WO2021170111A1 - 肿瘤免疫增强剂及其制法和应用 - Google Patents

肿瘤免疫增强剂及其制法和应用 Download PDF

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WO2021170111A1
WO2021170111A1 PCT/CN2021/078264 CN2021078264W WO2021170111A1 WO 2021170111 A1 WO2021170111 A1 WO 2021170111A1 CN 2021078264 W CN2021078264 W CN 2021078264W WO 2021170111 A1 WO2021170111 A1 WO 2021170111A1
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tumor
amino acid
polypeptide
immune enhancer
tumor immune
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PCT/CN2021/078264
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English (en)
French (fr)
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邓立
陈淑娴
曲秀霞
龚笑海
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无锡派列博生物医药科技有限公司
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Priority to AU2021226544A priority Critical patent/AU2021226544A1/en
Priority to US17/907,941 priority patent/US20230045104A1/en
Priority to CA3169907A priority patent/CA3169907A1/en
Priority to EP21761114.4A priority patent/EP4112636A4/en
Priority to JP2022552322A priority patent/JP2023515850A/ja
Publication of WO2021170111A1 publication Critical patent/WO2021170111A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants

Definitions

  • the present invention relates to the field of immunity, and more specifically to a tumor immune enhancer and its preparation method and application.
  • the root cause of tumor development is that the genetic material of tissue cells has mutations that promote abnormal cell growth. Although the number of gene mutations in tumor cells can be as many as thousands, the mutated genes (driver genes) that actually initiate tumorigenesis may only involve two to three. Part of the driver genes have been used as targets for tumor therapy drug design. Studies have found that the clinical efficacy of such targeted drugs depends on the expression of other non-driver mutant genes and untargeted driver genes in tumor cells, so the clinical efficacy of such drugs is very limited. The latest research progress of tumor vaccines shows that we can design vaccines that can cure tumors by using gene mutations that change the encoding of amino acid residues. This is an important milestone in tumor treatment.
  • the gene encoding the mutation may be broken down by the antigen-presenting cells to produce a tumor-specific antigen that can be recognized by T cells.
  • This antigen is called a tumor neoantigen. Since T cells that recognize neoantigens are not restricted by central tolerance, neoantigens are considered to have strong immunogenicity and can stimulate tumor-specific killing CD8+ T cell (CTL) responses. Clinical studies have confirmed that such neoantigen-specific CTLs do exist in tumor tissues, and the activity of such CTLs determines a variety of tumor treatment methods such as T cell growth factor IL-2, immune checkpoint inhibitors (PD-1 and PD-1). CTLA-4 monoclonal antibody) and the efficacy of infusion of expanded TILs in vitro.
  • neoantigens of most tumors are patient-specific, vaccines against neoantigens must be customized according to the genetic mutations of each patient.
  • genetic mutations in a single tumor have polyclonal properties.
  • tumor vaccines need to adopt a multi-target design strategy.
  • the number of neoantigen T epitopes used in the published clinical studies of tumor vaccines is more than 20.
  • the detection of CD4 and CD8 T cell responses revealed that only about one-third of the neoantigens can trigger anti-tumor T cell responses, and more than 90% of T cells are CD4 + .
  • Type I helper T cells are required for such cell activation and memory formation, as well as tumor infiltration. Therefore, it can be inferred that most of the neoantigens currently used in cancer vaccines cannot effectively trigger type I helper T cell responses.
  • T helper polypeptides For example, tetanus toxoid T helper peptide and pan-DR peptide (PARDE) have been used in clinical trials. As a result, it was found that these polypeptides could not effectively induce anti-tumor CD8+CTL responses.
  • PARDE pan-DR peptide
  • the purpose of the present invention is to provide an effective anti-tumor immune response immune enhancer and its preparation method and application.
  • a tumor immune enhancer which comprises one or more polypeptides having the structure of Formula I, or a pharmaceutically acceptable salt thereof:
  • Z0 is none, or a peptide consisting of 1-10 amino acid residues
  • Z2 is none, or a peptide consisting of 1-10 amino acid residues
  • Z1 is a peptide selected from the following group:
  • amino acid sequence of SEQ ID NO: 1-9 is formed by substitution, deletion or addition of one, two or three amino acid residues, and is capable of binding to human and mouse type II histocompatibility antigens. .
  • the derivative polypeptide retains ⁇ 70% of the activity of the polypeptide shown in SEQ ID NO: 1-9 to bind to human and mouse type II histocompatibility antigens.
  • the identity of the derivative polypeptide with the polypeptide shown in SEQ ID NO: 1-9 is ⁇ 80%, preferably ⁇ 90%, and more preferably ⁇ 95%.
  • the IC 50 of the binding of Z1 to the histocompatibility antigen is 5-50 nm.
  • Z0 and Z2 are not null at the same time
  • Z0 and Z2 contain at least 2-8, preferably 3-6, hydrophilic amino acid residues.
  • Z2 is none
  • Z0 is a peptide consisting of 2-8, preferably 3-6 hydrophilic amino acid residues.
  • Z0 is none
  • Z2 is a peptide consisting of 2-8, preferably 3-6, hydrophilic amino acid residues.
  • the hydrophilic amino acid is selected from the group consisting of arginine and lysine.
  • At least one of Z0 and Z2 contains (Arg)n structure, where n is a positive integer of 3-6.
  • the structure of Formula I is a structure from N-terminus to C-terminus.
  • the structure of Formula I is a structure from C-terminus to N-terminus.
  • "a” is a covalent bond (such as a peptide bond), or when Z0 is absent, then the "-" between Z0 and Z1 does not exist, or when Z2 is absent, then Z1 and The "-" between Z2 does not exist.
  • the tumor immunity enhancer has the activity of enhancing tumor immunity.
  • the tumor immune enhancer can induce a killer T cell response.
  • polypeptide having the structure of Formula I contains one or more CD4 + Th epitope peptides.
  • polypeptide having the structure of Formula I is a CD4+ T cell antigen receptor ligand.
  • the tumor immune enhancer comprises Z1 being the polypeptide of formula I shown in SEQ ID NO: 1, Z1 being the polypeptide of formula I shown in SEQ ID NO: 2, and Z1 being SEQ ID NO: A polypeptide of formula I shown in 3.
  • a multimer which is formed by m monomers in series and has the function of enhancing tumor immunity, wherein m is a positive integer ⁇ 2, and the Each monomer independently has the structure of Formula I:
  • Z0 is none, or a peptide consisting of 1-10 amino acid residues
  • Z2 is none, or a peptide consisting of 1-10 amino acid residues
  • Z1 is a peptide selected from the following group:
  • n 2, 3, 4, 5, or 6.
  • the two monomers are directly connected by a peptide bond or connected by a peptide linker.
  • the peptide linker is a flexible peptide linker, a rigid peptide linker, or a combination thereof.
  • the peptide linker is a peptide linker of 3-10 amino acids.
  • an isolated nucleic acid molecule which encodes a polypeptide, the polypeptide has the structure of Formula I, or the polypeptide is a multimer formed by m monomers in series, wherein each The monomers each independently have the structure of formula I, and m is a positive integer ⁇ 2.
  • the nucleic acid molecule is selected from the group consisting of DNA, RNA or a combination thereof.
  • a pharmaceutical composition which contains:
  • the dosage form of the composition is an injection.
  • the composition is a sustained-release dosage form.
  • the tumor immune enhancer according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, or the multimer according to the second aspect of the present invention contained in the composition can be selected from From the form of the next group:
  • Protein molecules (complete molecules, subunits, domains, polypeptides and recombinant engineering molecules), lipids, polysaccharides, lipids and polysaccharide complexes.
  • the pharmaceutical composition further contains:
  • the tumor antigen is a tumor neoantigen peptide.
  • the tumor antigen is an antigen that cannot effectively trigger an anti-tumor immune response.
  • the tumor antigen is natural, artificially synthesized, or a combination thereof.
  • the tumor antigen is selected from the group consisting of short peptides, intact proteins, tumor cell lysates, inactivated tumor cells, or a combination thereof.
  • the tumor antigens are derived from malignant tumors with non-synonymous mutations in the middle and high frequency encoding genes, including but not limited to:
  • Malignant melanoma breast cancer, lung cancer, pancreatic cancer, prostate cancer, bowel cancer, liver cancer, esophageal cancer, cervical cancer, bladder cancer, renal cell carcinoma, glioma multiforme.
  • the pharmaceutical composition further contains:
  • the DC activator is a nano-formulation, including but not limited to w/o agent and o/w agent.
  • the DC activator is a self-assembled nano-formulation.
  • the DC activator is selected from the group consisting of liposomes, viral outer capsids, and inactivated bacteria.
  • the pharmaceutical composition is a tumor vaccine composition.
  • the vaccine is selected from the following group: whole cell vaccine, cell lysate vaccine, tumor tissue split vaccine, tumor cell exosomal vaccine.
  • the polymer, or the isolated nucleic acid molecule of the third aspect of the present invention, and the substance is used to prepare drugs that improve the anti-tumor activity of tumor antigens, or to prepare anti-tumor vaccines (tumor vaccines) )combination.
  • the drug or vaccine composition is also used for one or more purposes selected from the following group:
  • helper T cell response preferably a type I helper T cell response
  • a medicine kit containing:
  • the drugs for treating tumors include immune checkpoint inhibitors, chemotherapeutic agents, radiotherapy agents, hyperthermia agents, oncolytic viruses, and immune cell therapeutic agents.
  • the immune cell therapeutic agent includes CAR-T cells, CAR-NK cells, TCR-T cells, DC cells or a combination thereof.
  • a method for treating cancer including the steps:
  • steps (i) and (ii) can be performed simultaneously or sequentially.
  • the object in need refers to a tumor patient.
  • step (ii) the drug for treating tumors is administered according to the usual dosage and frequency of administration.
  • the method is to combine existing tumor treatment methods such as immune checkpoint inhibitors, chemotherapy, radiotherapy, hyperthermia, and oncolytic viruses with the tumor immune enhancer of the present application.
  • existing tumor treatment methods such as immune checkpoint inhibitors, chemotherapy, radiotherapy, hyperthermia, and oncolytic viruses.
  • the method is to combine cell therapy with the tumor immune enhancer of the present application.
  • cell therapies include but are not limited to DC cell therapy, CAR-T cell therapy, CAR-NK cell therapy, etc. Cell therapy.
  • a method for preventing and/or treating tumors in mammals comprising the steps of: administering the tumor immune enhancer according to the first aspect of the present invention or its pharmaceutically acceptable Salt, the multimer according to the second aspect of the invention, the isolated nucleic acid molecule according to the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention.
  • the subject is a human.
  • the tumor antigens are derived from malignant tumors with non-synonymous mutations in the encoding subtypes of medium and high frequencies, such as: melanoma, pancreatic cancer, prostate cancer, breast cancer, lung cancer, colon cancer, liver cancer, esophageal cancer, Cervical cancer, bladder cancer, renal cell carcinoma, glioma multiforme.
  • the method further comprises: administering a tumor antigen to a subject in need.
  • the method further comprises: administering a drug for treating tumors to a subject in need.
  • Figure 1 shows that in one embodiment of the present invention, UThE enhances the anti-tumor immune response of the weak neoantigen of mouse melanoma.
  • Figure 2 shows that in another embodiment of the present invention, UThE enhances the anti-tumor immune response (tumor prevention) of the weak neoantigen of mouse melanoma.
  • Figure 2A and Figure 2B respectively show the change in tumor volume with the number of days after tumor injection.
  • FIG. 3 shows that in another embodiment of the present invention, UThE effectively enhances the therapeutic effect on mouse melanoma.
  • Figure 4 shows the results of detection of anti-UThE antibodies in mouse serum samples after vaccine immunization in an example.
  • Figure 5 shows the results of anti-UThE antibody detection in mouse serum samples after vaccine immunization in another embodiment.
  • Figure 6 shows the detection results of anti-neoantigen antibodies in mouse serum samples after vaccine immunization in another embodiment.
  • Fig. 7 shows the detection results of anti-neoantigen antibodies in mouse serum samples after vaccine immunization in another embodiment.
  • Figure 8 shows the results of specific CD8+CTL detection of mouse spleen tumors after vaccine immunization in another example.
  • Figure 9 shows the results of tumor-specific CD8+CTL detection in the spleen of immunized mice in another example.
  • Figure 10 shows the effective killing effect of the killing CD8+CTL on tumor target cells in another example.
  • FIG 11 shows that in another example, UThE effectively enhances the immunogenicity of mouse lung cancer (LLC) weak antigens and suppresses tumors.
  • LLC mouse lung cancer
  • FIG 12 shows another example, UThE effectively enhances the immunogenicity of the weak antigen of mouse melanoma (B16F10) and its inhibitory effect on tumors.
  • FIG 13 shows another example, UThE effectively enhances the immunogenicity of the weak antigen of mouse prostate cancer (RM-1) and its inhibitory effect on tumors.
  • FIG 14 shows another example, UThE effectively enhances the immunogenicity of mouse pancreatic cancer (PanC-02) weak antigen and its inhibitory effect on tumors.
  • Figure 15 shows the dose-efficacy relationship and safety of UThE in killing tumor cells in another example (mouse weight is stable).
  • Figure 16 shows the tumor suppressor effect of UThE candidate peptide, UThE1-7, in C57BL/6 mice and Balb/C mice in another example.
  • the inventors discovered for the first time a general Th epitope peptide derived from diphtheria toxin and tetanus toxin protein, which can enhance the tumor neoantigen vaccine-specific CD8 + CTL immune response.
  • the polypeptides of the present invention can enhance the anti-tumor immune response efficacy of tumor vaccines. On this basis, the present invention has been completed.
  • UThE1-UThE9 are polypeptide molecules composed of 15-21 amino acid residues, have a moderate affinity with mouse type II histocompatibility antigen molecules, and have an IC50 of 5-50 nm. Because these polypeptide molecules are too hydrophobic, it is necessary to add multiple hydrophilic amino acid residues (such as arginine residues or lysine residues) at the N-terminus or C-terminus during synthesis. These polypeptide molecules all carry one or more Th epitopes, which can support the function of inducing helper T cell responses in more than 80% of the population.
  • UThE1-UThE9 are polypeptide molecules composed of 15-21 amino acid residues, have a moderate affinity with mouse type II histocompatibility antigen molecules, and have an IC50 of 5-50 nm. Because these polypeptide molecules are too hydrophobic, it is necessary to add multiple hydrophilic amino acid residues (such as arginine residues or lysine residues) at the N-terminus
  • polypeptide of the present invention refers to a polypeptide having the structure of Formula I:
  • Z0 is none, or a peptide consisting of 1-10 amino acid residues
  • Z2 is none, or a peptide consisting of 1-10 amino acid residues
  • Z1 is a peptide selected from the following group:
  • a derivative polypeptide that is formed by substituting, deleting or adding one, two or three amino acid residues to the amino acid sequence of SEQ ID NO: 1-9 and can bind to type II histocompatibility antigen.
  • the polypeptide of the present invention includes a variant form of SEQ ID NO: 1-9 that has the function of enhancing tumor immunity.
  • variant forms include (but are not limited to): 1-4 (preferably 1-3, more preferably 1-2, most preferably 1) amino acid deletion, insertion and/or substitution, and One or several (usually within 4, preferably within 3, more preferably within 2) amino acids are added or deleted at the C-terminal and/or N-terminal.
  • amino acids with similar or similar properties are substituted
  • the function of the protein is usually not changed.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.
  • the term also includes the polypeptide of the invention in monomeric and multimeric forms.
  • the term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • polypeptide molecule of the present invention is too hydrophobic, it is necessary to add multiple hydrophilic amino acid residues (such as arginine residues or lysine residues) at the N-terminus or C-terminus during synthesis.
  • a typical polypeptide of the present invention is to add 3-6 arginines to the N-terminus or C-terminus of the polypeptide shown in SEQ ID NO: 1-9.
  • the present invention also includes active fragments, derivatives and analogs of UTHE polypeptides.
  • fragment refers to polypeptides that substantially maintain the function or activity of enhancing tumor immunity.
  • polypeptide fragments, derivatives or analogs of the present invention can be (i) a polypeptide with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusing a DTHE polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a protein fused to a leader sequence, secretory sequence, or 6His tag sequence). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of formula I, there are at most 4, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is composed of amino acids with similar or similar properties. Replaced to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
  • the present invention also provides analogs of DTHE polypeptides.
  • the difference between these analogs and the natural DTHE polypeptide may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.
  • the polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include: salts with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".
  • the present invention provides a tumor immune enhancer, which comprises one or more polypeptides having the structure of Formula I.
  • the tumor immune enhancer of the present invention can enhance the specific CD8 + CTL immune response of the tumor neoantigen vaccine, and has the activity of enhancing the anti-tumor immune response of the tumor vaccine.
  • the tumor immune enhancer of the present invention includes the polypeptide shown in SEQ ID NO.: 1-3 or a polypeptide derived therefrom.
  • Neoantigen The protein with specific amino acid sequence variation produced by cancer cells on the basis of gene variation is called "neoantigen" (NeoAg). This is because if there is no change in the amino acid sequence, these proteins should not be antigenic. Once mutated, these proteins will attract the attention of autoimmune cells and cause a series of immune responses.
  • the tumor immune enhancer of the present invention can enhance the immune response of tumor neoantigens, especially antigens that cannot effectively trigger anti-tumor immune responses.
  • the tumor antigen can be natural, artificially synthesized, or a combination thereof.
  • the tumor antigen can be a short peptide, an intact protein, a tumor cell lysate, or a combination thereof.
  • melanoma cells B16F10 neoantigen peptides (NeoAg) are used as vaccines, which cannot produce antigen-specific anti-tumor CTLs when administered alone.
  • the cell lysate involved in the present invention is to suspend the cells in an equal volume of phosphate buffer, freeze-thaw the cells to rupture, and centrifuge at 10,000 g for 10 minutes to remove the precipitate and use the supernatant as the cell lysate.
  • the present invention also relates to polynucleotides encoding DTHE polypeptides.
  • a preferred coding sequence encodes the short peptide shown in SEQ ID NO: 1-9.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the full-length sequence of the polynucleotide of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • the DNA sequence encoding the polypeptide (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or UTHE polypeptide coding sequence of the present invention.
  • the polypeptide of the present invention can be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptide of the present invention can be chemically synthesized or recombinant.
  • the polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.
  • the UThE polypeptide with 6 Arg at the end can be synthesized by chemical synthesis, the HPLC purification yield is greater than 20%, and the purity is greater than 99.9%.
  • a preferred method is to use liquid phase synthesis technology or solid phase synthesis technology, such as Boc solid phase method, Fmoc solid phase method or a combination of the two methods.
  • Solid-phase synthesis can quickly obtain samples, and an appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide.
  • the preferred solid-phase carrier in the Fmoc system is the Wang resin connected with the C-terminal amino acid in the peptide.
  • the Wang resin structure is polystyrene, and the arm between the amino acid is 4-alkoxybenzyl alcohol; 25% hexahydropyridine is used /Dimethylformamide is treated at room temperature for 20 minutes to remove the Fmoc protecting group and extend from the C-terminus to the N-terminus one by one according to the given amino acid sequence.
  • the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protective group is removed. After the resin is filtered off, the crude peptide can be separated by ether precipitation and separation.
  • the desired peptide is purified by gel filtration and reverse phase high pressure liquid chromatography.
  • the preferred resin is a PAM resin connected with the C-terminal amino acid in the peptide.
  • the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl benzene acetamide; synthesized in Boc
  • the protective group Boc is removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane.
  • the peptide chain condensation is completed Afterwards, use hydrogen fluoride (HF) containing p-cresol (5-10%) at 0°C for 1 hour to cut the peptide chain from the resin while removing the protective group.
  • HF hydrogen fluoride
  • acetic acid containing A small amount of mercaptoethanol
  • the solution is lyophilized and further separated and purified with molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide.
  • Various couplings known in the field of peptide chemistry can be used Reagents and coupling methods to couple each amino acid residue, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea hexafluorophosphate can be used (HBTU) for direct coupling.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU 1,1,3,3-tetraurea hexafluorophosphate
  • Another method is to use recombinant technology to produce the polypeptide of the present invention.
  • the polynucleotides of the present invention can be used to express or produce recombinant DTHE polypeptides.
  • the recombinant polypeptide can be expressed in the cell, on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • polypeptide of the present invention is relatively short, it can be considered to connect multiple polypeptides in series, obtain the expression product in the form of multimers after recombinant expression, and then form the required small peptides by methods such as restriction enzyme digestion.
  • the present invention also provides a pharmaceutical composition, which can be therapeutic or prophylactic (such as a vaccine).
  • the pharmaceutical composition of the present invention contains (a) a safe and effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient.
  • an effective dose is about 10 micrograms-100 mg/dose, preferably 100-1000 micrograms/dose of the polypeptide of the present invention administered to an individual.
  • the polypeptide of the present invention can be used alone or together with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the preventive pharmaceutical composition may be a vaccine composition, which comprises the polypeptide of the present invention and a tumor antigen, and is usually combined with a "pharmaceutically acceptable carrier".
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • the term refers to medicament carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration.
  • Such vectors are well known to those of ordinary skill in the art.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the (vaccine) composition of the present invention may also contain additional adjuvants.
  • vaccine adjuvants include (but are not limited to) the following types: inorganic adjuvants, such as aluminum hydroxide, alum, etc.; synthetic adjuvants, such as synthetic double-stranded polynucleotides (double-stranded polyadenosine Acid, uridine acid), levamisole, isoprinosine, etc.; oil agents, such as Freund’s adjuvant, peanut oil emulsification adjuvant, mineral oil, vegetable oil, etc.;
  • Adjuvants also include various new adjuvants and adjuvant components that are considered to be useful as vaccine adjuvants in current and future studies.
  • the vaccine composition or immunogenic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension or a liquid excipient before injection.
  • the preparation can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.
  • composition can be made into a unit or multiple dosage form.
  • Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, and appropriate pharmaceutical excipients.
  • composition of the invention can be administered by conventional routes, including (but not limited to): intravenous, intratumor, intramuscular, intraperitoneal, subcutaneous, intradermal, para-cancerous, or topical administration. medicine.
  • the object to be prevented or treated can be an animal; especially a human.
  • compositions of different dosage forms can be used according to the use situation.
  • These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a customary manner according to the dosage form.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation.
  • the short peptide DTHE or its salt can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the dosage of the short peptide DTHE or its pharmaceutically acceptable salt as the active ingredient can be adjusted according to the weight, age, sex, and degree of symptoms of each patient to be treated.
  • the present invention utilizes composite antigen carrier technology to recognize tumor neo-antigens to the greatest extent;
  • polypeptide of the present invention can improve the efficiency of immune cell activation in vitro.
  • neoAg neoantigenic peptide
  • amino acid sequences of the four neoantigen peptides are as follows:
  • the cells were suspended in an equal volume of phosphate buffer. After freezing and thawing to rupture the cells, the supernatant was used as cell lysate after centrifugation at 10,000 g for 10 minutes to remove the precipitate.
  • As tumor antigens B16F10 cell lysates, LLC cell lysates, and Hepa1-6 cell lysates were prepared respectively.
  • Example 1 UThE enhances the anti-tumor immune response of melanoma weak neoantigen
  • neoantigen peptides from the B16F10 cell line that have been shown to be ineffective in inducing anti-tumor immune responses in mice were selected by preclinical studies.
  • B16F10 is a mouse melanoma cell.
  • neoAg peptides #1, #2, #3, and #4 were mixed at a weight ratio of 1:1:1:1 and formulated into a vaccine together with UThE peptide and adjuvant. After immunizing mice, their anti-tumor effects were tested .
  • the specific method is as follows:
  • C57BL6 mice (6 weeks old, 7 in each experimental group) were immunized three times, one week apart. Each mouse used 200 microliters of vaccine for each immunization, and 50 microliters were injected subcutaneously near the side of the limbs.
  • the dosage of each component of 200 microliters vaccine is neoAg50 micrograms, DThE50 micrograms, Alum300 micrograms, and CpG20 micrograms.
  • the vaccine injection is prepared with PBS.
  • mice On the 7th day after inoculation, the mice began to develop tumors. On the 15th day, the average tumor volume (mean ⁇ SEM) of mice in each group of PBS, NeoAg, NeoAg+UThE was 785 ⁇ 153mm 3 , 890 ⁇ 80mm 3 , and 328 ⁇ 65mm 3 respectively .
  • Example 2 UThE enhances the anti-tumor immune response of melanoma weak neoantigen
  • the method of this example is basically the same as that of example 1, but the difference lies in the preparation of the vaccine and the number of immunizations, using B16F10 cell lysate or neoAg as the tumor antigen. Methods as below:
  • C57BL/6 mice (6 weeks old, 5 in each experimental group) were immunized four times with one week interval each time. Each mouse was injected with the vaccine at four sideways positions close to the extremities, each injection of 50 microliters.
  • the dosage of vaccine components for the first and second vaccines per 200 microliters is 25 micrograms of neoAg or B16F10 cell lysate, 25 micrograms of UThE 25 micrograms adjuvant (adj), and 100 microliters of MF59100 microliters.
  • Adjuvants include 12.5ugCpG, 12.5 ⁇ g PolyI:C, formulated with PBS.
  • the dosage of each component of the vaccine per 200 microliters is 12.5 micrograms of neoAg or B16F10 cell lysate, 12.5 micrograms of UThE, 12.5 micrograms of adjuvant (adj), and 59 microliters of MF.
  • Adjuvants include 6.25 micrograms of CpG and 6.25 micrograms of PolyI:C, formulated with PBS. The application status of each group is as follows:
  • mice were inoculated subcutaneously near the right armpit 105 B16F10 cells. The cells were suspended in 100 microliters of PBS. Then observe and record the tumor growth. After the tumor is visible, the tumor volume is measured every other day.
  • mice in the Lys+UThE+Adj group had no tumors on the 19th day.
  • mice in the Neo+UThE+Adj group also had smaller tumors and slower growth (Figure 2A).
  • the mean tumor volume (mean ⁇ SEM) of the adj group, UTEh+adj group, neo+adj group, neo+UThE+adj group and Lys+UThE+Adj group were 801.1 ⁇ 821.7mm 3 , respectively. 517.4 ⁇ 615.5mm 3 , 431.8 ⁇ 886.7mm 3 , 317.8 ⁇ 314.4mm 3 ( Figure 2B).
  • Example 3 effectively enhances the therapeutic effect on melanoma B16F10
  • Examples 1-3 it has been demonstrated that pre-administration of UThE peptide can be used as an immune enhancer to enhance the anti-tumor effect of tumor antigen peptides.
  • UThE peptide can be used as an immune enhancer to enhance the anti-tumor effect of tumor antigen peptides.
  • the method is as follows: simulate tumor treatment, first inoculate tumor cells, and then treat with UThE immunopotentiator.
  • B16F10 cells 1*10 5 were inoculated into the right forelimb of the mouse near the armpit, and 5 days later, the mice were immunized. Each mouse was vaccinated with 100 microliters, divided into two sideways close to the hindlimb injection site, each point was 50 microliters. The dosage of each component of the vaccine per 200 microliters is: 25 micrograms of UThE, 100 microliters of adjuvant Adj, and MF59. Adjuvants include CpG12.5 micrograms, PolyI:C25 micrograms, formulated with PBS. A total of four immunizations, one week apart each time. After the tumor began to appear, the size of the tumor was measured and the weight of the mouse was weighed.
  • Example 4 enhances the detection of humoral immune response of neoantigen peptide NeoAg by ELISA
  • the immunized serum of the mice was diluted with 0.1% BSA in PBS at a volume ratio of 1:100. Add 100 microliters of neoantigen peptide solution (10 micrograms/ml pH9.5 carbonic acid buffer) to the 96-well plate, and coat overnight at 4°C. Then, the 96-well plate was blocked with 0.1% BSA in PBS for 2 hours at room temperature.
  • Figure 4 shows the results of ELISA detection of UThE antibody in serum samples of immunized mice.
  • Mice were immunized with DThE+CpG+polyI:C+MF59 vaccine four times, with an interval of one week each time, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or on the 30th day after tumor inoculation.
  • the mice were sacrificed and blood was collected. Because IgG1 and IgG2 are present at the same time and their concentration ratio is greater than 1, the T helper cell response is a mixed type I + type II, and the type II response is stronger.
  • Figure 5 shows the results of ELISA detection of UThE antibodies in serum samples of immunized mice.
  • Mice were immunized four times with neoAg+UThE+CpG+polyI:C+MF59 vaccine, with an interval of one week each, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or the 30th after tumor inoculation. Day, the mice were sacrificed and blood was collected. Because IgG1 and IgG2 exist at the same time and their concentration ratio is greater than 1, the T helper cell response is type I + type II mixed, and type II response is stronger.
  • Figure 6 shows the results of ELISA detection of neoAg antibodies in serum samples of immunized mice.
  • Mice were immunized with neoAg+CpG+polyI:C+MF59 vaccine four times, with a one-week interval each time, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or on the 30th day after tumor inoculation.
  • the mice were sacrificed and blood was collected.
  • the antibodies produced are mainly IgG2, therefore, the T helper cell response is type I.
  • Figure 7 shows the results of ELISA detection of neoAg antibodies in serum samples of immunized mice.
  • Mice were immunized four times with neoAg+UThE+CpG+polyI:C+MF59 vaccine, with an interval of one week each, and vaccinated with 7.5*10 4 B16F10 cells 10 days after immunization.
  • the tumor volume of mice reached 1500mm 3 or the 30th after tumor inoculation. Day, the mice were sacrificed and blood was collected. Because IgG1 and IgG2 exist at the same time and their concentration ratio is greater than 1, the T helper cell response is type I + type II mixed, and type II response is stronger.
  • Example 5 UThE polypeptide promotes neo-Ag specific CD8+CTL cell response
  • the method is as follows: After immunized mouse spleen lymphocytes are incubated with UThE or neoAg antigen presenting cells for 5 days, the lymphocytes are purified using T lymphocytes or CD8+T separation kit. In the INF- ⁇ coated with the capture antibody immunoblot was added 100 microliters of a 96 well plate containing 10 4 lymphocytes purified broth. Incubate at 37°C and 5% CO 2 for 16 hours. Then the immunospot kit was used to detect the number of T cells secreting INF- ⁇ . After the T effector cells were co-cultured with tumor target cells at 20:1, LDH was used to detect cell killing.
  • UThE or neoAg antigen presenting cell dendritic cell preparation method Take healthy mice of the same strain after euthanasia, separate the cortex, take the front and rear leg bones, cut off both ends of the leg bones with scissors, and use PBS (containing 1%) FBS) 1ml syringe, aim the needle at the red spot on one end of the leg bone, and flush out the bone marrow cells inside with PBS. Use a pipette to gently blow away the tissue pieces, pass through a 200-mesh sieve and transfer to a 50ml centrifuge tube, and centrifuge at 400g for 10 minutes. Resuspend the cells in PBS, add red blood cell lysate, and centrifuge at 400g for 15 minutes.
  • PBS containing 1%) FBS 1ml syringe
  • Preparation method of mouse spleen lymphocytes Take the mouse spleen of the experimental group, grind and smash the culture medium and wash it through a 200-mesh screen, 400g, 8min, centrifuge to remove the supernatant, remove the red blood cells from the red blood cell lysate, resuspend the cells in PBS, count, and culture Add IL2 (20ng/ml) to the base to continue the culture.
  • the method of this example is basically the same as Example 1 and Example 2.
  • the difference lies in the addition of lung cancer cells LLC, prostate cancer cells RM-1, and pancreatic cancer cells PanC-02 based on the experimental basis of the protomelanoma cell B16F10.
  • the preparation and the number of immunizations are basically the same, and the tumor antigen of each cell lysate is used. Methods as below:
  • C57BL/6 mice (6 weeks old, 4-6 mice in each experimental group) were immunized four times with one week interval each time. Each mouse was injected subcutaneously at 2 sites on the roots of the hind limbs, with 50 microliters injected per point. The doses of the four immunizations were the same. The amount of vaccine components per 50 microliters was 25 micrograms of cell lysate and 25 micrograms of UThE. The adjuvants included 25 microliters of MF59, 25ugCpG, and 12.5 micrograms of PolyI:C, prepared with PBS.
  • mice On the third day after the fourth immunization, the mice were inoculated subcutaneously near the right side of the armpit.
  • LLC cell inoculation volume was 1*10 5
  • RM-1 cell inoculation volume was 1 *10 5 .
  • the cells were suspended in 100 microliters of PBS. Then observe and record the tumor growth. After the tumor is visible, the tumor volume is measured every other day.
  • the results of lung cancer LLC cell inoculation are shown in Figure 11 and Table 4.
  • the order of tumor growth rate and volume size was: Lys+adj group>Lys+Th+Adj group.
  • the tumor size on day 17 is shown in Figure 11.
  • the average tumor weight (mean ⁇ SEM) is 0.4475 ⁇ 0.04211g and 0.235 ⁇ 0.02958g, respectively.
  • melanoma B16F10 cell inoculation The results of melanoma B16F10 cell inoculation are shown in Figure 12 and Table 5. From the injection of tumor cells to the 24th day, the order of tumor growth rate and volume size was: Lys+adj group>Lys+Th+Adj group. Day 17 in tumor size 12, the mean tumor volume (mean ⁇ SEM) were: 419.9 ⁇ 165mm 3, 154.9 ⁇ 111.6mm 3.
  • the results of inoculation of prostate cancer RM-1 cells are shown in Figure 13 and Table 6.
  • the order of tumor growth rate and volume size was: Adj group>Th+Adj group>Lys+Adj group>Lys+Th+Adj group.
  • the tumor size on day 24 is shown in Figure 13.
  • the mean tumor volume (mean ⁇ SEM) is 1476 ⁇ 436.9mm 3 , 1457 ⁇ 377.1mm 3 , 1335 ⁇ 384.6mm 3 , and 881.3 ⁇ 301.3mm 3 respectively .
  • PanC-02 cells of pancreatic cancer are shown in Figure 14 and Table 7.
  • the order of tumor volume size was: Lys+adj group>Th+adj group>Lys+Th+Adj group.
  • the mean tumor volume (mean ⁇ SEM) was 0.4475 ⁇ 0.04211mm 3 and 0.235 ⁇ 0.02958mm 3 respectively .
  • the method of this embodiment is basically the same as that of Embodiment 1, Embodiment 2, and Embodiment 6.
  • the experimental protocol is a B16F10 tumor treatment model, and only low to high doses of UThE combined with the adjuvant Adj are used to treat tumors in mice.
  • Each mouse was injected subcutaneously at 2 sites on the roots of the hind limbs, with 50 microliters injected per point.
  • the doses of the four immunizations are the same, and the dosage of each 50 microliter vaccine component is adjuvant (including 25 microliters MF59, 25ugCpG, 12.5 microgram PolyI:C) and UThE, and the doses of UThE are 30 micrograms, 50ug and 80ug, respectively.
  • the results of tumor emergence in each treated group are shown in Figure 15 and Table 8.
  • the order of tumor growth rate and volume size was: PBS group>Adj group>Adj+Th-30ug group>Adj+Th -50ug group>Adj+Th-80ug group.
  • the mean tumor volume (mean ⁇ SEM) on day 18 was 480.4 ⁇ 118.6mm 3 , 309.9 ⁇ 139.6mm 3 , 253.3 ⁇ 70.59mm 3 , 115.8 ⁇ 45.05mm 3 , 5.753 ⁇ 5.743mm 3, respectively .
  • Example 8 The application method is the same as that of Example 7, but different from that of Example 7 to investigate the dose-effect relationship of UThE.
  • the purpose of Example 8 is to investigate the inhibitory effect of each polypeptide on tumors of different strains of mice at the same dose of 50 ug.
  • the dosage of each peptide of DThE1-7 is 50ug, and the dosage of adjuvant is the same as in Example 7.
  • C57BL/6 mice were inoculated with 10 5 B16F10 cells, Balb/C mice were inoculated with 10 4 4T1 cells, and immunized on the third day after tumor inoculation.
  • the tumor growth of mice in each group is shown in Figure 16.
  • mice have different anti-tumor effects on the polypeptides, which may be related to the MHC II type of the two strains of mice, suggesting that MHC-related typing should be considered in the later administration, and targeted immunization can be carried out.

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Abstract

提供一种肿瘤免疫增强剂、其药物组合物和制备方法，该增强剂包含一种或多种以SEQ ID NO.:1-9所示序列为核心结构的多肽，可以用于制备肿瘤疫苗。

Description

肿瘤免疫增强剂及其制法和应用 技术领域
本发明涉及免疫领域，更具体地涉及肿瘤免疫增强剂及其制法和应用。
技术背景
肿瘤发生发展的根本原因是组织细胞的遗传物质发生了促使细胞异常生长的突变。虽然肿瘤细胞基因突变数目可多达数千个，但是真正启动肿瘤发生的突变基因(驱动基因)可能只涉及两到三个。部分驱动基因已经被用于肿瘤治疗药物设计的靶点。研究发现这类靶向药物的临床疗效取决于肿瘤细胞的其它非驱动突变基因和未靶向的驱动基因的表达情况，因而这类药物临床疗效十分有限。肿瘤疫苗的最新研究进展表明，我们可以利用那些改变氨基酸残基编码的基因突变设计出可以治愈肿瘤的疫苗，这是肿瘤治疗上一个重要的里程碑。
一旦编码突变的基因表达成蛋白，就有可能被抗原呈递细胞分解产生可被T细胞识别的肿瘤特异抗原，这种抗原被称为肿瘤新生抗原。由于识别新抗原的T细胞不受中枢耐受限制，因此新抗原被认为具有强免疫原性，可以激发肿瘤特异杀伤性CD8+T细胞(CTL)应答。临床研究已经证实在肿瘤组织中确实存在这样的新生抗原特异的CTL，并且这类CTL的活性决定了多种肿瘤治疗方法如T细胞生长因子IL-2，免疫检查点抑制剂(PD-1和CTLA-4单克隆抗体)以及体外扩增TILs输注等的疗效。
由于绝大多数肿瘤的新生抗原是病人特有的，因此针对新生抗原的疫苗必须根据每个患者的基因突变来定制。另外，在单个肿瘤中遗传突变具有多克隆特性。为了解决肿瘤的这种异质性，肿瘤疫苗需要采用多靶点设计策略。在公布的肿瘤疫苗临床研究中使用的新生抗原T表位数在20个以上。通过CD4和CD8T细胞应答检测发现只有约1/3的新生抗原可以引发抗肿瘤T细胞应答，其中90％以上的T细胞是CD4 +。由于肿瘤疫苗要达到治疗功效，需要激发肿瘤特异杀伤性CD8 +T细胞应答。这类细胞激活和记忆形成，以及肿瘤浸润需要I型辅助T细胞。因此，可以推测，目前癌症疫苗中使用的大多数新生抗原都不能有效引发I型辅助T细胞应答。
增强I型辅助T细胞反应的方法之一是使用外源的T辅助多肽。例如破伤风类毒素T辅助肽和pan-DR肽(PARDE)已被用于临床试验。结果发现这些多肽不能有效诱导抗肿瘤的CD8+CTL应答。
综上所述，本领域尚缺乏令人满意的抗肿瘤的免疫增强剂。因此，本领域需要开发有效的抗肿瘤免疫应答的免疫增强剂。
发明内容
本发明的目的在于提供有效的抗肿瘤免疫应答的免疫增强剂及其制法和应用。
在本发明的第一方面，提供了一种肿瘤免疫增强剂，所述的肿瘤免疫增强剂包含一种或多种具有式I结构的多肽，或其药学上可接受的盐：
Z0-Z1-Z2   (I)
式中，
Z0为无，或1-10个氨基酸残基构成的肽段；
Z2为无，或1-10个氨基酸残基构成的肽段；
Z1为选自下组的肽段：
(a)如SEQ ID NO:1-9所示的多肽；
(b)将SEQ ID NO:1-9氨基酸序列经过一个、两个或三个氨基酸残基取代、缺失或添加而形成的，且能够与人和鼠II型组织相容性抗原结合的衍生多肽。
在另一优选例中，所述的衍生多肽保留了≥70％的SEQ ID NO:1-9所示多肽的与人和鼠的II型组织相容性抗原结合的活性。
在另一优选例中，所述的衍生多肽与SEQ ID NO:1-9所示多肽的相同性≥80％，较佳地≥90％，更佳地≥95％。
在另一优选例中，所述Z1与组织相容性抗原结合的IC 50为5-50nm。
在另一优选例中，Z0和Z2不同时为无；
在另一优选例中，Z0和Z2中至少含有2-8个，较佳地3-6个亲水性氨基酸残基。
在另一优选例中，Z2为无，且Z0为2-8个，较佳地3-6个亲水性氨基酸残基构成的肽段。
在另一优选例中，Z0为无，且Z2为2-8个，较佳地3-6个亲水性氨基酸残基构成的肽段。
在另一优选例中，所述的亲水性氨基酸选自下组：精氨酸、赖氨酸。
在另一优选例中，Z0和Z2中至少一个含有(Arg)n结构，其中，n为3-6的正整数。
在另一优选例中，所述的式I结构为N端至C端的结构。
在另一优选例中，所述的式I结构为C端至N端的结构。
在另一优选例中，“一”为共价键(如肽键)，或者当Z0为无时，则Z0和Z1之间的“－”不存在，或者当Z2为无时，则Z1与Z2之间的“－”不存在。
在另一优选例中，所述的肿瘤免疫增强剂具有增强肿瘤免疫的活性。
在另一优选例中，所述的肿瘤免疫增强剂能够诱导杀伤性T细胞应答。
在另一优选例中，所述的具有式I结构的多肽包含一个或多个CD4 +Th表位肽。
在另一优选例中，所述的具有式I结构的多肽为CD4+T细胞抗原受体配体。
在另一优选例中，所述的肿瘤免疫增强剂包含Z1为SEQ ID NO:1所示的式I结构的多肽、Z1为SEQ ID NO:2所示的式I结构的多肽和Z1为SEQ ID NO:3所示的式I结构的多肽。
在本发明的第二方面，提供了一种多聚体，所述多聚体由m个单体串联形成，并具有增强肿瘤免疫的功能，其中，m为≥2的正整数，而所述各个单体各自独立地具有式I结构：
Z0-Z1-Z2   (I)
式中，
Z0为无，或1-10个氨基酸残基构成的肽段；
Z2为无，或1-10个氨基酸残基构成的肽段；
Z1为选自下组的肽段：
(a)如SEQ ID NO:1-9所示的多肽；
(b)将SEQ ID NO:1-9氨基酸序列经过一个、两个或三个氨基酸残基取代、缺失或添加而形成的，且能够与组织相容性抗原结合的衍生多肽。
在另一优选例中，m为2、3、4、5、或6。
在另一优选例中，两个单体之间直接通过肽键连接、或者通过肽接头连接。
在另一优选例中，所述的肽接头为柔性肽接头、刚性肽接头、或其组合。
在另一优选例中，所述的肽接头为3-10个氨基酸的肽接头。
在本发明的第三方面，提供了一种分离的核酸分子，编码一多肽，所述多肽具有式I结构，或者所述多肽是由m个单体串联形成的多聚体，其中，各个单体各自独立地具有式I结构，m为≥2的正整数。
在另一优选例中，所述的核酸分子选自下组：DNA、RNA或其组合。
在本发明的第四方面，提供了一种药物组合物，它含有：
(a)本发明第一方面所述的肿瘤免疫增强剂或其药学上可接受的盐、本发明第二方面所述的多聚体、本发明第三方面所述的分离的核酸分子、和/或本发明第四方面所述的药物组合物；和
(b)药学上可接受的载体或赋形剂。
在另一优选例中，所述组合物的剂型为注射剂。
在另一优选例中，所述的组合物为缓释剂型。
另一优选例中，所述的组合物中含有的本发明第一方面所述的肿瘤免疫增强剂或其药学上可接受的盐、或本发明第二方面所述的多聚体可以以选自下组的形式存在：
蛋白质分子(完整分子、亚基、结构域、多肽及重组工程分子)、脂类、多糖类、脂类与多糖复合物。
在另一优选例中，所述的药物组合物还含有：
(c)肿瘤抗原。
在另一优选例中，所述的肿瘤抗原为肿瘤新生抗原肽。
在另一优选例中，所述的肿瘤抗原是原本无法有效引发抗肿瘤免疫反应的抗原。
在另一优选例中，所述的肿瘤抗原是天然的、人工合成的、或其组合。
在另一优选例中，所述的肿瘤抗原选自下组：短肽、完整蛋白、肿瘤细胞裂解物、灭活的肿瘤细胞、或其组合。
在另一优选例中，所述的肿瘤抗原来自中高度频率编码子非同义突变的恶性肿瘤，包括但不限于：
恶性黑色素瘤、乳腺癌、肺癌、胰腺癌、前列腺癌、肠癌、肝癌、食管癌、宫颈癌、膀胱癌、肾细胞癌、多形性胶质瘤。
在另一优选例中，所述的药物组合物还含有：
(d)DC激活剂
在另一优选例中，所述的DC激活剂为纳米制剂，包括但不限于w/o剂，o/w剂。
在另一优选例中，所述的DC激活剂为自组装纳米制剂。
在另一优选例中，所述的DC激活剂选自下组：脂质体、病毒外衣壳、灭活的细菌。
在另一优选例中，所述的药物组合物为肿瘤疫苗组合物。
在另一优选例中，所述的疫苗选自下组：全细胞疫苗、细胞裂解物疫苗、肿瘤组织裂解疫苗、肿瘤细胞外泌体疫苗。
在本发明的第五方面，提供了一种物质的用途，所述物质选自下组：本发明第一方面所述的肿瘤免疫增强剂或其药学上可接受的盐、本发明第二方面所述的多聚体、或本发明第三方面所述的分离的核酸分子，并且所述物质被用于制备提高肿瘤抗原的抗肿瘤活性的药物，或用于制备抗肿瘤的疫苗(瘤苗)组合物。
在另一优选例中，所述的药物或疫苗组合物还用于选自下组的一种或多种用途：
(a)诱导辅助性T细胞应答，较佳地为I型辅助型T细胞应答；
(b)诱导CD8+T细胞应答，较佳地为肿瘤特异杀伤性CD8+T细胞应答；
(c)诱导B细胞应答；
(d)抗肿瘤的细胞治疗；
(e)与免疫检查点抑制剂等其他治疗手段联用。
在本发明的第六方面，提供了一种药盒，所述药盒含有：
(i)第一容器，以及装于该第一容器中的本发明第一方面所述的肿瘤免疫增强剂，或含有所述肿瘤免疫增强剂的药物；
(ii)第二容器，以及装于该第二容器中的治疗肿瘤的药物；以及
(iii)说明书，所述说明书中记载了联合给予所述肿瘤免疫增强剂和所述治疗肿瘤的药物从而治疗癌症的说明。
在另一优选例中，所述的治疗肿瘤的药物包括免疫检查点抑制剂、化疗剂、放疗剂、热疗剂、溶瘤病毒、免疫细胞治疗剂。
在另一优选例中，所述的免疫细胞治疗剂包括CAR-T细胞、CAR-NK细胞、TCR-T细胞、DC细胞或其组合。
在本发明的第七方面，提供了一种治疗癌症的方法，包括步骤：
(i)给需要的对象施用本发明第一方面所述的肿瘤免疫增强剂或其药学上可接受的盐、本发明第二方面所述的多聚体、本发明第三方面所述的分离的核酸分子或本发明第四方面所述的药物组合物；和
(ii)给需要的对象施用治疗肿瘤的药物。
在另一优选例中，所述的步骤(i)和(ii)可以同时或先后进行。
在另一优选例中，所述需要的对象是指肿瘤患者。
在另一优选例中，在步骤(ii)中，按常规的施用剂量和施用频率施用所述治疗肿瘤的药物。
在另一优选例中，所述方法是将免疫检查点抑制剂、化疗、放疗、热疗、溶瘤病毒等现有肿瘤治疗手段与本申请的肿瘤免疫增强剂联用。
在另一优选例中，所述方法是将细胞治疗与本申请的肿瘤免疫增强剂联用，这些细胞治疗包括但不限于DC细胞治疗、CAR-T细胞治疗、CAR-NK细胞治疗等现有的细胞治疗手段。
在本发明的第八方面，提供了一种预防和/或治疗哺乳动物肿瘤的方法，包括步骤：给需要的对象施用本发明第一方面所述的肿瘤免疫增强剂或其药学上可接受的盐、本发明第二方面所述的多聚体、本发明第三方面所述的分离的核酸分子、或本发明第四方面所述的药物组合物。
在另一优选例中，所述的对象是人。
在另一优选例中，所述的肿瘤抗原来自中高度频率编码子非同义突变的恶性肿瘤，如：黑色素瘤、胰腺癌、前列腺癌、乳腺癌、肺癌、肠癌、肝癌、食管癌、宫颈癌、膀胱癌、肾细胞癌、多形性胶质瘤。
在另一优选例中，所述的方法还包括：向需要的对象施用肿瘤抗原。
在另一优选例中，所述的方法还包括：向需要的对象施用治疗肿瘤的药物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了在本发明的一个实施例中，UThE增强小鼠黑色素瘤弱新生抗原的抗肿瘤免疫应答。
图2显示了在本发明的另一个实施例中，UThE增强小鼠黑色素瘤弱新生抗原的抗肿瘤免疫应答(肿瘤预防)。图2A和图2B分别显示了随肿瘤注射后的天数变化的肿瘤体积变化。
图3显示了在本发明的另一个实施例中，UThE有效增强对小鼠黑色素瘤的治疗效果。
图4显示了一个实施例中，疫苗免疫后小鼠血清样品抗UThE抗体检测结果。
图5显示了另一个实施例中，疫苗免疫后小鼠血清样品抗UThE抗体检测结果。
图6显示了另一个实施例中，疫苗免疫后小鼠血清样品抗新生抗原抗体的检测结果。
图7显示了另一个实施例中，疫苗免疫后小鼠血清样品抗新生抗原抗体的检测结果。
图8显示了另一个实施例中，疫苗免疫后小鼠脾脏肿瘤特异CD8+CTL检测结果。
图9显示了另一个实施例中，免疫小鼠脾脏中肿瘤特异CD8+CTL检测结果。
图10显示了另一个实施例中，杀伤性CD8+CTL对肿瘤靶细胞的有效杀伤作用。
图11显示了另一个实施例中，UThE有效增强小鼠肺癌(LLC)弱抗原的免疫原性及对肿瘤抑制作用。
图12显示了另一个实施例中，UThE有效增强小鼠黑色素瘤(B16F10)弱抗原的免疫原性及对肿瘤的抑制作用。
图13显示了另一个实施例中，UThE有效增强小鼠前列腺癌(RM-1)弱抗原的免疫原性及对肿瘤的抑制作用。
图14显示了另一个实施例中，UThE有效增强小鼠胰腺癌(PanC-02)弱抗原的免疫原性及对肿瘤的抑制作用。
图15显示了另一个实施例中，UThE对肿瘤细胞杀伤的剂效关系以及安全性(小鼠体重平稳)。
图16显示了另一个实施例中，UThE候选肽，即UThE1-7在C57BL/6小鼠和Balb/C小鼠中的肿瘤抑制作用。
具体实施方式
本发明人经过广泛而深入地研究，首次发现一类衍生自白喉毒素和破伤风毒素蛋白的通用Th表位肽，其能够增强肿瘤新生抗原疫苗特异性CD8 +CTL免疫应答。实验表明，本发明的这类多肽能够增强肿瘤疫苗抗肿瘤免疫应答功效。在此基础上，完成了本发明。
具体地,本发明提供了UThE1-UThE9，其是由15-21个氨基酸残基组成的多肽分子，与小鼠II型组织相容性抗原分子有中度亲和力，IC50为5-50nm。由于这些多肽分子疏水性过强，合成时需要在N端或C端添加多个亲水性氨基酸残基(如精氨酸残基或赖氨酸残基)。这些多肽分子都带有一个或多个Th表位，可以支持在超过80％的人群中诱导辅助性T细胞应答的功能。
活性多肽
在本发明中，术语“本发明多肽”、“UThE多肽”、“Th表位肽”或“UThE1--UThE9”可互换使用，都指具有式I结构的多肽：
Z0-Z1-Z2   (I)
式中，
Z0为无，或1-10个氨基酸残基构成的肽段；
Z2为无，或1-10个氨基酸残基构成的肽段；
Z1为选自下组的肽段：
(a)如SEQ ID NO:1-9所示的多肽；
(b)将SEQ ID NO:1-9氨基酸序列经过一个、两个或三个氨基酸残基取代、缺失或添加而形成的，且能够与II型组织相容性抗原结合的衍生多肽。
本发明多肽包括具有增强肿瘤免疫的功能的、SEQ ID NO：1-9序列的变异形式。这些变异形式包括(但并不限于)：1-4个(较佳地1-3个，更佳地1-2个，最佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为4个以内，较佳地为3个以内，更佳地为2个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C端和/或N端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
由于本发明多肽分子的疏水性过强，合成时需要在N端或C端添加多个亲水性氨基酸残基(如精氨酸残基或赖氨酸残基)。一种典型的本发明多肽是在SEQ  ID NO：1-9所示多肽的N端或C端添加3-6个精氨酸。
本发明还包括UTHE多肽的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持增强肿瘤免疫的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)DTHE多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I的氨基酸序列相比，有至多4个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供DTHE多肽的类似物。这些类似物与天然DTHE多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这些盐包括(但不限于)与如下酸形成的盐：氢氯酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸、或羟乙磺酸。其他盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前体药物”的形式。
肿瘤免疫增强剂
本发明提供了一种肿瘤免疫增强剂，其包含一种或多种具有式I结构的多肽。
本发明的肿瘤免疫增强剂能够增强肿瘤新生抗原疫苗特异性CD8 +CTL免疫应答，具有增强肿瘤疫苗抗肿瘤免疫应答的活性。
在优选的实施方式中，本发明的肿瘤免疫增强剂包括SEQ ID NO.:1-3所示的多肽或其衍生多肽。
肿瘤新生抗原多肽
癌症细胞在基因变异的基础上产生的带有特异性氨基酸序列变异的蛋白被称为“新生抗原”(neoantigen，NeoAg)。这是因为如果没有氨基酸序列的改变，这些蛋白应该是没有抗原性的。而一旦发生变异，这些蛋白那就会引起自身免疫细胞的注意，并引起一系列的免疫反应。
本发明的肿瘤免疫增强剂能够增强肿瘤新生抗原，特别是原本无法有效引发抗肿瘤免疫反应的抗原的免疫应答。所述的肿瘤抗原可以是天然的、人工合成的、或其组合。所述的肿瘤抗原可以是：短肽、完整蛋白、肿瘤细胞裂解物、或其组合。
在优选的实施方式中，使用四个不同黑色素瘤细胞B16F10新生抗原肽 (NeoAg)，作为疫苗，其在单独施用时不能产生抗原特异抗肿瘤CTL。
本发明涉及的细胞裂解物是将细胞悬浮于等体积磷酸缓冲液中，冻融法使细胞破裂，10，000g离心10分钟去除沉淀后上清作为细胞裂解物。
编码序列
本发明还涉及编码DTHE多肽的多聚核苷酸。一种优选的编码序列编码SEQ ID NO:1-9所示的短肽。
本发明的多聚核苷酸可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。本发明多聚核苷酸的全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明多肽(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的多聚核苷酸的载体，以及用本发明的载体或UTHE多肽编码序列经基因工程产生的宿主细胞。
UThE多肽的制备
本发明多肽可以是重组多肽或合成多肽。本发明的多肽可以是化学合成的，或重组的。相应地，本发明多肽可用常规方法人工合成，也可用重组方法生产。
在优选的实施方式中，可以通过化学合成法合成末端带6个Arg的UThE多肽，HPLC纯化收率大于20％，纯度大于99.9％。
一种优选的方法是使用液相合成技术或固相合成技术，如Boc固相法、Fmoc固相法或是两种方法联合使用。固相合成可快速获得样品，可根据目的肽的序列特征选用适当的树脂载体及合成系统。例如，Fmoc系统中优选的固相载体如连接有肽中C端氨基酸的Wang树脂，Wang树脂结构为聚苯乙烯，与氨基酸间的手臂是4-烷氧基苄醇；用25％六氢吡啶/二甲基甲酰胺室温处理20分钟，以除去Fmoc保护基团，并按照给定的氨基酸序列由C端逐个向N端延伸。合成完成后，用含4％对甲基苯酚的三氟乙酸将合成的胰岛素原相关肽从树脂上切割下来并除去保护基，可过滤除树脂后乙醚沉淀分离得到粗肽。将所得产物的溶液冻干后，用凝胶过滤和反相高压液相层析法纯化所需的肽。当使用Boc系统进行固相合成时，优选树脂为连接有肽中C端氨基酸的PAM树脂，PAM树脂结构为聚苯乙烯，与氨基酸间的手臂是4-羟甲基苯乙酰胺；在Boc合成系统中，在去保护、中和、偶联的循环中，用TFA/二氯甲烷(DCM)除去保护基团Boc并用二异丙基乙胺(DIEA/二氯甲烷中和。肽链缩合完成后，用含对甲苯酚(5-10％)的氟化氢(HF),在0℃下处理1小时，将肽链从树脂上切下，同时除去保护基团。以50-80％乙酸(含少量巯基乙醇)抽提肽，溶液冻干后进一步用分子筛Sephadex G10或Tsk-40f 分离纯化，然后再经高压液相纯化得到所需的肽。可以使用肽化学领域内已知的各种偶联剂和偶联方法偶联各氨基酸残基，例如可使用二环己基碳二亚胺(DCC)，羟基苯骈三氮唑(HOBt)或1,1,3,3-四脲六氟磷酸酯(HBTU)进行直接偶联。对于合成得到的短肽，其纯度与结构可用反相高效液相和质谱分析进行确证。
另一种方法是用重组技术产生本发明多肽。通过常规的重组DNA技术，可利用本发明的多聚核苷酸可用来表达或生产重组的DTHE多肽。一般来说有以下步骤：
(1).用本发明的编码DTHE多肽的多聚核苷酸(或变异体)，或用含有该多聚核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
由于本发明多肽较短，因此可以考虑将多个多肽串联在一起，重组表达后获得多聚体形式的表达产物，然后通过酶切等方法形成所需的小肽。
药物组合物和施用方法
本发明还提供了一种药物组合物，其可以是治疗性的或预防性的(如疫苗)。本发明的药物组合物含有(a)安全有效量的本发明多肽或其药学上可接受的盐；以及(b)药学上可接受的载体或赋形剂。
为了本发明的目的，有效的剂量为给予个体约10微克-100毫克/剂，较佳地为100-1000微克/剂的本发明多肽。此外，本发明的多肽可以单用，也可与其他治疗剂一起使用(如配制在同一药物组合物中)。
在本发明中，预防性药物组合物可以是疫苗组合物，其包含本发明多肽和肿瘤抗原，并且通常与“药学上可接受的载体”组合。
术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡 萄糖、水、甘油、乙醇、佐剂及其组合。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
此外，本发明的(疫苗)组合物还可含有额外的佐剂。代表性的疫苗佐剂包括(但并不限于)以下种类：无机佐剂，如氢氧化铝，明矾等；合成佐剂，如人工合成的双链多聚核苷酸(双链多聚腺苷酸、尿苷酸)、左旋咪唑、异丙肌苷等；油剂，如弗氏佐剂、花生油乳化佐剂、矿物油、植物油等；
佐剂还包括目前及此后研究中被认为可以用于疫苗佐剂的各种新型佐剂及佐剂成分。
通常，可将疫苗组合物或免疫原性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液、液体赋形剂的固体形式。该制剂还可乳化或包封在脂质体中，以增强佐剂效果。
组合物可制成单元或多元剂型。各剂型包含为了产生所期望的治疗效应而计算出预定量的活性物质，以及合适的药剂学赋形剂。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：静脉内、瘤内、肌内、腹膜内、皮下、皮内、癌旁、或局部给药。待预防或治疗的对象可以是动物；尤其是人。
当本发明的组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，短肽DTHE或其盐可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的短肽DTHE或其药学上可接受的盐的剂量，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定
本发明的主要优点包括：
(a)本发明利用复合抗原载体技术，最大程度识别肿瘤新生抗原；
(b)利用本发明多肽制备的疫苗的纯度和质量更加容易控制，安全性高，毒副作用小；
(c)人体免疫响应率高，更高效；
(e)本发明多肽可以提高体外免疫细胞激活效率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
制备实施例1 UThE的合成
在本制备实施例中，采用化学合成方法，合成氨基酸序列如下表所示的UThE多肽，并将其中#1、#2、#3的组合用在以下实施案例中。
命名 氨基酸序列 SEQ ID NO.
UThE1 LSELKTVTGTNPVFAGANYAAWAV 1
UThE2 TGTNPVFAGANYAAWAVNVAQVID 2
UThE3 SIALSSLMVAQAIPLVGELVDIGFAAY 3
UThE4 ITAENTPLPIAGVLLPTIPGKLD 4
UThE5 TTAALSILPGIGSVMGIADGAV 5
UThE6 IVAQSIALSSLMVAQAIPLVGELV 6
UThE7 GELVDIGFAAYNFVESIINLFQVV 7
UThE8 TNSVDDALINSTKIYS 8
UThE9 KAIHLVNNESSEVIVH 9
制备实施例2 B16F10肿瘤新生抗原肽的制备
在本制备实施例中，采用化学合成方法，合成氨基酸序列如下的新生抗原肽(neoAg)：
四种新生抗原肽(neoAg)的氨基酸序列如下：
neoAg命名 氨基酸序列 SEQ ID No.
Pi4k2b WLPQAKVPFSEETQNLILPYISDMNFV 10
ddb1 LVLSFVGQTRVLMINGEEVEETELMGF 11
Pcdhga11 RGQSQLFSLNPRGRSLVTAGRIDREEL 12
Atp11a SSPDEVALVEGVQSLGFTYLRLKDNYM 13
制备实施例3肿瘤细胞裂解物的制备
细胞悬浮于等体积磷酸缓冲液中，冻融使细胞破裂后，10，000g离心10分 钟去除沉淀后上清作为细胞裂解物。作为肿瘤抗原，分别制得B16F10细胞裂解物、LLC细胞裂解物、和Hepa1-6细胞裂解物。
实施例1 UThE增强黑色素瘤弱新生抗原的抗肿瘤免疫应答
选择临床前研究已显示在诱导小鼠抗肿瘤免疫反应方面无效的来自B16F10细胞系的四种新生抗原肽(neoAg)。B16F10为小鼠黑色素瘤细胞。
将四种neoAg肽(#1、#2、#3和#4)按重量比1:1:1:1混合,与UThE肽和佐剂一起配制成疫苗，免疫小鼠后，检测其抗肿瘤效果。具体方法如下：
C57BL6小鼠(6周龄，每实验组7只)免疫三次，间隔一周。每只小鼠每次免疫使用200微升疫苗，分别在靠近四肢侧身位点皮下注射50微升。200微升疫苗各成分用量为neoAg50微克，DThE50微克，Alum300微克，CpG20微克。疫苗注射液用PBS配制完成。实验组为：(1)PBS组(Alum+CpG+PBS)；(2)neoAg组(neoAg+Alum+CpG+PBS)；(3)neoAg+UThE组(neoAg+UThE+Alum+CpG+PBS)。第三次免疫后第3天，小鼠右侧近腋处皮下接种10 5个B16F10细胞。细胞悬浮于100微升PBS中。然后观察记录肿瘤生长情况。
结果如图1和表1所示。接种后第7天，小鼠开始出瘤。第15天时，PBS,NeoAg,NeoAg+UThE各组小鼠出瘤的平均体积(mean±SEM)分别为：785±153mm 3,890±80mm 3,328±65mm 3。
表1
Figure PCTCN2021078264-appb-000001
结果表明，与PBS组相比，在NeoAg组中，施用neoAg和佐剂(Alum+CpG)并不能抑制肿瘤的形成。在NeoAg+UTh组中，肿瘤生长显著地被抑制了。
实施例2 UThE增强黑色素瘤弱新生抗原的抗肿瘤免疫应答
本实施例的方法与实施例1基本相同，不同点在于给疫苗的配制和免疫次数，使用B16F10细胞裂解物或neoAg作为肿瘤抗原。方法如下：
C57BL/6小鼠(6周龄，每实验组5只)免疫四次，每次间隔一周。每只小鼠分四个靠近四肢侧身位点注射疫苗，每点注射50微升。一免与二免每200微升疫苗组分用量为neoAg或B16F10细胞裂解物25微克，UThE25微克佐剂(adj)25微克， MF59100微升。佐剂包括12.5ugCpG，12.5微克PolyI:C，用PBS配制。三免与四免，每200微升疫苗各成分用量为neoAg或B16F10细胞裂解物12.5微克，UThE12.5微克，佐剂(adj)12.5微克，MF59微升。佐剂包括6.25微克CpG，6.25微克PolyI:C，用PBS配制。各组的施用情况如下：
第四次免疫后第三天，小鼠近右侧腋下皮下接种10 5个B16F10细胞。细胞悬浮于100微升PBS中。然后观察记录肿瘤生长情况。瘤可见后，每隔一天测量瘤体积。
结果如图2和表2所示。在注射肿瘤细胞后至第19天期间，肿瘤的生长快慢和体积大小顺序为：adj组＞UThE+adj组＞neo+adj组＞neo+UThE+adj组＞Lys+UThE+Adj组。其中，Lys+UThE+Adj组的小鼠在第19天时还没有出现肿瘤。此外，Neo+UThE+Adj组的小鼠瘤体也较小且生长较慢(图2A)。
在第19天时，adj组，UTEh+adj组，neo+adj组，neo+UThE+adj组和Lys+UThE+Adj组的瘤体积平均数(mean±SEM)分别为：801.1±821.7mm 3,517.4±615.5mm 3,431.8±886.7mm 3,317.8±314.4mm 3(图2B)。
表2
Figure PCTCN2021078264-appb-000002
上述结果表明，当原本无免疫反应的抗原肽(NeoAg或B16F10细胞裂解物)与UThE肽一起施用时，在接种疫苗的小鼠中显著的抑制肿瘤生长，采用肿瘤细胞裂解物+UThE肽时，可完全抑制肿瘤的生长。
实施例3 UThE有效增强对黑色素瘤B16F10的治疗效果
在实施例1-3中，已经证实了预先施用UThE肽可以作为免疫增强剂，增强肿瘤抗原肽的抗肿瘤效果。在本实施例中，进一步验证在荷瘤小鼠体内给予肿瘤免疫增强剂UThE肽，是否可以增强对肿瘤的治疗效果。
方法如下：模拟肿瘤治疗，先接种肿瘤细胞，再用UThE免疫增强剂进行治疗。
将B16F10细胞1*10 5接种于小鼠右侧前肢靠近腋下，5天后，对小鼠进行免 疫。每个小鼠接种疫苗100微升，分两个侧身靠近后肢注射位点，每点50微升。每200微升疫苗各成分用量为：UThE25微克，佐剂Adj，MF59 100微升。佐剂包括CpG12.5微克，PolyI:C25微克，用PBS配制。共免疫四次，每次间隔一周。肿瘤开始出现后，量取瘤体大小并称取小鼠体重。
结果如图3和表3所示。
表3
Figure PCTCN2021078264-appb-000003
结果表明，在荷瘤小鼠中，施用UThE可以有效增强小鼠的细胞免疫，抑制肿瘤细胞的生长。
实施例4 UThE增强新生抗原肽NeoAg的体液免疫反应ELISA法检测
小鼠免疫后的血清，用0.1％BSA的PBS按照1：100体积比进行稀释。96孔板中加入100微升新生抗原肽溶液(10微克/毫升pH9.5碳酸缓冲液)，4℃包被过夜。然后，96孔板用0.1％BSA的PBS室温封闭2小时。吸走封闭液后，加入100微升稀释后血清，室温孵育1小时，之后吸走血清，96孔板用0.05％Tween 20 PBS洗涤三次(每次孵育5分钟)，加入1：5000稀释后HRP-偶联的羊抗鼠IgG，IgG1，IgG2a，IgG2b。室温孵育1小时之后吸走抗体溶液。96孔板用0.05％Tween 20 PBS洗涤三次，重蒸水洗涤一次，按照说明书进行TMB显色。酶标仪上读取450nm的吸光度。
结果如图4-图7所示。
图4显示了免疫小鼠血清样品中UThE抗体的ELISA检测结果。小鼠经过四次免疫DThE+CpG+polyI:C+MF59疫苗，每次间隔一周，免疫后10天接种7.5*10 4个B16F10细胞，小鼠瘤体积达到1500mm 3或肿瘤接种后第30天，小鼠处死取血。由于IgG1和IgG2同时存在并且其浓度比率大于1，T辅助细胞应答是I型+II型混合型，II型应答较强。
图5显示了免疫小鼠血清样品中UThE抗体的ELISA检测结果。小鼠经过四次免疫neoAg+UThE+CpG+polyI:C+MF59疫苗，每次间隔一周，免疫后10天接种7.5*10 4个B16F10细胞，小鼠瘤体积达到1500mm 3或肿瘤接种后第30天，小鼠处死取血。由于IgG1和IgG2同时存在并且其浓度比率大于1，T辅助细胞应答是I型+II型混合型，II型应答较强。
图6显示了免疫小鼠血清样品中neoAg抗体的ELISA检测结果。小鼠经过四次免疫neoAg+CpG+polyI:C+MF59疫苗，每次间隔一周，免疫后10天接种7.5*10 4 个B16F10细胞，小鼠瘤体积达到1500mm 3或肿瘤接种后第30天，小鼠处死取血。产生的抗体主要是IgG2，因此，T辅助细胞应答是I型的。
图7显示了免疫小鼠血清样品中neoAg抗体的ELISA检测结果。小鼠经过四次免疫neoAg+UThE+CpG+polyI:C+MF59疫苗，每次间隔一周，免疫后10天接种7.5*10 4个B16F10细胞，小鼠瘤体积达到1500mm 3或肿瘤接种后第30天，小鼠处死取血。由于IgG1和IgG2同时存在并且其浓度比率大于1，T辅助细胞应答是I型+II型混合型，II型应答较强。
图4至图7的结果表明，UThE肽作为一种肿瘤免疫增强剂，可显著增强肿瘤新生抗原的体液免疫应答。
实施例5 UThE多肽促进neo-Ag特异CD8+CTL细胞应答
在本实施例中，验证UThE多肽是否促进neoAg特异CTL细胞应答。
方法如下：免疫后的小鼠脾脏淋巴细胞与UThE或neoAg抗原递呈细胞共孵育5天后，使用T淋巴细胞或CD8+T分离试剂盒纯化淋巴细胞。在包被了INF-γ捕捉抗体的免疫斑点96孔板中加入100微升含10 4个纯化的淋巴细胞培养液。37℃，5％CO 2培养16小时。然后用免疫斑点试剂盒检测分泌INF-γ的T细胞数量，T效应细胞以20：1和肿瘤靶细胞共同培养后，LDH检测细胞杀伤。
UThE或neoAg抗原递呈细胞树突状细胞制备方法：取同种系健康小鼠进行安乐死后，分离皮层，取前后腿骨，剪刀剪去腿骨的两端，用汲有PBS(含1％的FBS)的1ml注射器，针头对准腿骨的一端红点上，用PBS冲洗出里面的骨髓细胞。吸管轻轻吹散组织块，过200目的筛网转移到50ml的离心管，400g离心10min。PBS重悬细胞，加入红细胞裂解液，400g离心15min。细胞计数，接种24孔板(5*10 5-1*10 6个/ml最佳)；加入IL-4维持培养7天，加入25ng/ml TNFa,4h后加入10μg/ml相应的抗原(UThE和NeoAg)。
小鼠脾脏淋巴细胞制备方法：取实验组小鼠脾脏，研磨粉碎后培养基冲洗过200目筛网，400g，8min，离心除上清，红细胞裂解液去除红细胞，PBS重悬细胞并计数，培养基中加IL2(20ng/ml)继续培养。
ELIspot实验结果如图8和图9所示。CTL效应细胞对肿瘤靶细胞(20：1)的杀伤作用如图10所示。结果表明：UThE肽作为一种肿瘤免疫增强剂，可显著增强肿瘤新生抗原特异性的CD8+CTL细胞应答，因此可有效增强肿瘤疫苗的免疫效果。
实施例6 UThE增强肿瘤裂解物的抗肿瘤免疫应答
本实施例的方法与实施例1、实施例2基本相同，不同点在于在原黑色素瘤细胞B16F10的实验基础上，增加了肺癌细胞LLC、前列腺癌细胞RM-1、胰腺癌细 胞PanC-02，疫苗的配制和免疫次数基本相同，使用各细胞的裂解物肿瘤抗原。方法如下：
C57BL/6小鼠(6周龄，每实验组4-6只)免疫四次，每次间隔一周。每只小鼠后肢根部2个位点皮下注射疫苗，每点注射50微升。四次免疫剂量相同，每50微升疫苗组分用量为细胞裂解物25微克，UThE25微克，佐剂包括25微升MF59，25ugCpG，12.5微克PolyI:C，用PBS配制。
各组的施用情况如下：
第四次免疫后第三天，小鼠近右侧腋下皮下接种，基中：B16F10细胞接种量为7.5*10 4、LLC细胞接种量为1*10 5、RM-1细胞接种量为1*10 5。细胞悬浮于100微升PBS中。然后观察记录肿瘤生长情况。瘤可见后，每隔一天测量瘤体积。
肺癌LLC细胞结果接种如图11和表4所示。在注射肿瘤细胞后至第17天期间，肿瘤的生长快慢和体积大小顺序为：Lys+adj组＞Lys+Th+Adj组。在第17天时肿瘤的大小如图11所示，瘤平均重量(mean±SEM)分别为：0.4475±0.04211g,0.235±0.02958g。
表4
Figure PCTCN2021078264-appb-000004
黑色素瘤B16F10细胞接种结果如图12和表5所示。在注射肿瘤细胞后至第24天期间，肿瘤的生长快慢和体积大小顺序为：Lys+adj组＞Lys+Th+Adj组。在第17天时肿瘤的大小如图12所示，瘤平均体积(mean±SEM)分别为：419.9±165mm 3,154.9±111.6mm 3。
表5
Figure PCTCN2021078264-appb-000005
前列腺癌RM-1细胞接种结果如图13和表6所示。在注射肿瘤细胞后至第24天期间，肿瘤的生长快慢和体积大小顺序为：Adj组＞Th+Adj组＞Lys+Adj组＞Lys+Th+Adj组。在第24天时肿瘤的大小如图13所示，瘤平均体积(mean±SEM)分别为：1476±436.9mm 3,1457±377.1mm 3，1335±384.6mm 3,881.3±301.3mm 3。
表6
Figure PCTCN2021078264-appb-000006
Figure PCTCN2021078264-appb-000007
胰腺癌PanC-02细胞结果如图14和表7所示。在注射肿瘤细胞后第27天，肿瘤体积大小顺序为：Lys+adj组＞Th+adj组＞Lys+Th+Adj组。瘤平均体积(mean±SEM)分别为：0.4475±0.04211mm 3,0.235±0.02958mm 3。
表7
Figure PCTCN2021078264-appb-000008
结果表明免疫增强剂UThE对多种肿瘤具有抑制作用，限于实验室目前的肿瘤类别，我们可以推断，UThE可能对各种肿瘤均具抑制作用，即抑瘤广谱性较高。
实施例7 UThE的剂量和对肿瘤抑制作用的关系
本实施例的方法与实施例1、实施例2、实施例6基本相同。不同点在于该试验方案为B16F10肿瘤治疗模型，并只用由低到高剂量的UThE配合佐剂Adj对小鼠进行肿瘤治疗。每只小鼠后肢根部2个位点皮下注射疫苗，每点注射50微升。四次免疫剂量相同，每50微升疫苗组分用量为佐剂(包括25微升MF59，25ugCpG，12.5微克PolyI:C)和UThE，UThE的剂量分别为30微克，50ug和80ug。
经治疗的各组的出瘤结果如图15和表8所示。在注射肿瘤细胞后至第23天期间，各组小鼠的体重均正常呈稳步增加趋势，肿瘤的生长快慢和体积大小顺序为：PBS组＞Adj组＞Adj+Th-30ug组＞Adj+Th-50ug组＞Adj+Th-80ug组。在第18天时肿瘤平均体积(mean±SEM)分别为：480.4±118.6mm 3,309.9±139.6mm 3，253.3±70.59mm 3,115.8±45.05mm 3，5.753±5.743mm 3。
表8
Figure PCTCN2021078264-appb-000009
Figure PCTCN2021078264-appb-000010
结果表明，UThE配合适当的佐剂即可达到肿瘤抑制效果，这种抑制效果随着UThE施用量的增加而增强，直至UThE的含量增加到80ug，小鼠的体重未受影响，未见任何毒性，80ug时肿瘤抑制率已达98.8％。提示UThE进入体内之后，可以主动获取体内本就存在的肿瘤抗原，产生抗原特异性CTL，实现肿瘤杀伤，达到肿瘤抑制效果。
实施例8 UThE对不同种系小鼠肿瘤的抑制效果
施用方法同于实施例7，不同于实施例7考察UThE的剂效关系。实施例8的目的在于考察各多肽在50ug同剂量下的对不同种系小鼠肿瘤的抑制效果。DThE1-7各肽的剂量皆为50ug，佐剂的使用量同实施例7。C57BL/6小鼠接种10 5B16F10细胞，Balb/C小鼠接种10 4 4T1细胞，接瘤后第三天免疫，各组小鼠的肿瘤生长如图16所示。两种小鼠对各多肽的抑瘤效果不同，可能与两种品系的小鼠的MHC II类型有关，提示后期施用的时候可以考虑先做MHC相关分型，有针对性的进行免疫。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种肿瘤免疫增强剂，其特征在于，所述的肿瘤免疫增强剂包含一种或多种具有式I结构的多肽，或其药学上可接受的盐：
    Z0-Z1-Z2  (I)
    式中，
    Z0为无，或1-10个氨基酸残基构成的肽段；
    Z2为无，或1-10个氨基酸残基构成的肽段；
    Z1为选自下组的肽段：
    (a)如SEQ ID NO:1-9所示的多肽；
    (b)将SEQ ID NO:1-9氨基酸序列经过一个、两个或三个氨基酸残基取代、缺失或添加而形成的，且能够与人和鼠II型组织相容性抗原结合的衍生多肽。
  2. 如权利要求1所述的肿瘤免疫增强剂，其特征在于，Z0和Z2中至少含有2-8个，较佳地3-6个亲水性氨基酸残基。
  3. 如权利要求1所述的肿瘤免疫增强剂，其特征在于，Z0和Z2中至少一个含有(Arg)n结构，其中，n为3-6的正整数。
  4. 如权利要求1所述的肿瘤免疫增强剂，其特征在于，所述的肿瘤免疫增强剂具有增强肿瘤免疫的活性。
  5. 一种多聚体，其特征在于，所述多聚体由m个单体串联形成，并具有增强肿瘤免疫的功能，其中，m为≥2的正整数，而所述各个单体各自独立地具有式I结构：
    Z0-Z1-Z2  (I)
    式中，
    Z0为无，或1-10个氨基酸残基构成的肽段；
    Z2为无，或1-10个氨基酸残基构成的肽段；
    Z1为选自下组的肽段：
    (a)如SEQ ID NO:1-9所示的多肽；
    (b)将SEQ ID NO:1-9氨基酸序列经过一个、两个或三个氨基酸残基取代、缺失或添加而形成的，且能够与组织相容性抗原结合的衍生多肽。
  6. 一种分离的核酸分子，其特征在于，编码一多肽，所述多肽具有式I结构，或者所述多肽是由m个单体串联形成的多聚体，其中，各个单体各自独立地具有式 I结构，m为≥2的正整数。
  7. 一种药物组合物，其特征在于，它含有：
    (a)权利要求1所述的肿瘤免疫增强剂或其药学上可接受的盐、权利要求5所述的多聚体、和/或权利要求6所述的分离的核酸分子；和
    (b)药学上可接受的载体或赋形剂。
  8. 如权利要求7所述的药物组合物，其特征在于，所述的药物组合物还含有：
    (c)肿瘤抗原。
  9. 如权利要求8所述的药物组合物，其特征在于，所述的药物组合物还含有：
    (d)DC激活剂。
  10. 如权利要求7或8所述的药物组合物，其特征在于，所述的药物组合物为肿瘤疫苗组合物。
  11. 一种物质的用途，其特征在于，所述物质选自下组：权利要求1所述的肿瘤免疫增强剂或其药学上可接受的盐、权利要求5所述的多聚体、权利要求6所述的分离的核酸分子，并且所述物质被用于制备提高肿瘤抗原的抗肿瘤活性的药物，或用于制备抗肿瘤的疫苗组合物。
  12. 一种药盒，其特征在于，所述药盒含有：
    (i)第一容器，以及装于该第一容器中的权利要求1所述的肿瘤免疫增强剂，或含有所述肿瘤免疫增强剂的药物；
    (ii)第二容器，以及装于该第二容器中的治疗肿瘤的药物；以及
    (iii)说明书，所述说明书中记载了联合给予所述肿瘤免疫增强剂和所述治疗肿瘤的药物从而治疗癌症的说明。
  13. 一种治疗癌症的方法，其特征在于，包括步骤：
    (i)给需要的对象施用权利要求1所述的肿瘤免疫增强剂或其药学上可接受的盐、权利要求5所述的多聚体、权利要求6所述的分离的核酸分子或权利要求7所述的药物组合物；和
    (ii)给需要的对象施用治疗肿瘤的药物。
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