CN113106062A - Co-culture method of tumor neogenesis antigen specific tumor infiltrating lymphocytes - Google Patents
Co-culture method of tumor neogenesis antigen specific tumor infiltrating lymphocytes Download PDFInfo
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Abstract
The invention relates to the technical field of cell culture, in particular to a co-culture method of tumor neoantigen specific tumor infiltrating lymphocytes, which is characterized by comprising the following steps: s1, preparing a tumor neoantigen: the tumor cell is separated from the lung cancer (N1), pancreatic cancer (N2), bile duct cancer (N3), triple negative breast cancer (N4) and melanoma tumor (N5) tissues, the normal tissues of corresponding individuals are taken, sequencing is carried out by using a whole exome, the sequencing results of the normal tissues and the tumor tissues are compared to determine all amino acid mutation, then a gene construct containing all mutant peptide sequences is designed, and the long-chain polypeptide is synthesized according to the neoantigen coded by the mutant gene.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a co-culture method of tumor neoantigen specific tumor infiltrating lymphocytes.
Background
Tumor infiltrating lymphocyte immunotherapy, as early as 86 years, Steve Rosenberg of the National Cancer Institute (NCI) was developed and used for treating partial solid tumors (about 90% of cancers worldwide are solid tumors), clinical research results in 2019 show that the ORR (objective response rate) of 66 patients with unresectable melanoma is 38% (2 CR, 23 PR) and the DCR (disease control rate) is 80%, and the clinical efficacy of TIL therapy in melanoma, cervical cancer, head and neck cancer, lung cancer, breast cancer, cholangiocarcinoma and colorectal cancer is remarkable, and typical cases are as follows: 1 of the advanced breast cancers, 42 months without tumor survival, 1 of the advanced bile duct cancer with ineffective chemotherapy, survived for 10 years, 6 of 7 metastatic foci of the lung disappeared after 1 colorectal cancer case TIL treatment, the disease did not progress after 4 years, and 2 of 13 patients receiving TIL treatment, 2 of the non-small cell lung cancers with cancer progress after PD-1 treatment of tumors, were Completely Relieved (CR) for one year.
Gene mutations are often considered to be the root cause of cancer induction, for example, KRAS gene mutation is very persistent and few drugs can be reduced, and 22% of cancer patients have KRAS gene mutation, especially pancreatic cancer (68%), cholangiocarcinoma (27%) and lung cancer (17%), but TIL immune cell therapy brings eosin, so that chemotherapy-losers and newborn patients are obtained, so that Lovance, Calif., the U.S. Food and Drug Administration (FDA) awards LN-145, a candidate therapy, a breakthrough treatment prescription for treating recurrent metastatic or persistent cervical cancer after chemotherapy.
Previous researches show that TIL cells of all tumor species except melanoma have high specific recognition capability, the national TIL research follows the international trend in the nineties, although the research is early, most clinical researches do not achieve the expected effect for various reasons, the reasons are the same, the sampling is difficult (some tumor tissue samples are easy to be polluted, such as intestinal cancer is polluted by escherichia coli), the amount of the TIL cells separated from the tumor tissue is small (most of the TIL cells are collagen), the amplification time is long (more than 50 days), the cell amplification rate and the tumor killing activity are obviously reduced due to the long amplification time, the recognition capability of the TIL cells of most tumor sources is low, researchers additionally irradiate autologous tumor cells (used as antigens) with 100Gy into a culture system, the expansion rate of TIL cells and the killing activity to autologous tumor cells are obviously improved, and the original high tumor inhibition level can be restored.
In 5 months 2014, Science reported that a malignant advanced bile duct cancer patient was successfully cured by using in vitro expanded lymphocytes (TILs) capable of specifically recognizing cancer cells. After various chemotherapy schemes, the disease of the extremely refractory bile duct cancer still rapidly progresses, and the disease participates in a TIL cell treatment clinical test of the American national cancer center, a lung metastasis is taken out of the patient, a sufficient number of TIL cells are obtained, a certain number of tumor tissues and normal tissues are taken, complete exon gene sequencing comparison is carried out, 26 meaningful gene mutations are found, abnormal protein fragments caused by all the gene mutations are used for artificial synthesis, lymphocytes (TIL) separated from the tumor tissues of the patient are co-cultured with cells carrying the abnormal protein fragments, a part of T cells are found to be capable of identifying one of abnormal protein ERBB2IP and are re-infused into the body of the patient through in vitro activation and amplification, the TIL cells with specific identification capacity account for about 25 percent in the infusion cells in the first treatment course, the tumor of the patient begins to shrink after the cell infusion, after the disease condition is stabilized for 18 months, the tumor progresses again, a second treatment course is required, the TIL cells sampled again have the specific recognition capacity of 95%, all focuses in the body of the patient are found after cell feedback, the focus is detailed and reduced again, after a period of time, the focus is finally completely relieved, and then the curative effect of conventional immune Cells (CIK) is continued to be stabilized for years.
Tumor neoantigens (neoantigen) are ubiquitous in tumor cells: in the process of tumorigenesis, cancer cells generate a plurality of gene mutations, part of the gene mutations are not contained in normal tissues and cells, and the proteins can activate an immune system to cause the attack of the immune system to the cancer cells; therefore, the curative effect is not obvious, the tumor neoantigen is a somatic mutation product, does not exist in normal tissues and cells, has high immunogenicity and specificity, the preparation process of individual neoantigen vaccines is gradually systematized, the neoantigen is identified, firstly, the whole exon sequencing is used for comparing gene sequences of cancer and normal tissues to obtain cancer tissue mutation gene data, proper mutation neoantigens are selected according to the gene expression level, the bioinformatics or artificial intelligence algorithm is used for predicting the adhesion affinity of candidates and Human Leukocyte Antigens (HLA), therefore, the candidate antigen sequences and finally the neoantigens are selected, the neoantigens coded by the mutation genes are synthesized into long-chain polypeptide vaccines, the immunogenicity is further enhanced by being matched with immune adjuvants, the immune cells can be induced to have more targets, no cancer species restriction and immunological memory efficacy, and the prior method for culturing TIL cells lacks a target, the specificity of the compound is utilized to effectively kill autologous tumor target cells, and the compound can induce immune cells to have multiple target spots, has no cancer species limitation and has the immunological memory effect.
In conclusion, the present invention solves the existing problems by designing a tumor neoantigen specific tumor infiltrating lymphocyte co-culture method.
Disclosure of Invention
The invention aims to provide a co-culture method of tumor neoantigen specific tumor infiltrating lymphocytes, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a co-culture method of tumor neogenesis antigen specific tumor infiltrating lymphocytes comprises the following steps:
s1, preparing a tumor neoantigen: tumor cells are respectively taken from lung cancer (N1), pancreatic cancer (N2), bile duct cancer (N3), triple negative breast cancer (N4) and melanoma tumor (N5) tissues, normal tissues of corresponding individuals are taken, sequencing is carried out by using a whole exome, sequencing results of the normal tissues and the tumor tissues are compared to determine all amino acid mutations, then a gene construct containing all mutant peptide sequences is designed, long-chain polypeptides are synthesized according to neoantigens coded by the mutant genes, the mutant peptides are presented on a Major Histocompatibility Complex (MHC) of lymphocytes, then the sequences are combined into serial minigenes and inserted into TIL cells, and the synthesized tumor neopeptide antigens are respectively: n1 tumor neogenin peptide antigen, N2 tumor neogenin peptide antigen, N3 tumor neogenin peptide antigen, N4 tumor neogenin peptide antigen, N5 tumor neogenin peptide antigen;
s2, co-culture of TIL cells with tumor neoantigen:
1) extracting and activating TIL cells from tumor tissues;
2) large-volume culture expansion of IL cells;
3) harvested TIL cell assay: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
4) and (3) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 80-100 percent;
5) memory T cell assay in TIL cells: not less than 65 percent
As a preferred embodiment of the present invention, the steps of extracting TIL cells from tumor tissue and activating three dimensions in S2 are as follows:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the fragments into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method;
s02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN1 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
as a preferred embodiment of the present invention, the specific steps for large-volume culture and expansion of TIL cells in S2 are as follows:
s06: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s07: transferring to a T175 culture flask X1 on the 5 th day;
s08: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s09: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s10: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s11: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s12: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s13: cells can be collected at day 30, and the total amount of TIL cells is 5 × 1010 or more.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the TIL cells obtained by designing the co-culture method of the tumor neoantigen specific tumor infiltrating lymphocytes can effectively kill autologous tumor target cells, can induce multiple target spots of immune cells, has no cancer species limitation and has the immunological memory effect.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the protection scope of the present invention.
The invention provides a technical scheme that:
a co-culture method of tumor neogenesis antigen specific tumor infiltrating lymphocytes comprises the following steps:
s1, preparing a tumor neoantigen: tumor cells are respectively taken from lung cancer (N1), pancreatic cancer (N2), bile duct cancer (N3), triple negative breast cancer (N4) and melanoma tumor (N5) tissues, normal tissues of corresponding individuals are taken, sequencing is carried out by using a whole exome, sequencing results of the normal tissues and the tumor tissues are compared to determine all amino acid mutations, then a gene construct containing all mutant peptide sequences is designed, long-chain polypeptides are synthesized according to neoantigens coded by the mutant genes, the mutant peptides are presented on a Major Histocompatibility Complex (MHC) of lymphocytes, then the sequences are combined into serial minigenes and inserted into TIL cells, and the synthesized tumor neopeptide antigens are respectively: n1 tumor neogenin antigen, N2 tumor neogenin antigen, N3 tumor neogenin antigen, N4 tumor neogenin antigen, N5 tumor neogenin antigen.
S2, co-culture of TIL cells with tumor neoantigen:
1) extracting and activating TIL cells from tumor tissues, wherein the three-dimensional concrete steps of extracting and activating the TIL cells from the tumor tissues are as follows:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the fragments into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method;
s02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN1 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
2) large-capacity culture expansion of TIL cells, wherein the large-capacity culture expansion of TIL cells comprises the following specific steps:
s06: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s07: transferring to a T175 culture flask X1 on the 5 th day;
s08: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s09: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s10: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s11: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s12: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s13: collecting cells at 30 days, wherein the total amount of TIL cells is more than or equal to 5 multiplied by 1010;
3) harvested TIL cell assay: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
4) and (3) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 80-100 percent;
5) memory T cell assay in TIL cells: not less than 65 percent.
Detailed description of the preferred embodiments
Example 1
(I) co-culture of TIL cells with tumor neoantigen N1:
the extraction and activation of TIL cells from tumor tissue comprises the following steps:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the tissue blocks into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method.
S02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN1 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
(II) large-volume culture expansion of TIL cells:
s201: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s206: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s207: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s208: cells can be collected at day 30, and the total amount of TIL cells is 5 × 1010 or more.
(III) assay of harvested TIL cells:
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III.
Fourthly) detecting that the TIL cells kill autologous tumor target cells, wherein the killing rate is 95 percent when the TIL cells act for 24 hours at the ratio of 10:1 (effective target ratio);
and (V) detecting memory T cells in the TIL cells, wherein the detection result is more than or equal to 65 percent.
Example 2
(I) co-culture of TIL cells with tumor neoantigen N2:
the extraction and activation of TIL cells from tumor tissue comprises the following steps:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the tissue blocks into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method.
S02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN2 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
(II) large-volume culture expansion of TIL cells:
s201: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s206: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s207: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s208: cells can be collected at day 30, and the total amount of TIL cells is 5 × 1010 or more.
(III) assay of harvested TIL cells:
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III.
(IV) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 96 percent;
and (V) detecting memory T cells in the TIL cells, wherein the detection result is more than or equal to 92 percent.
Example 3
(I) co-culture of TIL cells with tumor neoantigen N3:
the extraction and activation of TIL cells from tumor tissue comprises the following steps:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the tissue blocks into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method.
S02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN3 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
(II) large-volume culture expansion of TIL cells:
s201: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s206: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s207: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s208: cells can be collected at day 30, and the total amount of TIL cells is 5 × 1010 or more.
(III) assay of harvested TIL cells:
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III.
(IV) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 92 percent;
(V) memory T cell assay in TIL cells: more than or equal to 88 percent.
Example 4
(I) co-culture of TIL cells with tumor neoantigen N4:
the extraction and activation of TIL cells from tumor tissue comprises the following steps:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the fragments into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method;
s02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN4 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
(II) large-volume culture expansion of TIL cells:
s201: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s206: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s207: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s208: cells can be collected at day 30, and the total amount of TIL cells is 5 × 1010 or more.
(III) assay of harvested TIL cells:
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III.
(IV) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 99 percent;
(V) memory T cell assay in TIL cells: not less than 65 percent.
Example 5
(I) co-culture of TIL cells with tumor neoantigen N5:
the extraction and activation of TIL cells from tumor tissue comprises the following steps:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the fragments into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method;
s02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN5 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
(II) large-volume culture expansion of TIL cells:
s201: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s206: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s207: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s208: collecting cells at 30 days, wherein the total amount of TIL cells is more than or equal to 5 multiplied by 1010;
(III) assay of harvested TIL cells:
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
(IV) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 90 percent;
(V) memory T cell assay in TIL cells: not less than 65 percent.
Example 6
(I) co-culture of peripheral blood lymphocyte cells and tumor polyneoantigen N1-5:
the isolation and activation of lymphocytes (TIL-cell-enriched) from the peripheral blood of a patient after completion of a course of TIL cell therapy comprises the steps of:
s01: drawing 50ml of anticoagulated peripheral blood: adopting 1ml of heparin sodium with 12,500 units for anticoagulation, and removing sodium citrate for anticoagulation; collecting lymphocyte-enriched cells (TILs) by density gradient centrifugation;
s02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: 30ml plasma (without inactivation) was collected in the density gradient tube;
s04: putting the separated lymphocytes (3-5 multiplied 107) into a coated T75 culture bottle, adding 30ml of complete culture solution (the AIM-V serum-free culture medium contains 0.1mgN1-5 tumor nascent peptide antigen, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% of autologous plasma (the cell factors are all 1000U/ml in a centrifuge tube);
s05: after 5 days, 0.1mgN1-5 tumor nascent peptide antigen is supplemented;
(II) large-volume culture expansion of TIL cells:
s201: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on the 10 th day, wherein the total amount of the supplementary solution reaches 1000 ml;
s205: transferring into culture bags with the volume of 2000ml multiplied by 2 at the 14 th day, wherein the total amount of the supplementary liquid in each bag reaches 1000 ml;
s206: on day 17, the total amount of the nutrient solution in each culture bag reaches 4000ml multiplied by 4;
s207: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 8000ml of culture bag multiplied by 4;
s208: cells can be collected at day 25, and the total amount of TIL cells is 5 × 1010 or more.
(III) assay of harvested TIL cells:
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
(IV) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 85 percent;
(V) memory T cell assay: not less than 65 percent.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (3)
1. A co-culture method of tumor neoantigen specific tumor infiltrating lymphocytes is characterized by comprising the following steps:
s1, preparing a tumor neoantigen: tumor cells are respectively taken from lung cancer (N1), pancreatic cancer (N2), bile duct cancer (N3), triple negative breast cancer (N4) and melanoma tumor (N5) tissues, normal tissues of corresponding individuals are taken, sequencing is carried out by using a whole exome, sequencing results of the normal tissues and the tumor tissues are compared to determine all amino acid mutations, then a gene construct containing all mutant peptide sequences is designed, long-chain polypeptides are synthesized according to neoantigens coded by the mutant genes, the mutant peptides are presented on a Major Histocompatibility Complex (MHC) of lymphocytes, then the sequences are combined into serial minigenes and inserted into TIL cells, and the synthesized tumor neopeptide antigens are respectively: n1 tumor neogenin peptide antigen, N2 tumor neogenin peptide antigen, N3 tumor neogenin peptide antigen, N4 tumor neogenin peptide antigen, N5 tumor neogenin peptide antigen;
s2, co-culture of TIL cells with tumor neoantigen:
1) extracting and activating TIL cells from tumor tissues;
2) large-volume culture expansion of TIL cells;
3) harvested TIL cell assay: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
4) and (3) detecting the killing of autologous tumor target cells by TIL cells: 10:1 (effective target ratio) for 24 hours, and the killing rate is 80-100 percent;
5) memory T cell assay in TIL cells: not less than 65 percent.
2. The method of claim 1, wherein the steps of extracting TIL cells from tumor tissue and activating three dimensions in S2 are as follows:
s01: obtaining tissues by operation, separating tissue blocks into 1mm fragments by using surgical scissors in a sterile culture dish, putting the fragments into a 50ml centrifugal tube, adding 25ml of 0.05 percent collagenase II type and 0.002 percent DNA enzyme solution I, digesting for 2-8 hours, removing undigested tissue blocks by using a sterile filter screen, cleaning and centrifuging for 2 times, and collecting a lymphocyte (TIL) layer and a tumor cell layer by using a Ficoll density gradient centrifugation method;
s02: coating a T75 culture bottle with 8ml of 'CD 3 monoclonal antibody (50 mu g)' for 24 hours at 4-8 ℃, and sucking out a coating solution for later use;
s03: separating 20ml of peripheral blood to obtain plasma (without inactivation);
s04: putting TIL cells (3-5 multiplied by 106) into a coated T75 culture bottle, adding 30ml of complete culture solution (0.1 mg of N1 tumor nascent peptide antigen in AIM-V serum-free culture medium, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human stem cell growth factor (SCF) adenosine disodium triphosphate injection (ATP), coenzyme A for injection and 10% of autologous plasma (the cell factors are all 1000U/ml in a centrifugal tube);
s05: after 5 days, 0.1mgN1-5 tumor neogenin antigen was added.
3. The method for co-culturing the tumor neoantigen-specific tumor-infiltrating lymphocytes according to claim 1, wherein the large-capacity culture and expansion of the TIL cells in S2 comprises the following steps:
s06: induced cell replacement 30ml complete medium and 10% autologous plasma after 72 hours;
s07: transferring to a T175 culture flask X1 on the 5 th day;
s08: transferring to a T175 culture flask multiplied by 2 on the 10 th day;
s09: transferring to a T175 culture flask multiplied by 4 on day 14, wherein the total amount of the supplementary solution reaches 1000 ml;
s10: transferring into culture bags with volume of 2000ml multiplied by 2 on day 17, wherein the total amount of the supplementary liquid in each bag is 1000 ml;
s11: the total amount of the fluid supplement of each culture bag on the 20 th day reaches 4000ml culture bags multiplied by 4;
s12: on the 25 th day, the total amount of the fluid supplement of each culture bag reaches 8000ml of culture bag multiplied by 4;
s13: cells can be collected at day 30, and the total amount of TIL cells is 5 × 1010 or more.
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