, salivary gland, thymus, adrenal gland, osteosarcoma , chondrosarcoma, lipoma, testicular cancer and malignant fibrous histiocytoma.
  • FIG. 1 is a structural diagram of an exemplary multifunctional fusion protein CCI taking Claudin18.2/CD3/IL-15 as an example.
  • Figure 2a is the CE-SDS image of CCI under non-reducing conditions
  • Figure 2b is the CE-SDS image of CCI under reducing conditions.
  • Figure 3 is the SEC-HPLC chart of the multifunctional fusion protein CCI after repeated freezing and thawing 5 times.
  • Figure 4 shows the binding activity of the multifunctional fusion protein CCI to Claudin18.2.
  • Figure 5 shows the binding activity of the multifunctional fusion protein CCI to the receptor IL-2R ⁇ .
  • Figure 6 shows the binding activity of the multifunctional fusion protein CCI to CD3.
  • Figure 7 is a graph showing the proliferation of PBMC cells by the multifunctional fusion protein CCI.
  • Figure 8 is a flow cytometric phenotyping diagram of PBMC proliferation experiment cells.
  • Figure 9 shows CCI-mediated killing of Claudin18.2-CHO-K1 cells.
  • Figure 10 shows the binding activity of the multifunctional fusion protein BCI to B7H3.
  • Figure 11 shows the binding activity of the multifunctional fusion protein BCI to CD3.
  • Figures 12a-12b show the detection of B7H3 expression abundance in MCF-7 cells by flow cytometry.
  • Figure 13 shows the binding activity of the multifunctional fusion protein BCI to the receptor IL-2R ⁇ .
  • Figure 14 shows the antitumor activity of the multifunctional fusion protein BCI.
  • the heavy chain amino acid sequence of the Claudin18.2 antibody is SEQ ID NO: 21, and the light chain amino acid sequence is SEQ ID NO: 22.
  • the heavy chain amino acid sequence of the B7H3 chimeric antibody is SEQ ID NO: 20
  • the light chain amino acid sequence is SEQ ID NO: 16.
  • the amino acid sequence information of the light chain and heavy chain of the multifunctional fusion protein CCI is selected from the published or self-developed Claudin18.2 (or B7H3) target monoclonal antibody sequence information, and the variable region and constant region of the sequence are obtained by analysis information.
  • the native IL-15 and IL-15R ⁇ complex variant sequence was inserted into the amino acid sequence of one heavy chain, and the CD3 antibody scFv sequence was inserted at the corresponding position in the other heavy chain.
  • the Fc of the amino acid sequence of the multifunctional fusion protein to other IgG types, such as IgG4, etc., and further design amino acid mutations in the desired form in each heavy chain, thereby obtaining the amino acid sequence of the target multifunctional fusion protein, for:
  • Multifunctional fusion protein CCI the first heavy chain is SEQ ID NO:1, the second heavy chain is SEQ ID NO:2, and the first light chain and the second light chain are SEQ ID NO:3;
  • Multifunctional fusion protein BCI the first heavy chain is SEQ ID NO:14, the second heavy chain is SEQ ID NO:15, and the first light chain and the second light chain are SEQ ID NO:16.
  • Each of the above target amino acid sequences was converted into nucleotide sequences and targeted for a series of parameters that may affect the expression of antibodies in mammalian cells: codon preference, GC content (ie, guanine G and cytosine in the four bases of DNA).
  • the final optimized antibody nucleotide sequence is:
  • the first heavy chain is SEQ ID NO:4, the second heavy chain is SEQ ID NO:5, and the first light chain and the second light chain are SEQ ID NO:6;
  • the first heavy chain is SEQ ID NO: 17
  • the second heavy chain is SEQ ID NO: 18
  • the first light chain and the second light chain are SEQ ID NO: 19.
  • the pcDNA3.1-G418 vector was used as a dedicated vector for expressing the light and heavy chains of the multifunctional antibody.
  • the pcDNA3.1-G418 vector contains the promoter CMVPromoter used for the heavy chain, the eukaryotic selection marker G418 tag and the prokaryotic selection tag Ampicilline.
  • the nucleotide sequences of the light chain and heavy chain of the antibody expression of the multifunctional fusion proteins CCI and BCI were obtained by gene synthesis, the vector and the target fragment were double-enzyme digested with HindIII and XhoI, and then enzymatically linked by DNA ligase after recovery, and transformed Escherichia coli competent cell DH5 ⁇ , select positive clones and carry out plasmid extraction and enzyme digestion verification to obtain full-length first heavy chain, second heavy chain, first light chain and second light chain containing the fusion protein CCI.
  • Recombinant plasmids respectively CCI-1 (first heavy chain), CCI-2 (second heavy chain) and CCI-3 (the same first light chain and second light chain); obtain the full length of the fusion protein BCI
  • the recombinant plasmids of the first heavy chain, the second heavy chain, the first light chain and the second light chain are BCI-1 (the first heavy chain), BCI-2 (the second heavy chain) and BCI-3 (the first heavy chain), respectively.
  • the first light chain and the second light chain are the same).
  • the recombinant plasmids containing the above target genes were transformed into E. coli competent cells DH5 ⁇ , and the transformed bacteria were spread on 100 ⁇ g/mL ampicillin-containing Cultivate on LB plate, select plasmid clones and culture in liquid LB medium, shake bacteria at 260 rpm for 14 hours, extract plasmids with endotoxin-free plasmid extraction kit, dissolve with sterile water and measure the concentration with nucleic acid protein quantifier.
  • ExpiCHO was grown at 37°C, 8% CO 2 , 100 rpm to a cell density of 6 ⁇ 10 6 cells/mL.
  • the constructed vectors CCI-1, CCI-2 and CCI-3; and BCI-1, BCI-2 and BCI-3 were respectively transfected into the above cells using liposomes, and the transfection plasmid concentration was 1 mg/ml, and the lipid
  • the plastid concentration was determined with reference to the ExpiCHO TM Expression System kit, and cultured at 32°C, 5% CO 2 , and 100 rpm for 7-10 days. Feeds were made between 18-22 h after transfection and between day 5.
  • the above cultured product was centrifuged at 4000 g, filtered through a 0.22 ⁇ m filter, and the supernatant of the medium was collected.
  • the obtained antibody 6 protein was purified by Protein A and ion column, and the eluate was collected.
  • the specific operation steps of ProteinA and ion column purification are as follows: after high-speed centrifugation of the cell culture fluid, the supernatant is taken, and affinity chromatography is performed using GE's ProteinA chromatography column.
  • the equilibration buffer for chromatography is 1 ⁇ PBS (pH 7.4), the cell supernatant is loaded and combined, washed with PBS until it returns to the baseline, and then the target protein is eluted with elution buffer 0.1M glycine (pH 3.0). , pH was adjusted to neutrality with Tris. Adjust the pH of the product obtained by affinity chromatography to 1-2 pH units below or above pI, and dilute appropriately to control the sample conductance below 5ms/cm.
  • pH buffers such as phosphate buffer, acetate buffer and other conditions
  • ion exchange chromatography methods in the field such as anion exchange or cation exchange to carry out NaCl gradient elution under corresponding pH conditions, select according to SDS-PAGE
  • the collection tubes where the target protein is located are combined and stored.
  • the CE-SDS assay proved that the non-reducing CE-SDS condition was 1 peak, and the target antibody was reduced to 3 peaks under reducing CE-SDS, that is, the positions were at LC, 1, and 2 positions, corresponding to the two peaks of the desired antibody. Different heavy chains as well as the same light chain. Therefore, after the plasmid transfection, transient expression and purification, it is proved that the obtained antibody fusion proteins CCI and BCI have correct structure and high purity.
  • the corresponding CE-SDS electrophoresis patterns of the multifunctional fusion protein CCI are shown in Figure 2a and Figure 2b, respectively. Then, the quality, in vitro binding activity and cell biological activity test analysis of the obtained antibody CCI was carried out.
  • the constructed Claudin18.2-CHO-K1 cells stably expressing the Claudin18.2 antigen grown in log phase were plated, plated in 96-well plates at 0.8 ⁇ 10 5 /well, and cultured in a carbon dioxide incubator at 37°C, 5% CO 2 .
  • Fixation Aspirate the excess medium, add 200 ⁇ L/well of 1X PBST to the well plate, wash twice, 4% paraformaldehyde 100 ⁇ L/well, place at -20°C for 15 minutes, remove the fixative with a pipette, use Wash twice with PBST; incubate at 37°C for 1 hour with blocking solution containing 2% BSA, and wash three times with PBST; dilute CCI with 0.5% BSA sample diluent to 0.3 ⁇ g/mL, which is the initial concentration , carry out 3-fold gradient dilution, a total of 7 gradients, and set up a negative control, 100 ⁇ L per well, incubate at 37 °C for 1 h; wash the plate 3 times with PBST, and use the HRP-labeled goat anti-human IgG Fc sample dilution solution at 1:1 20000 dilution, add 100 ⁇ L to each well, and incubate for 45 min at room temperature; after washing
  • the IL-2R ⁇ receptor was diluted to 4 ⁇ g/mL with PBS buffer pH 7.4, and 100 ⁇ L per well was added to a 96-well ELISA plate and coated overnight at 4 degrees. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the CCI was diluted to 4 ⁇ g/mL with 0.5% BSA sample diluent, taking this as the starting concentration, 3-fold gradient dilution was carried out, a total of 7 gradients, and a negative control was set, 100 ⁇ L per well, 37 Incubate at °C for 1 h.
  • the plate was washed three times with PBST, and the HRP-labeled goat anti-human IgG Fc was diluted 1:10000 with sample diluent, 100 ⁇ L was added to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, 100 ⁇ L of TMB substrate was added to each well, incubated at room temperature for 10 minutes in the dark, and 100 ⁇ L of 1M HCl solution was added to each well to stop the color reaction.
  • the logarithm of the concentration of CCI was taken as the abscissa, and the measured absorbance value of each well was the ordinate.
  • the Sigmoidaldose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, SanDiego, California) was used for nonlinear regression to obtain Binding curve of the multifunctional fusion protein CCI to IL-2R ⁇ .
  • the ELISA results of the multifunctional fusion protein CCI are shown in FIG. 5 .
  • the multifunctional fusion protein CCI can bind to IL-2R ⁇ at various concentrations, indicating that the CCI of the structure has good binding ability to IL-2R ⁇ .
  • the multifunctional fusion protein CCI, CD3 antibody, and irrelevant antibody IgG were diluted to 1.5 ⁇ g/mL with PBS buffer pH 7.4, and 100 ⁇ L per well was added to a 96-well ELISA plate, and coated overnight at 4 degrees. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the CD3 was diluted to 10 ⁇ g/mL with 1% BSA sample diluent, taking this as the starting concentration, 3-fold gradient dilution was carried out, a total of 7 gradients, and a negative control was set, 100 ⁇ L per well, 37 Incubate at °C for 1 h.
  • the plate was washed three times with PBST, and the HRP-labeled rabbit anti-6*His antibody was diluted 1:20000 with sample diluent, 100 ⁇ L was added to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 ⁇ L of TMB substrate to each well, incubate at room temperature for 10 minutes in the dark, and add 100 ⁇ L of 1M HCl solution to each well to stop the color reaction. Select the wavelength of 450nm on a multi-function microplate reader, and detect the absorbance value of each well at OD450nm.
  • the concentration of the multifunctional fusion protein CCI was taken as Log10 as the abscissa, and the measured absorbance value of each well was the ordinate.
  • the Sigmoidaldose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, SanDiego, California) was used for nonlinear Regression to obtain the binding curve of CCI and CD3.
  • the ELISA results of the multifunctional fusion protein CCI are shown in Figure 6.
  • the multifunctional fusion protein CCI can bind to CD3 at various concentrations, but IgG1 does not bind to CD3, which is consistent with the design effect.
  • the titer of monoclonal antibody CD3 is half of CCI.
  • PBMC cells Use commercial PBMC cells, after recovery, add 1 ⁇ 10 6 cells/mL to a 24-well plate, add anti-CD3 antibody OKT3 1 ⁇ g/mL to each well for activation, continue to culture, and add corresponding concentrations of different cells every 2 days. Antibodies (CCI or IL-15) continued to stimulate, and the total number of cells was counted each time.
  • CCI or IL-15 Antibodies
  • Flow antibodies include PerCp-cy5.5-CD3 (Cat: 552852; BD), APC-CD56 (Cat: 555518; BD), PE-CD4 (Cat: 550630; BD), PE-cy7-CD16 (557744; BD) ); at 1 ⁇ 10 6 /experiment, add flow antibody and incubate at 37°C for 1 h, centrifuge at 2000 r/min for 5 min, then resuspend and wash in 1 mL of PBS, repeat the washing operation twice, and finally use 200 ⁇ L of PBS to resuspend cells, Flow cytometry was used for analysis, with no added flow antibody as blank control, namely Blank.
  • A, D, G are the flow cytometry analysis of CD3 marker of PBMC proliferation under different stimulation conditions
  • B, E, H are flow cytometry analysis of CD16 marker of PBMC proliferation under different stimulation conditions
  • C, F, I are different stimulation conditions
  • Flow cytometry analysis of CD56 marker of PBMC proliferation under conditions, where A, B, C are CCI activation + no continuous stimulation, D, E, F are CCI activation + CCI continuous stimulation, G, H, L are OKT3 activation + IL-15 Continuous stimulation.
  • CD3 positive cells more than 90% of the cells that stimulate proliferation are CD3 positive cells, indicating that most of the proliferating cells are T cells, of which CD4+ and CD8+ are included in CD3 positive cells, and CD8+ cells are higher than CD4+ cells, of which CD4+ is Helper T cells, CD8+ are killer T cells, indicating that under the stimulation of CD3 antibody and IL-15, the proportion of killer T cells is greater than that of helper T cells; there is no significant difference between CD16 positive and CD56 positive conditions, indicating that the survival of NK cells is maintained.
  • CCI the cells maintained by CCI are basically T cells and NK cells, which is consistent with the design effect.
  • CCI not only has the function of CD3 to stimulate the activation of PBMC cells, but also can maintain the survival of T cells and NK cells.
  • CCI also has the function of CD3 antibody and IL-15 cytokine, which is consistent with the design effect.
  • the engineered cell line Claudin18.2-CHO-K1 was used, and 3 ⁇ 10 4 /well was plated in a 96-well plate. After culturing for 24 hours, 10 ⁇ g/mL of CCI antibody and irrelevant antibody were added to start with 10-fold dilution, with a total of 5 concentration gradients. , 1.5 ⁇ 10 5 /well of effector cells obtained by CCI activation and continuous stimulation of PBMC were added at the same time, so that the effector-target ratio was 5:1. After 24h incubation, PBS was rinsed for several times to wash away the effector cells.
  • the CCI antibody group has a killing effect on Claudin18.2-CHO-K1 cells, while the irrelevant antibody has no killing effect, indicating that the killing is target-specific; at the same time, the effect mediated by CCI
  • the cells were CCI-activated and continuously stimulated PBMC cells, indicating that CCI-activated and continuously stimulated PBMC cells could kill antibody-mediated target cells. Therefore, CCI can activate and continuously stimulate PBMC cells, and act as effector cells to specifically kill antibody-mediated target cells.
  • the huB7H3-his receptor was diluted to 0.5 ⁇ g/mL with pH 7.4 PBS buffer, 100 ⁇ L per well was added to a 96-well ELISA plate, and coated overnight at 4 degrees. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the multifunctional fusion protein BCI was diluted to 10 ⁇ g/mL with 0.5% BSA sample diluent, taking this as the starting concentration, 3-fold gradient dilution was carried out, a total of 11 gradients, and an irrelevant antibody negative control was set. Incubate with positive control B7H3 chimeric antibody, 100 ⁇ L per well, at 37°C for 1 h.
  • the plate was washed three times with PBST, and the HRP-labeled goat anti-human IgGFc was diluted 1:20000 with sample diluent, 100 ⁇ L was added to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 ⁇ L of TMB substrate to each well, incubate at room temperature for 10 minutes in the dark, and add 100 ⁇ L of 1M HCl solution to each well to stop the color reaction.
  • the logarithm of the antibody concentration was taken as the abscissa, and the measured absorbance value of each well was the ordinate.
  • the Sigmoidaldose-response (Variable Slope) method was used to perform nonlinear regression to obtain Binding curve of target antibody to B7H3 protein.
  • the binding activity of the multifunctional fusion protein BCI is shown in FIG. 10 .
  • the huCD3-his receptor was diluted to 1 ⁇ g/mL with PBS buffer pH 7.4, and 100 ⁇ L per well was added to a 96-well ELISA plate and coated overnight at 4°C. Block with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, dilute the BCI, CD3 antibody, and irrelevant antibody IgG sample dilutions to 10 ⁇ g/mL, using this as the starting concentration, carry out 3-fold gradient dilution, a total of 11 gradients, 100 ⁇ L per well, and incubate at 37 °C 1h.
  • the plate was washed three times with PBST, and the HRP-labeled goat anti-human IgG Fc was diluted 1:10000 with sample diluent, added 100 ⁇ L to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 ⁇ L of TMB substrate to each well, incubate at room temperature for 10 minutes in the dark, and add 100 ⁇ L of 1M HCl solution to each well to stop the color reaction.
  • the logarithm of the antibody concentration was taken as the abscissa, and the measured absorbance value of each well was the ordinate.
  • the Sigmoidaldose-response (Variable Slope) method was used to perform nonlinear regression to obtain Binding curve of BCI to CD3 protein.
  • the binding activity of the multifunctional fusion protein to IL-2R ⁇ was detected according to the method of Example 7, and the results are shown in FIG. 13 .
  • the multifunctional fusion protein can bind to IL-2R ⁇ at various concentrations, indicating that the multifunctional fusion protein has better binding ability to IL-2R ⁇ .
  • B7H3-positive breast cancer cells MCF-7 were plated in 96-well plates at 2 ⁇ 10 4 /well. After culturing for 24 h, 20 ⁇ g/mL of multifunctional fusion protein BCI and irrelevant antibodies were added to start with 5-fold dilution, with a total of 10 concentrations. Gradient, simultaneously add CIK (CD3+CD56+ cells) effector cells 4 ⁇ 10 4 /well, and set blank control (diluent), negative control (MCF-7+CIK, no antibody), irrelevant antibody group, in a cell incubator After incubation for 24 h, the cells were washed with PBS for several times to remove the effector cells.
  • BCI can kill B7H3-positive breast cancer cells MCF-7, while irrelevant antibodies have no killing effect, indicating that BCI mediates CIK cells to specifically kill B7H3-positive MCF-7 cells.

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Abstract

A multifunctional fusion protein targeting TAA and CD3 and having the biological effects of an IL-15/IL-15Rα complex. The multifunctional fusion protein can solve the drug-resistance and recurrence problem caused by a single-target antibody drug, reduce the effective dose, more effectively kill tumor cells, prolong the half-life of an IL-15/IL-15Rα serum and improve the tumor-targeting capability, and reduce the toxicity and side effects thereof. By means of using a combination of a CD3 antibody and a tumor-targeting antibody, a tumor can be directed and T cells can be recruited to get close to tumor cells, which plays a role in mediating T cells in order to kill tumor cells. The structure of the fusion protein is more stable in terms of structural design, for example, the formation of immune synapses and the relative distance between antibody functional sites and antigens. The IL-15/IL-15Rα complex promotes the persistent recruitment and activation of T cells and NK cells.

Description

一种多功能融合蛋白及其用途A kind of multifunctional fusion protein and use thereof 技术领域technical field
本发明属于生物技术领域，具体涉及一种多功能融合蛋白，尤其涉及靶向TAA与CD3，同时具有IL-15/IL-15Rα复合物的生物学效应的多功能融合蛋白。The invention belongs to the field of biotechnology, in particular to a multifunctional fusion protein, in particular to a multifunctional fusion protein targeting TAA and CD3 and having the biological effect of IL-15/IL-15Rα complex.
背景技术Background technique
肿瘤的发生和发展都伴随着对免疫***的入侵，免疫功能不全的个体常常有较高的癌症发生率和较差的预后。细胞因子(cytokine，CK)属于免疫调节分子，根据其性质、给药浓度和活性发挥部位的不同，对免疫***具有一定的激活或者抑制作用。因此给予免疫刺激性的细胞因子治疗的这种方式，可以达到提高癌症患者免疫功能的作用。Both tumor initiation and progression are accompanied by an invasion of the immune system, and immunocompromised individuals often have a higher incidence of cancer and a poorer prognosis. Cytokines (cytokine, CK) are immunomodulatory molecules, which have a certain activation or inhibition effect on the immune system according to their properties, administration concentrations and active sites. Therefore, this way of giving immunostimulatory cytokine therapy can improve the immune function of cancer patients.
细胞因子作为免疫疗法，虽然有一定益处，但在临床上的使用存在着单药给药靶向性差的缺点，只有高浓度给药才可以达到抗肿瘤作用，而高浓度给药会产生免疫抑制作用和高毒性。并且，非靶向性细胞因子对于免疫***的激活是***性的，免疫***被广泛的激活，具有致命的副作用。此外，由于细胞因子属于小分子量蛋白，不具备抗体的体内循环保护机制，单纯的细胞因子往往半衰期较短，需要短时间重复高剂量给药。目前临床研究药物多采用PEG化或者Fc融合来提高细胞因子的半衰期，虽然半衰期得以延长，但仍无法解决细胞因子的靶向性差的问题。目前已在多种小鼠肿瘤模型中验证了NK细胞和T细胞，尤其是细胞毒性T细胞在肿瘤免疫过程中的作用。多个临床试验正在评估某些细胞因子单用，或者与多种化疗药物和肿瘤靶向的单克隆抗体和其他细胞因子联用的抗癌疗效。但是，诸如IL-15等细胞因子存在的高剂量用药下的毒性以及半衰期短而导致的短时间重复给药问题仍然存在，成为联用策略的一大制约因素。As an immunotherapy, cytokines have certain benefits, but their clinical use has the disadvantage of poor targeting of single-drug administration. Only high-concentration administration can achieve anti-tumor effect, and high-concentration administration will produce immunosuppression. effects and high toxicity. Moreover, the activation of the immune system by non-targeted cytokines is systemic, and the immune system is widely activated with lethal side effects. In addition, because cytokines are small molecular weight proteins and do not have the protective mechanism of antibodies in vivo, simple cytokines often have a short half-life and require repeated high-dose administration in a short period of time. At present, most clinical research drugs use PEGylation or Fc fusion to improve the half-life of cytokines. Although the half-life is prolonged, it still cannot solve the problem of poor targeting of cytokines. The role of NK cells and T cells, especially cytotoxic T cells, in tumor immunity has been validated in a variety of mouse tumor models. Several clinical trials are evaluating the anticancer efficacy of certain cytokines alone or in combination with multiple chemotherapeutic agents and tumor-targeted monoclonal antibodies and other cytokines. However, the toxicity of cytokines such as IL-15 under high-dose administration and the short-term repeated administration caused by the short half-life still exist, which have become a major constraint to the combination strategy.
CD3是由肽链以非共价键组成的复合分子，在成熟T细胞表面表达，对阻断急性同种异体排斥反应起主要作用，且具有稳定TCR结构和传递活化信号的作用。针对CD3分子的单克隆抗体能够激活T细胞增殖及活化，在其他细胞因 子IL-15、IL-15Rα等共同作用下，产生具有增殖速度快、杀瘤活性高、杀瘤谱广及非MHC限制性杀瘤特点的CIK细胞，对癌症、慢性白血病、肝病及神经性疾病等多种疾病治疗具有显著疗效。因此利用CD3抗体与肿瘤靶向抗体进行组合，所构建的双特异性抗体可招募T细胞接近肿瘤细胞，起到介导T细胞杀伤肿瘤细胞的作用。CD3 is a complex molecule composed of peptide chains with non-covalent bonds. It is expressed on the surface of mature T cells and plays a major role in blocking acute allograft rejection. It also stabilizes the TCR structure and transmits activation signals. Monoclonal antibodies against CD3 molecules can activate T cell proliferation and activation. Under the combined action of other cytokines IL-15, IL-15Rα, etc., it produces fast proliferation, high tumoricidal activity, broad tumoricidal spectrum and non-MHC restriction. The CIK cells with tumor-killing characteristics have remarkable curative effect on the treatment of various diseases such as cancer, chronic leukemia, liver disease and neurological disease. Therefore, by combining CD3 antibody and tumor-targeting antibody, the constructed bispecific antibody can recruit T cells to approach tumor cells and play a role in mediating T cells to kill tumor cells.
双特异性抗体在临床上有望成为针对癌症、自身免疫和传染病的下一代生物疗法，具有单一靶点抗体无法达到的功能。双特性抗体为细化抗体药物作用机制、探索多机制协同效应提供了很好的途径。Bispecific antibodies are clinically expected to be the next generation of biotherapeutics for cancer, autoimmunity and infectious diseases, with functions that cannot be achieved by single-target antibodies. Bispecific antibodies provide a good way to refine the mechanism of action of antibody drugs and explore the synergistic effect of multiple mechanisms.
专利CN110023336A公开了包含至少三个结合结构域的结合剂，其中第一结合结构域与T细胞特异性抗原结合，并且第二结合结构域和第三结合结构域与密蛋白(Claudin6或Claudin18.2)结合。其首次报道了包含密蛋白的两个结合结构域和靶向T细胞特异性抗原(例如CD3)的另一个结合结构域的结合剂可诱导强效的T细胞介导的裂解并且在***疾病中有效。而此抗体结构没有细胞因子的加入，对T细胞和NK细胞的的激活作用不能达到很好的效果。Patent CN110023336A discloses a binding agent comprising at least three binding domains, wherein the first binding domain binds to T cell-specific antigen, and the second binding domain and the third binding domain bind to claudin (Claudin6 or Claudin18.2 ) combined. It is reported for the first time that a binding agent comprising two binding domains of claudin and another binding domain targeting T cell-specific antigens such as CD3 induces potent T cell-mediated lysis and is useful in the treatment of oncological diseases. effective in. However, this antibody structure does not have the addition of cytokines, so the activation of T cells and NK cells cannot achieve a good effect.
而随着抗体工程技术的发展和治疗机制研究的深入，三特异性抗体和多特异性抗体也在大量涌现。这些多功能抗体常常以细胞因子或CD3等靶点作为辅助抗肿瘤抗原的免疫刺激或抑制剂，达到单抗或联合用药不具备的机理或超过其疗效。With the development of antibody engineering technology and the in-depth study of therapeutic mechanisms, trispecific antibodies and multispecific antibodies are also emerging in large numbers. These multifunctional antibodies often use targets such as cytokines or CD3 as immune stimulators or inhibitors against tumor antigens, achieving mechanisms that are not available in monoclonal antibodies or combined drugs or exceeding their efficacy.
专利CN109496217A公开了一种包含至少两种可溶性融合蛋白的可溶性融合蛋白复合体，例如，第一融合蛋白是共价连接至白介素-15(IL-15)多肽或其功能性片段的抗-CD3抗体。第二融合蛋白包含识别疾病抗原的结合域，其中，这一域共价链接至可溶性白介素-15受体α(IL-15Rα)多肽或其功能性片段。虽然此结构抗体能够激活效应T细胞和NK细胞并引导其活性以对抗疾病细胞为目标，从而导致疾病特异性的细胞毒性。但是，由于IL-15和IL-15Rα很容易被体内酶降解，且在体外难表达，因此，此抗体在稳定性方面不尽人意。Patent CN109496217A discloses a soluble fusion protein complex comprising at least two soluble fusion proteins, for example, the first fusion protein is an anti-CD3 antibody covalently linked to an interleukin-15 (IL-15) polypeptide or a functional fragment thereof . The second fusion protein comprises a binding domain that recognizes a disease antigen, wherein this domain is covalently linked to a soluble interleukin-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof. Although this structural antibody is able to activate effector T cells and NK cells and direct their activity to target disease cells, resulting in disease-specific cytotoxicity. However, since IL-15 and IL-15Rα are easily degraded by enzymes in vivo and difficult to express in vitro, the stability of this antibody is not satisfactory.
因此靶点设计组合达到良好的多机制协同作用是优选的一种策略，然而多靶点设计同样带来结构设计上的难题。在行业经验中，不同的架构设计，对抗体的成药性乃至机制发挥有莫大的关系。免疫突触的形成，抗体功能位点与抗原之间 的相对距离等等都与架构设计息息相关。Therefore, target design combination to achieve good multi-mechanism synergy is a preferred strategy. However, multi-target design also brings difficulties in structural design. In industry experience, different architecture designs have a great relationship with the druggability and even mechanism of antibodies. The formation of immune synapses, the relative distance between antibody functional sites and antigens, etc., are closely related to architectural design.
发明内容SUMMARY OF THE INVENTION
为了克服现有技术的不足，解决现有技术中存在的上述问题，满足多功能融合蛋白架构设计需求，提高多功能融合蛋白的稳定性，改善肿瘤预后，兼具良好的成药性。本发明设计了一种结构，主要由抗TAA/CD3及IL-15/IL-15α复合物组成。本文叙述以多功能融合蛋白CCI(抗Claudin18.2与CD3，外加IL-15/IL-15α的多功能融合蛋白)和BCI(抗B7H3与CD3，外加IL-15/IL-15α的多功能融合蛋白)作为示例，使用该种架构设计与靶点组合，公开了通过基因工程技术获得的靶向Claudin18.2(或B7H3)与CD3，同时具有IL-15/IL-15Rα复合物的生物学效应的多功能融合蛋白，并公开了编码所述多功能融合蛋白的氨基酸序列、架构设计、以及包含所述重组载体的重组细胞以及所述多功能融合蛋白的制备方法及其医药用途。具体而言，本发明采用的技术方案如下：In order to overcome the deficiencies of the prior art, solve the above problems existing in the prior art, meet the needs of the multifunctional fusion protein architecture design, improve the stability of the multifunctional fusion protein, improve the tumor prognosis, and have good druggability. The present invention designs a structure mainly composed of anti-TAA/CD3 and IL-15/IL-15α complexes. This article describes the multifunctional fusion proteins CCI (anti-Claudin18.2 and CD3, plus IL-15/IL-15α) and BCI (anti-B7H3 and CD3, plus IL-15/IL-15α multifunctional fusion protein) protein) as an example, using this kind of framework design and target combination, discloses the biological effect of targeting Claudin18.2 (or B7H3) and CD3 obtained by genetic engineering technology, while having IL-15/IL-15Rα complex The multifunctional fusion protein is disclosed, and the amino acid sequence encoding the multifunctional fusion protein, the structural design, the recombinant cell comprising the recombinant vector, the preparation method of the multifunctional fusion protein, and the medical use thereof are disclosed. Specifically, the technical scheme adopted in the present invention is as follows:
本发明涉及一种多功能融合蛋白，其包含第一重链、第二重链和第一轻链、第二轻链，所述第一重链包含特异性结合靶标的VH和CH1，细胞因子和其受体，以及免疫球蛋白Fc部分；所述第二重链包含特异性结合靶标的VH和CH1，特异性结合靶标的scFv或Fab，以及免疫球蛋白Fc部分；第一轻链与第一重链，第二轻链与第二重链分别特异性配对。The present invention relates to a multifunctional fusion protein comprising a first heavy chain, a second heavy chain and a first light chain, a second light chain, the first heavy chain comprising VH and CH1 that specifically bind to a target, cytokines and its receptor, and the immunoglobulin Fc part; the second heavy chain comprises VH and CH1 that specifically bind to the target, scFv or Fab that specifically binds the target, and the immunoglobulin Fc part; the first light chain and the A heavy chain, a second light chain and a second heavy chain are specifically paired, respectively.
进一步地，所述第一重链和第二重链包含的VH和CH1可以特异性结合TAA抗原，所述TAA抗原为Claudin18.2、CA125、AFP、CEA、EGFR、HER2、B7H3、B7H6、MUC1、MUC16、GPC3、CD24其中的一种或几种，优选的，所述TAA抗原为Claudin18.2或B7H3。Further, the VH and CH1 contained in the first heavy chain and the second heavy chain can specifically bind to TAA antigens, and the TAA antigens are Claudin18.2, CA125, AFP, CEA, EGFR, HER2, B7H3, B7H6, MUC1 One or more of , MUC16, GPC3, and CD24, preferably, the TAA antigen is Claudin18.2 or B7H3.
进一步地，所述第一重链包含的细胞因子和其受体分别为IL-15和IL-15受体。Further, the cytokines and their receptors contained in the first heavy chain are IL-15 and IL-15 receptors, respectively.
进一步地，所述第二重链包含的scFv或Fab可以特异性激活T细胞、NK细胞和巨噬细胞，优选的，所述第二重链包含的scFv或Fab特异性靶向CD3。Further, the scFv or Fab contained in the second heavy chain can specifically activate T cells, NK cells and macrophages. Preferably, the scFv or Fab contained in the second heavy chain specifically targets CD3.
进一步地，所述第一重链和第二重链的免疫球蛋白Fc部分选自IgG1、IgG2、IgG3、IgG4的恒定区氨基酸序列，优选选自IgG1或IgG4的恒定区氨基酸序列。Further, the immunoglobulin Fc portion of the first heavy chain and the second heavy chain is selected from the constant region amino acid sequences of IgG1, IgG2, IgG3, and IgG4, preferably selected from the constant region amino acid sequences of IgG1 or IgG4.
进一步地，所述第一重链和第二重链的Fc部分还包含一个或多个选自由以下 组成的组的氨基酸替换：S228P、L234F、L235E、P331S、D356K、T366W、K392D、D399K、Y407A、和K409D，优选S228P、T366W和/或Y407A。Further, the Fc portion of the first and second heavy chains further comprises one or more amino acid substitutions selected from the group consisting of: S228P, L234F, L235E, P331S, D356K, T366W, K392D, D399K, Y407A , and K409D, preferably S228P, T366W and/or Y407A.
进一步地，所述第一重链中的IL-15与其受体和第二重链中的scFv或Fab可以分别嵌合于所述第一重链和所述第二重链的Fc部分内部，也可以存在于Fc部分外部，优选位于所述相应重链的CH1和CH2功能区之间。Further, the IL-15 in the first heavy chain and its receptor and the scFv or Fab in the second heavy chain can be chimeric inside the Fc portion of the first heavy chain and the second heavy chain, respectively, It may also be present outside the Fc portion, preferably between the CH1 and CH2 domains of the corresponding heavy chain.
进一步地，所述第一重链中的IL-15与其受体、第二重链中的scFv或Fab单独或与额外的连接肽一起共价结合于所述链中；所述连接肽包含甘氨酸(G)和丝氨酸(S)残基，优选包含GGGGS重复，更优选包含1-2个GGGGS重复。Further, the IL-15 in the first heavy chain is covalently bound to the chain with its receptor, scFv or Fab in the second heavy chain alone or together with an additional linking peptide; the linking peptide comprises glycine (G) and serine (S) residues, preferably comprising GGGGS repeats, more preferably 1-2 GGGGS repeats.
进一步地，所述IL-15选自天然的IL-15或其变体，所述变体包含选自N1D、N4D、D30N、E64Q、N65D、N72D、N79A、Q108E和N112A的组的一个或多个氨基酸突变，优选包含选自N4D、N65D、N72D、N79A和N112A的组的一个或多个氨基酸突变；所述IL-15受体片段选自IL-15Rα或其变体，优选IL-15Rα变体，更优选IL-15RαSushi结构域。Further, the IL-15 is selected from native IL-15 or a variant thereof comprising one or more selected from the group of N1D, N4D, D30N, E64Q, N65D, N72D, N79A, Q108E and N112A amino acid mutations, preferably comprising one or more amino acid mutations selected from the group of N4D, N65D, N72D, N79A and N112A; the IL-15 receptor fragment is selected from IL-15Rα or a variant thereof, preferably IL-15Rα mutation body, more preferably the IL-15RαSushi domain.
进一步地，所述第一重链氨基酸序列选自SEQ ID NO:1；所述多功能融合蛋白的第二重链氨基酸序列选自SEQ ID NO:2；所述多功能融合蛋白的第一轻链和第二轻链的氨基酸序列选自SEQ ID NO:3。Further, the amino acid sequence of the first heavy chain is selected from SEQ ID NO:1; the amino acid sequence of the second heavy chain of the multifunctional fusion protein is selected from SEQ ID NO:2; the amino acid sequence of the first heavy chain of the multifunctional fusion protein is selected from SEQ ID NO:2; The amino acid sequences of the chain and the second light chain are selected from SEQ ID NO:3.
进一步地，所述第一重链氨基酸序列选自SEQ ID NO:14；所述多功能融合蛋白的第二重链氨基酸序列选自SEQ ID NO:15；所述多功能融合蛋白的第一轻链和第二轻链的氨基酸序列选自SEQ ID NO:16。Further, the amino acid sequence of the first heavy chain is selected from SEQ ID NO: 14; the amino acid sequence of the second heavy chain of the multifunctional fusion protein is selected from SEQ ID NO: 15; the amino acid sequence of the first heavy chain of the multifunctional fusion protein is selected from SEQ ID NO: 15; The amino acid sequences of the chain and the second light chain are selected from SEQ ID NO:16.
本发明还涉及编码所述多功能融合蛋白的核酸分子，其包含编码第一轻链和第二轻链的核苷酸序列，或者包含编码第一重链的核苷酸序列，或者包含编码第二重链的核苷酸序列。The present invention also relates to a nucleic acid molecule encoding the multifunctional fusion protein, comprising a nucleotide sequence encoding a first light chain and a second light chain, or a nucleotide sequence encoding a first heavy chain, or a nucleotide sequence encoding a first light chain. Nucleotide sequence of the double chain.
进一步地，所述编码第一重链的核苷酸序列选自SEQ ID NO:4；所述编码第二重链的核苷酸序列选自SEQ ID NO:5；所述编码第一轻链和第二轻链的核苷酸序列选自SEQ ID NO:6。Further, the nucleotide sequence encoding the first heavy chain is selected from SEQ ID NO: 4; the nucleotide sequence encoding the second heavy chain is selected from SEQ ID NO: 5; the encoding first light chain and the nucleotide sequence of the second light chain is selected from SEQ ID NO:6.
进一步地，所述编码第一重链的核苷酸序列选自SEQ ID NO:17；所述编码第二重链的核苷酸序列选自SEQ ID NO:18；所述编码第一轻链和第二轻链的核苷酸序列选自SEQ ID NO:19。Further, the nucleotide sequence encoding the first heavy chain is selected from SEQ ID NO: 17; the nucleotide sequence encoding the second heavy chain is selected from SEQ ID NO: 18; the encoding first light chain and the nucleotide sequence of the second light chain is selected from SEQ ID NO:19.
进一步地，可以将此类核苷酸序列与编码对于初始抗体而言天然的信号肽或异源信号肽的多核苷酸融合。具体而言，所述核酸分子可在编码其轻链的核苷酸序列和编码其重链的核苷酸序列的5’端分别进一步包含编码信号肽的核苷酸序列，所述信号肽可以是天然的信号肽，也可以是异源信号肽；在编码轻链的核苷酸序列和编码重链的核苷酸序列的3’端分别进一步包含终止密码子。Further, such nucleotide sequences can be fused to a polynucleotide encoding a signal peptide native to the original antibody or a heterologous signal peptide. Specifically, the nucleic acid molecule may further comprise a nucleotide sequence encoding a signal peptide at the 5' ends of the nucleotide sequence encoding its light chain and the nucleotide sequence encoding its heavy chain, respectively, and the signal peptide may It is a natural signal peptide, and can also be a heterologous signal peptide; it further comprises a stop codon at the 3' end of the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain, respectively.
更进一步地，所述信号肽选自氨基酸序列SEQ ID NO:7和SEQ ID NO:9，编码所述信号肽的核苷酸序列选自SEQ ID NO:8和SEQ ID NO:10。Further, the signal peptide is selected from the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:9, and the nucleotide sequence encoding the signal peptide is selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:10.
本发明还涉及一种重组载体，例如表达载体，其包含编码所述多功能融合蛋白的第一重链、和/或第二重链、和/或第一轻链、和/或第二轻链的核苷酸序列。在此类载体中，本发明的核苷酸序列可以与一种或多种调节元件可操作连接。其中，所述调节元件选自表达调控序列，如启动子、增强子等。The present invention also relates to a recombinant vector, such as an expression vector, comprising a first heavy chain, and/or a second heavy chain, and/or a first light chain, and/or a second light chain encoding the multifunctional fusion protein The nucleotide sequence of the chain. In such vectors, the nucleotide sequences of the present invention may be operably linked to one or more regulatory elements. Wherein, the regulatory elements are selected from expression control sequences, such as promoters, enhancers and the like.
本发明的载体包括一段与编码所述多功能融合蛋白的第一重链、第二重链、第一轻链和/或第二轻链的核酸序列可操作性连接的调控元件(例如启动子或增强子)。“可操作性连接”是指所构成的核酸序列的布置使得可执行其正常功能。因此，与编码所述第一重链、第二重链、第一轻链或第二轻链的核苷酸序列可操作性连接的调控元件能够指导转录、复制和/或翻译而得到所述多功能融合蛋白。在一种实施方案中，该载体编码所述多功能融合蛋白的第一重链、第二重链、第一轻链或第二轻链的氨基酸序列。The vectors of the present invention include a regulatory element (eg, a promoter) operably linked to the nucleic acid sequence encoding the first heavy chain, second heavy chain, first light chain and/or second light chain of the multifunctional fusion protein or enhancer). "Operably linked" refers to an arrangement of nucleic acid sequences constructed such that their normal function is performed. Accordingly, a regulatory element operably linked to the nucleotide sequence encoding said first heavy chain, second heavy chain, first light chain or second light chain is capable of directing transcription, replication and/or translation to obtain said Multifunctional fusion protein. In one embodiment, the vector encodes the amino acid sequence of the first heavy chain, second heavy chain, first light chain or second light chain of the multifunctional fusion protein.
在本发明中，所述表达载体例如为原核表达载体、真核表达载体、噬菌体载体或病毒载体。进一步地，所述载体选自真核载体。融合蛋白的重链和轻链可以分别在载体中表达。In the present invention, the expression vector is, for example, a prokaryotic expression vector, a eukaryotic expression vector, a phage vector or a viral vector. Further, the vector is selected from eukaryotic vectors. The heavy and light chains of the fusion protein can be expressed separately in the vector.
在本发明的一个具体实施方案中，在编码第一轻链和/或第二轻链的核苷酸序列(SEQ ID NO:6)的5’端分别依次加上HindIII酶切位点、kozak序列和信号肽序列，在3’端加上终止密码子及XhoI酶切位点，通过酶切连接***到pcDNA3.4-G418；在编码第一重链的核苷酸序列(SEQ ID NO:4)和第二重链的核苷酸序列(SEQ ID NO:5)的5’端分别依次加上HindIII酶切位点、kozak序列和信号肽序列序列，在3’端加上终止密码子和XhoI酶切位点，通过酶切连接***到载体中。In a specific embodiment of the present invention, the 5' end of the nucleotide sequence encoding the first light chain and/or the second light chain (SEQ ID NO: 6) is sequentially added with a HindIII restriction site, a kozak cleavage site Sequence and signal peptide sequence, add a stop codon and XhoI restriction site at the 3' end, and insert into pcDNA3.4-G418 by restriction enzyme ligation; in the nucleotide sequence encoding the first heavy chain (SEQ ID NO: 4) and the 5' end of the nucleotide sequence (SEQ ID NO: 5) of the second heavy chain are respectively added with HindIII restriction site, kozak sequence and signal peptide sequence sequence, and a stop codon is added at the 3' end and XhoI restriction site, and inserted into the vector by restriction restriction.
本发明还涉及一种重组细胞，其含有本发明第三方面任一项的重组载体。进一步地，所述细胞包括人胚肾细胞HEK293或HEK293T、HEK293E、HEK293修饰的HEK293F、中国仓鼠卵巢细胞(CHO)、CHO-S、CHO-DHFR-、CHO/DG44、ExpiCHO、CHO修饰的ExpiCHO，和其组合。The present invention also relates to a recombinant cell comprising the recombinant vector of any one of the third aspect of the present invention. Further, the cells include human embryonic kidney cells HEK293 or HEK293T, HEK293E, HEK293F modified by HEK293, Chinese hamster ovary cells (CHO), CHO-S, CHO-DHFR-, CHO/DG44, ExpiCHO, and CHO-modified ExpiCHO, and its combination.
本发明还涉及所述多功能融合蛋白的制备方法，其具体包括：在足以表达本发明第一方面所述多功能融合蛋白的条件下，培养本发明第四方面所述的重组细胞；表达并纯化所述的多功能融合蛋白。The present invention also relates to a method for preparing the multifunctional fusion protein, which specifically includes: culturing the recombinant cell described in the fourth aspect of the present invention under conditions sufficient to express the multifunctional fusion protein described in the first aspect of the present invention; expressing and The multifunctional fusion protein was purified.
本发明还涉及一种含有所述多功能融合蛋白作为活性成分的药物，所述药物任选含有药学上可接受的载体或赋形剂。The present invention also relates to a medicament containing the multifunctional fusion protein as an active ingredient, the medicament optionally containing a pharmaceutically acceptable carrier or excipient.
本发明还涉及所述多功能融合蛋白在制备预防或治疗与TAA有关的疾病或病症如肿瘤的药物中的用途。The present invention also relates to the use of the multifunctional fusion protein in the preparation of a medicament for preventing or treating TAA-related diseases or disorders such as tumors.
优选地，所述肿瘤为对Claudin18.2单独治疗无效的肿瘤或晚期肿瘤，更优选为对抗Claudin18.2抗体单独治疗产生抗性或无效的肿瘤；进一步优选为胃癌、食道癌、胰腺癌等。Preferably, the tumor is a tumor that is ineffective against Claudin18.2 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against Claudin18.2 antibody monotherapy; further preferably, gastric cancer, esophageal cancer, pancreatic cancer, and the like.
优选地，所述肿瘤为对B7H3单独治疗无效的肿瘤或晚期肿瘤，更优选为对抗B7H3抗体单独治疗产生抗性或无效的肿瘤；进一步优选为胃癌、食道癌、胰腺癌等。Preferably, the tumor is a tumor that is ineffective against B7H3 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against anti-B7H3 antibody monotherapy; further preferably, gastric cancer, esophageal cancer, pancreatic cancer, and the like.
本发明还提供了一种***的方法，其包括向癌症患者施用治疗有效量的所述多功能融合蛋白。所述肿瘤为与发病机制相关的肿瘤，优选为对Claudin18.2单独治疗无效的肿瘤或晚期肿瘤，更优选为对抗Claudin18.2抗体单独治疗产生抗性或无效的肿瘤；进一步优选胃癌、食道癌、胰腺癌。The present invention also provides a method of treating tumors, comprising administering to a cancer patient a therapeutically effective amount of the multifunctional fusion protein. The tumor is a tumor related to pathogenesis, preferably a tumor that is ineffective against Claudin18.2 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against Claudin18.2 antibody monotherapy; more preferably gastric cancer, esophageal cancer , pancreatic cancer.
本发明还提供了一种***的方法，其包括向癌症患者施用治疗有效量的所述多功能融合蛋白。所述肿瘤为与发病机制相关的肿瘤，优选为对B7H3单独治疗无效的肿瘤或晚期肿瘤，更优选为对抗B7H3抗体单独治疗产生抗性或无效的肿瘤；进一步优选胃癌、食道癌、胰腺癌。The present invention also provides a method of treating tumors, comprising administering to a cancer patient a therapeutically effective amount of the multifunctional fusion protein. The tumor is a tumor related to the pathogenesis, preferably a tumor that is ineffective against B7H3 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against anti-B7H3 antibody monotherapy; further preferably gastric cancer, esophageal cancer, and pancreatic cancer.
本发明还涉及含有如上所述的多功能融合蛋白作为活性成分的药物制剂、药用组合物或试剂盒。The present invention also relates to a pharmaceutical preparation, pharmaceutical composition or kit containing the multifunctional fusion protein as described above as an active ingredient.
有益效果beneficial effect
本发明在已有的异源二聚体开发经验的基础上通过基因重组、密码子优化和分子生物学等技术，设计获得了一种靶向肿瘤相关抗原与CD3，同时具有IL-15/IL-15Rα复合物的生物学效应的多功能融合蛋白。该多功能融合蛋白在靶向肿瘤抗原的基础上，利用IL-15/IL-15Rα复合物可以有效地扩增和活化PMBC中的T细胞及NK细胞，并使得免疫细胞数目以及杀伤性细胞因子的释放增加，从而解决单靶点抗体药物的耐药和复发问题，同时也可降低有效剂量，更加有效地杀伤肿瘤细胞，且较IL-15或IL-15/IL-15受体复合物而言，延长了血清半衰期并提高了肿瘤靶向性，降低其毒副作用。同时利用CD3抗体与肿瘤靶向抗体的组合，可定向肿瘤并招募T细胞接近肿瘤细胞，起到介导T细胞杀伤肿瘤细胞的作用。On the basis of the existing heterodimer development experience, the present invention designs and obtains a tumor-related antigen and CD3 targeting tumor-related antigens and has IL-15/IL through gene recombination, codon optimization, molecular biology and other technologies. A multifunctional fusion protein for biological effects of the -15Rα complex. On the basis of targeting tumor antigens, the multifunctional fusion protein can effectively expand and activate T cells and NK cells in PMBC by using IL-15/IL-15Rα complex, and increase the number of immune cells and killer cytokines. IL-15 or IL-15/IL-15 receptor complex is more effective than IL-15 or IL-15/IL-15 receptor complex. In other words, the serum half-life is prolonged, the tumor targeting ability is improved, and its toxic and side effects are reduced. At the same time, the combination of CD3 antibody and tumor-targeting antibody can target tumors and recruit T cells to approach tumor cells, which can mediate T cells to kill tumor cells.
由于IL-15和IL-15Rα很容易被体内酶降解，且在体外难表达，因此，本发明人利用天然抗体在体内的稳定性，创新性的将IL-15和IL-15Rα的融合蛋白设计在多功能融合蛋白结构内部，将其被保护在多功能融合蛋白内部，免于暴露而被体内酶降解，同时降低了表达难度。此种设计的CCI多功能融合蛋白经实验证明具有极高的表达能力和稳定性。Since IL-15 and IL-15Rα are easily degraded by enzymes in vivo and difficult to express in vitro, the inventors innovatively designed a fusion protein of IL-15 and IL-15Rα using the stability of natural antibodies in vivo. Inside the multifunctional fusion protein structure, it is protected inside the multifunctional fusion protein from being exposed and degraded by enzymes in the body, and at the same time, the difficulty of expression is reduced. The designed CCI multifunctional fusion protein has been proved by experiments to have extremely high expression ability and stability.
同时，通过将IL-15和IL-15Rα通过接头(linker)连接，即IL-15的受体IL-15Rα内含一个sushi结构域，能与IL-15结合，使得CCI多功能融合蛋白结构更稳定。At the same time, by connecting IL-15 and IL-15Rα through a linker, that is, the receptor of IL-15, IL-15Rα, contains a sushi domain, which can bind to IL-15, making the structure of CCI multifunctional fusion protein more flexible. Stablize.
另外，本发明合理设计TAA、CD3和IL-15融合蛋白的相对距离，理论上为最佳接触距离，既保留了识别TAA端抗体的识别位点，又保证识别特异性抗体端与IL-15融合蛋白功能发挥，多功能融合蛋白CCI可以全方位接触抗原，并保持其他协同功能活性。In addition, the present invention rationally designs the relative distance of TAA, CD3 and IL-15 fusion protein, which is theoretically the optimal contact distance, which not only retains the recognition site of the antibody that recognizes the TAA end, but also ensures the recognition of the specific antibody end and IL-15. The fusion protein functions, and the multifunctional fusion protein CCI can contact the antigen in all directions and maintain other synergistic functional activities.
经所述示例试验验证，本发明所获得的多功能融合蛋白具有高效的Claudin18.2抗原亲和力和IL-15亲和力，以及与CD3较好的结合效果，此外还具有较好的纯度、稳定性以及生物活性，在常用的抗体表达宿主细胞CHO上，也可得到较好的表达量。It is verified by the example experiments that the multifunctional fusion protein obtained by the present invention has efficient Claudin18.2 antigen affinity and IL-15 affinity, as well as good binding effect to CD3, and also has good purity, stability and Biological activity, in the commonly used antibody expression host cell CHO, can also get a better expression level.
因此使用该靶点组合或结构设计，不影响功能域的功能发挥，增加了多功能融合蛋白结构的稳定性，且有良好协同作用机制，可应用于免疫或肿瘤治疗。Therefore, using the target combination or structure design does not affect the function of the functional domain, increases the stability of the multifunctional fusion protein structure, and has a good synergistic mechanism, which can be applied to immune or tumor therapy.
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件的它处另有明确定义，否则本文中使用的所有其他技术和科学术语都具 有本发明所属技术领域的一般技术人员通常理解的含义。For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
术语the term
在本说明书中，术语“抗体”是指天然的免疫球蛋白或者通过部分或完全合成而制备的免疫球蛋白。抗体可从天然存在该抗体的血浆或血清等的天然资源、或者产生抗体的杂交瘤细胞的培养上清中、动物免疫血清中、噬菌体文库筛选进行重建得到分离。备选地，可通过使用基因重组等的技术部分或完全地合成。优选的抗体包括，例如，免疫球蛋白的同种型或这些同种型的亚类的抗体。已知人免疫球蛋白包括IgGl、IgG2、IgG3、IgG4、IgAl、IgA2、IgD、IgE、IgM这9种类别(同种型)。在这些同种型中，本发明的抗体可以包括IgGl、IgG2、IgG3、IgG4。In the present specification, the term "antibody" refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be isolated from natural resources such as plasma or serum in which the antibodies are naturally present, or from culture supernatants of antibody-producing hybridoma cells, from animal immune sera, or from phage library screening. Alternatively, it may be partially or completely synthesized by techniques using genetic recombination or the like. Preferred antibodies include, for example, antibodies of immunoglobulin isotypes or subclasses of these isotypes. Human immunoglobulins are known to include 9 classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM. Among these isotypes, the antibodies of the invention may include IgGl, IgG2, IgG3, IgG4.
术语“多功能融合蛋白”是指包含两个或更多个抗原结合结构域，能够结合两个或更多个不同的表位(例如，两个、三个或更多个不同的表位)，表位可以在相同或不同的抗原上的融合蛋白，多功能融合蛋白还可以包含细胞因子(例如IL-15、IL-15Rα等)等。本发明中“多功能融合蛋白CCI”、“抗体CCI”、“CCI多功能融合蛋白”、“CCI抗体”、“CCI”并不矛盾，可互换使用。本发明中“多功能融合蛋白BCI”、“抗体BCI”、“BCI多功能融合蛋白”、“BCI抗体”、“BCI”并不矛盾，可互换使用。The term "multifunctional fusion protein" refers to a protein comprising two or more antigen binding domains capable of binding two or more different epitopes (eg, two, three or more different epitopes) , the epitopes can be fusion proteins on the same or different antigens, and the multifunctional fusion proteins can also contain cytokines (such as IL-15, IL-15Rα, etc.) and the like. In the present invention, "multifunctional fusion protein CCI", "antibody CCI", "CCI multifunctional fusion protein", "CCI antibody" and "CCI" are not contradictory and can be used interchangeably. In the present invention, "multifunctional fusion protein BCI", "antibody BCI", "BCI multifunctional fusion protein", "BCI antibody" and "BCI" are not contradictory and can be used interchangeably.
本发明中“Claudin18.2”和“CLDN18.2”具有相同的含义，可以互换使用。In the present invention, "Claudin18.2" and "CLDN18.2" have the same meaning and can be used interchangeably.
抗体的“可变区”或“可变域”指抗体重链或轻链的氨基末端结构域。重链和轻链的可变域可以分别称为“VH”和“VL”。这些结构域一般是抗体的最可变部分(相对于其他同类抗体)并含有抗原结合位点。The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding site.
术语“scFv”和“单链抗体”均指单个多肽链的抗体片段，其含有源自重链和轻链的可变区，但没有恒定区。通常，单链抗体还在VH和VL结构域之间含有多肽接头，这使得能够形成被认为允许抗原结合的所需结构。"The Pharmacology of Monoclonal Antibodies,Vol.113,Rosenburg and Moore,eds.,Springer-Verlag，New York,269-315(1994)"中详细讨论了单链抗体。还可参见国际专利WO1988/001649、美国专利US4946778和US5260203。在具体实施方案中，单链抗体可以是双特异性的和/或人源化的。The terms "scFv" and "single chain antibody" both refer to antibody fragments of a single polypeptide chain that contain variable regions derived from heavy and light chains, but no constant regions. Typically, single chain antibodies also contain a polypeptide linker between the VH and VL domains, which enables the formation of the desired structure thought to allow antigen binding. Single chain antibodies are discussed in detail in "The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994)". See also International Patent WO1988/001649, US Patents US4946778 and US5260203. In specific embodiments, single chain antibodies may be bispecific and/or humanized.
术语“多肽”是指任何长度的氨基酸链，而与修饰(例如磷酸化或糖基化)无关。 术语多肽包括蛋白质及其片段。多肽可以是“外源的”，意指它们是“异源的”，即是所利用的宿主细胞外来的，例如由细菌细胞产生的人多肽。本文将多肽公开为氨基酸残基序列。那些序列按氨基末端到羧基末端的方向从左到右书写。根据标准命名法，氨基酸残基序列以三字母或单字母代码命名，如下所示：丙氨酸(Ala，A)、精氨酸(Arg，R)、天冬酰胺(Asn，N)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酰胺(Gln，Q)、谷氨酸(Glu，E)、甘氨酸(Gly，G)、组氨酸(His，H)、异亮氨酸(Ile，I)、亮氨酸(Leu，L)、赖氨酸(Lys，K)、甲硫氨酸(Met，M)、苯丙氨酸(Phe，F)、脯氨酸(Pro，P)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、色氨酸(Trp，W)、酪氨酸(Tyr，Y)和缬氨酸(Val，V)。The term "polypeptide" refers to an amino acid chain of any length, regardless of modification (eg, phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. Polypeptides may be "foreign", meaning that they are "heterologous", ie foreign to the host cell being utilized, eg, human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written left to right in amino-terminal to carboxy-terminal direction. According to standard nomenclature, amino acid residue sequences are named with three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), Partic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F) , proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
术语“变体”是指与参与多肽或多核苷酸不同但保持基本特性的多肽或多核苷酸。多肽的典型变体在氨基酸序列方面与另一参考多肽不同。通常，差异是有限的，使得参与多肽和变体的序列总体上非常相似，并且在许多区域中是相同的。变体和参考多肽可以在氨基酸序列方面因一个或多个修饰(例如，取代、添加和/或缺失)而不同。取代或***的氨基酸残基可以是或可以不是由遗传密码编码的氨基酸残基。多肽的变体可以是天然存在的，诸如等位基因变体，或者可以是不知道会天然存在的变体。The term "variant" refers to a polypeptide or polynucleotide that differs from a participating polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Often, the differences are limited such that the sequences involved in polypeptides and variants are generally very similar and identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (eg, substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code. Variants of polypeptides may be naturally occurring, such as allelic variants, or may be variants not known to occur in nature.
术语“特异性”表示参与特异性结合的分子之一不显示任何与不同于结合分子中的一个或数个的分子的显著结合。此外，在含抗体可变区的结构域对抗原中的多个表位中的特定表位具有特异性时，也使用该术语。当含抗体可变区的结构域所结合的表位被包含在数个不同抗原中时，包含含抗体可变区的结构域的抗原结合分子可以结合具有所述表位的各种抗原。The term "specificity" means that one of the molecules involved in specific binding does not show any significant binding to molecules other than one or more of the binding molecules. In addition, the term is also used when the antibody variable region-containing domain is specific for a particular epitope of multiple epitopes in an antigen. When the epitope to which the antibody variable region-containing domain binds is contained in several different antigens, an antigen-binding molecule comprising the antibody variable region-containing domain can bind to various antigens having the epitope.
术语“肿瘤相关抗原(TAA)”优选涉及在正常条件下在有限数目的组织和/或器官中或在特定发育阶段中特异性表达以及在一个或更多个肿瘤或癌症组织中表达或异常表达的蛋白质。在本发明的上下文中，肿瘤相关抗原优选与癌细胞的细胞表面缔合，并且优选不在或仅很少在正常组织中表达。The term "tumor-associated antigen (TAA)" preferably relates to specific expression in a limited number of tissues and/or organs under normal conditions or in a specific developmental stage as well as expression or abnormal expression in one or more tumor or cancer tissues of protein. In the context of the present invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not or only rarely expressed in normal tissues.
本发明所用氨基酸三字母代码和单字母代码如J.Boil.Chem.,243,p3558(1968)中所述。本发明中第一重链的Fc或其变体和第二重链的Fc或其变体之间的“相互作用”指的是Fc间作用或Fc变体间作用。“Fc变体”指通过在Fc的合适位点处 存在一个或多个氨基酸替换、***或缺失突变引起Fc结构或功能的变化。“Fc变体间作用”指经突变设计的Fc变体之间可以形成空间填充效应、静电转向、氢键作用、疏水作用等。Fc变体间相互作用有助于形成稳定的异源二聚体。优选的突变设计为“Knob-in-hole”形式的突变设计。此外，本发明所述Fc上还可以存在其他导致其功能变化的突变，例如糖基化改造突变、FcγR结合区域突变(以调整ADCC活性)和改善抗体稳定性的氨基酸突变等。The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Boil. Chem., 243, p3558 (1968). The "interaction" between the Fc of the first heavy chain or its variant and the Fc of the second heavy chain or its variant in the present invention refers to an inter-Fc interaction or an inter-Fc variant interaction. An "Fc variant" refers to a change in Fc structure or function by the presence of one or more amino acid substitution, insertion or deletion mutations at appropriate sites in the Fc. "Interaction between Fc variants" refers to the formation of space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions and the like between Fc variants designed by mutation. Interaction between Fc variants contributes to the formation of stable heterodimers. Preferred mutagenesis designs are those in the "Knob-in-hole" format. In addition, the Fc of the present invention may also have other mutations that lead to changes in its function, such as glycosylation mutations, FcγR binding region mutations (to adjust ADCC activity), and amino acid mutations to improve antibody stability.
术语“IL-15”或“IL-15片段”可以是任何IL-15或其突变体，如人IL-15或非人哺乳动物或非哺乳动物的IL-15。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等；优选人的白介素15成熟分子(见数据库UniProtKB，登录号P40933，49-162aa)。术语“IL-15变体”指通过一个或多个氨基酸替换、增加或者缺失突变获得的对IL-15与其受体间亲和力提高或者降低，或其刺激T细胞或者NK细胞活性增加或者降低的突变体分子。本发明所述“IL-15片段”优选其变体形式，更优选为IL-15N72D(SEQ ID NO:11)。本发明“IL-15”和“IL-15片段”可以互换使用，并不矛盾。The term "IL-15" or "IL-15 fragment" can be any IL-15 or a mutant thereof, such as human IL-15 or non-human mammalian or non-mammalian IL-15. Exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, etc.; preferably human interleukin 15 mature molecules (see database UniProtKB, accession number P40933, 49-162aa). The term "IL-15 variant" refers to a mutation that increases or decreases the affinity between IL-15 and its receptor, or that stimulates T cells or NK cells, by one or more amino acid substitutions, additions, or deletions mutations that increase or decrease the activity of body molecules. The "IL-15 fragment" of the present invention is preferably a variant form thereof, more preferably IL-15N72D (SEQ ID NO: 11). "IL-15" and "IL-15 fragment" of the present invention can be used interchangeably and are not contradictory.
术语“IL-15Rα”可以是任何物种的IL-15Rα或者其功能性片段，如人IL-15Rα或非人哺乳动物IL-15Rα或非哺乳动物IL-15Rα。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等。优选人的IL-15Rα，更优选人IL-15Rα胞外域片段，简称IL-15RαECD(见数据库UniProtKB，登录号Q13261，31-205aa)。术语“IL-15Rα变体”指在IL-15Rα上通过一个或者多个氨基酸缺失、***或替换突变形成的具有与其配体分子如IL-15结合能力的功能性突变体，优选人的IL-15Rα分子更优选人的IL-15Rα胞外域片段的缩短形式，即从胞外域片段C端开始通过一个或多个氨基酸缺失突变所得的具有人IL-15受体α活性的分子，优选保留65-120个氨基酸的缺失突变形式，更优选保留65-102个氨基酸的缺失突变缩短形式，比如IL-15RαSushi(65)(SEQ ID NO:12)或IL-15RαSushi(77)(SEQ ID NO:13)。The term "IL-15Rα" can be IL-15Rα of any species or a functional fragment thereof, such as human IL-15Rα or non-human mammalian IL-15Rα or non-mammalian IL-15Rα. Exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, and the like. Preferably human IL-15Rα, more preferably human IL-15Rα extracellular domain fragment, abbreviated as IL-15Rα ECD (see database UniProtKB, accession number Q13261, 31-205aa). The term "IL-15Rα variant" refers to a functional mutant, preferably human IL- The 15Rα molecule is more preferably a shortened form of the human IL-15Rα extracellular domain fragment, that is, a molecule with human IL-15 receptor α activity obtained by one or more amino acid deletion mutations from the C-terminal of the extracellular domain fragment, preferably retaining 65- 120 amino acid deletion mutant forms, more preferably 65-102 amino acid deletion mutant shortened forms, such as IL-15RαSushi(65) (SEQ ID NO:12) or IL-15RαSushi(77) (SEQ ID NO:13) .
本发明中“与额外的连接肽一起共价结合”是指两个或多个基因的编码区之间可由编码连接肽的序列于一个或数个位置发生共价结合。In the present invention, "covalently bound together with an additional linker peptide" means that the coding regions of two or more genes can be covalently bound at one or several positions by the sequence encoding the linker peptide.
术语“免疫球蛋白”指具有抗体活性或化学结构与抗体分子相似的球蛋白，存在五种主要类别的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，这些中的几种可以进 一步划分为亚类(同种型)，例如IgG1、IgG2、IgG3和IgG4、IgA1和IgA2。对应于不同类别免疫球蛋白的重链恒定结构域分别称为α、δ、ε、γ和μ。The term "immunoglobulin" refers to a globulin with antibody activity or chemical structure similar to that of an antibody molecule. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into Subclasses (isotypes) such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
术语“免疫球蛋白Fc部分”指免疫球蛋白的C端区域，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位，是包含两个二硫化物连接的抗体重链Fc区多肽的二聚体分子。Fc区可以通过木瓜蛋白酶消化或IdeS消化成胰蛋白酶消化完整(全长)抗体来产生或可以重组产生。“Fc部分”优选包括至少一个免疫球蛋白铰链区，以及IgG的CH2和CH3区。The term "immunoglobulin Fc part" refers to the C-terminal region of immunoglobulins, which has no antigen-binding activity, is the site of interaction between antibody molecules and effector molecules and cells, and is an antibody heavy chain Fc region polypeptide comprising two disulfide linkages. of dimer molecules. Fc regions can be produced by papain digestion or IdeS digestion into trypsinization of intact (full length) antibodies or can be produced recombinantly. The "Fc portion" preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 regions of IgG.
Fc变体的突变设计技术在本领域内已经较为广泛的应用于制备双特异性抗体或者异源二聚的Fc融合蛋白形式。代表性的有Cater等人(Protein Engineering vol.9 no.7 pp617-621,1996)提出的“Knob-in-Hole”形式；Amgen公司技术人员利用静电转向(Electronic Steering)形成含Fc的异源二聚体形式(US2010286374A1)；Jonathan H.Davis等人(Protein Engineering,Design&Selection pp.1–8,2010)提出的通过IgG/IgA链交换形成的异源二聚体形式(SEEDbodies)；Genmab公司DuoBody(Science,2007.317(5844))平台技术形成的双特异性分子；Xencor公司的技术人员综合结构计算及Fc氨基酸突变，综合不同作用方式形成异源二聚体蛋白形式(mAbs3:6,546-557；November/December 2011)；苏州康宁杰瑞公司的基于电荷网络的Fc改造方法(CN201110459100.7)得到异源二聚体蛋白形式；以及其它基于Fc氨基酸变化或者功能改造手段，达到形成异源二聚体功能蛋白的基因工程方法。本发明所述的Fc变体片段上的Knob-in-Hole结构指两条Fc片段各自突变，突变后可以通过“Knob-in-Hole”形式进行结合。优选用Cater等人的“Knob-in-Hole”模型在Fc区上进行位点突变的改造，以使得到的第一Fc变体和第二Fc变体能以“Knob-in-Hole”的形式结合在一起形成异源二聚体。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。优选人类抗体IgG1、IgG2、IgG3、IgG4的Fc区，更优选人抗体IgG1和IgG4的Fc区。随机任选第一Fc变体或第二Fc变体中一个做knob的突变，另一个做hole的突变。在实施例中，所述的第一Fc变体做knob的突变；所述的第二Fc变体做hole的突变。The mutation design technology of Fc variants has been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative is the "Knob-in-Hole" form proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp617-621, 1996); Amgen company technicians use electrostatic steering (Electronic Steering) to form Fc-containing heterologous Dimeric form (US2010286374A1); heterodimeric form (SEEDbodies) formed by IgG/IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp. 1-8, 2010); DuoBody from Genmab (Science, 2007.317(5844)) platform technology to form a bispecific molecule; Xencor's technical staff integrated structural calculation and Fc amino acid mutation, and integrated different modes of action to form a heterodimeric protein form (mAbs3:6,546-557; November /December 2011); Suzhou Corning Jereh Company's Fc transformation method based on charge network (CN201110459100.7) to obtain the form of heterodimeric protein; and other methods based on Fc amino acid changes or functional transformation to achieve the formation of heterodimers Methods for genetic engineering of functional proteins. The Knob-in-Hole structure on the Fc variant fragment of the present invention refers to the mutation of the two Fc fragments, which can be combined in the form of "Knob-in-Hole" after the mutation. The "Knob-in-Hole" model of Cater et al. is preferably used for site mutation engineering in the Fc region, so that the resulting first and second Fc variants can be in the form of "Knob-in-Hole" bind together to form heterodimers. The selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is within the purview of those skilled in the art. The Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are preferred, and the Fc regions of human antibodies IgG1 and IgG4 are more preferred. One of the first Fc variant or the second Fc variant is randomly selected to mutate the knob and the other to mutate the hole. In an embodiment, the first Fc variant is mutated with knob; the second Fc variant is mutated with hole.
术语“连接肽”在本发明中用于将IL-15与IL-15Rα、CD3的VH与VL连接 至相应的重链中，以保证蛋白的正确折叠和肽稳定性。本发明的“连接肽”优选为(GGGGS)n，其中n可以为0、1、2、3、4、5或者更多，优选n为1-2。如果连接肽序列太短，可能影响两蛋白高级结构的折叠，从而相互干扰；如果连接肽序列太长，又涉及免疫原性的问题，因为连接肽序列本身就是新的抗原。The term "linker peptide" is used in the present invention to link IL-15 and IL-15Rα, VH and VL of CD3 into the corresponding heavy chains to ensure proper protein folding and peptide stability. The "linking peptide" of the present invention is preferably (GGGGS)n, wherein n can be 0, 1, 2, 3, 4, 5 or more, preferably n is 1-2. If the connecting peptide sequence is too short, it may affect the folding of the higher-order structures of the two proteins, thereby interfering with each other; if the connecting peptide sequence is too long, it will involve the problem of immunogenicity, because the connecting peptide sequence itself is a new antigen.
术语的“异源二聚体”优选为基因共表达的产物。如在原核细胞在大肠杆菌中共表达；或在真核细胞，如293、CHO中共表达。所述“共表达”指在一个细胞中多个基因一起表达，同时出现它们的产物。这些基因可以是同时存在而分别或共同地受控表达。在本发明中，优选在一个真核细胞中共表达三种基因。共表达得到的基因表达产物有利于高效、简单地形成复合物；在本发明中，有利于形成异源二聚体。The term "heterodimer" is preferably the product of gene co-expression. Such as co-expression in prokaryotic cells in E. coli; or co-expression in eukaryotic cells, such as 293, CHO. The "co-expression" refers to the co-expression of multiple genes in a cell and the simultaneous appearance of their products. These genes can be co-existing and individually or jointly controlled expression. In the present invention, three genes are preferably co-expressed in one eukaryotic cell. The gene expression product obtained by co-expression is favorable for the efficient and simple formation of complexes; in the present invention, it is favorable for the formation of heterodimers.
术语“核酸”旨在包括DNA和RNA，例如基因组DNA、cDNA、mRNA、重组产生和化学合成的分子。核酸可是单链或双链。RNA包括体外转录的RNA(IVT RNA)或合成的RNA。The term "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.
核酸可包含在载体中。本文中术语“载体”包括技术人员已知的任何载体，其包括质粒载体、黏粒载体、噬菌体载体(例如λ噬菌体)、病毒载体(例如腺病毒或杆状病毒载体)，或人工染色体载体(例如细菌人工染色体(BAC)、酵母人工染色体(YAC)或P1人工染色体(PAC))。所述载体包括表达载体以及克隆载体。表达载体包括质粒和病毒载体且一般含有用于在特定宿主生物体(例如，细菌、酵母、植物、昆虫或哺乳动物)或者在体外表达***中可操作地连接的编码序列之表达所必需的期望编码序列和合适的DNA序列。克隆载体一般用于改造和扩增某期望DNA片段，并可缺乏表达所期望DNA片段所需要的功能性序列。The nucleic acid can be contained in a vector. The term "vector" herein includes any vector known to the skilled artisan, including plasmid vectors, cosmid vectors, bacteriophage vectors (eg, lambda phage), viral vectors (eg, adenovirus or baculovirus vectors), or artificial chromosomal vectors ( For example bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or P1 artificial chromosome (PAC)). The vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired expectations for expression of operably linked coding sequences in a particular host organism (eg, bacteria, yeast, plants, insects, or mammals) or in in vitro expression systems Coding sequences and suitable DNA sequences. Cloning vectors are typically used to engineer and amplify a desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment.
本发明所述的“治疗有效量”是指达到治疗目的的疾病或病况(例如肿瘤，例如用于使肿瘤消退或减小其大小)所需的本发明的多功能融合蛋白，或者所述药物制剂、药用组合物、试剂盒中有效成分的量。可以通过实践、按照常规的方式出于特定的目的而确定所述有效量。特别地，所述治疗有效量可以是达到下述目的所需的量：减少癌细胞的数目；减少肿瘤大小；抑制(即减缓或停止)癌细胞浸润到外周器官中；抑制(即减缓或停止)肿瘤转移；抑制肿瘤生长；和/或缓解与癌症相关的一种或多种症状。A "therapeutically effective amount" as used herein refers to the multifunctional fusion protein of the present invention, or the drug, required to achieve the desired disease or condition (eg, a tumor, for example, to regress or reduce the size of a tumor). The amount of active ingredient in the formulation, pharmaceutical composition, and kit. The effective amount can be determined for a particular purpose by practice, in a conventional manner. In particular, the therapeutically effective amount may be that amount required to: reduce the number of cancer cells; reduce tumor size; inhibit (ie, slow or stop) infiltration of cancer cells into peripheral organs; inhibit (ie, slow or stop) ) tumor metastasis; inhibit tumor growth; and/or alleviate one or more symptoms associated with cancer.
本发明所述“肿瘤”可选自B细胞淋巴瘤、肺癌、支气管癌、结肠直肠癌、***癌、乳腺癌、胰腺癌、胃癌、卵巢癌、膀胱癌、脑或中枢神经***癌症、外周神经***癌症、食道癌、***、黑色素瘤、子宫或子宫内膜癌、口腔癌或喉癌、肝癌、肾癌、胆管癌、小肠癌或阑尾癌、唾液腺癌、胸腺癌、肾上腺癌、骨肉瘤、软骨肉瘤、脂肪瘤、睾丸癌以及恶性纤维组织细胞瘤等。The "tumor" of the present invention can be selected from B cell lymphoma, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nerve cancer Cancer of the system, esophagus, cervix, melanoma, uterine or endometrial, oral or laryngeal, liver, kidney, bile duct, small bowel or appendix, salivary gland, thymus, adrenal gland, osteosarcoma , chondrosarcoma, lipoma, testicular cancer and malignant fibrous histiocytoma.
附图说明Description of drawings
图1为以Claudin18.2/CD3/IL-15为例的示例性多功能融合蛋白CCI结构图。FIG. 1 is a structural diagram of an exemplary multifunctional fusion protein CCI taking Claudin18.2/CD3/IL-15 as an example.
图2a为非还原条件下CCI的CE-SDS图，图2b为还原条件下CCI的CE-SDS图。Figure 2a is the CE-SDS image of CCI under non-reducing conditions, and Figure 2b is the CE-SDS image of CCI under reducing conditions.
图3为多功能融合蛋白CCI反复冻融5次后的SEC-HPLC图。Figure 3 is the SEC-HPLC chart of the multifunctional fusion protein CCI after repeated freezing and thawing 5 times.
图4为多功能融合蛋白CCI与Claudin18.2结合活性。Figure 4 shows the binding activity of the multifunctional fusion protein CCI to Claudin18.2.
图5为多功能融合蛋白CCI与受体IL-2Rβ的结合活性。Figure 5 shows the binding activity of the multifunctional fusion protein CCI to the receptor IL-2Rβ.
图6为多功能融合蛋白CCI与CD3的结合活性。Figure 6 shows the binding activity of the multifunctional fusion protein CCI to CD3.
图7为多功能融合蛋白CCI对PBMC细胞的增殖图。Figure 7 is a graph showing the proliferation of PBMC cells by the multifunctional fusion protein CCI.
图8为PBMC增殖实验细胞流式鉴定分型图。Figure 8 is a flow cytometric phenotyping diagram of PBMC proliferation experiment cells.
图9为CCI介导的Claudin18.2-CHO-K1细胞杀伤。Figure 9 shows CCI-mediated killing of Claudin18.2-CHO-K1 cells.
图10为多功能融合蛋白BCI对B7H3的结合活性。Figure 10 shows the binding activity of the multifunctional fusion protein BCI to B7H3.
图11为多功能融合蛋白BCI对CD3的结合活性。Figure 11 shows the binding activity of the multifunctional fusion protein BCI to CD3.
图12a-12b为流式细胞术检测MCF-7细胞B7H3表达丰度。Figures 12a-12b show the detection of B7H3 expression abundance in MCF-7 cells by flow cytometry.
图13为多功能融合蛋白BCI与受体IL-2Rβ的结合活性。Figure 13 shows the binding activity of the multifunctional fusion protein BCI to the receptor IL-2Rβ.
图14为多功能融合蛋白BCI的抗肿瘤活性。Figure 14 shows the antitumor activity of the multifunctional fusion protein BCI.
具体实施方式detailed description
以下结合附图与具体实施例对本发明做进一步的描述，本发明的保护内容不局限于以下实施例。还应该理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所 附的权利要求及其任何等同物为本发明的保护范围。在本发明的说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域技术人员的普遍知识和公知常识，本发明没有特别限制内容。The present invention will be further described below with reference to the accompanying drawings and specific embodiments, and the protection content of the present invention is not limited to the following embodiments. It should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are all included in the present invention, and the appended claims and any equivalents thereof are the protection scope of the present invention. In the present specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are the general knowledge and common knowledge of those skilled in the art, and the present invention has no special limited contents.
本发明实施例中Claudin18.2抗体的重链氨基酸序列为SEQ ID NO:21，轻链氨基酸序列为SEQ ID NO:22。In the embodiment of the present invention, the heavy chain amino acid sequence of the Claudin18.2 antibody is SEQ ID NO: 21, and the light chain amino acid sequence is SEQ ID NO: 22.
本发明实施例中B7H3嵌合抗体的重链氨基酸序列为SEQ ID NO:20，轻链氨基酸序列为SEQ ID NO:16。In the embodiment of the present invention, the heavy chain amino acid sequence of the B7H3 chimeric antibody is SEQ ID NO: 20, and the light chain amino acid sequence is SEQ ID NO: 16.
实施例1核苷酸序列的获得与优化Example 1 Acquisition and Optimization of Nucleotide Sequences
多功能融合蛋白CCI(或BCI)的轻链和重链氨基酸序列信息选自公开的或自研Claudin18.2(或B7H3)靶点单抗序列信息，分析获得该序列的可变区和恒定区信息。将天然IL-15与IL-15Rα复合物变体序列***一条重链的氨基酸序列中，并在另一条重链的相应位置***CD3抗体的scFv序列。根据需要，调整所述多功能融合蛋白氨基酸序列的Fc为其他IgG类型，如IgG4等，并进一步在各重链中设计所需形式的氨基酸突变，由此得到目标多功能融合蛋白的氨基酸序列，为：The amino acid sequence information of the light chain and heavy chain of the multifunctional fusion protein CCI (or BCI) is selected from the published or self-developed Claudin18.2 (or B7H3) target monoclonal antibody sequence information, and the variable region and constant region of the sequence are obtained by analysis information. The native IL-15 and IL-15Rα complex variant sequence was inserted into the amino acid sequence of one heavy chain, and the CD3 antibody scFv sequence was inserted at the corresponding position in the other heavy chain. According to needs, adjust the Fc of the amino acid sequence of the multifunctional fusion protein to other IgG types, such as IgG4, etc., and further design amino acid mutations in the desired form in each heavy chain, thereby obtaining the amino acid sequence of the target multifunctional fusion protein, for:
多功能融合蛋白CCI—第一重链为SEQ ID NO:1，第二重链为SEQ ID NO:2，第一轻链和第二轻链为SEQ ID NO:3；Multifunctional fusion protein CCI—the first heavy chain is SEQ ID NO:1, the second heavy chain is SEQ ID NO:2, and the first light chain and the second light chain are SEQ ID NO:3;
多功能融合蛋白BCI—第一重链为SEQ ID NO:14，第二重链为SEQ ID NO:15，第一轻链和第二轻链为SEQ ID NO:16。Multifunctional fusion protein BCI—the first heavy chain is SEQ ID NO:14, the second heavy chain is SEQ ID NO:15, and the first light chain and the second light chain are SEQ ID NO:16.
将上述各目标氨基酸序列转化为核苷酸序列，并针对可能影响抗体在哺乳动物细胞中表达的一系列参数：密码子偏好性、GC含量(即DNA的4种碱基中鸟嘌呤G和胞嘧啶C所占的比率)、CpG岛(即CpG双核苷酸在基因组中密度较高的区域)、mRNA的二级结构、拼接位点、前成熟PolyA位点、内部Chi位点(基因组中一段短的DNA片段，在该位点附近发生同源重组的几率增加)或者核糖体结合位点、RNA不稳定序列、反向重复序列及可能干扰克隆的限制性酶切位点等进行优化；同时增加了可能会提高翻译效率的相关序列，例如Kozak序列、SD序列，以及终止密码子。设计得到分别编码上述多功能融合蛋白CCI的重链基因和 轻链基因，另外在重链和轻链的5’端分别设计上根据氨基酸序列优化而得的编码信号肽的核苷酸序列；此外，还对轻链和重链核苷酸序列的3’端分别加上终止密码子。Each of the above target amino acid sequences was converted into nucleotide sequences and targeted for a series of parameters that may affect the expression of antibodies in mammalian cells: codon preference, GC content (ie, guanine G and cytosine in the four bases of DNA). The ratio of pyrimidine C), CpG islands (that is, regions with high density of CpG dinucleotides in the genome), mRNA secondary structure, splice sites, pre-mature PolyA sites, internal Chi sites (a segment of the genome) Short DNA fragments, the probability of homologous recombination near this site is increased) or ribosome binding sites, RNA unstable sequences, inverted repeat sequences, and restriction enzyme sites that may interfere with cloning are optimized; at the same time Added related sequences that may improve translation efficiency, such as Kozak sequences, SD sequences, and stop codons. Design to obtain the heavy chain gene and light chain gene encoding the above-mentioned multifunctional fusion protein CCI respectively, and design the nucleotide sequence of the signal peptide encoding signal peptide optimized according to the amino acid sequence at the 5' end of the heavy chain and the light chain respectively; , and stop codons were added to the 3' ends of the light chain and heavy chain nucleotide sequences, respectively.
最终优化获得抗体核苷酸序列为：The final optimized antibody nucleotide sequence is:
CCI—第一重链为SEQ ID NO:4，第二重链为SEQ ID NO:5，第一轻链和第二轻链为SEQ ID NO:6；CCI—the first heavy chain is SEQ ID NO:4, the second heavy chain is SEQ ID NO:5, and the first light chain and the second light chain are SEQ ID NO:6;
BCI—第一重链为SEQ ID NO:17，第二重链为SEQ ID NO:18，第一轻链和第二轻链为SEQ ID NO:19。BCI—the first heavy chain is SEQ ID NO: 17, the second heavy chain is SEQ ID NO: 18, and the first light chain and the second light chain are SEQ ID NO: 19.
本发明设计的CCI的理论分子数据如表1所示。The theoretical molecular data of the CCI designed by the present invention are shown in Table 1.
表1 CCI的理论分子数据Table 1 Theoretical molecular data of CCI
Figure PCTCN2021114408-appb-000001
Figure PCTCN2021114408-appb-000001
实施例2基因合成与表达载体的构建Example 2 Gene synthesis and construction of expression vector
采用pcDNA3.1-G418载体作为表达所述多功能抗体的轻链和重链的专用载体。pcDNA3.1-G418载体含有重链所使用的启动子CMVPromoter、真核筛选标记G418标签和原核筛选标签氨苄西林(Ampicilline)。基因合成得到多功能融合蛋白CCI和BCI的抗体表达轻链和重链的核苷酸序列，用HindIII和XhoI对载体和目的片段进行双酶切，回收后通过DNA连接酶进行酶连，并转化大肠杆菌感受态细胞DH5α，挑选出阳性克隆并进行质粒提取和酶切验证，获得含所述融合蛋白CCI的全长第一重链、第二重链、第一轻链和第二轻链的重组质粒，分别为CCI-1(第一重链)、CCI-2(第二重链)和CCI-3(第一轻链和第二轻链相同)；获得所述融合蛋白BCI的全长第一重链、第二重链、第一轻链和第二轻链的重组质粒，分别为BCI-1(第一重链)、BCI-2(第二重链)和BCI-3(第一轻链和第二轻链相同)。The pcDNA3.1-G418 vector was used as a dedicated vector for expressing the light and heavy chains of the multifunctional antibody. The pcDNA3.1-G418 vector contains the promoter CMVPromoter used for the heavy chain, the eukaryotic selection marker G418 tag and the prokaryotic selection tag Ampicilline. The nucleotide sequences of the light chain and heavy chain of the antibody expression of the multifunctional fusion proteins CCI and BCI were obtained by gene synthesis, the vector and the target fragment were double-enzyme digested with HindIII and XhoI, and then enzymatically linked by DNA ligase after recovery, and transformed Escherichia coli competent cell DH5α, select positive clones and carry out plasmid extraction and enzyme digestion verification to obtain full-length first heavy chain, second heavy chain, first light chain and second light chain containing the fusion protein CCI. Recombinant plasmids, respectively CCI-1 (first heavy chain), CCI-2 (second heavy chain) and CCI-3 (the same first light chain and second light chain); obtain the full length of the fusion protein BCI The recombinant plasmids of the first heavy chain, the second heavy chain, the first light chain and the second light chain are BCI-1 (the first heavy chain), BCI-2 (the second heavy chain) and BCI-3 (the first heavy chain), respectively. The first light chain and the second light chain are the same).
实施例3质粒抽提Example 3 Plasmid extraction
根据《分子克隆实验指南》(2002年，科学出版社)所述方法将含有上述各目的基因的重组质粒转化至大肠杆菌感受态细胞DH5α中，将转化细菌涂布在含 100μg/mL氨苄青霉素的LB平板上培养，挑选质粒克隆至液体LB培养基中培养，260rpm摇菌14小时，由无内毒素质粒大抽试剂盒抽提质粒，用无菌水溶解并用核酸蛋白定量仪进行浓度测定。According to the method described in "Molecular Cloning Experiment Guide" (Science Press, 2002), the recombinant plasmids containing the above target genes were transformed into E. coli competent cells DH5α, and the transformed bacteria were spread on 100 μg/mL ampicillin-containing Cultivate on LB plate, select plasmid clones and culture in liquid LB medium, shake bacteria at 260 rpm for 14 hours, extract plasmids with endotoxin-free plasmid extraction kit, dissolve with sterile water and measure the concentration with nucleic acid protein quantifier.
实施例4质粒转染、瞬转表达与抗体纯化Example 4 Plasmid transfection, transient expression and antibody purification
在37℃、8％CO 2、100rpm下培养ExpiCHO至细胞密度6×10 6个/mL。使用脂质体分别将构建的载体CCI-1、CCI-2和CCI-3；以及BCI-1、BCI-2和BCI-3转染到上述细胞中，转染质粒浓度为1mg/ml，脂质体浓度参照ExpiCHO TM Expression System试剂盒确定，在32℃、5％CO 2，100rpm下培养7-10天。转染18-22h之后和第5天之间分别补料一次。4000g离心上述培养产物，0.22μm滤膜过滤并收集培养基上清液，采用ProteinA、离子柱纯化所得的抗体6蛋白并收集洗脱液。 ExpiCHO was grown at 37°C, 8% CO 2 , 100 rpm to a cell density of 6×10 6 cells/mL. The constructed vectors CCI-1, CCI-2 and CCI-3; and BCI-1, BCI-2 and BCI-3 were respectively transfected into the above cells using liposomes, and the transfection plasmid concentration was 1 mg/ml, and the lipid The plastid concentration was determined with reference to the ExpiCHO ™ Expression System kit, and cultured at 32°C, 5% CO 2 , and 100 rpm for 7-10 days. Feeds were made between 18-22 h after transfection and between day 5. The above cultured product was centrifuged at 4000 g, filtered through a 0.22 μm filter, and the supernatant of the medium was collected. The obtained antibody 6 protein was purified by Protein A and ion column, and the eluate was collected.
ProteinA、离子柱纯化的具体操作步骤为：细胞培养液经过高速离心后取上清，利用GE的ProteinA层析柱进行亲和层析。层析使用平衡缓冲液为1×PBS(pH7.4)，细胞上清上样结合后利用PBS洗涤至紫外线回到基线，然后利用洗脱缓冲液0.1M甘氨酸(pH3.0)洗脱目的蛋白，利用Tris调节pH至中性保存。将亲和层析所得产物调节pH至低于或者高于pI1-2个pH单位，适当稀释以控制样本电导在5ms/cm以下。利用合适的对应pH缓冲液如磷酸缓冲液、醋酸缓冲液等条件，利用本领域内常规的离子交换层析方法如阴离子交换或者阳离子交换进行对应pH条件下NaCl梯度洗脱，根据SDS-PAGE选择目的蛋白所在的收集管合并保存。The specific operation steps of ProteinA and ion column purification are as follows: after high-speed centrifugation of the cell culture fluid, the supernatant is taken, and affinity chromatography is performed using GE's ProteinA chromatography column. The equilibration buffer for chromatography is 1×PBS (pH 7.4), the cell supernatant is loaded and combined, washed with PBS until it returns to the baseline, and then the target protein is eluted with elution buffer 0.1M glycine (pH 3.0). , pH was adjusted to neutrality with Tris. Adjust the pH of the product obtained by affinity chromatography to 1-2 pH units below or above pI, and dilute appropriately to control the sample conductance below 5ms/cm. Use appropriate corresponding pH buffers such as phosphate buffer, acetate buffer and other conditions, use conventional ion exchange chromatography methods in the field such as anion exchange or cation exchange to carry out NaCl gradient elution under corresponding pH conditions, select according to SDS-PAGE The collection tubes where the target protein is located are combined and stored.
然后，将纯化后所得的洗脱液超滤换液至缓冲液中。通过SDS-聚丙烯酰胺凝胶电泳测定检测蛋白质。Then, the eluate obtained after purification was ultrafiltered and exchanged into buffer. Proteins were detected by SDS-polyacrylamide gel electrophoresis assay.
经CE-SDS测定证明，非还原CE-SDS条件为1个峰，还原CE-SDS下目标抗体被还原成3个峰，即位置在LC、1、2位置，对应于所需抗体的两个不同的重链以及相同的轻链。因此，经所述质粒转染、瞬转表达和纯化，证明所得到的抗体融合蛋白CCI和BCI结构正确，且纯度较高。多功能融合蛋白CCI相应的CE-SDS电泳图分别如图2a、图2b所示。然后对所得到的抗体CCI进行质量、体外结合活性、细胞生物活性试验分析。The CE-SDS assay proved that the non-reducing CE-SDS condition was 1 peak, and the target antibody was reduced to 3 peaks under reducing CE-SDS, that is, the positions were at LC, 1, and 2 positions, corresponding to the two peaks of the desired antibody. Different heavy chains as well as the same light chain. Therefore, after the plasmid transfection, transient expression and purification, it is proved that the obtained antibody fusion proteins CCI and BCI have correct structure and high purity. The corresponding CE-SDS electrophoresis patterns of the multifunctional fusion protein CCI are shown in Figure 2a and Figure 2b, respectively. Then, the quality, in vitro binding activity and cell biological activity test analysis of the obtained antibody CCI was carried out.
实施例5稳定性研究Example 5 Stability Study
将多功能融合蛋白CCI(或BCI)放于特定配方成分的制剂中，记为DR0， 放入-80℃1h后转至室温融化，反复操作两次视为冻融2次，本发明研究了DR0、DR2与DR5，即反复冻融2次与五次，结果如表2所示，反复冻融SEC数据均稳定在92％，表明该结构稳定，冻融第五次的SEC-HPLC数据如图3所示。Put the multifunctional fusion protein CCI (or BCI) in the preparation of specific formula ingredients, denoted as D0, put it at -80°C for 1 hour and then transfer to room temperature to thaw, and repeat the operation twice as freeze-thaw twice. DR0, DR2 and DR5, namely repeated freezing and thawing twice and five times, the results are shown in Table 2, the SEC data of repeated freezing and thawing are all stable at 92%, indicating that the structure is stable, and the SEC-HPLC data of the fifth freezing and thawing are as follows shown in Figure 3.
表2 CCI冻融稳定性SEC-HPLC数据Table 2 CCI freeze-thaw stability SEC-HPLC data
Figure PCTCN2021114408-appb-000002
Figure PCTCN2021114408-appb-000002
实施例6多功能融合蛋白CCI与Claudin18.2-CHO-K1细胞的结合活性Example 6 Binding activity of multifunctional fusion protein CCI to Claudin18.2-CHO-K1 cells
使用对数期生长的构建的稳定表达Claudin18.2抗原的Claudin18.2-CHO-K1细胞铺板，以0.8×10 5/孔铺96孔板，37℃、5％CO 2放入二氧化碳培养箱培养48h；固定：吸除多余培养基，使用1X PBST 200μL/孔加入孔板中，清洗2次，4％多聚甲醛100μL/孔，-20℃放置15分钟，移液枪吸除固定液，使用PBST清洗2次；用含2％BSA封闭液放入37℃孵育封闭1小时后，PBST洗3次后；将CCI用0.5％BSA样品稀释液稀释至0.3μg/mL，以此为起始浓度，进行3倍梯度稀释，共7个梯度，并设阴性对照，每孔100μL，37℃孵育1h；再用PBST洗板3次，将HRP标记的山羊抗人IgG Fc用样品稀释液按1:20000稀释，每孔加入100μL，室温孵育45min；PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10分钟，每孔加入100μL 1M盐酸液终止显色反应；在多功能酶标仪上选择波长450nm；将多功能融合蛋白CCI的浓度取对数后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidaldose-response(Variable Slope)方式(GraphPad Prism软件，GraphPad Software，SanDiego，California)进行非线性回归，得到CCI与Claudin18.2-CHO-K1的结合曲线。结果如图4所示，其中IgG1为无关抗体，Claudin18.2抗体为Claudin18.2阳性参照，结果表明CCI的结合能力与Claudin18.2抗体相当，该架构的CCI不会影响与Claudin18.2的结合能力。 The constructed Claudin18.2-CHO-K1 cells stably expressing the Claudin18.2 antigen grown in log phase were plated, plated in 96-well plates at 0.8×10 5 /well, and cultured in a carbon dioxide incubator at 37°C, 5% CO 2 . 48h; Fixation: Aspirate the excess medium, add 200 μL/well of 1X PBST to the well plate, wash twice, 4% paraformaldehyde 100 μL/well, place at -20°C for 15 minutes, remove the fixative with a pipette, use Wash twice with PBST; incubate at 37°C for 1 hour with blocking solution containing 2% BSA, and wash three times with PBST; dilute CCI with 0.5% BSA sample diluent to 0.3 μg/mL, which is the initial concentration , carry out 3-fold gradient dilution, a total of 7 gradients, and set up a negative control, 100 μL per well, incubate at 37 °C for 1 h; wash the plate 3 times with PBST, and use the HRP-labeled goat anti-human IgG Fc sample dilution solution at 1:1 20000 dilution, add 100 μL to each well, and incubate for 45 min at room temperature; after washing the plate 4 times with PBST, add 100 μL of TMB substrate to each well, incubate in the dark at room temperature for 10 minutes, and add 100 μL of 1M hydrochloric acid to each well to stop the color reaction; The wavelength of 450 nm was selected on the standard instrument; the logarithm of the concentration of the multifunctional fusion protein CCI was taken as the abscissa, and the measured absorbance value of each well was the ordinate, and the Sigmoidaldose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software , SanDiego, California) performed nonlinear regression to obtain the binding curve of CCI and Claudin18.2-CHO-K1. The results are shown in Figure 4, in which IgG1 is an irrelevant antibody, and Claudin18.2 antibody is a positive reference for Claudin18.2. The results show that the binding ability of CCI is comparable to that of Claudin18.2 antibody, and the CCI of this architecture will not affect the binding to Claudin18.2 ability.
实施例7多功能融合蛋白CCI与IL-2Rβ的结合活性Example 7 Binding activity of multifunctional fusion protein CCI and IL-2Rβ
采用pH7.4的PBS缓冲液将IL-2Rβ受体稀释至4μg/mL，每孔100μL加入到96孔ELISA板中，4度包被过夜。用1％BSA封闭液封闭1小时后。PBST洗 板3次后，将CCI用0.5％BSA样品稀释液稀释至4μg/mL，以此为起始浓度，进行3倍梯度稀释，共7个梯度，并设阴性对照，每孔100μL，37℃孵育1h。再用PBST洗板3次，将HRP标记的山羊抗人IgG Fc用样品稀释液按1:10000稀释，每孔加入100μL，室温孵育1小时。PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10分钟，每孔加入100μL 1M HCl液终止显色反应。在多功能酶标仪上选择波长450nm，参比波长570nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD 450nm-OD 570nm。将CCI的浓度取对数后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidaldose-response(Variable Slope)方式(GraphPad Prism软件，GraphPad Software，SanDiego，California)进行非线性回归，得到多功能融合蛋白CCI与IL-2Rβ的结合曲线。 The IL-2Rβ receptor was diluted to 4 μg/mL with PBS buffer pH 7.4, and 100 μL per well was added to a 96-well ELISA plate and coated overnight at 4 degrees. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the CCI was diluted to 4 μg/mL with 0.5% BSA sample diluent, taking this as the starting concentration, 3-fold gradient dilution was carried out, a total of 7 gradients, and a negative control was set, 100 μL per well, 37 Incubate at ℃ for 1 h. The plate was washed three times with PBST, and the HRP-labeled goat anti-human IgG Fc was diluted 1:10000 with sample diluent, 100 μL was added to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, 100 μL of TMB substrate was added to each well, incubated at room temperature for 10 minutes in the dark, and 100 μL of 1M HCl solution was added to each well to stop the color reaction. The wavelength of 450 nm was selected on a multi-function microplate reader, and the reference wavelength of 570 nm was used to measure the absorbance value of each well in the 96-well plate, and the absorbance value (OD) of each well = OD 450 nm -OD 570 nm . The logarithm of the concentration of CCI was taken as the abscissa, and the measured absorbance value of each well was the ordinate. The Sigmoidaldose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, SanDiego, California) was used for nonlinear regression to obtain Binding curve of the multifunctional fusion protein CCI to IL-2Rβ.
多功能融合蛋白CCI的ELISA结果如图5所示，所述多功能融合蛋白CCI在各浓度下均可与IL-2Rβ结合，表明所述结构的CCI与IL-2Rβ有较好的结合能力。The ELISA results of the multifunctional fusion protein CCI are shown in FIG. 5 . The multifunctional fusion protein CCI can bind to IL-2Rβ at various concentrations, indicating that the CCI of the structure has good binding ability to IL-2Rβ.
实施例8多功能融合蛋白CCI与CD3的结合活性Example 8 Binding activity of multifunctional fusion protein CCI and CD3
采用pH7.4的PBS缓冲液将多功能融合蛋白CCI、CD3抗体、无关抗体IgG稀释至1.5μg/mL，每孔100μL加入到96孔ELISA板中，4度包被过夜。用1％BSA封闭液封闭1小时后。PBST洗板3次后，将CD3用1％BSA样品稀释液稀释至10μg/mL，以此为起始浓度，进行3倍梯度稀释，共7个梯度，并设阴性对照，每孔100μL，37℃孵育1h。再用PBST洗板3次，将HRP标记的兔抗6*His抗体用样品稀释液按1:20000稀释，每孔加入100μL，室温孵育1小时。PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10分钟，每孔加入100μL1M HCl液终止显色反应。在多功能酶标仪上选择波长450nm，检测每孔吸光值OD450nm。将多功能融合蛋白CCI的浓度取Log10后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidaldose-response(Variable Slope)方式(GraphPad Prism软件，GraphPad Software，SanDiego，California)进行非线性回归，得到CCI与CD3的结合曲线。The multifunctional fusion protein CCI, CD3 antibody, and irrelevant antibody IgG were diluted to 1.5 μg/mL with PBS buffer pH 7.4, and 100 μL per well was added to a 96-well ELISA plate, and coated overnight at 4 degrees. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the CD3 was diluted to 10 μg/mL with 1% BSA sample diluent, taking this as the starting concentration, 3-fold gradient dilution was carried out, a total of 7 gradients, and a negative control was set, 100 μL per well, 37 Incubate at ℃ for 1 h. The plate was washed three times with PBST, and the HRP-labeled rabbit anti-6*His antibody was diluted 1:20000 with sample diluent, 100 μL was added to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 μL of TMB substrate to each well, incubate at room temperature for 10 minutes in the dark, and add 100 μL of 1M HCl solution to each well to stop the color reaction. Select the wavelength of 450nm on a multi-function microplate reader, and detect the absorbance value of each well at OD450nm. The concentration of the multifunctional fusion protein CCI was taken as Log10 as the abscissa, and the measured absorbance value of each well was the ordinate. The Sigmoidaldose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, SanDiego, California) was used for nonlinear Regression to obtain the binding curve of CCI and CD3.
多功能融合蛋白CCI的ELISA结果如图6所示，所述多功能融合蛋白CCI在各浓度下均可与CD3结合，IgG1与CD3无结合，与设计效果是一致的，理论 上同样浓度下，单抗CD3的效价为CCI的一半。The ELISA results of the multifunctional fusion protein CCI are shown in Figure 6. The multifunctional fusion protein CCI can bind to CD3 at various concentrations, but IgG1 does not bind to CD3, which is consistent with the design effect. In theory, at the same concentration, The titer of monoclonal antibody CD3 is half of CCI.
实施例9多功能融合蛋白CCI对PBMC增殖实验Example 9 Multifunctional fusion protein CCI on PBMC proliferation experiment
使用商品化的PBMC细胞，复苏后，以1×10 6个/mL加入24孔板中，每孔添加抗CD3抗体OKT3 1μg/mL进行激活，继续培养，每隔2天，加入相应浓度的不同抗体(CCI或IL-15)继续持续刺激，每次计数细胞总数。 Use commercial PBMC cells, after recovery, add 1×10 6 cells/mL to a 24-well plate, add anti-CD3 antibody OKT3 1 μg/mL to each well for activation, continue to culture, and add corresponding concentrations of different cells every 2 days. Antibodies (CCI or IL-15) continued to stimulate, and the total number of cells was counted each time.
结果如图7所示，无IL-15持续刺激下，PBMC无法存活；使用OKT3激活+IL-15持续刺激可使PBMC增殖。CCI激活+IL-15持续刺激或OKT3激活+CCI持续刺激与阳性对照OKT3激活+IL-15持续刺激有同等效果，细胞数差别不大；仅仅使用CCI进行激活，细胞数与增殖速度同样与阳性对照差别不大。实验证明，CCI同时具有CD3抗体与IL-15的功能活性。The results are shown in Figure 7. Without continuous stimulation with IL-15, PBMCs could not survive; activation of OKT3 + continuous stimulation with IL-15 could make PBMCs proliferate. CCI activation + IL-15 continuous stimulation or OKT3 activation + CCI continuous stimulation has the same effect as the positive control OKT3 activation + IL-15 continuous stimulation, and there is little difference in the number of cells; only using CCI for activation, the cell number and proliferation rate are the same as positive The comparison is not much different. Experiments show that CCI has the functional activity of CD3 antibody and IL-15 at the same time.
实施例10 PBMC增殖实验细胞分型流式分析Example 10 PBMC Proliferation Experiment Cell Type Flow Analysis
使用流式抗体对抗体刺激PBMC增殖的细胞进行细胞表面标记分析。流式抗体包含PerCp-cy5.5-CD3(Cat：552852；BD)、APC-CD56(Cat：555518；BD)、PE-CD4(Cat：550630；BD)、PE-cy7-CD16(557744；BD)；以1×10 6/实验，分别加入流式抗体孵育37℃孵育1h，2000r/分钟离心5分钟，而后使用1mL PBS重悬清洗，重复清洗操作两次，最后使用200μL PBS重悬细胞，使用流式细胞仪进行分析，以无添加流式抗体作为空白对照，即Blank。结果统计如表3所示，流式分析如图8所示。图8中，A、D、G为不同刺激条件下PBMC增殖的CD3标记流式分析，B、E、H为不同刺激条件下PBMC增殖的CD16标记流式分析，C、F、I为不同刺激条件下PBMC增殖的CD56标记流式分析，其中A、B、C为CCI激活+无持续刺激，D、E、F为CCI激活+CCI持续刺激，G、H、L为OKT3激活+IL-15持续刺激。 Cell surface marker analysis of antibody-stimulated PBMC-proliferated cells using flow antibodies. Flow antibodies include PerCp-cy5.5-CD3 (Cat: 552852; BD), APC-CD56 (Cat: 555518; BD), PE-CD4 (Cat: 550630; BD), PE-cy7-CD16 (557744; BD) ); at 1×10 6 /experiment, add flow antibody and incubate at 37°C for 1 h, centrifuge at 2000 r/min for 5 min, then resuspend and wash in 1 mL of PBS, repeat the washing operation twice, and finally use 200 μL of PBS to resuspend cells, Flow cytometry was used for analysis, with no added flow antibody as blank control, namely Blank. The statistics of the results are shown in Table 3, and the flow analysis is shown in Figure 8. In Figure 8, A, D, G are the flow cytometry analysis of CD3 marker of PBMC proliferation under different stimulation conditions, B, E, H are flow cytometry analysis of CD16 marker of PBMC proliferation under different stimulation conditions, C, F, I are different stimulation conditions Flow cytometry analysis of CD56 marker of PBMC proliferation under conditions, where A, B, C are CCI activation + no continuous stimulation, D, E, F are CCI activation + CCI continuous stimulation, G, H, L are OKT3 activation + IL-15 Continuous stimulation.
由数据分析得到，刺激增殖的细胞90％以上为CD3阳性细胞，表示增殖的细胞大部分为T细胞，其中CD4+与CD8+被包含在CD3阳性细胞中，其中CD8+细胞高于CD4+细胞，其中CD4+为辅助T细胞，CD8+为杀伤T细胞，表明CD3抗体与IL-15刺激下，杀伤T细胞比例大于辅助T细胞；CD16阳性与CD56阳性各条件下无明显区别，表示维持了NK细胞存活。From the data analysis, more than 90% of the cells that stimulate proliferation are CD3 positive cells, indicating that most of the proliferating cells are T cells, of which CD4+ and CD8+ are included in CD3 positive cells, and CD8+ cells are higher than CD4+ cells, of which CD4+ is Helper T cells, CD8+ are killer T cells, indicating that under the stimulation of CD3 antibody and IL-15, the proportion of killer T cells is greater than that of helper T cells; there is no significant difference between CD16 positive and CD56 positive conditions, indicating that the survival of NK cells is maintained.
由此证明，CCI维持存活的细胞基本为T细胞和NK细胞群体，与设计效果 一致。CCI既拥有CD3刺激PBMC细胞活化的功能，也能维持T细胞和NK细胞存活，CCI同时具有CD3抗体功能以及IL-15细胞因子功能，与设计效果一致。This proves that the cells maintained by CCI are basically T cells and NK cells, which is consistent with the design effect. CCI not only has the function of CD3 to stimulate the activation of PBMC cells, but also can maintain the survival of T cells and NK cells. CCI also has the function of CD3 antibody and IL-15 cytokine, which is consistent with the design effect.
表3细胞表面标记分析Table 3 Analysis of cell surface markers
Figure PCTCN2021114408-appb-000003
Figure PCTCN2021114408-appb-000003
实施例11 CCI介导的Claudin18.2-CHO-K1细胞杀伤实验Example 11 CCI-mediated killing of Claudin18.2-CHO-K1 cells
使用构建工程细胞株Claudin18.2-CHO-K1，3×10 4/孔铺于96孔板，培养24h后，加入CCI抗体和无关抗体10μg/mL起始，10倍稀释，共5个浓度梯度，同时加入由CCI激活与持续刺激PBMC得到的效应细胞1.5×10 5/孔，使效靶比为5：1，孵育24h后，PBS润洗次，洗去效应细胞。加入含10％CCK-8(Cat：CK04)培养基100uL，于培养箱孵育2h后，于酶标仪OD值450nm进行检测。计算细胞杀伤率，公式为细胞杀伤率(％)＝(样品孔OD值-blankOD值)/(阴性孔OD值-blankOD值)×100％。 The engineered cell line Claudin18.2-CHO-K1 was used, and 3×10 4 /well was plated in a 96-well plate. After culturing for 24 hours, 10 μg/mL of CCI antibody and irrelevant antibody were added to start with 10-fold dilution, with a total of 5 concentration gradients. , 1.5×10 5 /well of effector cells obtained by CCI activation and continuous stimulation of PBMC were added at the same time, so that the effector-target ratio was 5:1. After 24h incubation, PBS was rinsed for several times to wash away the effector cells. Add 100 uL of medium containing 10% CCK-8 (Cat: CK04), incubate for 2 h in an incubator, and detect at OD value of 450 nm on a microplate reader. The cell killing rate was calculated, and the formula was cell killing rate (%)=(OD value of sample well-blankOD value)/(OD value of negative well-blankOD value)×100%.
由图9可知，与无关抗体相比，CCI抗体组对Claudin18.2-CHO-K1细胞具有杀伤作用，而无关抗体不具有杀伤作用，说明此杀伤为靶点特异；同时，CCI介导的效应细胞为CCI激活与持续刺激PBMC的细胞，说明CCI激活与持续刺激PBMC的细胞可杀伤抗体介导的靶细胞。所以CCI可激活与持续刺激PBMC细胞，并且作为效应细胞特异杀伤抗体介导的靶细胞。It can be seen from Figure 9 that, compared with the irrelevant antibody, the CCI antibody group has a killing effect on Claudin18.2-CHO-K1 cells, while the irrelevant antibody has no killing effect, indicating that the killing is target-specific; at the same time, the effect mediated by CCI The cells were CCI-activated and continuously stimulated PBMC cells, indicating that CCI-activated and continuously stimulated PBMC cells could kill antibody-mediated target cells. Therefore, CCI can activate and continuously stimulate PBMC cells, and act as effector cells to specifically kill antibody-mediated target cells.
实施例12多功能融合蛋白BCI与B7H3的结合活性Example 12 Binding activity of multifunctional fusion protein BCI and B7H3
采用pH7.4的PBS缓冲液将huB7H3-his受体稀释至0.5μg/mL，每孔100μL加入到96孔ELISA板中，4度包被过夜。用1％BSA封闭液封闭1小时后。PBST洗板3次后，将多功能融合蛋白BCI用0.5％BSA样品稀释液稀释至10μg/mL，以此为起始浓度，进行3倍梯度稀释，共11个梯度，并设无关抗体阴性对照与阳性对照B7H3嵌合抗体，每孔100μL，37℃孵育1h。再用PBST洗板3次，将 HRP标记的山羊抗人IgGFc用样品稀释液按1:20000稀释，每孔加入100μL，室温孵育1小时。PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10分钟，每孔加入100μL 1M HCl液终止显色反应。在多功能酶标仪上选择波长450nm，参比波长570nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD570nm。将抗体的浓度取对数后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidaldose-response(Variable Slope)方式(GraphPad Prism软件，GraphPad Software，SanDiego，California)进行非线性回归，得到目标抗体与B7H3蛋白的结合曲线。多功能融合蛋白BCI的结合活性由图10所示。The huB7H3-his receptor was diluted to 0.5 μg/mL with pH 7.4 PBS buffer, 100 μL per well was added to a 96-well ELISA plate, and coated overnight at 4 degrees. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the multifunctional fusion protein BCI was diluted to 10 μg/mL with 0.5% BSA sample diluent, taking this as the starting concentration, 3-fold gradient dilution was carried out, a total of 11 gradients, and an irrelevant antibody negative control was set. Incubate with positive control B7H3 chimeric antibody, 100 μL per well, at 37°C for 1 h. The plate was washed three times with PBST, and the HRP-labeled goat anti-human IgGFc was diluted 1:20000 with sample diluent, 100 μL was added to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 μL of TMB substrate to each well, incubate at room temperature for 10 minutes in the dark, and add 100 μL of 1M HCl solution to each well to stop the color reaction. The wavelength of 450nm was selected on a multi-function microplate reader, and the reference wavelength of 570nm was used to measure the absorbance value of each well in the 96-well plate, and the absorbance value of each well (OD)=OD450nm-OD570nm. The logarithm of the antibody concentration was taken as the abscissa, and the measured absorbance value of each well was the ordinate. The Sigmoidaldose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, SanDiego, California) was used to perform nonlinear regression to obtain Binding curve of target antibody to B7H3 protein. The binding activity of the multifunctional fusion protein BCI is shown in FIG. 10 .
由图10可知，BCI在各浓度下均与B7H3有很好的结合能力。It can be seen from Figure 10 that BCI has good binding ability to B7H3 at all concentrations.
实施例13多功能融合蛋白BCI与CD3的结合活性Example 13 Binding activity of multifunctional fusion protein BCI and CD3
采用pH7.4的PBS缓冲液将huCD3-his受体稀释至1μg/mL，每孔100μL加入到96孔ELISA板中，4℃包被过夜。用1％BSA封闭液封闭1小时。PBST洗板3次后，将BCI、CD3抗体、无关抗体IgG样品稀释液稀释至10μg/mL，以此为起始浓度，进行3倍梯度稀释，共11个梯度，每孔100μL，37℃孵育1h。再用PBST洗板3次，将HRP标记的山羊抗人IgG Fc用样品稀释液按1:10000稀释，每孔加入100μL，室温孵育1小时。PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10分钟，每孔加入100μL 1M HCl液终止显色反应。在多功能酶标仪上选择波长450nm，参比波长570nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD570nm。将抗体的浓度取对数后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidaldose-response(Variable Slope)方式(GraphPad Prism软件，GraphPad Software，SanDiego，California)进行非线性回归，得到BCI与CD3蛋白的结合曲线。The huCD3-his receptor was diluted to 1 μg/mL with PBS buffer pH 7.4, and 100 μL per well was added to a 96-well ELISA plate and coated overnight at 4°C. Block with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, dilute the BCI, CD3 antibody, and irrelevant antibody IgG sample dilutions to 10 μg/mL, using this as the starting concentration, carry out 3-fold gradient dilution, a total of 11 gradients, 100 μL per well, and incubate at 37 °C 1h. The plate was washed three times with PBST, and the HRP-labeled goat anti-human IgG Fc was diluted 1:10000 with sample diluent, added 100 μL to each well, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 μL of TMB substrate to each well, incubate at room temperature for 10 minutes in the dark, and add 100 μL of 1M HCl solution to each well to stop the color reaction. The wavelength of 450nm was selected on a multi-function microplate reader, and the reference wavelength of 570nm was used to measure the absorbance value of each well in the 96-well plate, and the absorbance value of each well (OD)=OD450nm-OD570nm. The logarithm of the antibody concentration was taken as the abscissa, and the measured absorbance value of each well was the ordinate. The Sigmoidaldose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, SanDiego, California) was used to perform nonlinear regression to obtain Binding curve of BCI to CD3 protein.
由图11可知，BCI在各浓度下均与CD3有很好的结合能力。It can be seen from Figure 11 that BCI has good binding ability to CD3 at all concentrations.
实施例14流式细胞术检测MCF-7细胞B7H3表达丰度Example 14 Detection of B7H3 expression abundance in MCF-7 cells by flow cytometry
取细胞状态良好并处于对数期生长的MCF-7细胞悬液800g，室温离心3分钟，去掉上清，使用PBS洗涤细胞2次后，PBS重悬细胞至2×10 5/ml，按照1ml/离心管分成若干份。加入100μl使用PBS稀释至20ug/ml的B7H3抗体，并设空白对照组(Blank)，阴性对照组(NC)，充分混匀后，室温孵育半小时。800g室温离心5分钟，去掉含有抗体的上清，使用PBS洗涤细胞3次。取100ul重悬 细胞，加入0.5uL FITC标记的F(ab’)2山羊抗人IgG(Biolegend398006)，充分混匀后，室温避光孵育30min；800g室温离心5分钟，去掉含有二抗的上清，使用PBS洗涤细胞3次；使用100uLPBS重悬细胞，进行流式分析。结果如图12a-12b所示。 Take 800 g of MCF-7 cell suspension with good cell condition and grow in log phase, centrifuge at room temperature for 3 minutes, remove the supernatant, wash the cells twice with PBS, resuspend the cells in PBS to 2×10 5 /ml, according to 1ml / Centrifuge tube into several portions. 100 μl of B7H3 antibody diluted to 20ug/ml with PBS was added, and a blank control group (Blank) and a negative control group (NC) were set up, mixed thoroughly, and incubated at room temperature for half an hour. Centrifuge at 800g for 5 minutes at room temperature, remove the supernatant containing the antibody, and wash the cells 3 times with PBS. Take 100ul of resuspended cells, add 0.5uL of FITC-labeled F(ab')2 goat anti-human IgG (Biolegend398006), mix well, incubate in the dark at room temperature for 30 min; centrifuge at 800 g at room temperature for 5 min, remove the supernatant containing the secondary antibody , washed the cells 3 times with PBS; resuspended the cells with 100uL PBS for flow analysis. The results are shown in Figures 12a-12b.
由图12b可知，跟MCF-7阴性峰相比，MCF-7-B7H3抗体的峰沿X轴显著右移，表明MCF-7细胞高表达B7H3。As can be seen from Figure 12b, compared with the negative peak of MCF-7, the peak of MCF-7-B7H3 antibody was significantly shifted to the right along the X axis, indicating that MCF-7 cells highly expressed B7H3.
实施例15多功能融合蛋白与IL-2Rβ的结合活性Example 15 Binding activity of multifunctional fusion protein and IL-2Rβ
依照实施例7的方法检测多功能融合蛋白与IL-2Rβ的结合活性，结果如图13所示。所述多功能融合蛋白在各浓度下均可与IL-2Rβ结合，表明所述多功能融合蛋白与IL-2Rβ有较好的结合能力。The binding activity of the multifunctional fusion protein to IL-2Rβ was detected according to the method of Example 7, and the results are shown in FIG. 13 . The multifunctional fusion protein can bind to IL-2Rβ at various concentrations, indicating that the multifunctional fusion protein has better binding ability to IL-2Rβ.
实施例16多功能融合蛋白BCI的抗肿瘤活性Example 16 Antitumor activity of multifunctional fusion protein BCI
使用B7H3阳性的乳腺癌细胞MCF-7以2×10 4/孔铺于96孔板，培养24h后，加入多功能融合蛋白BCI和无关抗体20μg/mL起始，5倍稀释，共10个浓度梯度，同时加入CIK(CD3+CD56+细胞)效应细胞4×10 4/孔，并设置空白对照(稀释液)、阴性对照(MCF-7+CIK，无抗体)、无关抗体组，于细胞培养箱孵育24h后，PBS润洗次，洗去效应细胞。加入含10％CCK-8(Cat：CK04)培养基100μL，于培养箱孵育3h后，于酶标仪OD值450nm进行检测。计算细胞杀伤率，公式为细胞杀伤率(％)＝(样品孔OD值-空白OD值)/(阴性孔OD值-空白OD值)×100％。 B7H3-positive breast cancer cells MCF-7 were plated in 96-well plates at 2×10 4 /well. After culturing for 24 h, 20 μg/mL of multifunctional fusion protein BCI and irrelevant antibodies were added to start with 5-fold dilution, with a total of 10 concentrations. Gradient, simultaneously add CIK (CD3+CD56+ cells) effector cells 4×10 4 /well, and set blank control (diluent), negative control (MCF-7+CIK, no antibody), irrelevant antibody group, in a cell incubator After incubation for 24 h, the cells were washed with PBS for several times to remove the effector cells. Add 100 μL of medium containing 10% CCK-8 (Cat: CK04), incubate for 3 h in an incubator, and detect at OD value of 450 nm on a microplate reader. The cell killing rate was calculated, and the formula was cell killing rate (%)=(OD value of sample well-OD value of blank)/(OD value of negative well-OD value of blank)×100%.
由图14可知，BCI可杀伤B7H3阳性的乳腺癌细胞MCF-7，而无关抗体不具有杀伤作用，说明BCI介导CIK细胞特异性杀伤B7H3阳性的MCF-7细胞。It can be seen from Figure 14 that BCI can kill B7H3-positive breast cancer cells MCF-7, while irrelevant antibodies have no killing effect, indicating that BCI mediates CIK cells to specifically kill B7H3-positive MCF-7 cells.
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

Claims (24)

  1. 一种多功能融合蛋白，其特征在于，其包含第一重链、第二重链和第一轻链、第二轻链，所述第一重链包含特异性结合靶标的VH和CH1，细胞因子和其受体，以及免疫球蛋白Fc部分；所述第二重链包含特异性结合靶标的VH和CH1，特异性结合靶标的scFv或Fab，以及免疫球蛋白Fc部分；第一轻链与第一重链，第二轻链与第二重链分别特异性配对。A kind of multifunctional fusion protein, it is characterized in that, it comprises the first heavy chain, the second heavy chain and the first light chain, the second light chain, and the first heavy chain comprises the VH and CH1 of specific binding target, cell factor and its receptor, and an immunoglobulin Fc portion; the second heavy chain comprises VH and CH1 that specifically bind to a target, an scFv or Fab that specifically binds to a target, and an immunoglobulin Fc portion; the first light chain and The first heavy chain, the second light chain and the second heavy chain are specifically paired, respectively.
  2. 如权利要求1所述的多功能融合蛋白，其中所述第一重链和第二重链包含的VH和CH1可以特异性结合TAA抗原，所述TAA抗原为Claudin18.2、CA125、AFP、CEA、EGFR、HER2、B7H3、B7H6、MUC1、MUC16、GPC3、CD24其中的一种或几种，优选的，所述TAA抗原为Claudin18.2或B7H3。The multifunctional fusion protein of claim 1, wherein the VH and CH1 contained in the first heavy chain and the second heavy chain can specifically bind to TAA antigens, and the TAA antigens are Claudin18.2, CA125, AFP, CEA , one or more of EGFR, HER2, B7H3, B7H6, MUC1, MUC16, GPC3, CD24, preferably, the TAA antigen is Claudin18.2 or B7H3.
  3. 如权利要求1-2任一所述的多功能融合蛋白，其中所述第一重链包含的细胞因子和其受体分别为IL-15和IL-15受体。The multifunctional fusion protein of any one of claims 1-2, wherein the cytokine and its receptor contained in the first heavy chain are IL-15 and IL-15 receptor, respectively.
  4. 如权利要求1-3任一所述的多功能融合蛋白，其中所述第二重链包含的scFv或Fab可以特异性激活T细胞、NK细胞和巨噬细胞，优选的，所述第二重链包含的scFv或Fab特异性靶向CD3。The multifunctional fusion protein according to any one of claims 1-3, wherein the scFv or Fab contained in the second heavy chain can specifically activate T cells, NK cells and macrophages, preferably, the second heavy chain can specifically activate T cells, NK cells and macrophages. The scFv or Fab contained in the chain specifically targets CD3.
  5. 如权利要求1-4任一所述的多功能融合蛋白，其中所述第一重链和第二重链的免疫球蛋白Fc部分选自IgG1、IgG2、IgG3、IgG4的恒定区氨基酸序列，优选选自IgG1或IgG4的恒定区氨基酸序列。The multifunctional fusion protein of any one of claims 1-4, wherein the immunoglobulin Fc portion of the first heavy chain and the second heavy chain is selected from the constant region amino acid sequences of IgG1, IgG2, IgG3, IgG4, preferably A constant region amino acid sequence selected from IgG1 or IgG4.
  6. 如权利要求1-5任一所述的多功能融合蛋白，其中所述第一重链和第二重链的Fc部分还包含一个或多个选自由以下组成的组的氨基酸替换：S228P、L234F、L235E、P331S、D356K、T366W、K392D、D399K、Y407A、和K409D，优选S228P、T366W和/或Y407A。The multifunctional fusion protein of any one of claims 1-5, wherein the Fc portion of the first and second heavy chains further comprises one or more amino acid substitutions selected from the group consisting of: S228P, L234F , L235E, P331S, D356K, T366W, K392D, D399K, Y407A, and K409D, preferably S228P, T366W and/or Y407A.
  7. 如权利要求3所述的多功能融合蛋白，其中所述第一重链中的IL-15与其受体和第二重链中的scFv或Fab可以分别嵌合于所述第一重链和所述第二重链的Fc部分内部，也可以存在于Fc部分外部，优选位于所述相应重链的CH1和CH2功能区之间。The multifunctional fusion protein of claim 3, wherein the IL-15 in the first heavy chain and its receptor and the scFv or Fab in the second heavy chain can be chimeric in the first heavy chain and the scFv or Fab, respectively The Fc portion of the second heavy chain may also be present outside the Fc portion, preferably between the CH1 and CH2 domains of the corresponding heavy chain.
  8. 如权利要求3所述的多功能融合蛋白，其中所述第一重链中的IL-15与其受体、第二重链中的scFv或Fab单独或与额外的连接肽一起共价结合于所述链中；所述连接肽包含甘氨酸(G)和丝氨酸(S)残基，优选包含GGGGS重复，更优选包含1-2个GGGGS重复。The multifunctional fusion protein of claim 3, wherein the IL-15 in the first heavy chain is covalently bound to its receptor, scFv or Fab in the second heavy chain alone or with an additional linking peptide In the chain; the linking peptide comprises glycine (G) and serine (S) residues, preferably GGGGS repeats, more preferably 1-2 GGGGS repeats.
  9. 如权利要求3所述的多功能融合蛋白，其中所述IL-15选自天然的IL-15 或其变体，所述变体包含选自N1D、N4D、D30N、E64Q、N65D、N72D、N79A、Q108E和N112A的组的一个或多个氨基酸突变，优选包含选自N4D、N65D、N72D、N79A和N112A的组的一个或多个氨基酸突变；所述IL-15受体选自IL-15Rα或其变体。The multifunctional fusion protein of claim 3, wherein the IL-15 is selected from native IL-15 or a variant thereof, and the variant comprises N1D, N4D, D30N, E64Q, N65D, N72D, N79A , one or more amino acid mutations of the group of Q108E and N112A, preferably comprising one or more amino acid mutations selected from the group of N4D, N65D, N72D, N79A and N112A; the IL-15 receptor is selected from IL-15Rα or its variants.
  10. 如权利要求1-9任一所述的多功能融合蛋白，其中所述第一重链氨基酸序列选自SEQ ID NO:1；所述多功能融合蛋白的第二重链氨基酸序列选自SEQ ID NO:2；所述多功能融合蛋白的第一轻链和第二轻链的氨基酸序列选自SEQ ID NO:3。The multifunctional fusion protein of any one of claims 1-9, wherein the first heavy chain amino acid sequence is selected from SEQ ID NO: 1; the second heavy chain amino acid sequence of the multifunctional fusion protein is selected from SEQ ID NO:2; the amino acid sequences of the first light chain and the second light chain of the multifunctional fusion protein are selected from SEQ ID NO:3.
  11. 如权利要求1-9任一所述的多功能融合蛋白，其中所述第一重链氨基酸序列选自SEQ ID NO:14；所述多功能融合蛋白的第二重链氨基酸序列选自SEQ ID NO:15；所述多功能融合蛋白的第一轻链和第二轻链的氨基酸序列选自SEQ ID NO:16。The multifunctional fusion protein of any one of claims 1-9, wherein the first heavy chain amino acid sequence is selected from SEQ ID NO: 14; the second heavy chain amino acid sequence of the multifunctional fusion protein is selected from SEQ ID NO: 15; the amino acid sequences of the first light chain and the second light chain of the multifunctional fusion protein are selected from SEQ ID NO: 16.
  12. 一种编码如权利要求1-11任一项所述多功能融合蛋白的核酸分子，其特征在于包含编码第一轻链和第二轻链的核苷酸序列，或者包含编码第一重链的核苷酸序列，或者包含编码第二重链的核苷酸序列。A nucleic acid molecule encoding a multifunctional fusion protein according to any one of claims 1-11, characterized in that it comprises a nucleotide sequence encoding a first light chain and a second light chain, or a nucleotide sequence encoding the first heavy chain. The nucleotide sequence, or comprising the nucleotide sequence encoding the second heavy chain.
  13. 如权利要求12所述的核酸分子，其中所述编码第一重链的核苷酸序列选自SEQ ID NO:4；所述编码第二重链的核苷酸序列选自SEQ ID NO:5；所述编码第一轻链和第二轻链的核苷酸序列选自SEQ ID NO:6。The nucleic acid molecule of claim 12, wherein the nucleotide sequence encoding the first heavy chain is selected from the group consisting of SEQ ID NO:4; the nucleotide sequence encoding the second heavy chain is selected from the group consisting of SEQ ID NO:5 ; The nucleotide sequences encoding the first light chain and the second light chain are selected from SEQ ID NO: 6.
  14. 如权利要求12所述的核酸分子，其中所述编码第一重链的核苷酸序列选自SEQ ID NO:17；所述编码第二重链的核苷酸序列选自SEQ ID NO:18；所述编码第一轻链和第二轻链的核苷酸序列选自SEQ ID NO:19。The nucleic acid molecule of claim 12, wherein the nucleotide sequence encoding the first heavy chain is selected from SEQ ID NO: 17; the nucleotide sequence encoding the second heavy chain is selected from SEQ ID NO: 18 ; The nucleotide sequences encoding the first light chain and the second light chain are selected from SEQ ID NO: 19.
  15. 如权利要求12所述的核酸分子，其中所述核酸分子可在编码其轻链的核苷酸序列和编码其重链的核苷酸序列的5’端分别进一步包含编码信号肽的核苷酸序列，在编码轻链的核苷酸序列和编码重链的核苷酸序列的3’端分别进一步包含终止密码子。The nucleic acid molecule of claim 12, wherein the nucleic acid molecule further comprises a nucleotide encoding a signal peptide at the 5' end of the nucleotide sequence encoding its light chain and the nucleotide sequence encoding its heavy chain, respectively The sequence further comprises a stop codon at the 3' end of the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain, respectively.
  16. 如权利要求15所述的核酸分子，其中所述信号肽选自氨基酸序列SEQ ID NO:7和SEQ ID NO:9，编码所述信号肽的核苷酸序列选自SEQ ID NO:8和SEQ ID NO:10。The nucleic acid molecule of claim 15, wherein the signal peptide is selected from the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:9, and the nucleotide sequence encoding the signal peptide is selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:8 ID NO: 10.
  17. 一种重组载体，其包含编码权利要求1-11任一项所述多功能融合蛋白的第一重链、和/或第二重链、和/或第一轻链、和/或第二轻链的核苷酸序列。A recombinant vector comprising the first heavy chain, and/or the second heavy chain, and/or the first light chain, and/or the second light chain of the multifunctional fusion protein of any one of encoding claims 1-11 The nucleotide sequence of the chain.
  18. 一种重组细胞，其含有权利要求17所述的重组载体，优选地，所述细胞包括人胚肾细胞HEK293或HEK293T、HEK293E、HEK293修饰的HEK293F、中国仓鼠卵巢细胞(CHO)、CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO、CHO修饰的ExpiCHO，和其组合。 A kind of recombinant cell, it contains the described recombinant vector of claim 17, preferably, described cell comprises human embryonic kidney cell HEK293 or HEK293T, HEK293E, HEK293F modified by HEK293, Chinese hamster ovary cell (CHO), CHO-S, CHO - dhfr-, CHO/DG44, ExpiCHO, CHO-modified ExpiCHO, and combinations thereof.
  19. 一种制备如权利要求1-11任一项所述多功能融合蛋白的方法，其具体包括：A method for preparing a multifunctional fusion protein as described in any one of claims 1-11, which specifically comprises:
    在足以表达权利要求1-11任一项所述多功能融合蛋白的条件下，培养权利要求18所述的重组细胞，表达并纯化所述的多功能融合蛋白。The recombinant cell of claim 18 is cultured under conditions sufficient to express the multifunctional fusion protein of any one of claims 1-11, and the multifunctional fusion protein is expressed and purified.
  20. 如权利要求1-11任一项所述多功能融合蛋白作为活性成分的药物，所述药物任选含有药学上可接受的载体或赋形剂。The medicament using the multifunctional fusion protein as an active ingredient according to any one of claims 1-11, the medicament optionally contains a pharmaceutically acceptable carrier or excipient.
  21. 如权利要求1-11任一项所述多功能融合蛋白在制备预防或治疗与TAA有关的疾病或病症如肿瘤的药物中的用途。Use of the multifunctional fusion protein according to any one of claims 1-11 in the preparation of a medicament for preventing or treating TAA-related diseases or disorders such as tumors.
  22. 如权利要求21所述的用途，所述肿瘤为对Claudin18.2单独治疗无效的肿瘤或晚期肿瘤，更优选为对抗Claudin18.2抗体单独治疗产生抗性或无效的肿瘤；进一步优选为胃癌、食道癌、胰腺癌等。The use according to claim 21, wherein the tumor is a tumor that is ineffective against Claudin18.2 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against Claudin18.2 antibody monotherapy; more preferably gastric cancer, esophagus cancer, pancreatic cancer, etc.
  23. 如权利要求21所述的用途，所述肿瘤为对B7H3单独治疗无效的肿瘤或晚期肿瘤，更优选为对抗B7H3抗体单独治疗产生抗性或无效的肿瘤；进一步优选为胃癌、食道癌、胰腺癌等。The use according to claim 21, wherein the tumor is a tumor that is ineffective against B7H3 monotherapy or an advanced tumor, more preferably a tumor resistant or ineffective against anti-B7H3 antibody monotherapy; further preferably gastric cancer, esophageal cancer, pancreatic cancer Wait.
  24. 如权利要求1-11任一项所述的多功能融合蛋白作为活性成分的药物制剂、药用组合物或试剂盒。A pharmaceutical preparation, a pharmaceutical composition or a kit using the multifunctional fusion protein according to any one of claims 1 to 11 as an active ingredient.
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