CN111647606B - DC cell and CTL cell of targeted AFP whole antigen, and preparation method and application thereof - Google Patents

DC cell and CTL cell of targeted AFP whole antigen, and preparation method and application thereof Download PDF

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CN111647606B
CN111647606B CN202010780655.0A CN202010780655A CN111647606B CN 111647606 B CN111647606 B CN 111647606B CN 202010780655 A CN202010780655 A CN 202010780655A CN 111647606 B CN111647606 B CN 111647606B
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CN111647606A (en
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张欢
程铧
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Shanghai Qiyun Fangke Biotechnology Co ltd
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Beijing Yibo Puhui Biotechnology Development Co ltd
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Abstract

The invention provides a DC cell and a CTL cell of a targeted AFP holoantigen, and a preparation method and application thereof. The AFP whole antigen of the invention is a composition comprising more than two protein fragments and/or polypeptide fragments, wherein the amino acid sequence of all possible polypeptides binding to different MHC in the AFP protein is completely contained in at least one of the protein fragments or polypeptide fragments. In the invention, a gene for coding AFP holoprotein antigen is cloned to a lentiviral vector to transfect dendritic cells to obtain DC cells presenting AFP holoantigen, and the DC cells and autologous PBMC cells are mixed and cultured to obtain a large amount of CTL cells. Killing experiments show that the group of CTL cells can efficiently kill target cells carrying AFP antigens, and the killing effect of the CTL cells is positively correlated with the matching degree of HLA molecules.

Description

DC cell and CTL cell of targeted AFP whole antigen, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a DC cell and a CTL cell targeting AFP holoantigen, and a preparation method and application thereof.
Background
In recent years, cellular immunotherapy has been a great leap in cancer treatment. T cell therapy, including CAR-T, TCR-T, is the most promising approach to specific T cell therapy. The common point of the two is that T cells are used as a carrier, and the engineered T cells targeting a certain cancer antigen individual site are prepared by gene modification. To increase the specificity of T cells, finding suitable antigenic targets is the most challenging task. In addition, current clinical trial data show that T cells targeting a single site on a cancer antigen are prone to tumor cell escape in the face of highly heterogeneous solid tumor treatments, eventually leading to cancer recurrence.
The search for new antigens to prepare peptide vaccines or DC therapeutic vaccines after DC cell loading by means of next generation sequencing and computer simulation calculation has become a recent research hotspot. The method is highly dependent on computer algorithm, and the combination of the neogenetic antigen peptides obtained by different research and development teams aiming at a specific case can be greatly different, which leads to uncertainty of curative effect. Therefore, the clinical need for highly specific, multi-antigen target cell therapy methods or techniques remains urgent.
Disclosure of Invention
It is an object of the present invention to provide a novel DC cell for cellular immunotherapy.
To achieve the above objects, the present invention provides a DC cell targeting an alpha-fetoprotein (AFP) whole antigen.
Alpha-fetoprotein (AFP) is a glycoprotein derived from embryonic endoderm tissue cells. Fetal serum is very high in AFP, while adult serum is very low in AFP. AFP has many important physiological functions including transport function, bidirectional regulatory function as a growth regulator, immunosuppression, T lymphocyte induced apoptosis, etc. AFP was found to be highly expressed in liver cancer. AFP is mainly used as a serum marker of primary liver cancer clinically at present and is used for diagnosis and curative effect monitoring of the primary liver cancer. Meanwhile, AFP has become a target for liver cancer immunotherapy. Although it is reported that it is weak in immunogenicity and has an effect of inhibiting dendritic cells, NK cells and T cells, the technologies of cellular vaccine against AFP, peptide vaccine, CAR-T and TCR-T are still in clinical research. There is increasing evidence that AFP is also frequently in an elevated state in gastric cancer, colorectal cancer, pancreatic cancer and malignancies of the reproductive system, and therefore the above mentioned vaccine or technique targeting AFP may be extended in the future for application to other cancer species.
The invention firstly provides a method for targeting AFP holoprotein antigen, which divides an AFP gene sequence into more than two segments, and the amino acid sequences of all possible polypeptides combined with different MHC in the AFP protein are completely contained in the amino acid segment coded by at least one gene segment, thereby covering all possible polypeptide-MHC segments of the whole AFP protein antigen, avoiding the difficulty of the prior art in antigen target selection of cellular immunotherapy and the problem of immune escape possibly caused by a single target, and simultaneously reducing or eliminating the immunosuppressive action of the AFP holoantigen on DC cells, T cells and NK cells as much as possible. According to some embodiments of the invention, the AFP gene sequence is divided into two fragments: AFP-1 segment (the nucleotide sequence of which is shown as SEQ ID number 1) and AFP-2 segment (the nucleotide sequence of which is shown as SEQ ID number 3), the amino acid sequences of the antigen fragments coded by the segments are respectively shown as SEQ ID number 2 and SEQ ID number 4.
According to a further embodiment of the invention, DC cells are efficiently loaded with AFP whole antigen by means of lentivirus transfection, processed by digestion and presented with various polypeptide fragments of AFP, and then co-cultured with HLA partially-matched or fully-matched PBMC cells to obtain a population of AFP antigen-specific T cells, which is a composition of T cells encompassing multiple antigenic peptide sites, demonstrated to have a highly efficient killing effect.
Thus, in one aspect, the invention provides an AFP whole antigen, which is a composition comprising two or more protein fragments and/or polypeptide fragments, wherein the amino acid sequence of all possible polypeptides binding to different MHC in the AFP protein is completely comprised in at least one of said protein fragments or polypeptide fragments.
According to a particular embodiment of the invention, in the AFP whole antigen of the invention, the sum of the amino acid sequences of the individual protein fragments or polypeptide fragments covers the full-length amino acid sequence of the AFP protein, and the end of the preceding protein fragment or polypeptide fragment overlaps the beginning of the following protein fragment or polypeptide fragment by at least 9 amino acid residues, in the order of the amino acid sequence of the AFP protein.
According to a preferred embodiment of the invention, the AFP whole antigen of the invention comprises two protein fragments, the sum of the amino acid sequences of the two protein fragments covering the full-length amino acid sequence of the AFP protein, and the end of the preceding protein fragment overlaps the beginning of the following protein fragment by at least 9, preferably 9-50, more preferably 30-40 amino acid residues, in the amino acid sequence order of the AFP protein. Preferably, the former protein fragment is a fragment consisting of 280-330 amino acid residues, more preferably a fragment consisting of the amino acid sequence shown in SEQ ID number 2.
Still more preferably, the AFP whole antigen comprises a protein fragment consisting of an amino acid sequence shown in SEQ ID number 2 and a protein fragment consisting of an amino acid sequence shown in SEQ ID number 4.
According to some embodiments of the invention, the AFP autoantigen comprises a protein fragment consisting of the amino acid sequence shown in SEQ ID number 2 and a protein fragment consisting of the amino acid sequence shown in SEQ ID number 4.
In another aspect, the invention also provides a gene encoding said AFP whole antigen. That is, the gene encoding the AFP autoantigen of the present invention is a composition comprising a polynucleotide fragment encoding each protein or polypeptide fragment of the AFP autoantigen of the present invention.
According to a preferred embodiment of the invention, the gene encoding said AFP whole antigen according to the invention comprises two polynucleotide fragments, the end of the preceding polynucleotide fragment overlapping the start of the following polynucleotide fragment by at least 27bp bases, for example by 27-150, more preferably by 90-120 bases, in sequence order of the AFP gene. Preferably, the former polynucleotide fragment is a polynucleotide consisting of the sequence shown in SEQ ID number 1.
According to some embodiments of the invention, the gene encoding said AFP autoantigen of the invention is a composition comprising a polynucleotide consisting of the sequence shown in SEQ ID number 1 and a polynucleotide consisting of the sequence shown in SEQ ID number 3.
In another aspect, the invention also provides the use of an AFP autoantigen of the invention or a gene encoding the same in the preparation of AFP autoantigen loaded DC cells and/or target cells.
In another aspect, the invention also provides a DC cell (DC-AFP cell) loaded with the AFP whole antigen of the invention. The AFP whole antigen is divided into more than two sections to be simultaneously expressed in DC cells, so that the immunosuppression effect of the whole AFP protein on the DC and effector cells thereof can be reduced or avoided.
In another aspect, the invention also provides a method for preparing the AFP whole antigen loaded DC cell, comprising:
constructing a lentivirus vector containing the gene for coding the whole antigen, transfecting DC cells, and obtaining the DC cells presenting AFP whole antigen.
According to a specific embodiment of the present invention, the lentiviral vector used in the present invention may be, for example, a pCDH series, pLVX series vector.
According to a particular embodiment of the invention, the DC cells used in the invention are expandable dendritic cells. The DC cells having amplifiable properties may be obtained by commercially available DC cells having amplifiable properties, or may be prepared by the method described in the prior art, for example, by stimulating PBMC with PHA, transducing PBMC with the Tax gene of any of the STLV1-4 viruses, and screening the cells to obtain DC cells, which may be further amplified by culture. In the invention, other properties of the DC cell are not particularly required.
According to a particular embodiment of the present invention, stable, high-efficiency expression of the AFP whole antigen of the present invention in DC cells is shown. The method disclosed by the invention covers all possible polypeptide-MHC combinations of all HLA molecules of the DC cell aiming at the AFP protein through polypeptide-MHC compounds expressed, processed and formed by the DC cell, so that the DC vaccine or CTL cells generated by induction of the DC vaccine have target diversity, and the DC vaccine or the CTL cells are favorable for completely eliminating cancer cells.
The DC-AFP cell prepared by the method can be used for inducing autologous T cells and allogenic T cells. In some embodiments of the invention, two types of HLA-0201 and HLA-A2402 are identified, wherein the proportion of HLA-A0201 in the Chinese population is 18-45%, the proportion of HLA-2402 in the population is 25%, and the proportion of the population expressing both HLA-A0201 and HLA-A240 is about 11.5%. The allogeneic DC cells in the method should be applied to the population to achieve certain effects.
In some embodiments of the invention, the AFP-whole antigen-bearing DC cells of the invention can be prepared as cancer therapeutic vaccines, for the treatment of AFP-positive cancers, mainly primary liver cancer.
In some embodiments of the invention, the DC cells carrying AFP whole antigen of the invention, AFP-specific cytotoxic T cells that can be induced in vitro, are used for adoptive T cell therapy for cancer.
On the other hand, the invention also provides application of the AFP whole antigen loaded DC cell in preparing an AFP protein antigen specific immune cell composition.
In another aspect, the invention also provides an immune cell induced by the AFP whole antigen loaded DC cells of the invention.
According to some embodiments of the invention, the immune cells comprise CTL cells;
according to some embodiments of the invention, the immune cells are obtained by co-culturing the AFP whole antigen-loaded DC cells of the invention with autologous PBMC cells.
According to some embodiments of the invention, the AFP-holoantigen-loaded DC cells are co-cultured with autologous PBMC cells according to a DC cell: PBMC =1: 5-1: 500. More preferably, 200-1000 unit/ml of interleukin 2 can be added into the culture system.
In another aspect, the invention also provides the use of said immune cell in the preparation of a reagent for killing target cells carrying an AFP antigen.
In another aspect, the present invention also provides a kit comprising: the whole antigen, the gene for coding the whole antigen or the primer for amplifying the gene, the lentiviral vector containing the gene and/or the DC cell loaded with the AFP whole antigen. The kit may also include transfection reagents or the like conventional in the art.
In conclusion, the invention provides a DC cell and a CTL cell targeting AFP holoantigen, and preparation methods and applications thereof. In the invention, the AFP holoantigen is divided into more than two sections to be simultaneously expressed in DC cells, so that the immunosuppression effect of the full AFP protein on the DC and effector cells thereof is reduced or avoided. In the invention, a gene for coding AFP holoprotein antigen is cloned to a lentiviral vector and is used for transfecting a dendritic cell capable of amplifying high activity to obtain a DC cell presenting the AFP holoantigen, and the DC cell is mixed and cultured with PBMC cells from self sources to obtain a large amount of CTL cells capable of amplifying. Killing experiments show that the group of CTL cells can efficiently kill target cells carrying AFP antigens, and the killing effect of the CTL cells is positively correlated with the matching degree of HLA molecules. In the method, only two HLA-A matched target cells are selected during verification, and CTL cells generated by a DC-CTL whole body system can play a role and also comprise HLA-B, C and other sites. Thus, in theory, the killing effect produced would be higher than that already produced by the present method.
Drawings
FIG. 1 shows the results of a DC cell loaded with and expressing AFP antigen detection of the present invention.
FIG. 2 shows the results of the detection of immune cell types induced by DC-AFP.
FIG. 3 shows the results of the measurement of the number of immune cells induced by DC-AFP.
FIG. 4 shows the results of immunophenotypic identification of cells after sorting.
FIG. 5 shows the results of a target cell flow analysis experiment.
FIG. 6 shows the results of detection of AFP antigen expression by target cells.
FIG. 7 shows the results of experiments on the killing effect of CTL cells induced by DC-AFP on target cells.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. The experimental methods in the examples, in which specific conditions are not noted, are conventional methods and conventional conditions well known in the art, or conditions as recommended by the manufacturer.
Example 1 AFP antigen loaded DC cells and target cells
Target cell construction, DC cells and target cell antigen Loading
1. Designing a primer, cloning an AFP gene by PCR, and constructing a lentiviral vector
(1) According to the AFP nucleic acid sequence published in NCBI nucleotide, in the invention, two segments of AFP nucleic acid sequences with overlapping parts are designed: AFP-1 nucleic acid sequence (the nucleotide sequence is shown as SEQ ID number 1, and the coded amino acid sequence is shown as SEQ ID number 2) and AFP-2 nucleic acid sequence (the nucleotide sequence is shown as SEQ ID number 3, and the coded amino acid sequence is shown as SEQ ID number 4), and a third party is entrusted to carry out primer synthesis (the primer sequences are shown as SEQ ID number 5, SEQ ID number 6, SEQ ID number 7 and SEQ ID number 8).
AFP-1 nucleic acid sequence (SEQ ID number 1):
Agaacactgcatagaaatgaatatggaatagcttccatattggattcttaccaatgtactgcagagataagtttagctgacctggctaccatattttttgcccagtttgttcaagaagccacttacaaggaagtaagcaaaatggtgaaagatgcattgactgcaattgagaaacccactggagatgaacagtcttcagggtgtttagaaaaccagctacctgcctttctggaagaactttgccatgagaaagaaattttggagaagtacggacattcagactgctgcagccaaagtgaagagggaagacataactgttttcttgcacacaaaaagcccactccagcatcgatcccacttttccaagttccagaacctgtcacaagctgtgaagcatatgaagaagacagggagacattcatgaacaaattcatttatgagatagcaagaaggcatcccttcctgtatgcacctacaattcttctttgggctgctcgctatgacaaaataattccatcttgctgcaaagctgaaaatgcagttgaatgcttccaaacaaaggcagcaacagttacaaaagaattaagagaaagcagcttgttaaatcaacatgcatgtgcagtaatgaaaaattttgggacccgaactttccaagccataactgttactaaactgagtcagaagtttaccaaagttaattttactgaaatccagaaactagtcctggatgtggcccatgtacatgagcactgttgcagaggagatgtgctggattgtctgcaggatggggaaaaaatcatgtcctacatatgttctcaacaagacactctgtcaaacaaaataacagaatgctgcaaactgaccacgctggaacgtggtcaatgtataattcatgcagaaaatgat
AFP-1 stretch amino acid sequence (SEQ ID number 2):
RTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKITECCKLTTLERGQCIIHAEND
AFP-2 nucleic acid sequence (SEQ ID number 3):
tcctacatatgttctcaacaagacactctgtcaaacaaaataacagaatgctgcaaactgaCcacgctggaacgtggtcaatgtataattcatgcagaaaatgatgaaaaacctgaaggtctAtctccaaatctaaacaggtttttaggagatagagattttaaccaattttcttcaggggaaAaaaatatcttcttggcaagttttgttcatgaatattcaagaagacatcctcagcttgctgtCtcagtaattctaagagttgctaaaggataccaggagttattggagaagtgtttccagactgAaaaccctcttgaatgccaagataaaggagaagaagaattacagaaatacatccaggagagcCaagcattggcaaagcgaagctgcggcctcttccagaaactaggagaatattacttacaaaaTgcgtttctcgttgcttacacaaagaaagccccccagctgacctcgtcggagctgatggccaTcaccagaaaaatggcagccacagcagccacttgttgccaactcagtgaggacaaactattgGcctgtggcgagggagcggctgacattattatcggacacttatgtatcagacatgaaatgactCcagtaaaccctggtgttggccagtgctgcacttcttcatatgccaacaggaggccatgcttCagcagcttggtggtggatgaaacatatgtccctcctgcattctctgatgacaagttcatttTccataaggatctgtgccaagctcagggtgtagcgctgcaaacgatgaagcaagagtttctcAttaaccttgtgaagcaaaagccacaaataacagaggaacaacttgaggctgtcattgcagatTtctcaggcctgttggagaaatgctgccaaggccaggaacaggaagtctgctttgctgaagagggacaaaaactgatttcaaaaactcgtgctgctttgggagtttaa
AFP-2 stretch amino acid sequence (SEQ ID number 4):
SYICSQQDTLSNKITECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGV*
AFP-1 segment primer sequence:
5’cgcggatccagaacactgcatagaaatgaatat（SEQ ID No. 5）
3’gagtctagattaatcattttctgcatgaattat（SEQ ID No. 6）
AFP-2 primer sequence:
5’gcgggatcctcctacatatgttctcaacaagac（SEQ ID No. 7）
3’ tgttctagattaaactcccaaagcagcacgagt（SEQ ID No. 8）。
(2) dissolving the primers in TE buffer solution, and diluting to working concentration for later use. cDNA templates were obtained after reverse transcription with mRNA from HepG2 cells. Setting PCR system to carry out PCR to two segments of AFP gene (AFP-1 segment nucleic acid sequence and AFP-2 segment nucleic acid sequence).
(3) The PCR product was mixed with 6 × loading buffer, loaded on 1% agarose gel, 130V, 35 min. The target strip is cut and glue is recovered.
(4) The recovered gel product and the vector plasmid were digested simultaneously with BamHI and XbaI sites at 37 ℃ for 2 h.
(5) And (3) running glue on the product after enzyme digestion, respectively cutting the PCR product fragment and the carrier to obtain a large fragment, recovering the glue, and carrying out carrier addition according to the molar ratio: fragment =1: 3, connecting and standing for 2 hours at normal temperature.
(6) The ligation product was added to 50. mu.l of stbl3 and mixed well and allowed to stand on ice for 20 min. The heat was applied at 42 ℃ for 90 s. Quickly putting back on ice, and standing for 2 min.
(7) To the EP tube, 500. mu.l of LB liquid medium was added, and the mixture was placed in a shaker at 37 ℃ for 30min at 180 rpm.
(8) The transformed competent cells were removed at 5000rpm for 3 min. Most of the supernatant was discarded, and 100. mu.l of LB medium at the bottom was left to resuspend the bacteria. And transferring the bacterial liquid to an LB solid culture medium plate, and uniformly coating.
(9) Incubate at 37 ℃ overnight.
(10) The next day, monoclonal shake bacteria were picked.
(11) Collecting thalli, and extracting plasmids according to the instruction of the plasmid miniextraction kit.
(12) The resulting plasmid, along with a commercial pCDH lentiviral backbone vector, was double digested with BamH1 and XbaI, respectively, and verified by running the gel.
(13) Taking the clone plasmid with the correct band position, and sending the clone plasmid to a third party for sequencing.
(14) After the sequencing shows that the sequence is correct, extracting the target plasmid greatly for later use.
2. Packaging and concentrating molecular lentivirus such as AFP antigen, luciferase (luciferase), HLA
(1) Recovering 293 cells, and culturing and subculturing.
(2) One day before transfectionPancreatin digestion according to 5X106The cells/dish were seeded in 10cm dishes and incubated overnight at 37 ℃.
(3) Transfection: and mixing the lentivirus packaging plasmid and the target gene vector according to the mass ratio of 1:1, adding a transfection reagent according to the mass ratio of plasmid =3:1, and standing at room temperature for 30 min.
(4) Replacing the culture medium in the culture dish with the RPMI1640 culture medium, and gently adding the transfection mixed solution into the culture dish and gently mixing the transfection mixed solution and the culture dish.
(5) After 4 hours, the transfection supernatant was discarded and replaced with FBS-containing medium to continue cell culture.
(6) The viruses were collected at 24 hours, 48 hours and 72 hours, respectively.
(7) Adding the virus concentrated solution, uniformly mixing, and standing overnight in a refrigerator at 4 ℃.
(8) 3000rpm, 4 ℃, 1 hour, abandoning the supernatant, adding fresh culture medium according to the amount of 1/10 of original volume to resuspend the virus, subpackaging and storing in a refrigerator at-80 ℃.
3. Target cell construction, DC cells and target cells loaded with AFP antigen
(1) NIH3T3 cells at 5X105Individual cells were seeded in 6-well plates and attached for 3 hours. Preparing 500 mu l of concentrated luciferase virus solution, 500 mu l of complete culture medium and 1 mu l of polybrene (polybrene) mixed solution with the concentration of 10mg/ml, and adding the mixed solution into cells for culturing overnight;
(2) the next morning, cells were cultured by changing fresh medium. In the afternoon, the medium was replaced again and secondary transfection with luciferase virus mixture was continued.
(3) Beta 2M virus was transfected again according to the procedures (1) and (2) to give 3T3L cells.
(4) Digested and according to 5X1053T3L cells were inoculated into 6-well plates, and were transfected with HLA-2402, HLA-0201, and HLA2402+ HLA A0201 viruses, respectively, 3 times, and HLA molecule expression was detected by flow assay.
(5) Respectively taking 2-5 × 106DC cells (Celartics-DC cells) or 5X 10T5Target cells (3T 3L, 3T3L HLA-A2402, 3T3L HLA-A0201 and 3T3L HLA-A224) Seeded in 6-well plates.
(6) Prepare the AFP virus liquid after concentration and 1 mul 10mg/ml polybrene mixed liquid, add into the cell and culture overnight.
(7) The next morning, cells were cultured by changing fresh medium. In the afternoon, the medium was replaced again and secondary transfection with AFP virus mixture was continued.
(8) And (5) repeating the step (6) and culturing the cells to a sufficient number.
(9) Prepare 10% SDS PAGE gel for use.
(10) Target cells were collected after lentiviral transfection, while 3T3L cells before AFP transfection were collected as controls.
(11) Adding a certain proportion of protein lysate according to the total amount of the cells, and cracking the cells on ice.
(12) The protein after cracking is boiled at the high temperature of 95 ℃ for 20min for standby.
(13) Protein loading, 80V, 30 min. 120V, 60 min.
(14) And (5) rotating the membrane, 300mA and 60 min.
(15) The primary antibody was incubated on a shaker at 4 ℃ overnight.
(16) The next day, PBST washed the membrane 3 times, incubated with secondary antibody, room temperature, 1 hour.
(17) PBST thin membrane 3 times. Adding ECL luminous liquid and exposing.
(18) And (5) data sorting and analyzing.
In this example, to verify the expression of DC loaded with AFP antigen, a portion of primitive DC cells and antigen-transferred DC cells were lysed and the expression of the AFP protein in the cells was detected by western blot. The results, shown in FIG. 1, show that DC cells express AFP protein (the two polypeptide sequences of AFP are similar in size and cannot be distinguished clearly in the figure), and can be used for subsequent validation.
The results of flow analysis of HLA molecule expression in target cells are shown in figure 5. After the NICH 3T3 is sequentially put into the Luciferase gene and the beta 2M gene, the genes such as HLA2402, HLA0201 and the like are respectively or jointly put into the genes, and the expression conditions of the HLA-A2402 and the HLA-A0201 are detected by flow analysis. As can be seen, all target cells expressed HLA-2402 molecules and/or HLA-A0201 molecules after lentivirus transfection.
In order to verify the AFP antigen expression of target cells, a part of 3T3L series target cells are taken, and after cracking, the expression condition of the AFP protein in each cell is detected by western blot. Results referring to fig. 6, the results show that each target cell expresses AFP protein, and that the expression level of AFP between target cells is comparable, which can be used for subsequent validation.
Example 2 DC-AFP Induction of expansion of immune cells
In the implementation, the DC-AFP is adopted to induce and amplify immune cells, and AFP protein antigen specific CTL cells are prepared. The preparation process mainly comprises the following steps:
(1) antigen-loaded DC cells and autologous PBMC cells were taken and counted.
(2) Using X-VIVO medium containing 5% human serum, as per DC cells: PBMC =1:5 to 1:500, and the DC cells and PBMC cells were mixed and cultured overnight under static conditions.
(3) And on the next day, adding 200-1000 unit/ml interleukin 2 into the culture system, and mixing and culturing.
(4) According to the growth condition of the cells, a proper amount of culture medium and interleukin 2 are added.
(5) On days 12-14, a portion of the cells were collected for flow analysis.
(6) Taking 1-5X 105Cells were centrifuged at 2500rpm for 5 min.
(7) PBS wash 2 times, 2500rpm, 5min centrifugation.
(8) 100ul of blocking solution containing BSA was added to the cells and blocked on ice for 10-15 min.
(9) The cells were split into two aliquots, one to which was added the CD3+ CD56 antibody and the other to which was added an isotype control antibody for both antibodies, and incubated on ice for 30 min.
(10) PBS wash 3 times, 2500rpm, 5 min.
(11) 500ul PBS resuspended cells, filtered and detected by flow cytometry.
(12) When the detection result shows that the number of cells in the CD3-CD 56-region is less than 3 percent, collecting a part of cells to carry out magnetic bead sorting, continuing culturing another part of cells for 21 days, and recording the expansion multiple of immune cells.
(13) According to the cell density and the proportion of CD3 cells, enough cells were taken and placed in a 15ml centrifuge tube at 1000rpm for 3 min.
(14) Discard the supernatant, wash with PBS, 1000rpm, 3 min.
(15) Discard the supernatant, suspend the cells in 500ul X-VIVO15 medium, add 100ul CD3 magnetic beads, and mix well.
(16) The cell suspension was placed on a shaker and run at room temperature for 15min at low speed.
(17) Adding 5ml PBS into the cell suspension, fully reversing and uniformly mixing, placing on a magnetic frame, and standing for 2 min.
(18) The supernatant was carefully transferred to a clean 15ml centrifuge tube, the original tube removed from the magnetic rack, 5ml PBS was added and the beads washed by inversion.
(19) Repeat (18) step 1 time.
(20) The beads were resuspended in X-VIVO15 medium containing 5% human serum, 500unit/ml IL2 was added, and the cells were incubated overnight in 6-well plates.
(21) And collecting the magnetic beads in the pore plate in a centrifugal tube, fully blowing the magnetic beads, and putting the magnetic beads in a magnetic force to separate the T cells from the magnetic beads.
(22) Supernatants were collected and pooled, 1000rpm, 5 min.
(23) And continuously culturing the cells in the X-VIVO15 culture medium containing 5% human serum for 3-5 days to obtain the T cells.
(24) After the T cells are sufficient in number, they are used for subsequent studies.
In this example, immune cell types induced by DC-AFP were examined. The DC-AFP cells (HLA-A0201 + HLA-A2402) and autologous PBMC cells are co-cultured according to the ratio of 1:300, recombinant IL2 is added into the culture medium according to the ratio of 500unit/ml, a small amount of cells are taken for flow analysis on the 14 th day and the 21 st day after induction, and the types of the amplified immune cells are detected. The results are shown in figure 2, at day 14, the immune cells contained CD3+ T cells, CD56+ NK cells, and a portion of CD3+ CD56+ cells. At day 21, the proportion of NK cells increased significantly and the cell status was good.
In this example, DC-AFP cells were mixed with autologous PBMC cells according to a 1: the culture was carried out at a ratio of 300 in a medium of X-VIVO15 containing 5% autologous plasma, and in which IL2 was added at a ratio of 500unit/ml per day based on the total volume of the medium. The experimental results are shown in figure 3, and the results show that the immune cells are well proliferated, full and healthy in cell morphology under the culture system. On day 21, cells expanded more than 1000-fold.
In this example, DC-AFP was co-cultured with PBMC at a ratio of 1:300 in a medium of X-VIVO15 containing 5% autologous plasma, and the culture was performed by adding IL2 at a concentration of 500unit/ml per day based on the total volume of the medium. By day 11, a portion of the immune cells were taken and sorted with CD3 magnetic beads to obtain T cells. The T cells on the beads were isolated and cultured, and the purity of the sorted T cells was measured by flow assay on day 14 of the western blot. As can be seen from FIG. 4, the proportion of CD3+ cells after sorting was 95%, which was useful for subsequent studies.
Example 3 killing of target cells by AFP antigen-specific CTL cells
In this example, the killing effect of AFP antigen-specific CTL cells on target cells was examined. The related method comprises the following operations:
(1) digesting target cells 3T3L/AFP, 3T3L/2402/AFP and 3T3L/224/AFP, counting, inoculating the cells/hole of 5x104 cells/hole into a 24-well plate, and attaching the cells to the wall for 2-3 hours;
(2) cell count, according to effector cell: adding effector cells into target cells at the ratio of 1:1,2:1,5:1 and 10:1 respectively, and performing mixed culture for 4 hours;
(3) the suspended T cells are aspirated, the cells are washed gently with PBS for 1-3 times to remove floating dead target cells, and the liquid in the pores is aspirated;
(4) adding 100 mul of cell lysis solution into each well, and carrying out ice lysis to release luciferase protein from the cells;
(5) transferring the lysate to a 1.5ml EP tube, centrifuging at 3000rpm for 3 min;
(6) adding 100 mul of luciferase reporter gene substrate into an enzyme label plate, and respectively adding 20 mul of lysate supernatant into the substrate;
(7) and (3) detecting the sample on a machine, and calculating the killing efficiency of the target cells according to the fluorescence intensity.
According to the technical scheme, the 3T3L cells can be used for detecting the killing effect of the T cells after expression detection of AFP, HLA A0201 and HLA-A2402. According to the effective target ratio of 1:1,2:1,5:1 and 10:1, the results of the killing test of the three target cells are shown in fig. 7, the killing effect of the CTL cells on the 3T3L/AFP cells which do not express HLA molecules is weaker, and the effective target ratio is 10: killing under condition 1 may be caused by a small number of NK cells in the CTL cells. CTL cells exhibit strong killing against HLA-a2402 and AFP expressing target cells, 10: the killing effect under the proportion of 1 can reach 70 percent, and the killing effect is strong. Furthermore, when HLA-A2402 and HLA-0201 are expressed simultaneously, it is found that the killing effect of CTL cells on target cells is stronger, and the statistical difference is provided. This result indicates that the higher the number of HLA molecule matches, the stronger the killing effect of CTL cells.
Sequence listing
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Claims (9)

1. An AFP whole antigen loaded DC cell prepared by a method comprising the steps of:
two vectors were constructed: a lentiviral vector into which a gene encoding a protein fragment consisting of the amino acid sequence shown in SEQ ID number 2 is inserted, and a lentiviral vector into which a gene encoding a protein fragment consisting of the amino acid sequence shown in SEQ ID number 4 is inserted;
co-transfecting the two vectors to amplify the DC cell to obtain the DC cell presenting AFP whole antigen.
2. The AFP full antigen-loaded DC cell according to claim 1, wherein the gene encoding the protein fragment consisting of the amino acid sequence represented by SEQ ID number 2 is a polynucleotide consisting of the sequence represented by SEQ ID number 1, and the gene encoding the protein fragment consisting of the amino acid sequence represented by SEQ ID number 4 is a polynucleotide consisting of the sequence represented by SEQ ID number 3.
3. A method of preparing a DC cell loaded with AFP autoantigen according to claim 1 or 2, the method comprising:
two vectors were constructed: a lentiviral vector into which a gene encoding a protein fragment consisting of the amino acid sequence shown in SEQ ID number 2 is inserted, and a lentiviral vector into which a gene encoding a protein fragment consisting of the amino acid sequence shown in SEQ ID number 4 is inserted;
co-transfecting the two vectors to amplify the DC cell to obtain the DC cell presenting AFP whole antigen.
4. Use of an AFP whole antigen loaded DC cell according to claim 1 or 2 for the preparation of an immune cell composition specific for an AFP protein antigen.
5. An immune cell induced by the AFP whole antigen loaded DC cells of claim 1 or 2, said immune cell comprising CTL cells, wherein the cellular proportion of CD3+ is up to 95%.
6. The immune cell of claim 5, which is obtained from co-culturing the AFP whole antigen-loaded DC cell of claim 1 or 2 with autologous PBMC cells; when the AFP holoantigen-loaded DC cells and autologous PBMC cells are co-cultured, the ratio of the DC cells: PBMC =1: 5-1: 500.
7. The immune cell according to claim 6, wherein when the AFP full antigen-loaded DC cell is co-cultured with autologous PBMC cell, interleukin 2 of 200-1000 unit/ml is added into the culture system for mixed culture.
8. Use of an immune cell according to any one of claims 5 to 7 in the preparation of a medicament having the ability to kill target cells bearing an AFP antigen.
9. A kit, comprising: the AFP whole antigen-loaded DC cell of claim 1 or 2.
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