CN111518216B

CN111518216B - Polypeptide, composition containing polypeptide and application of composition in tumor immunity

Info

Publication number: CN111518216B
Application number: CN202010077682.1A
Authority: CN
Inventors: 莫南德·阿德维希; 陈楚蒙; 金华君; 刘祥箴; 钱其军
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2019-02-02
Filing date: 2020-01-31
Publication date: 2024-02-02
Anticipated expiration: 2040-01-31
Also published as: CN111518216A

Abstract

The present invention provides a polypeptide composition comprising peptide fragments derived from one or more selected from eEF2, HSP105, MCL-1, MUC1, PSF1 and Survivin and an isolated fusion polypeptide comprising covalently linked peptide fragments derived from two or more selected from eEF2, HSP105, MCL-1, MUC1, PSF1 and Survivin. The polypeptide and the polypeptide composition provided by the invention have high immunogenicity, can effectively induce DC maturation, can obviously stimulate proliferation of T cells and secretion of IFN-gamma after being incubated with the DC so as to improve the killing power of the T cells, can not induce the DC to undergo apoptosis, can not induce the expression up-regulation of an immunosuppressive receptor of the DC, and has very excellent immune cell treatment potential and potential anti-tumor effect.

Description

Polypeptide, composition containing polypeptide and application of composition in tumor immunity

Technical Field

The invention relates to the field of medical immunology, in particular to a polypeptide, a composition containing the polypeptide and application of the polypeptide in tumor vaccines.

Background

Dendritic Cells (DCs) are considered a unique class of immune Cells for their ability to initiate and regulate innate and acquired immunity. DCs themselves play a very critical role in the immune monitoring of tumors. Under normal circumstances, DCs are typically maintained in an immature and unactivated state until they are exposed to a preferred immunostimulation, such as inflammatory cytokines, microbial factors, or endogenous sirens (Nie, y.et al. (2016) Alarmins and antitumor immunity.clin.ther.38, 1042-1053). Once activated, DCs will rapidly mature and process antigens and present the processed antigens to T cells via major histocompatibility complex (Major Histocompatibility Complex, MHC) molecules on their surface. Although all DCs are specialized Antigen Presenting Cells (APCs), specific DC subsets have specific Antigen processing mechanisms and are good at activating cd4+ or cd8+ somatic cells, respectively. Classical conventional DCs are largely divided into two subgroups, cd1c+ DCs and cd141+ DCs. Cd1c+ DCs are migratory DCs that are primarily thought to activate cd4+ T cells. Whereas cd141+ DCs are predominantly lymph node resident cells, have greater antigen cross-presentation capacity and are primarily responsible for the activation of cd8+ T cells. The DC further transfers antigen to resident lymph node DC by the "cross-loading" process (Allan, R.S. et al (2006) Migratory Dendritic cells transfer antigen to a lymph node-resident Dendritic cell population for efficient CTL priority 25,153-162;Carbone,F.R.et al (2004) Transfer of antigen between migrating and lymph node-resident DCs in peripheral T-cell tolerance and priority immunol.25, 655-658), or acquires antigen from exosomes (exosomes), infected APCs or apoptotic cells (Pitt, J.M.et al (2016) Denditic cell-derived exosomes for cancer therapy.J.Clin. Invest.126,1224-1232;Thery,C.et al (2002) Indirect activation of naive CD4+ T cells by Dendritic cell-determined exosomes. Nat. Immunol.3, 1156-1162), thereby further expanding the range of immune responses.

Tumor immunotherapy has now become an attractive cancer treatment modality, being considered a fourth cancer treatment modality in addition to surgery, radiation and chemotherapy. Tumor vaccines are considered to be a positive form of tumor immunotherapy capable of activating the immune system, selectively destroying tumor cells by targeting TAA expressed on tumor cells with polypeptides derived from Tumor Associated Antigens (TAA), recombinant TAA proteins, viral vectors encoding TAA or TAA-loaded DCs. Active tumor immunotherapy includes several strategies: gene therapy, modified tumor whole cell immunity, DC-based vaccines and tumor-associated antigen-derived polypeptide vaccines. DC vaccines targeting tumor antigens have been an important branch in immunotherapy, with the aim of raising the patient's own immune response against tumors in itself. In addition, cell-based therapies are particularly desirable because of their low risk of toxicity and the potential for activation of other immune-modulating cells, such as NK cells, in addition to T cells, in anticancer mechanisms. Preclinical studies in the nineties of the twentieth century were the first to introduce the use of autologous Bone marrow derived DCs as a viable vaccine option (portador, a.and gilbo a, e. (1995) Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes.j. Exp. Med.182, 255-260). Tumor vaccines are further classified into prophylactic vaccines and therapeutic vaccines according to their action. The design of the preventive tumor vaccine aims at removing the cancerogenic risk factors from healthy individuals, further preventing the generation and development of cancers and reducing the treatment rate and death rate of the cancers; early diagnosis and treatment of pre-symptomatic cancer carriers, or prevention of recurrence and metastasis in survivors of the occult tumor lesions. Therapeutic vaccines, on the other hand, are designed to clear the etiology of the disease, whose activity is primarily dependent on antigen-specific cd8+ T cell-forming cytotoxic T lymphocytes (Cytotoxic T Lympocytes, CTLs) producing killing of tumor cells. Ideally, a therapeutic tumor vaccine should be able to activate not only the naive T cells, but also to modulate the memory T cells already present, i.e. induce non-protective cd8+ T cells to healthy cd8+ T cells that are able to form effector CTLs. Clinical studies evaluating the efficacy of tumor vaccine treatment over the last two decades have determined that the need for cd8+ T cell-related anti-cancer properties induced by the vaccine include: 1) TCR or T cells have high affinity for polypeptide-MHC complexes; 2) Highly expressed granzyme and perforin (Appay, v., douek, d.c., and Price, d.a. (2008) & cd8+ T cell efficacy in vaccination and disease. Nat. Med.14, 623-628); 3) The expression of cell surface molecules into tumors (such as CXCR 3) (mullens, d.w., sheasley, s.l., r.m., patent, buclok, t.n., 35.n., 35.35.92.35.198, and Engelhard, 2003.35.35.35.2.198, and 35.35.9.9.570 and CD (medium.4.35.35.2) and the expression of molecules (such as standard protocols, 2.35.2.35.2.2.m., 35.35.1.35.35.2.35.35.35.9) in a tumor (see also 35.35.2.35.2.2.1.35.2.35.35.2.1) and (see also 2.35.35.6.35.2.35.2.35.35.2.1) in a. 35.6.35.2.2.35.35.6.2.35.2.7.35.7 (see.35.35.7.2.35.5.35.2.35.6.5.35.5.35.2.2.35.2) in a.6.5.6.2.35.2.3 (see.35.5.35.2.2.35.2.2.3) of 35.2.6.35.2.2.2.2.2.35.2.2.2.3 (see.35.35.35.35.35.2.35.2.3.2.35.35.2.3) of (see.m. patent for (see.m. patent, see.7.7.8.7.7.7.7.7.7.8) of such patent application, so as so., g.j., wherry, e.j., ahmed, r., and sharp, a.h. (2006) Reinvigorating exhausted HIV-specific T cells via PD-1-PD-1ligand blockade.J.Exp.Med.203,2223-2227).

For DC-based tumor vaccines, autologous whole tumor lysis solution is still used in most current clinical trials to load the DCs, specifically by lysing the tumor tissue from the patient's own by multiple cycles of freeze thawing, pulsing the DC cells with the lysis solution. The freeze-thaw cycle induces tumor cell necrosis, but freeze-thaw induced tumor cell necrosis is not immunogenic and even inhibits Toll-like receptor (TLR) -induced DC cell maturation and normal function (Hatgeld P, merrick AE, west E, O' Donnell D, selby P, vile R, et al optimization of dendritic cell loading with tumor cell lysates for cancer immunology J Immunother (2008) 31 (7): 620-32). And the patient's tumor tissue is not always readily available. Tumor cell lysates, purified tumor-associated antigens and tumor-derived mRNA have also been demonstrated to be useful as a source of DC-loaded antigens. Tumor cell lysates can provide multiple antigens for DC loading, and can induce CD4+ and CD8+ T cell responses and confer a different Damage-associated molecular pattern to the DC (Damage-Associated Molecular Patterns, DAMP) to ensure maturation of the DC, but also can provide immune-modulating cytokines to the DC, induce tolerance transformation of the DC cells (Guida M, pisconte S, colucci G.Metastatic melana: the new era of targeted therapy.Expert Opin Ther Targets (2012); 16Suppl 2: S61-70); purified tumor-associated antigen-loaded DCs are capable of activating antigen-specific T cell responses and inducing cd4+ and cd8+ T cell responses, but have a limited number of different antigen species for single use. Tumor derived mRNA can be transferred into tumor associated antigens and costimulatory molecules, ensuring antigen presentation by MHC class I, and does not require cross presentation (Robbins PF, morgan RA, feldman SA, yang JC, shermy RM, dudley ME, wunderlich JR, nahvi AV, helman LJ, mackall CL, et al Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytesreactive with NY-ESO-1.J Clin Oncol (2011); 29:917-24), but does not induce DC cell maturation nor effective CD4+ immune responses, while the number of different antigen species used in a single pass is limited.

Another common approach is to load DCs with short peptides (typically 9-10aa in length). However, it has also been found in recent years that the use of 9-10aa of short peptides for loading DCs in tumor vaccines has also encountered limitations in clinical trials (Rosenberg SA, shermry RM, morton KE, scharfman WJ, yang JC, topalian SL, royal RE, kamma U, restifo NP, hughes MS, schwartzentruber D, berman DM, schwarz SL, ngo LT, mavroukakis SA, white DE, steinberg SM.Tumor progression can occur despite the induction of very high levels of self/thermo anti-specific CD8+ T cells in patients with melanoma.J. immunol. (2005) Nov 1;175 (9): 6169-76.). One possible reason for this phenomenon is the lack of assistance by CD4+ T cells, which are known to be necessary for the production of effector CTLs and long-term CD8+ memory T cells (Janssen EM, droin NM, et al CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-reduced cell de-ath. Nature.2005Mar 3;434 (7029): 88-93.; filipazzi P, pila L, et al Limited induction of tumor cross-reactive T cells without a measurable clinical benefit in early melanoma patients vaccinated with human leukocyte antigen class I-modified peptides. Clin Cancer Res. (2012) Dec 1;18 (23): 6485-96.). In addition, short peptides are prone to degradation, have low immunogenicity, require multiple doses, have limited maturation-inducing effects on DCs, may induce tolerance, and may have transient and/or low levels of immune responses (Berzofsky JA, et al, progress on new vaccine strategies for the immunotherapy and prevention of cancer J Clin invest (2004) Jun;113 (11): 1515-25). There is thus a need for better DC-based tumor vaccines that activate immune responses over a longer period of time and at a higher level of immune response.

Disclosure of Invention

The invention aims to solve the technical problems that the current method for loading DC by using short peptide in a DC-based tumor vaccine encounters limitation in clinic, the generated effect CTL and CD8+ memory T cells are not high in quantity, the short peptide is easy to degrade, the immunogenicity is low, multiple administration is needed, tolerance is possibly induced, and the immune response is instantaneous and/or low in level and poor in-vivo and in-vitro effects are caused, and provides a polypeptide and polypeptide composition and application thereof in tumor immunity. The polypeptide and the polypeptide composition provided by the invention have high immunogenicity, can effectively induce DC maturation, can obviously stimulate proliferation of T cells and secretion of IFN-gamma after being incubated with the DC so as to improve the killing power of the T cells, can not induce the DC to undergo apoptosis, can not induce the expression up-regulation of an immunosuppressive receptor of the DC, and has very excellent immune cell treatment potential and potential anti-tumor effect.

In one aspect, the invention provides a polypeptide composition comprising a peptide fragment derived from one or more selected from eEF2, HSP105, MCL-1, MUC1, PSF1 and Survivin.

In a preferred embodiment, the peptide fragment derived from PSF1 is shown as SEQ ID NO.1, or is a variant sequence of SEQ ID NO.1, which has at least 77% identity, preferably at least 88% identity, with SEQ ID NO. 1.

In a preferred embodiment, the peptide fragment derived from eEF2 is as shown in SEQ ID No.2, or is a variant sequence of SEQ ID No.2, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 2.

In a preferred embodiment, the peptide fragment derived from MCL-1 is shown as SEQ ID No.3, or is a variant sequence of SEQ ID No.3, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 3.

In a preferred embodiment, the peptide fragment derived from HSP105 is shown as SEQ ID No.4, or is a variant sequence of SEQ ID No.4, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 4.

In a preferred embodiment, the MUC-1 derived peptide fragment is shown as SEQ ID No.5, or is a variant sequence of SEQ ID No.5, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 5.

In a preferred embodiment, the Survivin-derived peptide fragment is depicted as SEQ ID No.6 or is a variant sequence of SEQ ID No.6 having at least 77% identity, preferably at least 88% identity with SEQ ID No. 6.

In a preferred embodiment, the polypeptide composition comprises peptide fragments derived from PSF1, eEF2 and MCL-1. Preferably, the peptide stretch derived from PSF1 is shown as SEQ ID NO.1, or is a variant sequence of SEQ ID NO.1, which has at least 77% identity, preferably at least 88% identity, with SEQ ID NO. 1; preferably, the peptide fragment derived from eEF2 is as shown in SEQ ID No.2, or is a variant sequence of SEQ ID No.2, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 2; preferably, the MCL-1 derived peptide fragment is shown as SEQ ID No.3, or is a variant sequence of SEQ ID No.3, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 3.

In a preferred embodiment, the polypeptide composition comprises peptide fragments derived from HSP105, MUC-1 and Survivin. Preferably, the peptide fragment derived from HSP105 is shown as SEQ ID No.4, or is a variant sequence of SEQ ID No.4, which has at least 77% identity, preferably at least 88% identity, to SEQ ID No. 4; preferably, the MUC-1 derived peptide fragment is as shown in SEQ ID No.5, or is a variant sequence of SEQ ID No.5, which has at least 77% identity, preferably at least 88% identity, to SEQ ID No. 5; preferably, the Survivin-derived peptide fragment is shown as SEQ ID No.6 or is a variant sequence of SEQ ID No.6 having at least 77% identity, preferably at least 88% identity with SEQ ID No. 6.

In a preferred embodiment, the polypeptide composition binds to an HLA class I molecule and/or an HLA class II molecule.

In a preferred embodiment, the polypeptide composition binds to a HLA-A02 subtype molecule in an HLA type I molecule.

In a preferred embodiment, the polypeptide composition binds to a HLA-A03 subtype molecule in an HLA type I molecule.

In a preferred embodiment, the polypeptide composition binds to a HLA-A11 subtype molecule in an HLA type I molecule.

In a preferred embodiment, the polypeptide composition binds to a HLA-A24 subtype molecule in an HLA type I molecule.

In another preferred embodiment, the polypeptide composition may further comprise an immunopotentiator. Preferably, the immunopotentiator comprises a compound selected from the group consisting of polyIC: IL, complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum salt, aluminum hydroxide nanoparticles, prostaglandin E2, interferon-alpha and HB _100-108 One or more of the polypeptides.

In another aspect, the invention provides an isolated fusion polypeptide comprising covalently linked peptide fragments derived from two or more selected from eEF2, HSP105, MCL-1, MUC1, PSF1 and Survivin.

In a preferred embodiment, the isolated fusion polypeptide comprises covalently linked peptide fragments derived from 3 selected from eEF2, HSP105, MCL-1, MUC1, PSF1 and Survivin.

In a preferred embodiment, the peptide fragments are covalently linked directly to each other.

In a preferred embodiment, the peptide fragments are covalently linked by a linker; preferably, the linker is selected from (GS ₄ ) ₃ One or more of Furin2A peptide and twin arginine (RR); more preferably, the linker is a double arginine.

In a preferred embodiment, the isolated fusion polypeptide further comprises a covalently linked immunopotentiator polypeptide; preferably, the immunopotentiator polypeptide is selected from the group consisting of a prostaglandin E2-derived polypeptide, an interferon-alpha-derived polypeptide and HB _100-108 One or more of the peptides, more preferably HB _100-108 A peptide; preferably, the immunopotentiator polypeptide is located at the N-terminus, C-terminus or between any two of the peptide stretches of the isolated fusion polypeptide.

In a preferred embodiment, the isolated fusion polypeptide comprises covalently linked peptide fragments derived from PSF1, eEF2 and MCL-1. Preferably, the peptide stretch derived from PSF1 is shown as SEQ ID NO.1, or is a variant sequence of SEQ ID NO.1, which has at least 77% identity, preferably at least 88% identity, with SEQ ID NO. 1; preferably, the peptide fragment derived from eEF2 is as shown in SEQ ID No.2, or is a variant sequence of SEQ ID No.2, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 2; preferably, the MCL-1 derived peptide fragment is shown as SEQ ID No.3, or is a variant sequence of SEQ ID No.3, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 3.

In a preferred embodiment, the isolated fusion polypeptide comprises a polypeptide formed by covalent attachment of the peptide fragments shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.

In a preferred embodiment, the isolated fusion polypeptide comprises, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID NO.1, an RR linker, a peptide fragment shown in SEQ ID NO.2, an RR linker and a peptide fragment shown in SEQ ID NO.3, which are covalently linked, the sequences of which are shown in SEQ ID NO. 8.

In a preferred embodiment, the isolated fusion polypeptide further comprises an immunopotentiator polypeptide; preferably, the immunopotentiator polypeptide is located at the N-terminus of the isolated fusion polypeptide, covalently linked to the peptide stretch shown in SEQ ID No. 8. Preferably, the immunopotentiator polypeptide is covalently linked to the peptide stretch shown in SEQ ID NO.8 via an RR linker. Preferably, the immunopotentiator polypeptide is HB _100-108 The peptide has a sequence shown in SEQ ID NO. 7.

In a preferred embodiment, the sequence of the isolated fusion polypeptide is shown in SEQ ID NO. 9.

In another preferred embodiment, the isolated fusion polypeptide comprises covalently linked peptide fragments derived from HSP105, MUC-1 and Survivin. Preferably, the peptide fragment derived from HSP105 is shown as SEQ ID No.4, or is a variant sequence of SEQ ID No.4, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 4; preferably, the MUC-1 derived peptide fragment is as shown in SEQ ID No.5, or is a variant sequence of SEQ ID No.5, which has at least 77% identity, preferably at least 88% identity, to SEQ ID No. 5; preferably, the Survivin-derived peptide fragment is shown as SEQ ID No.6 or is a variant sequence of SEQ ID No.6 having at least 77% identity, preferably at least 88% identity with SEQ ID No. 6.

In a preferred embodiment, the isolated fusion polypeptide comprises a polypeptide formed by covalent linkage of the peptide fragments shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

In a preferred embodiment, the isolated fusion polypeptide comprises, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID NO.4, an RR linker, a peptide fragment shown in SEQ ID NO.5, an RR linker and a peptide fragment shown in SEQ ID NO.6, which are covalently linked, and the sequence of the isolated fusion polypeptide is shown in SEQ ID NO. 10.

In a preferred embodiment, the isolated fusion polypeptide further comprises an immunopotentiator polypeptide; preferably, the immunopotentiator polypeptide is located at the N-terminus of the isolated fusion polypeptide, covalently linked to the peptide stretch shown in SEQ ID No. 10. Preferably, the immunopotentiator polypeptide is covalently linked to the peptide stretch shown in SEQ ID NO.10 via an RR linker. Preferably, the immunopotentiator polypeptide is HB _100-108 The peptide has a sequence shown in SEQ ID NO. 7.

In a preferred embodiment, the sequence of the isolated fusion polypeptide is shown in SEQ ID NO. 11.

In another preferred embodiment, the isolated fusion polypeptide comprises covalently linked peptide fragments derived from PSF1, MCL-1 and HSP 105. Preferably, the peptide stretch derived from PSF1 is shown as SEQ ID NO.1, or is a variant sequence of SEQ ID NO.1, which has at least 77% identity, preferably at least 88% identity, with SEQ ID NO. 1; preferably, the MCL-1 derived peptide fragment is shown as SEQ ID No.3, or is a variant sequence of SEQ ID No.3, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 3; preferably, the peptide fragment derived from HSP105 is shown as SEQ ID No.4, or is a variant sequence of SEQ ID No.4, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 4.

In a preferred embodiment, the isolated fusion polypeptide comprises a polypeptide formed by covalent attachment of the peptide fragments shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO. 4.

In a preferred embodiment, the isolated fusion polypeptide comprises, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID NO.1, an RR linker, a peptide fragment shown in SEQ ID NO.3, an RR linker and a peptide fragment shown in SEQ ID NO.4, which are covalently linked, and the sequence of the isolated fusion polypeptide is shown in SEQ ID NO. 12.

In a preferred embodiment, the isolated fusion polypeptide further comprises an immunopotentiator polypeptide; preferably, the HB _100-108 The peptide is located at the N-terminus of the isolated fusion polypeptide and is covalently linked to the peptide stretch shown in SEQ ID NO. 12. Preferably, the immunopotentiator polypeptide is covalently linked to the peptide stretch shown in SEQ ID NO.12 via an RR linker. Preferably, the immunopotentiator polypeptide is HB _100-108 The peptide has a sequence shown in SEQ ID NO. 7.

In a preferred embodiment, the sequence of the isolated fusion polypeptide is shown in SEQ ID NO. 13.

In another preferred embodiment, the isolated fusion polypeptide comprises covalently linked peptide fragments derived from eEF2, MUC-1 and Survivin. Preferably, the peptide fragment derived from eEF2 is as shown in SEQ ID No.2, or is a variant sequence of SEQ ID No.2, which has at least 77% identity, preferably at least 88% identity, with SEQ ID No. 2; preferably, the MUC-1 derived peptide fragment is as shown in SEQ ID No.5, or is a variant sequence of SEQ ID No.5, which has at least 77% identity, preferably at least 88% identity, to SEQ ID No. 5; preferably, the Survivin-derived peptide fragment is shown as SEQ ID No.6 or is a variant sequence of SEQ ID No.6 having at least 77% identity, preferably at least 88% identity with SEQ ID No. 6.

In a preferred embodiment, the isolated fusion polypeptide comprises a polypeptide formed by covalent linkage of the peptide fragments shown in SEQ ID NO.2, SEQ ID NO.5 and SEQ ID NO. 6.

In a preferred embodiment, the isolated fusion polypeptide comprises, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID NO.2, an RR linker, a peptide fragment shown in SEQ ID NO.5, an RR linker and a peptide fragment shown in SEQ ID NO.6, which are covalently linked, and the sequence of the isolated fusion polypeptide is shown in SEQ ID NO. 14.

In a preferred embodiment, the isolated fusion polypeptide further comprises an immunopotentiator polypeptide; preferably, the immunopotentiator polypeptide is located at the N-terminus of the isolated fusion polypeptide, covalently linked to the peptide stretch shown in SEQ ID No. 14. Preferably, the immunopotentiator polypeptide is covalently linked to the peptide stretch shown in SEQ ID NO.14 via an RR linker. Preferably, the immunopotentiator polypeptide is HB _100-108 The peptide has a sequence shown in SEQ ID NO. 7.

In a preferred embodiment, the sequence of the isolated fusion polypeptide is shown in SEQ ID NO. 15.

In another aspect, the invention provides an isolated nucleic acid encoding the aforementioned isolated fusion polypeptide.

In a preferred embodiment, the isolated nucleic acid is single-stranded DNA or double-stranded DNA, preferably double-stranded DNA.

In a preferred embodiment, the isolated nucleic acid is unmodified or chemically modified RNA.

In a preferred embodiment, the chemical modification on the chemically modified RNA includes ribose ring modification, phosphodiester backbone modification, and base modification. Preferably, the ribonucleoside modification is a 2 '-oxyalkyl modification and a 2' -F modification. Preferably, the phosphodiester backbone modification is a Locked Nucleic Acid (LNA) modification. Preferably, the base modifications are 4-thiourea pyrimidine modifications, 2-thiourea pyrimidine modifications and C-linked pseudo-uracil modifications.

In a preferred embodiment, the chemically modified RNA comprises one or more of the above chemical modifications.

In another aspect, the invention provides an immunopeptides composition comprising an isolated fusion polypeptide as described above and an immunopotentiator.

In a preferred embodiment, the immunopotentiationThe agent comprises a compound selected from polyIC: IL, complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum salt, aluminum hydroxide nanoparticle, prostaglandin E2, interferon alpha and HB _100-108 One or more of the peptides; preferably HB _100-108 A peptide.

In another aspect, the invention provides an immunomodulator comprising one or more of the foregoing polypeptide compositions, isolated fusion polypeptides, isolated nucleic acids and immune polypeptide compositions.

In another aspect, the invention provides a polypeptide vaccine comprising: a) One or more of the foregoing polypeptide compositions, isolated fusion polypeptides, isolated nucleic acids, and immune polypeptide compositions, and b) a pharmaceutically acceptable carrier.

In a preferred embodiment, the polypeptide vaccine further comprises an adjuvant. Preferably, the adjuvant is selected from the group consisting of, preferably, the adjuvant is selected from aluminum adjuvants (e.g. aluminum hydroxide), freund's adjuvants (e.g. complete Freund's adjuvant and incomplete Freund's adjuvant), prostaglandin E2, interferon-alpha, corynebacterium parvum, lipopolysaccharide, cytokines, oil-in-water emulsions, water-in-oil emulsions, nanoemulsions, microparticle delivery systems, liposomes, microspheres, biodegradable microspheres, plaque virions, proteoliposomes, proteasomes, immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric micro/nanoparticles, polymeric Lamellar Substrate Particles (PLSP), microparticle resins, nanoliposome polymeric gels (nanonolipogel) synthetic/biodegradable and biocompatible semisynthetic or natural polymers or dendrimers (e.g. PLG, PLGA, PLA, polycaprolactone, silicon polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyl trimethyl ammonium chloride, polystyrene divinylbenzene resins, polyphosphazenes, poly- [ di- (carboxyacetyl phenoxy) phosphazene (PCPP), poly- (methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and derivatives, chitosan and derivatives thereof, polysaccharides, delta inulin polysaccharides, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (e.g. polyamino acids, poly- (gamma-glutamic acid), poly-arginine-HCl, poly-L-lysine), polypeptides, biopolymers), cationic Dimethyl Dioctadecyl Ammonium (DDA), alpha-galactosyl ceramide and derivatives thereof, archaebacteria lipids and derivatives, lactams, bellens, glycerides, phospholipids and spirochetes.

In another aspect, the present invention provides a method of preparing an antigen-loaded, activated antigen presenting cell comprising contacting the antigen presenting cell with an immunomodulator and/or polypeptide vaccine as described above.

In a preferred embodiment, the antigen presenting cells are lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or any combination thereof, preferably any one or more selected from monocytes, macrophages and dendritic cells. Preferably, the population of antigen presenting cells is autologous or allogeneic.

In a preferred embodiment, the antigen presenting cells are dendritic cells and the contacting is incubation.

In a preferred embodiment, the incubation time is 2-10 hours, preferably 3-8 hours, more preferably 3-6 hours, such as 6 hours.

In a preferred embodiment, the ratio of the amount of said immunomodulator and/or polypeptide vaccine to said antigen presenting cells is (10-120. Mu.g/10 ⁶ Individual cells), preferably 20-90. Mu.g/10 ⁶ Individual cells), more preferably 20-40. Mu.g/10 ⁶ Individual cells, e.g. 40. Mu.g/10 ⁶ Individual cells.

In a preferred embodiment, the temperature of the incubation is 37 ℃.

In a preferred embodiment, the incubated CO ₂ The concentration was 5%.

In another aspect, the invention provides a tumor vaccine comprising antigen presenting cells prepared by the aforementioned preparation method.

In a preferred embodiment, the tumor vaccine further comprises the aforementioned immunomodulator.

In a preferred embodiment, the tumor vaccine further comprises a pharmaceutically acceptable carrier and/or adjuvant. Preferably, the method comprises the steps of, the adjuvant is selected from aluminium adjuvants (e.g. aluminium hydroxide), freund's adjuvants (e.g. complete Freund's adjuvant and incomplete Freund's adjuvant), corynebacterium parvum, lipopolysaccharide, cytokine, oil-in-water emulsion, water-in-oil emulsion, nanoemulsion, microparticle delivery system, liposome, microsphere, biodegradable microsphere, plaque virion, proteoliposome, proteasome, immunostimulatory complex (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric microparticles/nanoparticles, polymeric Lamellar Substrate Particles (PLSP), microparticle resins, nanoliposome polymeric gels (nanotechnology), synthetic/biodegradable and biocompatible semisynthetic or natural polymers or dendrimers (e.g. PLG, PLGA, PLA) polycaprolactone, silicone polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyl trimethyl ammonium chloride, polystyrene divinylbenzene resins, polyphosphazenes, poly- [ bis- (carboxyacetaminophenoxy) phosphazenes (PCPP), poly- (methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and derivatives, chitosan and derivatives thereof, polysaccharides, delta inulin polysaccharides, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (such as polyamino acids, poly- (gamma-glutamic acid), poly-arginine-HCl, poly-L-lysine, polypeptides, biopolymers), cationic Dimethyl Dioctadecyl Ammonium (DDA), alpha-galactosyl ceramide and derivatives thereof, archaebacteria lipids and derivatives, lactam, bellen, glyceride, phospholipid and one or more of spirochete.

In another aspect, the invention provides a method of activating immune effector cells comprising contacting the immune effector cells with antigen presenting cells treated with one or more of the foregoing polypeptide compositions, isolated fusion polypeptides, isolated nucleic acids, immune polypeptide compositions, immunomodulators, polypeptide vaccines, and tumor vaccines.

In a preferred embodiment, the antigen presenting cell is a Dendritic Cell (DC).

In a preferred embodiment, the immune effector cell is a T cell.

In a preferred embodiment, the treatment is incubation; preferably, the temperature of the incubation is 37 ℃; preferably, the incubated CO ₂ The concentration is 5%; preferably the incubation time is 3-6 hours, more preferably 6 hours.

In a preferred embodiment, the contacting is co-cultivation. Preferably, the temperature of the co-cultivation is 37 ℃; preferably, the CO-cultured CO ₂ The concentration is 5%; preferably, the co-cultivation takes from 12 to 96 hours, more preferably 96 hours; preferably, the ratio of antigen presenting cells to immune effector cells in the co-culture is 1:1-1:5, more preferably 1:5.

In another aspect, the invention provides the use of one or more of the aforementioned polypeptide compositions, isolated fusion polypeptides, isolated nucleic acids, immune polypeptide compositions, immunomodulators, polypeptide vaccines and tumor vaccines for the manufacture of a medicament for the prevention and/or treatment of cancer.

In another aspect, the invention provides a method of preventing and/or treating cancer comprising administering to a cancer patient one or more of the foregoing peptide compositions, isolated fusion polypeptides, immune polypeptide compositions, immune modulators, polypeptide vaccines, and tumor vaccines.

In a preferred embodiment, the cancer is a cancer whose cancer cells abnormally express one or more of eEF2, HSP105, MCL-1, MUC1, PSF1, and Survivin. Preferably, the cancer comprises one or more selected from adenocarcinoma, lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, melanoma, non-small cell lung cancer, renal cell carcinoma/cervical cancer, gastric cancer, cholangiocarcinoma, gall bladder cancer, esophageal cancer, pancreatic cancer, and prostate cancer.

The aforementioned polypeptide compositions, isolated fusion polypeptides, isolated nucleic acids, immune polypeptide compositions and immunomodulators of the invention may be obtained by methods conventional in the art, such as by conventional chemical synthesis or genetic engineering methods.

The polypeptide and the polypeptide composition provided by the invention have the positive effects that the polypeptide and the polypeptide composition have high immunogenicity, can effectively induce DC maturation, can obviously stimulate proliferation of T cells and secretion of IFN-gamma after being incubated with the DC so as to improve the killing power of the T cells, can not induce the DC to undergo apoptosis, can not induce the expression up-regulation of an immunosuppressive receptor of the DC, and has excellent immune cell treatment potential and potential anti-tumor effect.

Drawings

Fig. 1: schematic structural representation of the polypeptide constructs used in the present invention. The upper and lower panels show schematic structures of polypeptide constructs used in the present invention that do not comprise and comprise, respectively, an immunopotentiator polypeptide.

Fig. 2: polypeptide composition 1, MPC1 and HB _100-108 Effect of MPC1 on DC aggregation effect.

Fig. 3: polypeptide composition 2, MPC2 and HB _100-108 Effect of MPC2 on DC aggregation effects.

Fig. 4: absorption levels of DCs after 3 hours incubation for different concentrations of MPC 1.

Fig. 5: DC vs. HB at different concentrations _100-108 Absorption level after 3 hours incubation of MPC 1.

Fig. 6: absorption levels of DCs after 6 hours incubation for different concentrations of MPC 1.

Fig. 7: DC vs. HB at different concentrations _100-108 Absorption level after 6 hours incubation of MPC 1.

Fig. 8A: DC and MPC1, MPC1+HB respectively _100-108 Peptides and HB _100-108 Flow cytometry detection peak profile of absorbance levels after MPC1 incubation.

Fig. 8B: DC and MPC1, MPC1+HB respectively _100-108 Peptides and HB _100-108 Flow cytometry detection of absorbance levels after MPC1 incubation.

Fig. 9A: DC and MPC2, MPC2+HB respectively _100-108 Peptides and HB _100-108 Flow cytometry detection peak profile of absorbance levels after MPC2 incubation.

Fig. 9B: DC and MPC2, MPC2+HB respectively _100-108 Peptides and HB _100-108 Flow cytometry detection of absorbance levels after MPC2 incubation.

Fig. 10A: peak flow cytometry detection of absorbance levels after 3 hours incubation of DCs with MPC 3.

Fig. 10B: DC and different concentrations HB _100-108 Flow cytometry detection peak profile of absorbance levels after 3 hours incubation with 6 hours of MPC 3.

Fig. 11: DC and different concentrations of MPC4 or HB _100-108 Flow cytometry detection peak profile of absorbance levels after 6 hours incubation of MPC4, respectively.

Fig. 12: DC and HB at different concentrations _100-108 -flow cytometry detection results of PI-Annexin apoptosis double staining after MPC1 incubation.

Fig. 13A: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -CCR 7 positive cell flow cytometry detection peak profile after MPC1 incubation.

Fig. 13B: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -CCR 7 positive cell flow cytometry detection quantification results after MPC1 incubation.

Fig. 14A: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -CCR 7 positive cell flow cytometry detection peak profile after MPC2 incubation.

Fig. 14B: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -CCR 7 positive cell flow cytometry detection quantification results after MPC2 incubation.

Fig. 15: DC and HB respectively _100-108 -CCR 7 positive cell flow cytometry detection peak profile after MPC3 incubation.

Fig. 16A: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -peak pattern of CD80 positive cell flow cytometry detection after MPC1 incubation.

Fig. 16B: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -CD 80 positive cell flow cytometry detection quantification results after MPC1 incubation.

Fig. 17A: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -peak pattern of CD80 positive cell flow cytometry detection after MPC2 incubation.

Fig. 17B: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -CD 80 positive cell flow cytometry detection quantification results after MPC2 incubation.

Fig. 18A: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -peak pattern of CD86 positive cell flow cytometry detection after MPC1 incubation.

Fig. 18B: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -CD 86 positive cell flow cytometry detection quantification results after MPC1 incubation.

Fig. 19A: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -peak pattern of CD86 positive cell flow cytometry detection after MPC2 incubation.

Fig. 19B: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -CD 86 positive cell flow cytometry detection quantification results after MPC2 incubation.

Fig. 20A: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -peak pattern of CD40 positive cell flow cytometry detection after MPC1 incubation.

Fig. 20B: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 -quantitative results of CD40 positive cell flow cytometry detection after MPC1 incubation.

Fig. 21A: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -peak pattern of CD40 positive cell flow cytometry detection after MPC2 incubation.

Fig. 21B: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 -figure of quantitative results of CD40 positive cell flow cytometry detection after MPC2 incubation.

Fig. 22A: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 Incubation of MPC1Post HLA-ABC positive cell flow cytometry detection peak pattern.

Fig. 22B: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 HLA-ABC positive cell flow cytometry detection quantification results after MPC1 incubation.

Fig. 23A: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 HLA-DR positive cell flow cytometry detection peak pattern after MPC1 incubation.

Fig. 23B: DC and polypeptide composition 1, MPC1, MPC1+HB, respectively _100-108 Peptides and HB _100-108 HLA-DR positive cell flow cytometry detection quantitative result graph after MPC1 incubation.

Fig. 24A: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 HLA-ABC positive cell flow cytometry detection peak profile after MPC2 incubation.

Fig. 24B: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 HLA-ABC positive cell flow cytometry detection quantification results after MPC2 incubation.

Fig. 25A: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 HLA-DR positive cell flow cytometry detection peak patterns after MPC2 incubation.

Fig. 25B: DC and polypeptide composition 2, MPC2, MPC2+HB, respectively _100-108 Peptides and HB _100-108 HLA-DR positive cell flow cytometry detection quantitative result graph after MPC2 incubation.

Fig. 26: DC in MPC3 and HB, respectively _100-108 HLA-ABC positive cell flow cytometry detection peak profile after MPC3 incubation.

Fig. 27: DC in MPC3 and HB, respectively _100-108 HLA-DR positive cell flow cytometry detection peak pattern after MPC3 incubation.

Fig. 28: DC in MPC4 and HB, respectively _100-108 HLA-ABC positive cell flow cytometry detection peak profile after MPC4 incubation.

Fig. 29: DC in MPC4 and HB, respectively _100-108 HLA-DR positive cell flow cell after incubation of MPC4The detected peak pattern was counted.

Fig. 30A: the DCs are treated by maturation cocktail (IFN-gamma, poly (I: C), R848) and then respectively mixed with MPC1 or HB with different concentrations _100-108 Quantitative determination of IL-6 secretion levels after incubation of MPC 1.

Fig. 30B: the DCs are treated by maturation cocktail (IFN-gamma, poly (I: C), R848) and then respectively mixed with MPC1 or HB with different concentrations _100-108 Quantitative determination of TNF- α secretion levels after MPC1 incubation.

Fig. 31A: the DCs are treated by maturation cocktail (IFN-gamma, poly (I: C), R848) and then respectively mixed with MPC2 or HB with different concentrations _100-108 Quantitative determination of IL-6 secretion levels after MPC2 incubation.

Fig. 31B: the DCs are treated by maturation cocktail (IFN-gamma, poly (I: C), R848) and then respectively mixed with MPC2 or HB with different concentrations _100-108 Quantitative determination of TNF- α secretion levels after MPC2 incubation.

Fig. 32A: by MPC1 and HB, respectively _100-108 CFSE assay of the effect of MPC 1-treated DCs on T cell proliferation.

Fig. 32B: by MPC2 and HB respectively _100-108 CFSE assay of the effect of MPC 2-treated DCs on T cell proliferation.

Fig. 33A: by MPC1 and HB, respectively _100-108 Quantitative determination of IFN-gamma secretion levels in T cells after co-culture of MPC 1-treated DCs with T cells.

Fig. 33B: by MPC2 and HB respectively _100-108 Quantitative determination of IFN-gamma secretion levels in T cells after co-culture of MPC 2-treated DCs with T cells.

Fig. 34A: HB (high-molecular-weight HB) _100-108 PD-L1, PD-L2, HVEM and ILT4 positive cell flow cytometry detection peak patterns of MPC1 treated DCs.

Fig. 34B: HB (high-molecular-weight HB) _100-108 PD-L1, PD-L2, HVEM and ILT4 positive cell flow cytometry detection peak patterns of MPC2 treated DCs.

Fig. 34C: HB (high-molecular-weight HB) _100-108 -PD-L1 positive cell flow cytometry detection peak patterns of DCs treated with MPC4 and MPC4, respectively.

Detailed Description

The following is a description of some of the terms involved in the present invention. Unless otherwise defined below, the terms herein are used in the manner commonly used in the art.

In the present invention, the term "polypeptide" is intended to cover both singular "polypeptides" and plural "polypeptides" and refers to a molecule consisting of monomers (amino acids) that are linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of a product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other term used to refer to one or more chains having two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to products of modification of a polypeptide after expression, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner, including by chemical synthesis.

The term "tumor-associated antigen" or "TAA" refers to an antigen that is specifically expressed by tumor cells or expressed by tumor cells at a higher frequency or density than non-tumor cells of the same tissue type. The tumor-associated antigen may be an antigen that is not normally expressed by the host; they may be displayed abnormally by mutation, truncation, misfolding, or other means of the molecule normally expressed by the host; they may be identical to normally expressed molecules but expressed at abnormally high levels; or they may be expressed in an abnormal situation or environment. The tumor-associated antigen may be, for example, a protein or protein fragment, complex carbohydrate, ganglioside, hapten, nucleic acid, or a combination of these or other biomolecules.

The term "vaccine" refers to an immunogenic composition for administration to a mammal for eliciting an immune response in the mammal against a specific antigen. Vaccines typically contain agents (known as "antigens" or "immunogens") that are similar to or derived from the target of the immune response, such as a disease causing microorganism or tumor cell. Vaccines intended for the treatment of tumors, such as cancers, typically contain an antigen derived from a TAA found on the tumor of interest and capable of eliciting immunogenicity to the TAA on the tumor of interest.

The term "immunopotentiator", as used herein, means a substance that when mixed with an immunogen is capable of eliciting a stronger immune response than when the immunogen alone is present. For example, an immunopotentiator can enhance immunogenicity and provide an excellent immune response. For another example, an immunopotentiator can function by increasing the expression of co-stimulatory factors on macrophages and other antigen presenting cells.

The terms "cancer," "tumor," "cancerous," and "malignant" refer to or describe physiological conditions in mammals that are typically characterized by uncontrolled cell growth. Examples of cancers include, but are not limited to, epithelial cancers, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small-cell lung carcinoma, non-small cell lung carcinoma, gastrointestinal carcinoma, hodgkin's lymphoma and non-hodgkin's lymphoma, pancreatic carcinoma, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (such as liver cancer and hepatoma), bladder cancer, breast cancer (including hormone-mediated breast cancer), colon cancer, colorectal cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland cancer, renal cancer (such as renal cell carcinoma and wilms ' tumor), basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, blood cancers (including but not limited to Acute Myelogenous Leukemia (AML) and Multiple Myeloma (MM)), various types of head and neck cancer (including but not limited to squamous cell carcinoma), and cancers of mucous origin (such as mucinous ovarian cancer), cholangiocarcinoma (liver), and papillary renal carcinoma. In certain embodiments, the blood cancer is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and chronic myelogenous leukemia.

The term "linker" or hinge is a polypeptide fragment that connects between different proteins or polypeptides in order to maintain the connected proteins or polypeptides in their respective spatial conformations in order to maintain the function or activity of the protein or polypeptide. Exemplary linkers include a linker comprising G and/or S, or a linker comprising two R.

The term "adjuvant" refers to a non-specific immunopotentiator that, when delivered with an antigen or pre-delivered into an organism, can enhance the organism's immune response to the antigen or alter the type of immune response. There are many adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in current animal trials. Aluminum hydroxide adjuvants are used more in clinical trials.

The term "antigen presenting cells" (APC) refers to a class of cells capable of expressing Major Histocompatibility Complex (MHC) type I or II, and capable of forming MHC-antigen peptide complexes by MHC binding to antigen peptides and further binding to receptors on the surface of T cells, thereby activating T cells, including but not limited to Dendritic Cells (DCs), monocytes/macrophages, B cells, langerhans cells.

The term "antigen-loaded APC" includes APCs that have been exposed to and activated by an antigen. For example, APCs can be loaded with antigen in vitro (e.g., during culture in the presence of antigen). APCs can also be loaded in vivo by exposure to antigen. "antigen-loaded APCs" are typically prepared in one of two ways: (1) Small fragments called antigenic peptides are "pulsed" directly outside of the APC to bind to MHC molecules; (2) The APC is incubated with a large fragment of the polypeptide, the complete protein or the protein particle, and then the large fragment of the polypeptide, the complete protein or the protein particle is taken up by the APC. These large fragments of polypeptides or protein molecules are digested into small peptide fragments by the APC and eventually transported and presented on the APC surface. In addition, antigen-loaded APCs can also be produced by introducing a polynucleotide encoding an antigen into a cell.

The term "variant" refers to a peptide or polypeptide that has been altered in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant may also refer to a polypeptide having an amino acid sequence that is substantially identical to the sequence of a reference polypeptide. The reference polypeptide has an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., substitution of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree of dotted region, and distribution), are generally recognized in the art as involving minor changes. These minor changes can be identified in part by considering the hydropathic index of amino acids.

High mobility group protein B1 (High mobility group box, hmgb1) is a highly conserved protein in mammals that can be transferred to the nucleus to regulate gene expression and released during cellular injury and inflammation. HMGB1 consists of two DNA binding motifs: a frame and B frame, and C tail. The research shows that HP91 short peptide in the B frame corresponding to amino acids 91-108 of HMGB1 can promote the maturation and activation of DC, induce the secretion of proinflammatory cytokines such as IL-6, IL-12 and the like and trigger the polarization of Th1 cells. Sanez r. and his team demonstrated that HP91 may act as an adjuvant in vivo by enhancing cellular and humoral immune responses. Further studies showed that the immunostimulatory domain of HP91 is located at the C-terminus and at amino acids 100-108 of HMGB 1. In the invention, the short peptide corresponding to the 100 th to 108 th amino acids of HMGB1 is named HB _100-108 。

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The practical flow cytometer of the invention is purchased from Beckman-Coult, model: navios ^TM Flow Cytometer；

The term "room temperature" as used in the examples herein refers to the temperature between the operations in which the test is conducted, typically 25 ℃.

"overnight" as used in the examples herein refers to 8 hours or more.

The enzymes used in the examples of the present invention are derived from PSF1, eEF2, MCL-1, HSP105, MUC-1, survivin and HB _100-108 The polypeptide sequences are shown as SEQ ID NO.1, 2, 3, 4, 5, 6 and 7, and are hereinafter abbreviated as PSF1 peptide, eEF2 peptide, MCL-1 peptide, HSP105 peptide, MUC-1 peptide, survivin peptide and HB, respectively _100-108 A peptide. Wherein HB _100-108 The peptides are used as immunopotentiator polypeptides. The polypeptide composition comprising SEQ ID NOS.1, 2 and 3 is hereinafter referred to as polypeptide composition 1; the polypeptide composition comprising SEQ ID NOS.4, 5 and 6 is hereinafter referred to as polypeptide composition 2.

SEQ ID NO.1(PSF1)：YLYDRLLRI；

SEQ ID NO.2(eEF2)：LILDPIFKV；

SEQ ID NO.3(MCL-1)：AVLPLLELV；

SEQ ID NO.4(HSP105)：RLMNDMTAV；

SEQ ID NO.5(MUC-1)：SLAPPVHNV；

SEQ ID NO.6(Survivin)：LTLGEFLKL；

SEQ ID NO.7(HB100-108)：SAFFLFCSE。

A schematic representation of a polypeptide construct (multiple peptide construct, MPC) represented by the fused polypeptide used in the examples of the present invention is shown in FIG. 1, and both schematic representations respectively represent schematic structures of polypeptide constructs used in the present invention that do not comprise and comprise an immunopotentiator polypeptide, the specific sequences of which are shown in SEQ ID NO.8, 9, 10, 11, 12, 13, 14 and 15, respectively, comprising the following components (from N-terminus to C-terminus): 1-RR-SEQ ID No.2-RR-SEQ ID No.3, 7-RR-SEQ ID No.1-RR-SEQ ID No.2-RR-SEQ ID No.3, 4-RR-SEQ ID No.5-RR-SEQ ID No.6, 7-RR-SEQ ID No.4-RR-SEQ ID No.5-RR-SEQ ID No.6, 1-RR-SEQ ID No.3-RR-SEQ ID No.4, 7-RR-SEQ ID No.1-RR-SEQ ID No.3-RR-SEQ ID No.4, 2-RR-SEQ ID No.5-RR-SEQ ID No.6 and 5-RR-SEQ ID No. 2-SEQ ID No.5 RR-SEQ ID NO.6, the above polypeptide constructs were designated MPC1, HB, respectively, in sequence _100-108 -MPC1、MPC2、HB _100-108 -MPC2、MPC3、HB _100-108 -MPC3, MPC4 and HB _100-108 -MPC4。

The above PSF1 peptide, eEF2 peptide, MCL-1 peptide, HSP105 peptide, MUC-1 peptide, survivin peptide and HB _100-108 Peptides and polypeptide constructs MPC1, HB _100-108 -MPC1、MPC2、HB _100-108 -MPC2、MPC3、HB _100-108 -MPC3, MPC4 and HB _100-108 MPC4 was synthesized by Shanghai Taogu Biotechnology Co. The fluorescent dye labeling of each polypeptide construct is performed by methods conventional in the art after the synthesis of the polypeptide construct is completed.

In the examples of the present invention, DCs were cultured using a serum-free AIM-V medium.

The donor subjects of blood origin used in the examples of the present invention were healthy adults.

EXAMPLE 1 isolation of PBMC cells and Dendritic Cells (DCs)

The isolation of PBMC cells was performed as follows:

1. from the donor subject blood draw 200 ~ 400 u L with white blood cell analyzer determination of white blood cell concentration.

2. Ficoll is preheated in water bath at 20 ℃ for more than 20 minutes.

3. Based on the measured leukocyte concentration, each blood sample was diluted with PBS to a leukocyte concentration of 5 to 9X 10 ⁹ and/L, the dilution factor is calculated according to the situation.

4. Ficoll separation

Calculating the dosage of Ficoll according to the ratio of the volume ratio of the blood sample to the ratio of Ficoll=4:3, slowly adding the diluted blood sample into the Ficoll along the wall of the centrifuge tube at a constant speed, slightly holding the centrifuge tube after the completion, and putting the centrifuge tube into a centrifuge. The centrifugal machine is rotated for 25min at 800g, the rising speed is 1g/s, the falling speed is 0, and the centrifugal machine starts to centrifuge.

5. Harvesting white blood cells

After the centrifugation in the last step is finished, the centrifuge tube is gently taken out, and whether layering is uniform or not is observed, and whether abnormality exists or not is observed. Transferring the centrifuge tube to an ultra-clean workbench, firstly sucking the plasma layer, discarding, lightly sucking the white membrane layer by using a pipette tip, collecting the white membrane layer into a new centrifuge tube, adding physiological saline for washing, and putting the centrifuge tube into a centrifuge with the volume which is 3 times of the volume of the white cell quantity. The rotational speed of the centrifugal machine is adjusted to 400g, the lifting speed is 9g/s, and the centrifugal machine is centrifuged for 10min.

6. Washing white blood cells

After the centrifugation in the last step is finished, observing whether the supernatant is clear or not, if so, dumping and discarding the supernatant, adding physiological saline again, mixing uniformly, and centrifuging again, wherein the rotating speed and the time are the same. And if the supernatant is turbid, sucking the supernatant into a new centrifugal tube, and continuing to centrifuge, wherein the rotation speed and the time are the same as those of cell precipitation.

7. Harvesting white blood cells

Discarding supernatant in centrifuge tube, combining 2 cell pellet, counting, adding AIM-V culture solution, and mixing at a ratio of 6X10 ⁷ The culture flask was then covered with/mL for 4h or overnight.

8. Collection of T cells

After the PBMC is attached, collecting the cells still suspended in the cell suspension into a centrifuge tube, centrifuging at 1200rpm for 5min, discarding the upper culture medium, adding a certain volume of physiological saline for washing, sampling, counting and centrifuging to obtain T cells, and carrying out subsequent experiments or freezing storage according to the requirement;

9. T cell cryopreservation (optional)

Adding the calculated frozen stock solution according to the total number of the T cells harvested in the step 8, and adding the frozen stock solution according to the ratio of 2 multiplied by 10 ⁷ 1 mL/mL.

The separation of DC is carried out according to the following steps:

d0: adding AIM-V culture medium without serum into PBMC, adhering for 2h, and separating suspension cells from adherent cells; suspension cells were counted, 10 ⁶ Cell assay HLA-A typing; the adherent cells were continued to be cultured using AIM-V medium without serum, with GM-CSF added to a final concentration of 50ng/mL and IL-4 added to a final concentration of 1000U/mL;

d3& D5: half-volume liquid exchange of adherent cells is carried out, GM-CSF and IL-4 are supplemented to a final concentration of 50ng/mL and IL-4 to a final concentration of 1000U/mL;

d6: collecting non-adherent cells without EDTA or pancreatin;

the collected DCs were assayed following incubation with different polypeptide constructs or polypeptide compositions in the experimental and control settings in the examples below.

Example 2 detection of DC aggregation Effect

1) The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ⁶ DC suspension/mL;

2) The wells plated with DC were divided into 7 groups, DC control, DC+ polypeptide composition 1, DC+ MPC1, DC+ HB, respectively _100-108 -MPC1, dc+polypeptide composition 2, dc+mpc2 and dc+hb _100-108 MPC2, 3 parallel secondary wells per group, to each experimental group was added the respective corresponding polypeptide composition or polypeptide construct (MPC), wherein the final concentration of each of the polypeptide compositions 1 and 2 was 60. Mu.g/mL (the concentration of each polypeptide in the polypeptide composition was 20. Mu.g/mL), all the polypeptide constructs (MPC 1, HB _100-108 -MPC1、MPC2、HB _100-108 The final concentrations of the MPC 2) were 40. Mu.g/mL, 5% CO at 37 ℃ ₂ Incubating for 6 hours;

3) Cells were washed with PBS, and each group was selected for observation under the microscope field in 3 sub-wells, and the results are shown in fig. 2 and 3.

FIG. 2 shows that polypeptide composition 1 comprising PSF1 peptide, eEF2 peptide and MCL-1 peptide showed only a slight increase in DC cell aggregation after incubation with DC; the DC after incubation with MPC1 (DC+MPC1 group) significantly increased the aggregated cells compared to the DC control; with HB _100-108 post-MPC 1 incubation DC (DC+HB) _100-108 -MPC1 group) the cell aggregation phenomenon is very pronounced compared to the DC control and the cell aggregation is larger compared to the cell aggregation in the dc+mpc1 group. FIG. 3 shows results similar to FIG. 2, FIG. 3 showing that there is only a slight increase in DC cell aggregation after incubation of polypeptide composition 2 comprising HSP105 peptide, MUC1 peptide and Survivin peptide with DC; the DC after incubation with MPC2 (DC+MPC2 group) significantly increased the aggregated cells compared to the DC control; with HB _100-108 post-MPC 2 incubation DC (DC+HB) _100-108 -MPC 2) the cell aggregation phenomenon is very pronounced compared to the DC control and the aggregated cells compared to the cell aggregation in the DC+MPC2 groupThe clusters are larger.

According to the prior art, it has been reported that DC aggregation is related to its maturation (Delemare FG, hoogeveen PG, de han-Meulman M, simons PJ, drexhage HA. Homotypic cluster formation of dendritic cells, a close correlate of their state of formation. Defects in the biobreeding diabetes-process. J Leukoc biol.2001Mar;69 (3): 373-80.), the above results indicate that HB is included or not included as an immunopotentiator with the present invention _100-108 Following incubation of the peptide polypeptide construct, the DC is induced to produce a distinct aggregation effect and comprises HB _100-108 The effect of DC aggregation induced by the polypeptide constructs of the peptides is more pronounced, demonstrating that the polypeptide constructs of the invention are capable of significantly promoting DC maturation.

EXAMPLE 3 fluorescent microscopy of DC absorption levels of polypeptide constructs

2) FITC-labeled different polypeptide constructs (MPC 1 and HB) were added at 10. Mu.g, 20. Mu.g, 40. Mu.g and 80. Mu.g to different wells, respectively _100-108 -MPC1)，37℃5％CO ₂ The level of uptake of the polypeptide construct by DCs was observed under fluorescent microscopy, incubated for 3 hours and 6 hours, respectively.

The results are shown in FIGS. 4-7. FIGS. 4-7 show DC versus different concentrations of MPC1 and HB, respectively _100-108 Absorption level of MPC1 after 3h, 6h incubation. Figures 4 and 6 show the absorption levels of DC after incubation for 3h and 6h, respectively, for different concentrations of MPC 1. Figures 4, 6 show that the level of uptake of MPC1 by DC after 6h incubation was significantly higher than 3h incubation. FIGS. 5 and 7 are DC for HB at different concentrations, respectively _100-108 Absorption level of MPC1 after 3h, 6h incubation. Similar to the uptake of MPC1 by DC, DC vs HB after 6h incubation _100-108 The absorption level of MPC1 is significantly higher than that of incubation for 3h.

By contrast of FIGS. 4-5 and 6-7, it was found that DC vs HB were found in the same concentration of polypeptide constructs and with the same incubation period _100-108 The absorption level of MPC1 was significantly higher than that of MPC1, indicating HB _100-108 The presence of peptides can be advancedThe uptake of the polypeptide construct of the invention by the DC is increased in one step.

Furthermore, the higher the concentration of the polypeptide construct, the higher the level of uptake of DC by it, on the premise of incubation for the same period of time, which trend is reflected in fig. 4-7.

Taken together, FIGS. 4-7 demonstrate that DCs are able to efficiently absorb the polypeptide constructs of the present invention at levels significantly higher than 3 hours at 6 hours, and that an increase in the concentration of the polypeptide constructs aids in the absorption of the DCs. Simultaneous immunopotentiator polypeptide HB _100-108 Is effective to promote the uptake of the polypeptide construct by the DC.

EXAMPLE 4 polypeptide construct MPC1, HB _100-108 -MPC1, MPC2 and HB _100-108 Flow cytometer detection of DC absorption level of-MPC 2

1) The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ^6/ mL of DC suspension;

2) 40. Mu.g MPC1, 40. Mu.g MPC1+30. Mu.g HB were added to the different wells, respectively _100-108 Peptide, 40 μg HB _100-108 -MPC1、40μg MPC2、40μg MPC2+30μg HB _100-108 Peptide and 40 μg HB _100-108 -MPC2，MPC1、HB _100-108 MPC1, MPC2 and HB _100-108 MPC2 is FITC labeled, 5% CO at 37 DEG C ₂ FITC was detected by flow cytometry after 6 hours of incubation ⁺ And (3) cells.

The results are shown in fig. 8A, 8B, 9A and 9B.

FIGS. 8A and 8B show DC vs FITC-labeled MPC1, MPC1+HB _100-108 Peptides and HB _100-108 FITC after incubation of-MPC 1 ⁺ Variation of cell proportion. FIG. 8A shows FITC after incubation of DCs with MPC1 ⁺ The proportion of cells is obviously increased; DC and MPC1 and HB _100-108 FITC after incubation of the peptide mixture ⁺ The proportion of cells was further increased compared to after incubation with MPC 1; and DC and HB _100-108 FITC after incubation of-MPC 1 ⁺ The cell proportion is the highest in lift, significantly higher than the former two. FIG. 8B shows DC and FITC labeled MPC1, MPC1+HB _100-108 Peptides and HB _100-108 Cell fluorescence intensity values after incubation of MPC1, three experimentsThe average value of the results is consistent with the trend shown in fig. 10A.

FIGS. 9A and 9B show DC vs FITC-labeled MPC2, MPC2+HB _100-108 Peptides and HB _100-108 FITC after incubation of-MPC 2 ⁺ The results show a trend similar to that of fig. 8A and 8B with a change in cell ratio. FIG. 9A shows FITC after incubation of DC with MPC2 ⁺ The proportion of cells is obviously increased; DC and MPC2 and HB _100-108 FITC after incubation of the peptide mixture ⁺ The proportion of cells was further increased compared to after incubation with MPC 2; and DC and HB _100-108 FITC after incubation of-MPC 2 ⁺ The cell proportion is the highest in lift, significantly higher than the former two. FIG. 9B shows DC and FITC labeled MPC2, MPC2+HB _100-108 Peptides and HB _100-108 Cell fluorescence intensity values after incubation of MPC2, the average of the results of three experiments were consistent with the trend shown in fig. 9A.

The results of FIGS. 8A, 8B, 9A and 9B show that the polypeptide constructs of the invention are efficiently absorbed by DC and when HB _100-108 Peptides, when present in covalently or non-covalently linked form, can further increase the level of uptake of the polypeptide construct by DCs.

EXAMPLE 5 polypeptide construct MPC3, HB _100-108 -MPC3, MPC4 and HB _100-108 Flow cytometer detection of DC absorption level of-MPC 4

2) 40. Mu.g MPC3, 20. Mu.g HB were added to the different wells, respectively _100-108 -MPC3、40μgHB _100-108 -MPC3、20μg MPC4、20μg HB _100-108 -MPC4、30μg MPC4、30μg HB _100-108 -MPC4, 40. Mu.g MPC4 and 40. Mu.g HB _100-108 -MPC4，MPC3、HB _100-108 -MPC3, MPC4 and HB _100-108 MPC4 is FITC labeled, MPC3 and HB are added _100-108 Wells of-MPC 3 at 37 ℃ 5% co ₂ Incubation was performed for 3 hours and 6 hours, respectively, and MPC4 and HB were added _100-108 Wells of-MPC 4 at 37 ℃ 5% co ₂ Incubation for 6 hours, FITC was detected by flow cytometry after incubation ⁺ And (3) cells.

The results are shown in FIGS. 10A, 10B and 11. FIG. 10A shows FITC after 3 hours incubation of MPC3 with DC ⁺ The proportion of cells reaches 47.0%, and the ratio is obviously improved compared with DC contrast; FITC after 6 hours incubation of MPC3 with DC ⁺ The proportion of cells reaches 82.4 percent, and the cells are further remarkably improved after being incubated for 3 hours; FIG. 10B shows 20 μg HB _100-108 FITC after 3 hours incubation of-MPC 3 with DC ⁺ The proportion of cells reaches 88.7%, and the ratio is greatly improved compared with DC contrast; FITC after 6 hours of incubation ⁺ The proportion of cells reaches 94.4%, and the cells are further improved on the basis of incubation for 3 hours; when HB _100-108 DC and HB when the amount of MPC3 was increased to 40. Mu.g _100-108 FITC after 3 and 6 hours incubation of-MPC 3 ⁺ Ratio of cells compared to FITC after the same incubation period at 20. Mu.g ⁺ The proportion of cells was further increased to 93.4% and 98.1%, respectively. And under the condition of the same dose and the same incubation time, HB _100-108 FITC in MPC3 well ⁺ The proportion of cells was significantly higher than that of MPC3 wells.

FIG. 11 shows different doses of MPC4 and HB _100-108 FITC after 6 hours incubation of-MPC 4 with DC ⁺ Proportion of cells. The left column of FIG. 11 shows FITC after incubation of 20. Mu.g, 30. Mu.g and 40. Mu.g of MPC4 with DC for 6 hours ⁺ The proportion of the cells reaches 28.9%, 51.2% and 76.3%, respectively, and the cells are remarkably improved compared with the control DC; the right column of FIG. 11 shows HB at 20 μg, 30 μg and 40 μg _100-108 FITC after 6 hours incubation of-MPC 4 with DC ⁺ The proportion of cells reached 98.9%, 99.6% and 99.7%, respectively, was further greatly improved compared to cells incubated with MPC4, which was nearly 100%, and when HB was used _100-108 FITC at a dose of 20 μg when MPC4 was incubated with DC ⁺ The proportion of cells is already very high, approaching 100%.

The results of FIGS. 10A, 10B and 11 show that the same as the MPC1, MPC2 and HB described above _100-108 MPC1 and HB _100-108 MPC2 and DC incubation results were similar, MPC3, MPC4, HB _100-108 -MPC3 and HB _100-108 MPC4 is highly absorbed by DC and the absorption level is dose dependent within a certain rangeSex, and HB _100-108 Is capable of further increasing the level of uptake of the polypeptide construct by the DC.

Example 6 detection of DC apoptosis Induction by polypeptide constructs

Whether the polypeptide constructs of the invention induce apoptosis in DC was detected by PI and Annexin V-FITC double staining.

The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ^6/ mL of DC suspension; 0 μg, 10 μg, 20 μg and 40 μg HB were added to the different wells, respectively _100-108 -MPC1，37℃5％CO ₂ Incubation for 6 hours, washing 2 times with AIM-V, incubation overnight, and double staining for PI and Annexin-FITC apoptosis were performed as described in Annexin V-FITC apoptosis detection kit (Kaiyi Biocat# KGA105-KGA 108) and the results were detected by flow cytometry.

The results are shown in FIG. 12. The results showed that for DC with polypeptide constructs of 0 μg, 10 μg, 20 μg and 40 μg, the cell ratios of all 4 quadrants were essentially identical, representing the cell ratios in the lower left quadrant (Q4) of living cells, 0 μg, 10 μg, 20 μg and 40 μg HB were added _100-108 Four wells of-MPC 1 were all around 98%; whereas the cell ratios representing late apoptosis and early apoptosis in the upper right quadrant (Q2) and lower right quadrant (Q3) were added with 0 μg, 10 μg, 20 μg and 40 μg HB _100-108 Four wells of MPC1 were all around 1%.

The above results indicate that the addition of the polypeptide construct of the invention is in the same state as DC without addition, i.e.the addition of the polypeptide construct of the invention does not induce DC apoptosis.

EXAMPLE 7 Effect of polypeptide constructs and polypeptide compositions on expression levels of the chemokine receptor CCR7 of DC

The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ^6/ mL of DC suspension; 60. Mu.g of polypeptide composition 1 (PSF 1 peptide, eEF2 peptide and MCL-1 peptide each in a dose of 20. Mu.g), 60. Mu.g of polypeptide composition 2 (HSP 105 peptide, MUC-1 peptide and Survivin peptide each in a dose of 20. Mu.g), 40. Mu.g of MPC1, 40. Mu.g of MPC2, 40. Mu.g of MPC1+30. Mu.g were added to the wells, respectivelyg HB _100-108 Peptide, 40. Mu.g MPC2+30. Mu.g HB _100-108 Peptide, 40 μg HB _100-108 MPC1, 40. Mu.g HB _100-108 MPC2 and 40. Mu.g HB _100-108 -MPC3，37℃5％CO ₂ After 6 hours incubation, washing with PBS, then incubation overnight, digestion of DCs, washing with PBS containing 5% FBS (from Gibco), incubation with FITC-labeled CCR7 antibody (from BD) and isotype control antibody (murine IgG) in the dark for 30 minutes, detection of FITC again with flow cytometer after washing with PBS containing 5% FBS ⁺ The results of the cells are shown in FIGS. 13-15.

FIG. 13A shows FITC after incubation of DC with polypeptide composition 1 ⁺ The ratio of cells did not change significantly from the DC control; FITC after incubation of DCs with MPC1 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC1+HB _100-108 Peptides or HB _100-108 FITC after incubation of-MPC 1 ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC1 group ratio MPC1+HB _100-108 Group FITC ⁺ The proportion of cells is also significantly higher. Fig. 13B is an average of quantification after 3 replicates of the experiment in fig. 13A, 3 replicates of each group of samples per experiment, consistent with the results of fig. 13A. FIG. 13 shows that MPC1 is capable of significantly increasing the CCR7 expression level of DC and when HB _100-108 The presence of the peptide (covalently linked or non-covalently linked) can even further increase the expression level of CCR 7.

FIG. 14A is a result similar to FIG. 13A, showing FITC after incubation of DC with polypeptide composition 2 ⁺ The ratio of cells was not significantly changed; with MPC2+HB _100-108 Peptides or HB _100-108 FITC after incubation of-MPC 2 ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC2 group ratio MPC2+HB _100-108 Group FITC ⁺ The proportion of cells is also significantly higher. Fig. 14B is an average of quantification after three replicates of the experiment in fig. 14A, consistent with the results of fig. 14A. FIG. 14 shows that MPC2 is capable of significantly increasing the CCR7 expression level of DC, and when HB _100-108 The presence of the peptide (covalently linked or non-covalently linked) can even further increase the expression level of CCR 7.

FIG. 15 shows DC and HB _100-108 FITC after incubation of-MPC 3 ⁺ Proportion of cells. DC and HB _100-108 FITC after incubation of-MPC 3 ⁺ The proportion of cells was significantly increased compared to the DC control, indicating HB _100-108 MPC3 was able to significantly increase CCR7 expression levels of DCs.

Figures 13-15 illustrate that the polypeptide constructs of the invention are capable of significantly increasing CCR7 expression levels in DCs, and that CCR7 expression levels are significantly higher in the presence of immunopotentiator polypeptides. When receiving an infection or danger signal, DCs are generally thought to leave peripheral tissues during which the DCs begin to mature and the expression level of CCR7 increases (Chan, V.W., S.Kothakota, M.C.Rohan, L.Panganiban-Lustan, J.P.Gardner, M.S.Wachowicz, J.A.Winter, L.T.Williams.1999.Secondary lyshoid-tissue chemical (SLC) is chemotactic for mature dendritic cells 93:3610-3616; dieu, M.C., B.Vanbervliet, A.Vicari, J.M.Bridon, E.Oldham, S.Ait-Yahia, F.Briere, A.Zlotnik, S.Lebecque, C.Caux.1998.Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites.J.exp. Med.188:373-386; sallucon, F., P.Schaerli, P.Loetscher, C.Schaniel, D.Lenig, C.R.Mackay, S.Qin, A.Lanzavecchia.1998.Rapid and coordinated switch in chemokine receptor expression during dendritic cell adaptation. Eur. J.Immunol.28:2760-2769.). Thus an increase in CCR7 expression levels suggests maturation of DCs. And the process of homing (home) of DCs into the T cell region of lymph nodes and antigen presentation relies on CCR7 at its ligands CCL19 and CCL21, thus elevation of CCR7 expression levels of DCs shows a significant enhancement in the potential for DC homing.

Example 8 Effect of polypeptide constructs and polypeptide compositions on expression levels of DC maturation-associated costimulatory molecules

The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ^6/ mL of DC suspension; 60. Mu.g of polypeptide composition 1 (PSF 1 peptide, eEF2 peptide and MCL-1 peptide each in a dose of 20. Mu.g), 60. Mu.g of polypeptide composition 2 (HSP 105 peptide, MUC-1 peptide and Survivin peptide each in a dose of 20. Mu.g), 40. Mu.g of MPC1, 40. Mu.g of MPC2, 40. Mu.g of MPC1+30. Mu.g of HB were added to the different wells, respectively _100-108 Peptide, 40. Mu.g MPC2+30. Mu.g HB _100-108 Peptide, 40 μg HB _100-108 MPC1 and 40. Mu.g HB _100-108 -MPC2，37℃5％CO ₂ After 6 hours incubation, washing with PBS, then incubation overnight, digestion of DCs, washing with PBS containing 5% FBS (purchased from Gibco), incubation of each group of cells with FITC-labeled CD80 antibody, APC-labeled CD86 antibody and Alexa Fluor 700 (AF 700) -labeled CD40 antibody (all purchased from BD) for 30 minutes, respectively, in the absence of light, incubation of isotype control antibody (murine IgG) as control, detection of FITC by flow cytometry after washing again with PBS containing 5% FBS after incubation ⁺ The results of the cells are shown in FIGS. 16-21.

FIG. 16A shows FITC after incubation of DC with polypeptide composition 1 ⁺ The ratio of cells did not change significantly from the DC control; FITC after incubation of DCs with MPC1 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC1+HB _100-108 Peptides or HB _100-108 FITC after incubation of-MPC 1 ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC1 group ratio MPC1+HB _100-108 Group FITC ⁺ The proportion of cells is also significantly higher. Fig. 16B is an average of quantification after three replicates of the experiment in fig. 16A, consistent with the results of fig. 16A. FIG. 16 shows that MPC1 is capable of significantly increasing CD80 expression levels of DC, and when HB _100-108 The presence of the peptide (covalently linked or non-covalently linked) can even further increase the expression level of CD 80.

FIG. 17A shows FITC after incubation of DC with polypeptide composition 2 ⁺ The ratio of cells did not change significantly from the DC control; FITC after incubation of DC with MPC2 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC2+HB _100-108 Peptides or HB _100-108 FITC after incubation of-MPC 2 ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC2 group ratio MPC2+HB _100-108 Group FITC ⁺ The proportion of cells is also significantly higher. Fig. 17B is an average of quantification after three replicates of the experiment in fig. 17A, consistent with the results of fig. 17A. FIG. 17 shows that MPC2 is capable of significantly increasing CD80 expression levels of DC, and when HB _100-108 The presence of the peptide (covalently linked or non-covalently linked) can even further increase the expression level of CD 80.

FIG. 18A shows APC after incubation of DC with polypeptide composition 1 ⁺ The ratio of cells did not change significantly from the DC control; post-incubation APC of DC with MPC1 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC1+HB _100-108 Peptides or HB _100-108 After incubation of-MPC 1, APC ⁺ The proportion of cells is obviously improved. Fig. 18B is an average of quantification after three replicates of the experiment in fig. 18A, consistent with the results of fig. 18A. FIG. 18 shows that MPC1 is at HB _100-108 Peptides can significantly increase CD86 expression levels in DCs either in the presence (covalent or non-covalent linkage) or in the absence.

FIG. 19A shows APC after incubation of DC with polypeptide composition 2 ⁺ The ratio of cells did not change significantly from the DC control; post-incubation APC of DC and MPC2 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC2+HB _100-108 Peptides or HB _100-108 After incubation of-MPC 2, APC ⁺ The proportion of cells is obviously improved. Fig. 19B is an average of quantification after three replicates of the experiment in fig. 19A, consistent with the results of fig. 19A. FIG. 19 shows MPC2 at HB _100-108 Peptides can significantly increase CD86 expression levels in DCs either in the presence (covalent or non-covalent linkage) or in the absence.

FIG. 20A shows AF700 after incubation of DC with polypeptide composition 1 ⁺ The ratio of cells did not change significantly from the DC control; AF700 after incubation of DC with MPC1 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC1+HB _100-108 Peptides or HB _100-108 AF700 after incubation of-MPC 1 ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC1 group ratio MPC1+HB _100-108 Group AF700 ⁺ The proportion of cells is also significantly higher. Fig. 20B is an average of quantification after three replicates of the experiment in fig. 20A, consistent with the results of fig. 20A. FIG. 20 shows that MPC1 is capable of significantly increasing CD40 expression levels of DC, and when HB _100-108 The presence of the peptide (covalently linked or non-covalently linked) can even further increase the expression level of CD 40.

FIG. 21A shows AF700 after incubation of DC with polypeptide composition 2 ⁺ The ratio of cells did not change significantly from the DC control; AF700 after incubation of DC with MPC2 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC2+HB _100-108 Peptides or HB _100-108 AF700 after incubation of-MPC 2 ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC2 group ratio MPC2+HB _100-108 Group AF700 ⁺ The proportion of cells is also significantly higher. Fig. 21B is an average of quantification after the experiment in fig. 21A was repeated three times, consistent with the results of fig. 21A. FIG. 21 shows that MPC2 is capable of significantly increasing CD40 expression levels of DC, and when HB _100-108 The presence of the peptide (covalently linked or non-covalently linked) can even further increase the expression level of CD 40.

The results in FIGS. 16-21 demonstrate that the expression levels of CD80, CD86 and CD40 of DC were significantly increased after incubation of DC with the polypeptide constructs of the invention. CD80, CD86 and CD40 are costimulatory molecules associated with DC maturation and activation, and increased expression of CD80, CD86 and CD40 indicates that the polypeptide constructs of the invention are capable of significantly enhancing DC maturation.

Example 9 Effect of polypeptide constructs and polypeptide compositions on the expression level of type I and type II HLA of DCs

The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ^6/ mL of DC suspension; 60. Mu.g of polypeptide composition 1 (PSF 1 peptide, eEF2 peptide and MCL-1 peptide each in a dose of 20. Mu.g), 60. Mu.g of polypeptide composition 2 (HSP 105 peptide, MUC-1 peptide and Survivin peptide each in a dose of 20. Mu.g), 40. Mu.g of MPC1, 40. Mu.g of MPC2, 40. Mu.g of MPC1+30. Mu.g of HB were added to the different wells, respectively _100-108 Peptide, 40. Mu.g MPC2+30. Mu.g HB _100-108 Peptide, 40 μg HB _100-108 MPC1, 40. Mu.g HB _100-108 MPC2, 40. Mu.g of MPC3, 40. Mu.g of HB _100-108 MPC3, 40. Mu.g of MPC4 and 40. Mu.g of HB _100-108 -MPC4，37℃5％CO ₂ After 6 hours incubation, washing with PBS, then incubation overnight, digestion of DCs, washing with PBS containing 5% v/v FBS (purchased from Gibco), incubation of each group of cells with PE-labeled HLA-ABC antibody or PE-labeled HLA-DR antibody, respectively, for 30 minutes in the absence of light, incubation of isotype control antibody (murine IgG) as control, detection of PE by flow cytometry after incubation again with PBS containing 5% FBS ⁺ And (3) cells. The results are shown in FIGS. 22-29.

FIG. 22A shows the expression levels of HLA-ABC for different sets of DC surfaces. Following incubation of DC with polypeptide composition 1, PE ⁺ The ratio of cells did not change significantly from the DC control; PE after incubation of DC with MPC1 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC1+HB _100-108 Peptides or HB _100-108 After incubation of-MPC 1 PE ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC1 group ratio MPC1+HB _100-108 Group PE ⁺ The proportion of cells is also higher. Fig. 22B is an average of quantification after three replicates of the experiment in fig. 22A, consistent with the results of fig. 22A. FIGS. 22A and 22B show that MPC1 is capable of significantly increasing the HLA-ABC expression level of DC, and when HB _100-108 The expression level of HLA-ABC can be increased even further in the presence of the peptide (covalently linked or non-covalently linked).

FIG. 23A shows the expression levels of HLA-DR on DC surfaces of different sets. Following incubation of DC with polypeptide composition 1, PE ⁺ The ratio of cells did not change significantly from the DC control; PE after incubation of DC with MPC1 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC1+HB _100-108 Peptides or HB _100-108 After incubation of-MPC 1 PE ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC1 group ratio MPC1+HB _100-108 Group PE ⁺ The proportion of cells is also significantly increased. Fig. 23B is an average of quantification after three replicates of the experiment in fig. 23A, consistent with the results of fig. 23A. FIGS. 23A and 23B show that MPC1 is capable of significantly increasing HLA-DR expression levels of DC, and when HB _100-108 The peptides, in the presence of the peptides (covalently or non-covalently linked), are able to increase the level of HLA-DR expression even further.

FIG. 24A shows the expression levels of HLA-ABC for different sets of DC surfaces. Following incubation of DC with polypeptide composition 2, PE ⁺ The ratio of cells did not change significantly from the DC control; PE after incubation of DC with MPC2 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC2+HB _100-108 Peptides or HB _100-108 After incubation of-MPC 2 PE ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC2 group ratio MPC2+HB _100-108 Group PE ⁺ Proportion of cellsAnd also higher. Fig. 24B is an average of quantification after three replicates of the experiment in fig. 24A, consistent with the results of fig. 24A. FIGS. 24A and 24B show that MPC2 is capable of significantly increasing the HLA-ABC expression level of DC, and when HB _100-108 The expression level of HLA-ABC can be increased even further in the presence of the peptide (covalently linked or non-covalently linked).

FIG. 25A shows the expression levels of HLA-DR on DC surfaces of different sets. Following incubation of DC with polypeptide composition 2, PE ⁺ The ratio of cells did not change significantly from the DC control; PE after incubation of DC with MPC2 ⁺ The proportion of cells is improved to a certain extent; while when DC and MPC2+HB _100-108 Peptides or HB _100-108 After incubation of-MPC 2 PE ⁺ The proportion of cells is obviously improved, and HB _100-108 MPC2 group ratio MPC2+HB _100-108 Group PE ⁺ The proportion of cells is also significantly increased. Fig. 25B is an average of quantification after three replicates of the experiment in fig. 25A, consistent with the results of fig. 25A. FIGS. 25A and 25B show that MPC2 is capable of significantly increasing HLA-DR expression levels of DC, and when HB _100-108 The peptides, in the presence of the peptides (covalently or non-covalently linked), are able to increase the level of HLA-DR expression even further.

FIG. 26 shows the expression levels of HLA-ABC for different sets of DC surfaces. DC with MPC3 or HB _100-108 After incubation of-MPC 3 PE ⁺ The proportion of cells is obviously improved compared with the DC control, which indicates that the MPC3 can obviously improve the expression level of HLA-ABC of DC.

FIG. 27 shows the expression levels of HLA-DR on DC surfaces of different sets. DC with MPC3 or HB _100-108 After incubation of-MPC 3 PE ⁺ The ratio of cells is obviously improved compared with the DC control, and is compared with HB _100-108 PE after incubation of MPC3 ⁺ The ratio of cells was significantly higher than after incubation with MPC3, indicating that MPC3 was able to significantly increase the HLA-DR expression level of DC and when incubated with HB _100-108 The expression level of HLA-DR was increased more in the case of peptide ligation.

FIG. 28 shows the expression levels of HLA-ABC for different sets of DC surfaces. DC with MPC4 or HB _100-108 After incubation of-MPC 4 PE ⁺ The proportion of cells is obviously improved compared with the DC control, From 42.6% to 69.5% and 77.7%, respectively, indicating that MPC4 is capable of significantly increasing the expression level of HLA-ABC of DC and when compared to HB _100-108 The expression level of HLA-ABC was increased more in the case of peptide ligation.

FIG. 29 shows the expression levels of HLA-DR on different sets of DC surfaces. DC with MPC4 or HB _100-108 After incubation of-MPC 4 PE ⁺ The ratio of cells is obviously improved compared with the DC control, and is respectively improved from 12.2% to 32.6% and 69.2%, which shows that the MPC4 can obviously improve the expression level of HLA-ABC of DC and when compared with HB _100-108 The expression level of HLA-ABC was increased more in the case of peptide ligation.

FIGS. 22-29 show that incubation of DCs with the polypeptide constructs of the present invention with DCs significantly increases the expression levels of HLA-type I and type II molecules on DC cells. The elevated levels of HLA-type I and type II molecule expression are correlated with maturation of DCs, and the results of figures 22-29 demonstrate that the polypeptide constructs of the present invention are capable of significantly promoting maturation of DCs.

Example 10 Effect of polypeptide constructs on DC cytokine secretion

The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ^6/ mL of DC suspension, 20. Mu.g of MPC1, 20. Mu.g of MPC2, 40. Mu.g of MPC1, 40. Mu.g of MPC2, 20. Mu.g of HB were added to the different wells, respectively _100-108 MPC1, 20 μg HB _100-108 MPC2, 40. Mu.g HB _100-108 MPC1 and 40. Mu.g HB _100-108 -MPC2，37℃5％CO ₂ After 6 hours incubation, washing with PBS, IFN-. Gamma.and poly (I: C) and R848 were added to each well to give final concentrations of 100IU/mL, 30. Mu.g/mL and 5. Mu.g/mL, respectively, while DC control wells stimulated by incubation with IFN-. Gamma.and poly (I: C) and R848 but not with the polypeptide construct were kept, and after further incubation for 24 hours, the secretion levels of cytokines IL-6 and TNF-. Alpha.in the culture supernatant were detected by flow cytometry using a CBA kit (purchased from BD), and the above procedure was repeated 3 times for each sample, 3 multiplex wells at a time, and the results are shown in FIGS. 30-31.

FIG. 30A shows that IL-6 secretion levels after incubation with DC with 20. Mu.g of MPC1 did not change significantly compared to the DC control; after addition of 40. Mu.g of MPC1 and incubation with DC, IThe secretion level of L-6 is obviously improved compared with that of a DC control group and 20 mu g MPC1 group, and the concentration reaches more than 1000pg/mL; when 20. Mu.g HB was added _100-108 After incubation of MPC1 with DC, the secretion level of IL-6 was further significantly increased, exceeding 1500pg/mL; when 40. Mu.g HB was added _100-108 IL-6 secretion levels were slightly lower than 20. Mu.g HB after incubation of MPC1 with DC _100-108 The MPC1 group is still significantly higher than the 20. Mu.g and 40. Mu.g MPC1 groups.

FIG. 30B shows that after incubation with DC with 20. Mu.g of MPC1, the secretion level of TNF-. Alpha.was not significantly changed compared to the DC control group; after 40 mug of MPC1 is added for incubation with DC, the secretion level of TNF-alpha is obviously improved compared with that of a DC control group and 20 mug of MPC1, and the concentration reaches 400pg/mL; when 20. Mu.g HB was added _100-108 After incubation of MPC1 with DC, the secretion level of TNF-alpha was further significantly increased, exceeding 500pg/mL; when 40. Mu.g HB was added _100-108 After incubation of MPC1 with DC, the secretion level of TNF-alpha was below 20. Mu.g HB _100-108 MPC1 group, still higher than 20. Mu.g and 40. Mu.g MPC1 group, exceeding 400pg/mL.

FIG. 31A shows that IL-6 secretion levels after incubation with DC with 20. Mu.g of MPC2 were not significantly altered from that of the DC control group; after 40 mug of MPC2 is added for incubation with DC, the secretion level of IL-6 is obviously improved compared with that of a DC control group and 20 mug of MPC2 group, and the concentration reaches 900pg/mL; when 20. Mu.g HB was added _100-108 After incubation of MPC2 with DC, the secretion level of IL-6 was further significantly increased to 1400pg/mL; when 40. Mu.g HB was added _100-108 IL-6 secretion levels were below 20. Mu.g HB after incubation of MPC2 with DC _100-108 The MPC2 group, still significantly higher than the 20. Mu.g and 40. Mu.g MPC2 group, reached about 1200pg/mL.

FIG. 31B shows that TNF- α secretion levels were slightly elevated after addition of 20 μg of MPC2 incubated with DC compared to the DC control group; after 40 μg of MPC2 is added for incubation with DC, the secretion level of TNF-alpha is obviously improved compared with that of a DC control group and 20 μg of MPC2 group, and the concentration is close to 500pg/mL; when 20. Mu.g HB was added _100-108 After incubation of MPC2 with DC, the secretion level of TNF-alpha was further significantly increased, exceeding 600pg/mL; when 40. Mu.g HB was added _100-108 TNF-after incubation of MPC2 with DCSecretion level of alpha with 20. Mu.g HB _100-108 The MPC2 group was comparable, still above 20. Mu.g and 40. Mu.g MPC2 group, about 600pg/mL.

Taken together, the results of FIGS. 30-31 demonstrate that the polypeptide constructs of the present invention are capable of significantly increasing secretion levels of cytokines IL-6 and TNF- α following incubation with DCs. IFN-gamma, poly (I: C) and R848 are capable of stimulating DC maturation and secretion of pro-inflammatory factors including IL-6 and TNF-alpha. The addition of the polypeptide construct of the present invention can significantly increase secretion levels of IL-6 and TNF-alpha, indicating that the polypeptide construct of the present invention can effectively promote DC maturation.

EXAMPLE 11 Effect of polypeptide construct treated DCs on proliferation of T cells and IFN-y secretion

The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ^6/ mL of DC suspension, 40. Mu.g of MPC1, 40. Mu.g of MPC2, 40. Mu.g of HB were added to the different wells, respectively _100-108 MPC1 and 40. Mu.g HB _100-108 -MPC2，37℃5％CO ₂ After 6 hours incubation, wash with PBS. IFN-. Gamma., poly (I: C) and R848 were added to each well to give final concentrations of 100IU/mL, 30. Mu.g/mL and 5. Mu.g/mL, respectively, and after further culturing for 24 hours, each group of DC cells was collected and counted in 1X 10 ^6/ mL cell density 24 well plates were re-plated with 1mL per well.

Proliferation of T cells was detected by CFSE. CFSE (purchased from sceleck china) was dissolved in DMSO to 50 μm and stored as a mother liquor, diluted to 5 μm with PBS prior to use. Incubating PBMC cells with diluted CFSE at 37deg.C for 10-15 min, adding AIM-V culture medium containing 10% FBS to 10 times of initial volume, stopping reaction, centrifuging to remove supernatant, adding obtained T cells into each of the above wells containing DC at a ratio of DC to T cells of 1:5, providing a group of T cells not co-cultured with DC as non-stimulated control, co-culturing for 96 hr, collecting T cells and supernatant, and detecting CFSE by flow cytometry ⁺ T cell numbers and the concentration of IFN- γ in the supernatant was measured using CBA kit (purchased from BD). The concentration of IFN-gamma was measured by 3 replicates, with 3 replicates per group of samples in parallel. The results are shown in FIGS. 32-33.

Figure 32A shows a schematic representation of the process,no significant proliferation was found in the unstimulated T cell group; after co-cultivation with DC not treated with the polypeptide construct, T cells proliferated to a low level, with a proportion of proliferated T cells of 10.2%; after the cell is co-cultured with the DC treated by the MPC1, the proliferation level of T cells is obviously improved, and the proportion of the proliferated T cells reaches 41.3 percent; and pass through HB _100-108 After the co-culture of the DCs treated with MPC1, the proliferation level of T cells is further remarkably improved, and the proportion of the proliferated T cells is 53.1 percent.

FIG. 32B shows that no significant proliferation was found in the unstimulated T cell group; following co-culture with DC not treated with the polypeptide construct, T cells proliferated to a low level, with a proportion of proliferated T cells of 5.59%; after the cell is co-cultured with the DC treated by the MPC2, the proliferation level of the T cells is obviously improved, and the proportion of the proliferated T cells reaches 33.3 percent; and pass through HB _100-108 After the co-culture of the DC treated with the MPC2, the proliferation level of the T cells is further remarkably improved, and the proportion of the proliferated T cells is 44.2 percent.

FIG. 33A shows that IFN- γ concentration in supernatant after co-culture of non-polypeptide construct treated DC and T cells (T cell+DC) is raised to about 1000pg/mL as compared to IFN- γ concentration in supernatant of non-stimulated T cell group; after the DC is co-cultured by MPC1 treatment (T cell+DC+MPC1 group), the concentration of IFN-gamma in the T cell supernatant is obviously improved by more than 2000pg/mL compared with the concentration of IFN-gamma in the T cell+DC group supernatant; and pass through HB _100-108 After the MPC 1-treated DC co-culture (T cell +DC +HB) _100-108 -MPC1 group), the concentration of IFN- γ in T cell supernatants was very significantly elevated compared to the concentration of IFN- γ in the supernatants of the other groups, reaching approximately 5000pg/mL.

FIG. 33B shows that IFN- γ concentration in supernatants after co-culture of non-polypeptide construct-treated DCs with T cells (T cell + DC) was elevated to approximately 1000pg/mL as compared to IFN- γ concentration in supernatants from non-stimulated T cell groups; after the DC is co-cultured by MPC2 treatment (T cell+DC+MPC2 group), the concentration of IFN-gamma in the T cell supernatant is obviously improved by more than 1500pg/mL compared with the concentration of IFN-gamma in the T cell+DC group supernatant; and pass through HB _100-108 -MPC2 treatmentAfter DC co-cultivation (T cell +DC +HB) _100-108 -MPC2 group), the concentration of IFN- γ in T cell supernatants was very significantly elevated compared to the concentration of IFN- γ in the supernatants of the other groups, reaching approximately 3000pg/mL.

The results in FIGS. 32-33 demonstrate that the polypeptide constructs of the invention significantly enhance stimulation and proliferation of T cells by DCs, while DCs treated with the polypeptide constructs of the invention significantly enhance IFN-gamma secretion by T cells.

Example 12 Effect of polypeptide constructs on expression of DC surface immunosuppressive molecular receptors

The DC from example 1 was plated in 24 well plates with 1mL cell density of 1X 10 ⁶ DC suspension/mL; 40 μg HB was added to each well _100-108 MPC1, 40. Mu.g HB _100-108 MPC2, 40. Mu.g of MPC4 and 40. Mu.g of HB _100-108 -MPC4, reserving DCs without added polypeptide construct as DC controls for different groups, incubating for 6 hours, washing 3 times with PBS containing 5% v/v fbs, adding fresh medium for overnight incubation, digesting and harvesting DCs, washing 2 times with PBS, HB _100-108 -MPC1 group DCs were stained with fluorescent-labeled monoclonal antibodies targeting PD-L1, PD-L2, HVEM and ILT4, respectively; HB (high-molecular-weight HB) _100-108 -MPC2 group DCs were also stained with fluorophore-labeled monoclonal antibodies targeting PD-L1, PD-L2, HVEM and ILT4, respectively; MPC4 group DC and HB _100-108 MPC4 group DCs were stained with fluorescent group-labeled monoclonal antibodies targeting PD-L1 (the above PD-L1-targeting antibodies were labeled with PE, purchased from BD; PD-L2, HVEM and ILT 4-targeting monoclonal antibodies were labeled with FITC, APC and PE, purchased from biotechnology ltd, p.s., the concentrations of antibodies used were determined as indicated), isotype control groups (isotype control antibodies were conventional murine IgG), incubated at 4 ℃ for 30 minutes in the absence of light, washed again with PBS containing 5% v/v fbs 3 times, and the proportion of PD-L1, PD-L2, HVEM and ILT4 positive cells was detected by flow cytometry. The results are shown in FIGS. 34A-34C.

FIG. 34A shows the passage HB _100-108 The proportion of PD-L1, PD-L2, HVEM and ILT4 positive cells in the MPC1 treated DC was not significantly compared to control DCThe distinction is made.

FIG. 34B shows the passage HB _100-108 The proportion of PD-L1, PD-L2, HVEM and ILT4 positive cells in MPC2 treated DCs was not significantly different from that in control DCs.

FIG. 34C shows that the treated MPC4 or HB _100-108 The proportion of PD-L1 positive cells in the MPC4 treated DCs was 2.8% and 3.6%, respectively, with no significant difference compared to the proportion of positive cells of 2.6% in the DC control group.

PD-L1, PD-L2, HVEM and ILT4 are immunosuppressive molecular receptors, and binding to their corresponding ligands on T cells inhibits T cell activation and proliferation. The results of FIG. 34 show that the polypeptide constructs of the invention do not increase the expression level of immunosuppressive molecular receptors of DCs, and therefore DCs treated with the polypeptide constructs of the invention do not potentially exert an inhibitory effect on T cells.

All documents referred to herein are incorporated by reference in this application as if each was individually incorporated by reference. Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the foregoing description of the invention, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Shanghai cell therapy group Co., ltd

SHANGHAI CELL THERAPY Research Institute

<120> polypeptide, composition containing the same and use thereof in tumor immunity

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Claims

1. An isolated fusion polypeptide comprising, in order from N-terminus to C-terminus, a covalently linked peptide fragment selected from any one of the following groups: (1) a peptide fragment derived from PSF1, a peptide fragment derived from eEF2 and a peptide fragment derived from MCL-1, (2) a peptide fragment derived from HSP105, a peptide fragment derived from MUC-1 and a peptide fragment derived from Survivin, (3) a peptide fragment derived from PSF1, a peptide fragment derived from MCL-1 and a peptide fragment derived from HSP105, and (4) a peptide fragment derived from eEF2, a peptide fragment derived from MUC-1 and a peptide fragment derived from Survivin,

The peptide fragment derived from PSF1 is shown as SEQ ID NO.1,

the peptide fragment derived from eEF2 is shown as SEQ ID NO.2,

the peptide fragment derived from MCL-1 is shown as SEQ ID NO.3,

the peptide fragment derived from HSP105 is shown in SEQ ID NO.4,

the peptide fragment derived from MUC-1 is shown in SEQ ID NO.5,

the peptide fragment derived from Survivin is shown as SEQ ID NO. 6.

2. The isolated fusion polypeptide of claim 1, wherein the peptide segments are covalently linked directly or through a linker.

3. The polypeptide composition of claim 2, wherein the linker is selected from (GS ₄ ) ₃ One or more of Furin2A peptide and twin arginine.

4. The polypeptide composition of claim 3, wherein the linker is a double arginine.

5. The isolated fusion polypeptide of claim 4, comprising, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID No.1, a double arginine linker, a peptide fragment shown in SEQ ID No.2, a double arginine linker, and a peptide fragment shown in SEQ ID No.3, which are covalently linked, wherein the sequence of the isolated fusion polypeptide is shown in SEQ ID No. 8.

6. The isolated fusion polypeptide of claim 5, further comprising an immunopotentiator polypeptide which is HB _100-108 Peptides, the sequence thereofThe sequence is shown as SEQ ID NO. 7.

7. The isolated fusion polypeptide of claim 6, wherein the immunopotentiator polypeptide is located at the N-terminus of the isolated fusion polypeptide and is covalently linked to the peptide stretch depicted in SEQ ID No. 8.

8. The isolated fusion polypeptide of claim 7, wherein the immunopotentiator polypeptide is covalently linked to the peptide stretch depicted in SEQ ID No.8 via a double arginine linker.

9. The isolated fusion polypeptide of claim 4, wherein the sequence of the isolated fusion polypeptide is set forth in SEQ ID No. 9.

10. The isolated fusion polypeptide of claim 4, comprising, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID No.4, a double arginine linker, a peptide fragment shown in SEQ ID No.5, a double arginine linker, and a peptide fragment shown in SEQ ID No.6, which are covalently linked, wherein the sequence of the isolated fusion polypeptide is shown in SEQ ID No. 10.

11. The isolated fusion polypeptide of claim 10, further comprising an immunopotentiator polypeptide which is HB _100-108 The peptide has a sequence shown in SEQ ID NO. 7.

12. The isolated fusion polypeptide of claim 11, wherein the immunopotentiator polypeptide is located at the N-terminus of the isolated fusion polypeptide, covalently linked to the peptide stretch depicted in SEQ ID No. 10.

13. The isolated fusion polypeptide of claim 12, wherein the immunopotentiator polypeptide is covalently linked to the peptide stretch depicted in SEQ ID No.10 via a double arginine linker.

14. The isolated fusion polypeptide of claim 4, wherein the sequence of the isolated fusion polypeptide is set forth in SEQ ID No. 11.

15. The isolated fusion polypeptide of claim 4, comprising, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID No.1, a double arginine linker, a peptide fragment shown in SEQ ID No.3, a double arginine linker, and a peptide fragment shown in SEQ ID No.4, which are covalently linked, wherein the sequence of the isolated fusion polypeptide is shown in SEQ ID No. 12.

16. The isolated fusion polypeptide of claim 15, further comprising an immunopotentiator polypeptide which is HB _100-108 The peptide has a sequence shown in SEQ ID NO. 7.

17. The isolated fusion polypeptide of claim 16, wherein the immunopotentiator polypeptide is located at the N-terminus of the isolated fusion polypeptide, covalently linked to the peptide fragment set forth in SEQ ID No. 12.

18. The isolated fusion polypeptide of claim 17, wherein the immunopotentiator polypeptide is covalently linked to the peptide stretch depicted in SEQ ID No.12 via a double arginine linker.

19. The isolated fusion polypeptide of claim 4, wherein the sequence of the isolated fusion polypeptide is set forth in SEQ ID No. 13.

20. The isolated fusion polypeptide of claim 4, comprising, in order from the N-terminus to the C-terminus, a peptide fragment shown in SEQ ID No.2, a double arginine linker, a peptide fragment shown in SEQ ID No.5, a double arginine linker, and a peptide fragment shown in SEQ ID No.6, which are covalently linked, wherein the sequence of the isolated fusion polypeptide is shown in SEQ ID No. 14.

21. The isolated fusion polypeptide of claim 20, further comprising an immunopotentiator polypeptide, which is HB _100-108 The peptide has a sequence shown in SEQ ID NO. 7.

22. The isolated fusion polypeptide of claim 21, wherein the immunopotentiator polypeptide is located at the N-terminus of the isolated fusion polypeptide, covalently linked to the peptide stretch depicted in SEQ ID No. 14.

23. The isolated fusion polypeptide of claim 22, wherein the immunopotentiator polypeptide is covalently linked to the peptide stretch depicted in SEQ ID No.14 via a double arginine linker.

24. The isolated fusion polypeptide of claim 4, wherein the sequence of the isolated fusion polypeptide is set forth in SEQ ID No. 15.

25. An isolated nucleic acid encoding the isolated fusion polypeptide of any one of claims 1-24.

26. The isolated nucleic acid of claim 25, which is an unmodified or chemically modified RNA.

27. The isolated nucleic acid of claim 26, wherein the chemical modification on the chemically modified RNA is selected from the group consisting of ribose ring modification, phosphodiester backbone modification, and base modification.

28. The isolated nucleic acid of claim 27, wherein the ribosyl modification is a 2 '-oxyalkyl modification and a 2' -F modification.

29. The isolated nucleic acid of claim 27, wherein the phosphodiester backbone modification is a locked nucleic acid modification.

30. The isolated nucleic acid of claim 27, wherein the base modification is a 4-thiouracil modification, a 2-thiouracil modification, and a C-linked pseudo-uracil modification.

31. An immunopeptidic composition comprising the isolated fusion polypeptide of any one of claims 1-24 and an immunopotentiator.

32. The immunopeptides composition of claim 31, wherein the immunopotentiator comprises a polypeptide selected from the group consisting of polyIC: IL, complete freund's adjuvant, incomplete freund's adjuvant, aluminum salts, aluminum hydroxide nanoparticles, prostaglandin E2, interferon alpha, and HB _100-108 One or more of the peptides.

33. The immunopotentiator composition of claim 32, wherein the immunopotentiator is HB _100-108 A peptide.

34. The immune polypeptide composition of claim 33, wherein the HB _100-108 The peptide sequence is shown in SEQ ID NO. 7.

35. An immunomodulator comprising one or more of the isolated fusion polypeptide of any of claims 1-24, the isolated nucleic acid of any of claims 25-30, and the immune polypeptide composition of any of claims 31-34.

36. A polypeptide vaccine comprising a) the isolated fusion polypeptide of any one of claims 1-24, the isolated nucleic acid of any one of claims 25-30, and one or more of the immune polypeptide compositions of any one of claims 31-34, and b) a pharmaceutically acceptable carrier.

37. The polypeptide vaccine of claim 36, further comprising an adjuvant.

38. A method of preparing an antigen-loaded, activated antigen presenting cell comprising contacting the antigen presenting cell with the immunomodulator of claim 35 and/or the polypeptide vaccine of any one of claims 36 and 37.

39. The method of claim 38, wherein the antigen presenting cells are lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or any combination thereof.

40. The method of claim 39, wherein the antigen presenting cells are any one or more selected from the group consisting of monocytes, macrophages and dendritic cells.

41. The method of claim 38, wherein the antigen presenting cells are autologous or allogeneic.

42. The method of claim 38, wherein the contacting is incubating.

43. The method of claim 42, wherein the incubation is for a period of 2 to 10 hours.

44. The method of claim 42, wherein the incubation is for a period of 3 to 8 hours.

45. The method of claim 42, wherein the incubation is for a period of 3 to 6 hours.

46. The system of claim 38The preparation method is characterized in that the dosage ratio of the immunomodulator and/or polypeptide vaccine to the antigen presenting cells is 10-120 mug/10 ⁶ Individual cells.

47. The method of claim 46, wherein the ratio of the immunomodulator and/or polypeptide vaccine to the antigen presenting cells is 20-90 μg/10 ⁶ Individual cells.

48. The method of claim 47, wherein the ratio of the immunomodulator and/or polypeptide vaccine to the antigen presenting cells is 20-40 μg/10 ⁶ Individual cells.

49. The method of claim 42, wherein the incubation is at a temperature of 37 ℃.

50. The method of claim 42, wherein the incubated CO ₂ The concentration was 5%.

51. A tumor vaccine comprising antigen presenting cells produced by the method of any one of claims 38-50.

52. The tumor vaccine of claim 51, further comprising an immunomodulatory agent of claim 35.

53. The tumor vaccine of claim 51, further comprising a pharmaceutically acceptable carrier and/or adjuvant.

54. A method of preparing an activated immune effector cell comprising contacting the immune effector cell with an antigen presenting cell treated with one or more of the isolated fusion polypeptide of any one of claims 1-24, the isolated nucleic acid of any one of claims 25-30, the immune polypeptide composition of any one of claims 31-34, the immunomodulator of claim 35, the polypeptide vaccine of claim 36 or 37, and the tumor vaccine of any one of claims 51-53.

55. The method of claim 54, wherein the antigen presenting cells are dendritic cells.

56. The method of claim 54, wherein the immune effector cell is a T cell.

57. The method of claim 54, wherein the treatment is incubation.

58. The method of claim 47, wherein the incubation is at a temperature of 37 ℃.

59. The method of claim 47, wherein the incubated CO ₂ The concentration was 5%.

60. The method of claim 47, wherein the incubation is for a period of 3-6 hours.

61. The method of claim 54, wherein the contacting is co-cultivation.

62. The method of claim 61, wherein the co-cultivation is at a temperature of 37 ℃.

63. The method of claim 61, wherein the CO-cultured CO ₂ The concentration was 5%.

64. The method of claim 61, wherein the co-cultivation is for a period of 12-96 hours.

65. The method of claim 61, wherein the ratio of antigen presenting cells to immune effector cells in the co-culture is 1:1-1:5.

66. The method of claim 65, wherein the ratio of antigen presenting cells to immune effector cells in the co-culture is 1:5.