CN117586353A - Liver cancer related polypeptide, compound, pharmaceutical composition and application - Google Patents

Liver cancer related polypeptide, compound, pharmaceutical composition and application Download PDF

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CN117586353A
CN117586353A CN202210967650.8A CN202210967650A CN117586353A CN 117586353 A CN117586353 A CN 117586353A CN 202210967650 A CN202210967650 A CN 202210967650A CN 117586353 A CN117586353 A CN 117586353A
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polypeptide
cells
liver cancer
cell
polypeptides
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孟颂东
鞠莹
王九儒
张含
王皓宇
王子豪
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Beijing Rexiu Biotechnology Co ltd
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Abstract

The invention belongs to the field of biological medicine, and relates to liver cancer related polypeptides, a compound, a pharmaceutical composition and application. The invention also relates to a group of polypeptide libraries and application thereof in treating hepatocellular carcinoma. In particular, the invention relates to polypeptides or combinations of polypeptides selected from any one or more of the polypeptides shown in SEQ ID NOs: 1-14. The compound or sensitized DC vaccine of the invention can activate tumor specific T cell response, effectively kill liver cancer cells and obviously inhibit liver tumor growth, thereby achieving the purpose of treating hepatocellular carcinoma. The invention has wide application prospect as a tumor therapeutic vaccine or immune cell therapy for treating malignant tumors such as hepatocellular carcinoma and the like.

Description

Liver cancer related polypeptide, compound, pharmaceutical composition and application
Technical Field
The invention belongs to the field of biological medicine, and relates to liver cancer related polypeptides, a compound, a pharmaceutical composition and application. The invention also relates to a group of polypeptide libraries and application thereof in treating hepatocellular carcinoma.
Background
Liver cancer refers to malignant tumors occurring in the liver, including primary liver cancer and metastatic liver cancer, most of which belong to hepatocellular carcinoma. Primary liver cancer is one of the most common malignant tumors in clinic. In male cancer patients worldwide, liver cancer rates are sixth and mortality rates are second; in female cancer patients, liver cancer rates are seventh and mortality rates are sixth. In 2008, there were 748,300 new liver cancer cases worldwide, and 695,900 liver cancer patients died. And half of these newly increased liver cancer cases and death cases are in china. The regions with highest liver cancer incidence are mainly in eastern asia, southeast asia, middle africa and western africa. The higher incidence of liver cancer in parts of asia and saharan africa is probably due to the prevalence of HBV in these areas, because 8% of the population in these areas chronically infects HBV, and 60% of liver cancer patients in developing countries are infected with HBV.
Vaccines composed of TAAs, TSAs, oncogenes or tumor suppressor muteins and polypeptides are currently classified as tumor polypeptide vaccines. T cells predominantly bind presented antigen polypeptides by recognition of major histocompatibility complex (major histocompatibility complex, MHC) molecules. The synthetic polypeptides can be directly conjugated to MHC class I molecules without the need for Antigen Presenting Cells (APCs) processing. The tumor polypeptide vaccine is mostly tumor antigen peptide fragments which are prepared by tumor cell cleavage or artificial synthesis, and is used for tumor patients singly or in combination with an adjuvant, wherein the tumor antigen peptide fragments enter the body to directly induce the response of in vivo cytotoxic T lymphocytes (cytotoxic lymphocyte, CTL) and excite the specific tumor immune response of the body.
Glypican 3 (gpc 3), also known as MXR7, OCI-5, gtr2-2, is a member of the heparan sulfate glycoprotein (heparan sulfate proteoglycan, HSPG) family. The human GPC3 gene is located on chromosome xq26.10, an HSPG glycoprotein anchored to the cell membrane by Glycosyl Phosphatidylinositol (GPI). GPC3 encodes 580 amino acids with a relative molecular mass of the core protein of about 70kDa. The Furin protease cleavage site is in the middle. Studies have suggested that GPC3 core protein acts by first cleaving Arg 358 and Cys 359 by Furin protease to form an N-terminal protein of about 40kDa and a C-terminal protein containing two Heparan Sulfate (HS) chains at about 30kDa, thereby participating in the regulation of transmission of the relevant signal pathway. GPC3 was found to be associated with a variety of cancers and to have a differential expression in tumor tissues. High expression in liver cancer, non-small cell lung cancer, melanoma, ovarian yolk sac tumor, ovarian clear cell carcinoma and colon cancer.
Paternally expressed gene 10 (PEG 10) is located on human chromosome 7q21.3 and is derived from the Ty3/Gypsy retrotransposon family, and is an oncogene involved in proliferation, apoptosis and metastasis of tumors. The abnormal expression of PEG10 is closely related to cell proliferation, apoptosis and the occurrence and development of malignant tumors. PEG10 overexpression is associated with high incidence of solid tumors, low degree of differentiation, and increased lymph node metastasis.
Heat shock proteins (Heat shock protein, HSP) are a class of proteins that are highly conserved in biological evolution and widely present in prokaryotes and eukaryotes. HSPs can be divided into subfamilies of HSP110, HSP90, HSP70, HSP60, HSP40, small molecule HSPs, ubiquitin, and the like, depending on the degree of homology and molecular weight. Heat shock protein (Heat shock protein, HSP) gp96 belongs to a member of the HSP90 subfamily, which is the most abundant heat shock protein on the cytoplasmic reticulum. The heat shock protein gp96 protein has polypeptide binding properties and is capable of receiving polypeptide fragments from the TAP complex in the endoplasmic reticulum, facilitating its assembly to MHC class I molecules for presentation on the cell membrane. The heat shock protein gp96 from different tissues can carry polypeptide fragments specifically expressed in the tissues from which the heat shock protein gp96 is derived.
Disclosure of Invention
The present inventors have made intensive studies and creative efforts to obtain a polypeptide or a combination of polypeptides. The inventors have surprisingly found that the polypeptide or combination of polypeptides is capable of forming a complex with gp96, sensitizing DC cells, activating T cells, and is effective in the treatment and/or prevention of liver cancer. The following invention is thus provided:
one aspect of the invention relates to a polypeptide or combination of polypeptides selected from any one or more of the polypeptides shown in SEQ ID NOs: 1-14.
The amino acid sequences of the polypeptides of the present invention are shown in table 1 below.
TABLE 1
In some embodiments of the invention, the polypeptide or combination of polypeptides comprises a polypeptide as set forth in SEQ ID NOs 1-14; preferably, it consists of the polypeptides shown in SEQ ID NOs.1-14 (also referred to herein as polypeptide libraries).
Another aspect of the invention relates to gp 96-polypeptide complexes, wherein the polypeptide is one or more of the polypeptides of the invention.
In some embodiments of the invention, the gp 96-polypeptide complex, wherein the mass ratio of gp96 to polypeptide is from (1:5) to (5:1), preferably from (1:2) to (2:1) or from (1:1.5) to (1.5:1), more preferably 1:1, a step of;
preferably, the gp96 is a recombinant human gp96 protein; preferably, the sequence of gp96 is shown in SEQ ID NO. 64.
MDDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAE(SEQ ID NO:64)
When the polypeptides are of a plurality, e.g., using the aforementioned polypeptide libraries (SEQ ID NOs: 1-14), one gp96 molecule may form a complex with the same or different polypeptides, e.g., the polypeptides in a gp 96-polypeptide complex may be the same or different.
The polypeptide and heat shock protein gp96 form a complex in vitro in a natural adsorption or heat shock mode.
The polypeptide of the invention is respectively and chemically synthesized, preferably mixed according to the equal mass ratio, polypeptide solution with the concentration of 20mg/mL is prepared by DMSO, polypeptide and heat shock protein gp96 are mixed according to the mass ratio of 1:1 or 1:10, PBS buffer solution with the pH of 7.4.01 mol/L is used for dissolving the polypeptide to the total volume of 4mL, the solution is placed at the temperature of 20 ℃ or 4 ℃ for 30 minutes or is thermally shocked at the temperature of 55 ℃ for 10 minutes, the solution is cooled at room temperature for 30 minutes, and finally unbound polypeptide is washed out by a 50kD ultrafilter tube, thus obtaining the gp 96-polypeptide compound.
In the present invention, gp 96-polypeptide complex is also denoted as gp96+ polypeptide complex.
A further aspect of the invention relates to a gp 96-polypeptide complex combination consisting of at least two gp 96-polypeptide complexes according to any of the invention;
preferably, the gp 96-polypeptide complex combination consists of 14 gp 96-polypeptide complexes formed by polypeptides shown in SEQ ID NOs 1-14 and gp96 respectively;
preferably, the mass ratio between the 14 gp 96-polypeptide complexes, calculated as the mass of polypeptides therein, is from (1:5) to (5:1), preferably from (1:2) to (2:1) or from (1:1.5) to (1.5:1), more preferably 1:1.
in the present invention, gp 96-polypeptide library complex is also denoted as gp96+ polypeptide library complex.
A further aspect of the invention relates to a sensitized DC cell obtained from treatment of a DC cell with a gp 96-polypeptide complex according to any one of the invention or with a gp 96-polypeptide complex combination according to the invention;
preferably, the DC cells are human DC cells or mouse DC cells;
the concentration of gp 96-polypeptide complex in the DC cell culture is 50 μg/mL to 200 μg/mL calculated as the mass of gp96 protein; preferably 80 μg/mL to 120 μg/mL; more preferably 100. Mu.g/mL.
In some embodiments of the invention, the sensitized DC cells are obtained by:
(1) Isolating human PBMC at a concentration of 2 to 4X10 6 Laying into culture flask, 40ml each flask, placing at 37deg.C and 5% CO 2 An incubator;
(2) After 2 hours incubation, shake the flask slightly, collect the suspension cells for culturing T cells, wash the flask 1-2 times with PBS, add 40ml of DC culture solution (1640 culture medium +20ng/ml IL-4+50ng/ml GM-CSF) and then continue to place at 37℃and 5% CO 2 An incubator;
(3) On day 3 40 ml/bottle DC medium was added;
(4) TNF- α was added at a final concentration of 20ng/ml on day 6;
(5) Collecting 1X 10 on day 7-8 7 DC was dissolved in 1ml of DC culture medium in a 6-well plate, 50. Mu.g of gp 96-polypeptide complex of the present invention was added, and the mixture was placed at 37℃in 5% CO 2 Co-stimulating in an incubator for 4 hours;
(6) DC were collected in 50ml centrifuge tubes and washed 2 times with 0.9% physiological saline at 1500rpm/min for 5min.
In yet another aspect, the invention relates to an activated human T cell obtained by co-culturing the sensitized DC cell of the invention with a human T cell; preferably, the activated human T cells are activated human cytotoxic T lymphocytes (Cytotoxic T Lymphocyte, tc cells); preferably, the primed DC cells are primed human DC cells; preferably, the ratio of sensitized human DC cells to human T cells is 1:10.
in some embodiments of the invention, the sensitized DC cells are obtained by:
according to 1:10, transferring the sensitized DC together with the culture medium into a T cell culture bag (the number of T cells reaches 3×10) 8 ) Performing combined culture;
collecting at least 3×10 on day 14 9 Is washed by centrifugation at 1500rpm/min for 3 times, 5min each, to obtain activated human T cells.
A further aspect of the invention relates to a pharmaceutical composition comprising a polypeptide or combination of polypeptides according to any one of the invention, a gp 96-polypeptide complex combination according to the invention, a primed DC cell according to the invention or an activated CTL cell according to the invention, and one or more pharmaceutically acceptable excipients.
A further aspect of the invention relates to a vaccine composition comprising the primed DC-cells of the invention and a cell culture medium,
preferably, the cell culture medium is serum-free RPMI 1640;
preferably, the vaccine composition is a vaccine composition for treating or preventing liver cancer;
preferably, the liver cancer is hepatocellular carcinoma;
preferably, the liver cancer is primary liver cancer or metastatic liver cancer.
After the T cells activated by the medicine (vaccine) are returned to the nude mice, tumor-specific T cell immunity can be provided for the nude mice, and tumor cells in vivo and in situ transplanted tumor cells can be removed, so that the purpose of treating cancers can be achieved. The invention can be used as a tumor therapeutic vaccine (medicine) or a cell therapy and a cell medicine, is expected to effectively help patients to reduce tumor load, and can effectively prevent metastasis and recurrence by immediate treatment after operation.
A further aspect of the invention relates to the use of a polypeptide according to any one of the invention or a combination thereof, a gp 96-polypeptide complex according to any one of the invention, a gp 96-polypeptide complex combination, a primed DC cell according to the invention or an activated CTL cell according to the invention for the manufacture of a medicament for the treatment or prophylaxis of liver cancer;
preferably, the liver cancer is hepatocellular carcinoma;
preferably, the liver cancer is primary liver cancer or metastatic liver cancer.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (e.g., liver cancer); a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.
In the present invention, the term "sensitization" refers to a treatment of an organism or a cell, the reactivity of which is enhanced, and is called sensitization. Specifically, sensitization is a state in which the body or cell is automatically produced by contact with an antigenic substance, or the immunoreactivity is increased by passive production after receiving an antibody or immunocompetent lymphocyte.
Advantageous effects of the invention
The invention achieves one or more of the following technical effects:
(1) The complex formed by the polypeptide and gp96 or the sensitized DC vaccine can activate tumor-specific T cell response, effectively kill liver cancer cells and obviously inhibit liver tumor growth.
(2) The polypeptide of the invention can be obtained by in vitro chemical synthesis, forms a complex with recombinant gp96, and solves the problem of source limitation of human placenta-derived gp96 complex.
(3) The polypeptide of the invention contains a plurality of HLA class I molecule restricted epitopes, basically covers the typing of human main HLA, and has broad spectrum. One of the most important and central works in the neoantigen prediction process is to determine the subtype of HLA class I genes, and only tumor antigen epitopes matched with the subtype can be identified by CD8+ T cells, so that tumor cells are killed. The common HLA-I genotypes in Chinese population are mainly HLA-A2, HLA-A11, HLA-A24 and HLA-A33, so that the invention selects four HLA restriction study objects and covers most of Chinese population. In use, if all polypeptides are mixed and used as a polypeptide library for treating liver cancer, a complex procedure for genotyping can be avoided.
Drawings
Fig. 1: analysis of GEO public transcriptome dataset data against hepatocellular carcinoma (HCC), human placenta, against protein to which placenta gp96 binding polypeptide belongs. Wherein GSE1133 and GSE101685 are human tissue related transcriptome database and liver cancer related transcriptome database, respectively, both from GEO (Gene Expression Omnibus data base) public dataset of NCBI.
Fig. 2A to 2D: IFN-gamma+ELISPOT detection of the number of plaques in T lymphocytes of the polypeptide-gp 96 complex immunized transgenic mice, PBS group was used as a negative control. Data in the figures are mean ± SD of 3 replicates. Wherein, FIGS. 2A-2D used HLA-A2 transgenic mice, HLA-A24 transgenic mice, HLA-A11 transgenic mice, and HLA-A33 transgenic mice, respectively.
Fig. 3A to 3D: IFN-gamma+ELISPOT detection of the number of plaques in T lymphocytes of mice immunized with the polypeptide-gp 96 complex-activated mouse BMDC vaccine. Data in the figures are mean ± SD of 3 replicates. Wherein, FIGS. 3A to 3D used HLA-A2 transgenic mice, HLA-A24 transgenic mice, HLA-A11 transgenic mice, and HLA-A33 transgenic mice, respectively.
Fig. 4A to 4D: the polypeptide-gp 96 complex activated mouse BMDC vaccine induces the killing effect of specific killer T Cells (CTL) of transgenic mice on target cells. Data in the figures are mean ± SD of 3 replicates. Among them, the target cells of FIGS. 4A to 4D are HepG2 cell, SK-HEP-1 cell, huh-7 cell and PLC/PRF/5 cell, respectively.
Fig. 5A to 5D: inhibition of liver cancer tumor growth in mice by lymphocytes of mice after immunization with BMDCs activated by the reinfusion polypeptide-gp 96 complex. Data in the figure are mean ± SD of 5 mice. Among them, FIGS. 5A to 5D are lymphocyte reinfusion of a mice vaccinated with HepG2 cells and transgenic with HLA-A2, SK-HEP-1 cells and transgenic with HLA-A24, huh-7 cells and transgenic with HLA-A11, and PLC/PRF/5 cells and lymphocyte reinfusion of a mice vaccinated with HLA-A33, respectively.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The quantitative tests in the following examples were all set up in triplicate and the results averaged.
HLA-A2 and nude mice are products of Beijing vitamin Torilhua laboratory animal Limited liability company. HLA-A24, HLA-A11 and HLA-A33 transgenic mice were constructed by the laboratory themselves. Human Leukocyte Antigens (HLA) are human MHC molecules that present antigens of human cells to T cells, mediate activation of T cells, and by transgenesis of HLA molecules into mice, human antigens can be presented and activated in mice and antigen-specific T cells can be activated. HLA-A2, HLA-A24, HLA-A11 or HLA-A33, respectively, represent the major four alleles of the A locus on human HLA. Transgenic mice of chimeric HLA-A2, HLA-A24, HLA-A11 or HLA-A33 class molecules can mediate efficient positive selection of mouse T cells, capable of recognizing the complete T cell pool molecules of peptides presented by human HLA-A2, HLA-A24, HLA-A11 or HLA-A33 class, respectively. The peptide epitope presented and recognized by the transgenic mouse T cells is identical to that presented by HLA-A2+, HLA-A24+, HLA-A11+ or HLA-A33+ humans.
The polypeptides were synthesized by Shanghai Jier Biochemical Co.
HepG2 cells (human hepatoma cells) were purchased from ATCC (American type culture Collection) under the product catalog number HB-8065, HLA-A2 restriction cell line.
SK-HEP-1 cells (human hepatoma cells) were purchased from ATCC (American type culture Collection) under the product catalog number HTB-52, HLA-A24 restricted cell line.
Huh-7 cells (human liver cancer cells) were purchased from the cell resource center of basic medical institute of China medical sciences, and the product number is 1101HUM-PUMC000679, HLA-A11 restricted cell line.
PLC/PRF/5 (human hepatoma cell) was purchased from ATCC (American type culture Collection) under the product catalog number CRL-8024, HLA-A33 restricted cell line.
Example 1: establishment of liver cancer antigen polypeptide library
1. Mass spectrometry and identification of human placenta-derived gp96 binding polypeptides
Human placenta derived gp96 references were prepared (Meng S, song J, rao Z, tien P, gao G.2002.Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp-bound peptides. Journal of Immunological Methods,264 (1-2): 29-35.). Gp96 extracted by this method naturally binds to various specific antigens in placenta, and heat shock protein gp96-bound polypeptide is isolated using acid stripping: 5mg of heat shock protein gp96 was taken separately and the concentrations were adjusted to 1mg/mL, and an appropriate amount of HPLC grade trifluoroacetic acid (TFA) was added to give a final concentration of 0.2%. The reaction was carried out at 4℃for one hour to allow the polypeptide bound to the heat shock protein gp96 to be dissociated sufficiently. The protein-polypeptide mixture was centrifuged using a 3kD ultrafiltration tube to pass the free polypeptide through the ultrafiltration membrane, while heat shock protein gp96 was trapped outside the ultrafiltration membrane. The polypeptide mixture passing through the ultrafiltration membrane was lyophilized and subjected to HPLC purification and analysis. After all the polypeptide elution peaks are pooled, polypeptide sequence analysis and protein identification are performed by using an Orbitrap fusion liquid chromatograph (Thermo Scientific).
2. Screening and identification of placenta gp 96-combined liver cancer related polypeptides
Human tissue transcriptome data (GSE 1133, human tissue related transcriptome database) were analyzed, and placenta tissue expressed gene relative to liver tissue was crossed with liver cancer expressed gene relative to liver tissue and protein to which peptide fragment bound in placenta gp96 (i.e. gp96 bound polypeptide eluted by the above acid) by transcriptome data processing (FIG. 1).
Two proteins of GPC3 and PEG10 were obtained, and further, by analyzing liver cancer transcriptome data of different GEO data sets (GSE 105130, GSE121248 are two liver cancer related transcriptome databases), the results showed that these two genes were stably and highly expressed in liver cancer, and therefore, it was determined that two proteins of GPC3 and PEG10 were liver cancer related antigens bound in placenta gp 96. The placenta-derived heat shock protein gp96 is combined with all polypeptides derived from GPC3 and PEG10 to build a liver cancer antigen-related polypeptide library, as shown in Table 2. Epitopes were predicted and scored by Immune Epitope Database (IEDB) website.
Table 2: liver cancer antigen polypeptide library
3. Preparation and identification of recombinant gp96 protein-polypeptide complexes
Expression and preparation of recombinant gp96 protein are described in the literature (Liu W, chen M, li X, zhao B, hou J, zheng H, qia L, li Z, meng S.interface of Toll-Like Receptors with the Molecular Chaperone Gp96 Is Essential for Its Activation of Cytotoxic T Lymphocyte response.PLoS one.2016 May 16;11 (5): e0155202.Doi: 10.1371/journ.fine.0155202. PMID:27183126; PMCID: PMC 4868323.). The polypeptides 1-14 shown in Table 1 were chemically synthesized, respectively, and a polypeptide solution having a concentration of 20mg/mL was prepared with DMSO, or a polypeptide mixed solution having a concentration of 20mg/mL was prepared with DMSO by mixing at equal mass ratio, and the mass of the total polypeptide was calculated for the mixed polypeptide. Dissolving polypeptide and heat shock protein gp96 according to a mass ratio of 1:1, using PBS buffer solution with pH of 7.4.01 mol/L to a total volume of 4mL, standing at 20 ℃ or 4 ℃ for 30 minutes, or heat-shocking at 55 ℃ for 10 minutes (the effect is to combine the polypeptide with gp96 protein), cooling at room temperature for 30 minutes, and finally washing unbound polypeptide by using a 50kD ultrafiltration tube to obtain gp 96-polypeptide complexes, wherein the steps are as follows:
recombinant gp 96-peptide 1 complex, recombinant gp 96-peptide 2 complex, recombinant gp 96-peptide 3 complex, recombinant gp 96-peptide 4 complex, recombinant gp 96-peptide 5 complex, recombinant gp 96-peptide 6 complex, recombinant gp 96-peptide 7 complex, recombinant gp 96-peptide 8 complex, recombinant gp 96-peptide 9 complex, recombinant gp 96-peptide 10 complex, recombinant gp 96-peptide 11 complex, recombinant gp 96-peptide 12 complex, recombinant gp 96-peptide 13 complex, recombinant gp 96-peptide 14 complex, recombinant gp 96-peptide library complex. Wherein the polypeptide library in the recombinant gp 96-polypeptide library complex is formed by mixing peptides 1 to 14 according to the equal mass ratio.
Heat shock allows for a transient conformational change in gp96 that can bind to polypeptides to form gp 96-polypeptide complexes. The 15 gp 96-polypeptide complexes prepared above were identified: the unbound polypeptide is first removed by ultrafiltration, the gp96-bound polypeptide is then isolated by the acid elution method described above, and the bound polypeptide is identified by liquid chromatography, which proves that the product obtained by the heat shock method described above is a gp 96-polypeptide complex, but not a mixture of gp96 and polypeptide.
Example 2: evaluation of the activity of the prepared gp 96-polypeptide complex sensitized DC induced mouse specific CTL cells Killing effect on liver cancer target cells
1. Culture of mouse bone marrow derived DC (BMDC)
(1) HLA-A2, HLA-A24, HLA-A11 and HLA-A33 mice were sacrificed by cervical removal, all femur and tibia were surgically removed, and excess muscle was cut off with scissors after removal of the pelt.
(2) The two ends of the femur are cut off by a sterile scissors, PBS is extracted by a syringe to flush the bone marrow cavity, and cells in the bone marrow are flushed into the PBS until the bone is completely whitened.
(3) The washed cells were loaded into a 15mL centrifuge tube and centrifuged at 2000 rpm for 10min.
(4) Collecting bone marrow suspension, filtering fragments and muscle tissue with 200 mesh nylon filter screen
(5) GM-CSF cytokine was added at a ratio of 1:1000 for differentiation (using a concentration of 10 ng/mL).
(6) Placing at 37deg.C, containing 5% CO 2 Is cultured in an incubator of (a).
(7) After two days of incubation, the solution was changed and 10mL of fresh 10% fetal bovine serum in RPMI 1640 was added, and GM-CSF cytokine was added to the dish at a concentration of 10ng/mL continuing on a culture dish with 5% CO 2 Is cultured in an incubator at 37 ℃.
(8) On day 6, a liquid change was performed again.
(9) On day 7, cells differentiated completely, the dishes were gently shaken, the supernatant was aspirated, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cell pellet was fully resuspended in fresh medium, counted and the cells were adjusted to 100 ten thousand/mL.
2. Preparation of BMDC vaccine sensitized with polypeptide-gp 96 Complex (if a human vaccine, human PBMC may be isolated, DC cells induced for vaccine preparation)
(1) BMDC cells cultured to day 7 were removed, washed twice with PBS, and counted.
(2) BMDC were resuspended to a concentration of 200 ten thousand/mL.
(3) BMDCs were distributed into 2mL tubes of cell cryopreservation, 1mL each was added, for a total of 200 ten thousand BMDC cells.
(4) Different antigens were added to each tube: human placental gp96, recombinant gp 96-polypeptide complexes, and recombinant gp96 protein alone. The final concentration of heat shock protein gp96 was set to 100 μg/mL (calculated as gp96 protein).
(5) At 37deg.C, containing 5% CO 2 Is cultured in an incubator for 4 hours.
(6) The frozen tube was centrifuged at 1500rpm for 5 minutes at 4℃and the supernatant was discarded.
(7) Then, PBS was added for resuspension, and the mixture was centrifuged at 1500rpm for 5 minutes at 4℃to discard the supernatant.
(8) To the cell pellet, 1mL of serum-free RPMI 1640 was added, and the cell pellet was homogenized by blowing, thereby preparing immunized mice.
3. Mouse immunization program
1. Four transgenic mice, HLA-A2, HLA-A24, HLA-A11 and HLA-A33, from 6 to 8 weeks, were each divided into 17 groups (10 per group) and were immunized subcutaneously on day 0, day 7, day 21, respectively: recombinant gp 96-individual polypeptide complexes (complexes of recombinant gp96 with peptides 1 to 14, respectively), recombinant gp 96-polypeptide library complexes or human placenta-derived gp96 (in fact, complexes of gp96 with polypeptides) were used as negative controls with individual recombinant gp96 vaccines. Each mouse was immunized with 100 μg protein (calculated as gp96 protein).
2. Four transgenic mice, HLA-A2, HLA-A24, HLA-A11 and HLA-A33, from 6 to 8 weeks, were each divided into 17 groups (10 per group) and were immunized subcutaneously on day 0, day 7, day 21, respectively: recombinant gp 96-individual polypeptide complex-activated BMDC vaccine (complexes of recombinant gp96 with peptides 1 to 14, respectively), recombinant gp 96-polypeptide library complex-activated BMDC vaccine or human placenta-derived gp 96-activated BMDC vaccine, with individual recombinant gp 96-activated BMDC vaccine as negative control. Each mouse was immunized with 50 ten thousand BMDC cells.
4. BMDC immunization sensitized with polypeptide-gp 96 complex induces HepG2, SK-HEP-1, huh7 and PLC/PRF/5 cell-specific T cell immune responses
One week after the last immunization, mice were sacrificed, and spleen lymphocytes were isolated from the mice and prepared for the later operations of this example and the operations of example 3.
1. Detection of lymphocyte immune response to polypeptide using IFN-gamma+ ELISPOT
The main operation steps are as follows:
(1) And (3) pre-coating: the coated antibody was diluted to 20mg/mL (1:200 dilution) with sterile PBS (pH 7.4), the diluted coated antibody was added to the ELISPOT plate, 100. Mu.L per well was added, and incubated overnight at 4 ℃. The above operations should be performed in an ultra clean bench.
(2) Closing: the coating solution in the ELISPOT plates was discarded, and washed 3 times with 200 μl per well with sterile PBS. After the last wash, the wells were then dried by patting, 200. Mu.L of RPMI 1640 medium containing 10% serum was added to each well and incubated for 30 minutes at room temperature.
(3) An appropriate amount of mouse lymphocytes was diluted to a concentration of 350 ten thousand/mL in RPMI 1640 medium containing 10% serum. IL-2 and anti-mouse CD28 antibody are added simultaneously to make the final concentration of the antibody be 20U/mL and 1 mug/mL respectively.
(4) Cells were added separately to each well, 200 μl per well, so that eventually each well contained 50 ten thousand cells. The individual polypeptide fragments were used as stimulators, and PMA and ionomycin were stimulated as positive controls.
(5) Placing at 37deg.C and 5% CO 2 Is cultured in a cell culture incubator for 24 hours.
(6) After the incubation, the ELISPOT plates were removed, the cells and medium were discarded and the plates were dried.
(7) 200 μl of pre-chilled deionized water was added to each well, left for 3 minutes, discarded, and the procedure repeated once more, and the swatter dried.
(8) 200. Mu.L of PBST was added to each well, left for 3 minutes, and discarded. Washing for 5 times, and finally beating on absorbent paper.
(9) The biotin-labeled anti-IFN-gamma monoclonal antibody is prepared by PBS and diluted 1:200. mu.L of each well was added and incubated at room temperature for 2 hours.
(10) The antibody was discarded, 200. Mu.L of PBST was added to each well, and the mixture was left for 3 minutes and discarded. Washing for 5 times, and finally beating on absorbent paper.
(11) Avidin-labeled HRP was formulated with PBS and diluted 1:100. mu.L of each well was added and incubated at room temperature for 1 hour.
(12) The liquid was discarded, 200. Mu.L of PBST was added to each well, and the mixture was left for 3 minutes and discarded. Washing for 5 times, washing twice with PBS, and drying on absorbent paper for the last time.
(13) AEC substrate was formulated and added to ELISPOT plates at 100 μl per well.
(14) The mixture was left at room temperature for 10 minutes to develop color.
(15) After the spots developed to the appropriate size, the development reaction was terminated by washing 3 times with deionized water. And then the plate is reversely buckled on the water absorbing paper, the tiny water drops are beaten to dry, the front surface of the plate is placed in a ventilated place, and the plate is naturally dried at room temperature.
(16) The ELISPOT plate spots were counted with an image autoanalyzer and various parameters of the spots were recorded for statistical analysis.
The experimental results are shown in fig. 2A to 2D and fig. 3A to 3D. The results show that placenta-extracted gp96 and recombinant gp 96-polypeptide complexes immunized or sensitized with DC vaccines both activate polypeptide-specific T cells, whereas recombinant gp96 protein alone does not activate specific T cells.
2. UsingNon-radioactive cytotoxicity assay (Promega, catalog No. G1780) for cytotoxic activity assays, the main steps are as follows (see kit instructions for more details): />
(1) Respectively taking HepG2 cells as target cells, inoculating the target cell numberFor purposes of 5X 10 3 Adding the lymphocytes as effector cells according to the ratio of 5:1, 10:1 and 20:1 of the effective target ratio, and inoculating the effector cells into a 96-well culture plate at 50 mu 1/well to obtain a final volume of 100 mu 1;
in addition, an effector spontaneous LDH (lactate dehydrogenase) release group was additionally provided for calibrating the spontaneous LDH release of effector cells (each group of effector cells was added to a 96-well plate at 50. Mu.1/well, supplemented with 50. Mu.1 of RPMI-1640 medium containing 5% fetal bovine serum to a final volume of 100. Mu.1). Target cells spontaneous LDH release groups were used to correct for spontaneous LDH release from target cells (each group of target cells was added to 96-well plates at 50 μ1/well, supplemented with 50 μ1 RPMI-1640 medium containing 5% fetal bovine serum to a final concentration of 100 μ1). The maximum LDH release group of target cells was used as a reference for determining 100% LDH release (spontaneous release group of cells loaded with the same target cells) at the time of calculation. Volume correction control group was used to correct for volume changes due to addition of lysate (addition of RPMI-1640 medium with 5% fetal bovine serum 100. Mu.1). Culture background control group was used to correct for background absorption by LDH activity produced by serum in the culture (addition of RPMI-1640 medium 100. Mu.1 containing 5% fetal bovine serum) and phenol red.
(2) After cell inoculation, centrifugation was performed for 4min using 250g followed by incubation in an incubator at 37 ℃ for 4h; lysates (10×), 10 μ1/well were added to the target cell maximum LDH release group 45min before harvest of the supernatant; then, the supernatant was harvested by centrifugation at 250g for 4 min;
(3) Transferring 50 mu 1 supernatant to an ELISA plate, preparing a substrate by using a detection buffer solution, adding the prepared substrate into the ELISA plate at a ratio of 50 mu 1/hole, covering the plate, performing light-proof reaction at room temperature for 30min, adding 50 mu 1 stop solution into each space, and detecting the light absorption value OD at 490nm in an ELISA instrument within 1 h.
(4) Calculating cell killing rate
Killing rate (%) = [ (experimental group OD value-effector cell spontaneous release group OD value-target cell spontaneous release group OD value)/(target cell maximum release group OD value-target cell spontaneous release group OD value) ] ×100%
The experimental results are shown in fig. 4A to 4D. The results show that, at an effective target ratio of 10:1, the recombinant gp 96-polypeptide complex and placenta-derived gp 96-sensitized DC vaccine-activated CTL had significant killing effect on HepG2, SK-HEP-1, huh-7 and PLC/PRF/5 cells, whereas immunization with the recombinant gp 96-sensitized DC vaccine alone had substantially no killing activity.
Examples3: polypeptides-gp96Complex sensitizedDCSpecificity of activationCTLTherapeutic action on liver cancer
Taking 30 female nude mice of 6-8 weeks, subcutaneously inoculating 5×10 mice 6 HEPG2 liver cancer cells (HLA-A 2 restricted cell line); mice were divided into 3 groups on day 2 post tumor inoculation, 10 each, each treated as follows:
a first group: human placental gp 96-induced immune activation of DC vaccine by T lymphocytes from mice, fed back (0.5 ml each time) on days 2, 3, and 4, at a single dose of 10 7 Only;
second group: tail vein reinfusion recombinant gp 96-polypeptide library complex induced immune activation of DC vaccine by HLA-A2 mouse T lymphocyte, reinfusion on days 2, 3 and 4 (0.5 ml each time), single reinfusion dose of 10 7 Only;
third group: the lymphocytes of the DC vaccine immune activated HLA-A2 mice induced by the recombinant gp96 alone are infused back by tail vein, and the single infusion dose is 10 after 2, 3 and 4 days (0.5 ml each time) 7 Only;
the other three liver cancer cells SK-HEP-1 (HLA-A 24 restricted cell line), huh-7 (Huh-7 cells are HLA-A11 restricted cell line) and PLC/PRF/5 (PLC/PRF/5 are HLA-A33 restricted cell line) were inoculated as described above and the tail vein reinfusion protocol was followed.
In the above 12 groups: reinfusion was performed 2, 3, 4 days after tumor cell inoculation. Starting from the first day of reinfusion, tumor growth was observed daily, tumor size was recorded, and tumor volume was calculated according to the following formula: v=ab2/2 (V-volume, a-tumor long diameter, b-tumor short diameter).
The tumor volume changes are shown in fig. 5A-5D.
The results show that the CTL activated by the DC vaccine sensitized by the polypeptide library-recombinant gp96 complex can obviously inhibit the growth of liver tumor, and has no obvious difference with the DC vaccine sensitized by the placenta gp 96.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. A polypeptide or a combination of polypeptides selected from any one or more of the polypeptides shown in SEQ ID NOs 1-14.
2. The polypeptide or combination of polypeptides according to claim 1 comprising the polypeptides shown in SEQ ID NOs 1-14; preferably, it consists of the polypeptides shown in SEQ ID NOs 1-14.
A gp 96-polypeptide complex, wherein the polypeptide is one or more polypeptides according to any one of claims 1 to 2.
4. A gp 96-polypeptide complex according to claim 3, wherein the mass ratio of gp96 to polypeptide is from (1:5) to (5:1), preferably from (1:2) to (2:1) or from (1:1.5) to (1.5:1), more preferably 1:1, a step of;
preferably, the gp96 is a recombinant human gp96 protein; preferably, the sequence of gp96 is shown in SEQ ID NO. 64.
A gp 96-polypeptide complex combination consisting of at least two gp 96-polypeptide complexes according to any one of claims 3 to 4;
preferably, the gp 96-polypeptide complex combination consists of 14 gp 96-polypeptide complexes formed by polypeptides shown in SEQ ID NOs 1-14 and gp96 respectively;
preferably, the mass ratio between the 14 gp 96-polypeptide complexes, calculated as the mass of polypeptides therein, is from (1:5) to (5:1), preferably from (1:2) to (2:1) or from (1:1.5) to (1.5:1), more preferably 1:1.
6. a sensitized DC cell obtained from the gp 96-polypeptide complex of any one of claims 3 to 4 or the gp 96-polypeptide complex combination of claim 5 treated DC cells;
preferably, the DC cells are human DC cells or mouse DC cells;
the concentration of gp 96-polypeptide complex in the DC cell culture is 50 μg/mL to 200 μg/mL calculated as the mass of gp96 protein; preferably 80 μg/mL to 120 μg/mL; more preferably 100. Mu.g/mL.
7. An activated human T cell obtained by co-culturing the sensitized DC cell of claim 6 with a human T cell;
preferably, the activated human T cells are activated human cytotoxic T lymphocytes;
preferably, the primed DC cells are primed human DC cells;
preferably, the ratio of sensitized human DC cells to human T cells is 1:10.
8. a pharmaceutical composition comprising the polypeptide or combination of polypeptides of any one of claims 1 to 2, the gp 96-polypeptide complex of any one of claims 3 to 4, the gp 96-polypeptide complex combination of claim 5, the primed DC cell of claim 6 or the activated CTL cell of claim 7, and one or more pharmaceutically acceptable excipients.
9. A vaccine composition comprising the primed DC cells of claim 6 and a cell culture medium,
preferably, the cell culture medium is serum-free RPMI 1640;
preferably, the vaccine composition is a vaccine composition for treating or preventing liver cancer;
preferably, the liver cancer is hepatocellular carcinoma;
preferably, the liver cancer is primary liver cancer or metastatic liver cancer.
10. Use of the polypeptide of any one of claims 1 to 2 or a combination thereof, the gp 96-polypeptide complex of any one of claims 3 to 4, the gp 96-polypeptide complex combination of claim 5, the primed DC cell of claim 6 or the activated CTL cell of claim 7 in the manufacture of a medicament for the treatment or prevention of liver cancer;
preferably, the liver cancer is hepatocellular carcinoma;
preferably, the liver cancer is primary liver cancer or metastatic liver cancer.
CN202210967650.8A 2022-08-12 2022-08-12 Liver cancer related polypeptide, compound, pharmaceutical composition and application Pending CN117586353A (en)

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