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WO2022042576A1 - Protéine de fusion multifonctionnelle et son utilisation - Google Patents

Protéine de fusion multifonctionnelle et son utilisation Download PDF

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Publication number
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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Prior art keywords
heavy chain
fusion protein
multifunctional fusion
seq
cells
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Chinese (zh)
Inventor
周冲
吴崇兵
王艺臻
姜晓玲
殷刘松
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Sunho China Biopharmaceutical Co Ltd
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Sunho China Biopharmaceutical Co Ltd
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Priority claimed from CN202110971791.2A external-priority patent/CN114106195B/zh
Application filed by Sunho China Biopharmaceutical Co Ltd filed Critical Sunho China Biopharmaceutical Co Ltd
Publication of WO2022042576A1 publication Critical patent/WO2022042576A1/fr
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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

La présente invention concerne une protéine de fusion multifonctionnelle ciblant TAA et CD3 et ayant les effets biologiques d'un complexe IL-15/IL-15Rα. La protéine de fusion multifonctionnelle peut résoudre le problème de résistance aux médicaments et de récurrence provoqué par un médicament à base d'anticorps à une seule cible, peut réduire la dose efficace, détruire de manière plus efficace des cellules tumorales, prolonger la demi-vie d'un sérum IL-15/IL-15Rα, améliorer la capacité de ciblage de tumeur, et réduire la toxicité et les effets secondaires de celle-ci. L'utilisation d'une combinaison d'un anticorps CD3 et d'un anticorps ciblant une tumeur, permet à une tumeur d'être dirigée et à des lymphocytes T d'être recrutés pour se rapprocher des cellules tumorales, ce qui joue un rôle dans la médiation des lymphocytes T afin de détruire les cellules tumorales. La structure de la protéine de fusion est plus stable en termes de conception structurale, par exemple la formation de synapses immunitaires et la distance relative entre des sites fonctionnels d'anticorps et des antigènes. Le complexe IL-15/IL-15Rα favorise le recrutement et l'activation persistants des lymphocytes T et des cellules NK.
PCT/CN2021/114408 2020-08-27 2021-08-25 Protéine de fusion multifonctionnelle et son utilisation Ceased WO2022042576A1 (fr)

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WO2024197709A1 (fr) * 2023-03-30 2024-10-03 中国科学院深圳先进技术研究院 Activateur de lymphocytes t, son procédé de préparation et son utilisation

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