CN116948967A

CN116948967A - Genetically modified dendritic cell and application thereof

Info

Publication number: CN116948967A
Application number: CN202310901890.2A
Authority: CN
Inventors: 丁平; 蒋金凤; 雷霆钧; 付婷婷; 徐柳
Original assignee: Sichuan Kantsai Medical Technology Co ltd
Current assignee: Sichuan Kantsai Medical Technology Co ltd
Priority date: 2022-07-21
Filing date: 2023-07-21
Publication date: 2023-10-27
Also published as: CN117089527A

Abstract

The present invention relates to a genetically modified dendritic cell and uses thereof, the genetic modification comprising introducing into the dendritic cell an RNA sequence encoding a tumor antigen comprising an ovarian, liver, breast, sarcoma and/or acute leukemia associated antigen. The genetically modified dendritic cells are useful as pharmaceutical compositions for treating tumors and are useful inAcclimatization of CD8 ⁺ T cells, further, acclimatized CD8 ⁺ T cells can be used to prepare tumor-bearing non-human animal models or drugs.

Description

Genetically modified dendritic cell and application thereof

The present invention claims that in China, the application number is "2022108689483", the application date is 2022, month 07, and the application of the invention name is "a genetically modified tumor cell line, a method for constructing a humanized animal tumor model, and an application thereof", which are all incorporated in the present invention as part of the present invention as priority.

Technical Field

The invention belongs to the field of animal genetic engineering, and in particular relates to a genetically modified tumor cell line, a construction method of a humanized animal tumor model and application of the humanized animal tumor model in the field of medical biology.

Background

With the progressive control of diseases such as infectious diseases, the incidence of malignant tumors (cancers) has become relatively prominent and increased in recent years, and the number of people dying from malignant tumors has increased significantly. Treatment of malignant tumors generally includes surgical treatment, anticancer drug treatment, radiation treatment, biological treatment, and combination therapy of traditional Chinese and western medicine.

Immunotherapy in biological therapy represents a significant leap forward in successful treatment of cancer, with unprecedented long-term survival rates under different indications. However, many patients still cannot benefit from these immunotherapies, resulting in increased attention to identify new immunotherapies or combinations that can prolong or alter the patient's response. For this reason, there is an increasing need for more predictive preclinical models to drive rational immunotherapeutic drug/combination development and minimize clinical trial failure. Mouse animal models have long been a key tool in biomedical research. Four main methods for evaluating mouse models of immunotherapy are: (1) Isogenic mouse tumor model with fully immunocompetent host; (2) a genetically engineered mouse model; (3) a chemical induction model; (4) other models. Although the first three methods are widely used, one major disadvantage is that they rely on the murine immune system, which however does not fully reproduce the human immune response. Thus, there is a great need to reproduce preclinical models of the functional human immune system.

The humanized mouse model for cancer immunotherapy studies includes three elements: (1) an immunodeficient host mouse; (2) human immune cells; (3) human tumor cells. Recently, a variety of humanized mice have been developed that can study the human immune system in vivo. The most commonly used humanized mouse model is Il2rγ ^null Mouse strains, e.g. NOG (NOD/Shi-Prkdc ^scid Il2rγ ^tm1Sug Mice or NSG (NOD/Ltsz-Prkdc) ^scid Il2rγ ^tm1Wjl J) mice, transplanted human tissue, human hematopoietic cells (HSCs) or human Peripheral Blood Mononuclear Cells (PBMC) reconstitute the human immune system. The simplest and most economical method is to transplant human PBMCs into immunodeficient mice. However, there is severe xenograft versus host disease (xeno-GVHD) in these mice, all transplanted T cells are activated by GVHD, and finally all mice die from xenogeneic GVHD, resulting in a limited experimental window, which makes long-term and accurate analysis of human immune responses difficult. To avoid xenogeneic GVHD in Hu-PBMC mice, different strategies were developed based on genetic manipulation of MHC molecules. Transplantation of PBMCs into NSG or NOG mice knocked out of MHC class I and class II molecules reduces and prolongs xenogeneic GvHD production. Researchers have also found that CD4 in PBMC was removed prior to implantation ⁺ T cell clearance is effective in reducing the occurrence of GVHD.

In tumor immunology studies, human cancer cells, either tumor cell lines or patient tumor cells, can be transplanted into the humanized mice described above to study interactions between human immune cells and human tumor cells. While the humanized mouse model described above provides unique opportunities for in vivo studies of human immune cells and human tumor cells, traditional models are limited to assessing non-specific T cell interactions due to the lack of antigen presenting cells. In recent years, professional antigen presenting cell-Dendritic Cell (DC) vaccines have high therapeutic potential in inducing anti-tumor immunity in cancer patients. However, there are few reports of evaluating immune responses after vaccination with DCs based on humanized tumor cell lines or mouse models.

Disclosure of Invention

Therefore, the invention develops an in vitro or in vivo system, provides a genetically modified tumor cell line, application of the tumor cell line in preparation of a tumor-bearing non-human animal model, application or method of the prepared tumor-bearing non-human animal model in screening vaccines or medicaments, and the screened vaccines or medicaments. In particular, the method comprises the steps of,

In a first aspect of the invention, there is provided a genetically modified tumour cell line, the genetic modification comprising introducing into the tumour cell line a nucleotide sequence encoding a human leukocyte antigen (human leucocyte antigen, HLA).

Preferably, the HLA gene comprises an HLA-I gene, an HLA-II gene, or an HLA-III gene, more preferably, the HLA-I gene comprises HLA-A, HLA-B, HLA-C; the HLA-II gene comprises HLA-DR, HLA-DP and HLA-DQ; further preferred, the HLA genes comprise HLA-a 02:01.

Preferably, the tumor cell line is derived from any malignancy, more preferably, the malignancy includes a malignancy of the reproductive system, respiratory system, cardiovascular system, digestive system, urinary system, endocrine system, nervous system, blood system, skeletal muscle system, and the like. Further preferred, the malignancy includes malignancy of the reproductive system, digestive system, blood system, such as ovarian cancer, cervical cancer, liver cancer, acute leukemia, and the like.

In a specific embodiment, the tumor cell line comprises OVCAR8, hepG2, MDA-MB-453 or MV4-11.

Preferably, the genetic modification further comprises introducing into the tumor cell line a nucleotide sequence of a tumor gene encoding a tumor antigen. The tumor gene is determined by the tumor cell line.

More preferably, the tumor antigen is a relatively specific antigen of a tumor cell line, can be used as a judgment mark for tumor diagnosis, tumor invasion and/or metastasis, treatment effect and prognosis, and further preferably comprises a tumor-associated antigen and a tumor neogenesis antigen;

preferably, the tumor-associated antigen comprises CA125, claudin 6, NY-NSO-1, AFP, MAGEA3, HSP70, BAGE, PRTN3, PRAME, MART and/or WT1 proteins; tumor neogenesis antigen is obtained from tumor tissues of patients through biological information analysis.

The specific assay may be a TSA assay. The specific operation is as follows:

the tumor samples from the patient were subjected to high throughput sequencing (NGS) separately from normal control samples, which were subjected to whole-exome sequencing (WES) and transcriptome sequencing (RNA-seq), and normal control samples were subjected to whole-exome sequencing (WES) alone. And filtering the raw data of the sequencing machine to remove low-quality sequences, and obtaining clean data. Then, the clean data is compared to a human reference genome, and indexes such as sequencing coverage and the like are controlled in quality. Detecting somatic mutation in tumor according to the comparison result, and filtering to obtain high-quality mutation; detecting HLA typing of a tumor sample and a normal control sample; detecting the gene expression level of the tumor sample, and analyzing the fusion gene. And translating the mutation annotation result to obtain peptide fragments, intercepting the alternative mutant peptide by taking the mutation amino acid locus as a center, and removing the peptide fragments with the affinity of more than 500nm to obtain candidate neoantigens. The nascent antigens with high immunogenicity are scored based on various biological factors such as homology, binding affinity of peptides to HLA class I, proteasome C-terminal cleavage, transport efficiency of the Transporter (TAP) associated with antigen processing, antigen presentation capacity, expression abundance, clonality, etc. Screening out top10 neoantigen peptide fragments of the non-homologous peptide with the RNA-seq supported by mutation sequences, TPM expression quantity of >5, affinity of <500nm, mutation frequency of more than or equal to 2%.

In a specific embodiment, the CA125, claudin 6, NY-NSO-1, MART, WT1 is a tumor-associated antigen that is relatively specific for ovarian cancer;

in a specific embodiment, the AFP, MAGEA3, HSP70, CA125 are tumor-associated antigens that are relatively specific for liver cancer;

in a specific embodiment, the BAGE, PRTN3, PRAME, WT1 is a tumor associated antigen that is relatively specific for acute leukemia.

In a specific embodiment, the tumor neoantigen is a tumor antigen that is relatively specific for breast cancer.

In a second aspect of the present invention, there is provided a method of preparing the above-described genetically modified tumor cell line, the method comprising introducing into the tumor cell line a nucleotide sequence encoding a human leukocyte antigen (human leucocyte antigen, HLA).

Preferably, the method of preparation further comprises introducing a nucleotide sequence encoding a tumor antigen into the tumor cell line.

Preferably, the introducing comprises introducing a nucleotide sequence encoding a tumor antigen into a tumor cell line using gene editing techniques including embryonic stem cell-based gene homologous recombination techniques, CRISPR/Cas9, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases, or other molecular biology techniques; further preferred, gene transfer is performed using CRISPR/Cas 9-based gene editing techniques.

More preferably, the introducing comprises introducing the above nucleotide sequence into a tumor cell line using a targeting vector.

In a third aspect of the invention there is provided the use of a genetically modified tumour cell line as described above, including in the screening of drugs or the preparation of disease models.

Preferably, the medicament is for the treatment of tumors.

The disease model includes a tumor disease model.

In a fourth aspect of the invention, there is provided a genetically modified Dendritic Cell (DC) comprising introducing into the Dendritic cell an RNA sequence encoding a tumor antigen.

Preferably, the tumor antigen is derived from any malignancy, more preferably, the malignancy comprises a malignancy of the reproductive system, respiratory system, cardiovascular system, digestive system, urinary system, endocrine system, nervous system, blood system, skeletal muscle system, and the like. Further preferred, the malignant tumor includes malignant tumors of the reproductive system, digestive system, blood system, such as ovarian cancer antigen, cervical cancer antigen, liver cancer antigen, acute leukemia antigen, etc.

More preferably, the method further comprises the steps of, the "tumor-associated antigens" described herein include, but are not limited to, AFP, CA125, CA199, NY-ESO-1, ALK, BAGE protein, BIRC5 (survivin), BIRC7, CA9, CALR, CCR5, CD19, CD20 (MS 4A 1), CD22, CD27, CD30, CD33, CD38, CD40L, CD, CD52, CD56, CD79, CDK4, CEACAM3, CEACAM5, CLEC12A, EGFR, EGFR variant III, ERBB2 (HER 2), ERBB3, ERBB4, EPCAM, EPHA2, EPHA3, FCRL5, FLT3, FOLR1, GAGE protein, GCP3, GD2 one or more of GD3, GPNMB, GM3, GPR112, IL3RA, KIT, KRAS, LGR5, EBV derived LMP2, L1CAM, MAGE protein, MLANA, MSLN, MUC1, MUC2, MUC3, MUC4, MUC5, MUC16, MUM1, ANKRD30A, NY-ESO1 (CTAG 1B), OX40, PAP, PAX3, PAX5, PLAC1, PRLR, PMEL, PRAME, PSMA (FOLH 1), RAGE protein, RET, RGS5, ROR1, SART3, SLAMF7, SLC39A6 (LIV 1), STEAP1, STEAP2, TERT, TMPRSS2, thompson-noville antigen, TNFRSF17, TYR, UPK3A, VTCN1, or WT 1.

Further preferred, the tumor antigen comprises an ovarian, liver, breast, sarcoma and/or acute leukemia associated antigen, such as CA125, claudin 6, NY-NSO-1, AFP, MAGEA3, HSP70, BAGE, PRTN3, PRAME and/or WT1 protein.

In a specific embodiment, the amino acid structure of the tumor antigen and the RNA sequence encoding the tumor antigen are shown in the following table:

table 1: amino acid sequence and RNA sequence of tumor antigen

Or the amino acid sequence or RNA sequence has at least 90% identity with the amino acid or RNA sequence shown in Table 1 and has the function of a tumor antigen or a function of encoding a tumor antigen.

Preferably, the genetic modification further comprises introducing an RNA sequence encoding an immunoadjuvant into the dendritic cell.

Preferably, the immunoadjuvant comprises a cytokine, a chemokine, a second signal chimeric molecule, a third signal molecule antibody, a third signal multimer, and/or an immune checkpoint antibody chimeric molecule.

Preferably, the cytokine is IL-7, IL-15 and/or IL-23; the chemokine is CXCL16; the second signal chimeric molecule is MICA-CD28, CD80, CD86, CD40, CD70 and/or ICOSL; the third signal chimeric molecule is CD40L-CD40; the third signal molecule antibody is anti CD40; the third signal multimer is CD40L-MTQ or CD40-MTQ; the immune checkpoint antibody chimeric molecule is anti PD-L1, anti PD-1 or anti CTLA4-CD28-41BBL.

Preferably, the immunoadjuvant comprises CD40L, more preferably, the amino acid sequence of the CD40L is from accession number NCBI Reference Sequence:NP-000065.1.

The RNA sequence encoding CD40L comprises: NM-000074.3.

Preferably, the DCs comprise HLA identical to that of the tumor cell lines described above.

In a fifth aspect of the invention, there is provided a method of constructing a genetically modified Dendritic Cell (DC) as described above, the method comprising introducing RNA encoding a tumor antigen into a DC locus.

Preferably, the introducing comprises introducing a nucleotide sequence encoding a tumor antigen into the DC using gene editing techniques including embryonic stem cell-based gene homologous recombination techniques, CRISPR/Cas9, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases or other molecular biology techniques; further preferred, construction of DCs is performed using CRISPR/Cas 9-based gene editing techniques.

More preferably, the construction method comprises introducing a nucleotide sequence encoding a tumor antigen into the above-described locus of the DC using a targeting vector.

In a sixth aspect of the invention, a CD8 is provided ⁺ A method for T cell acclimation, which comprises combining the above-described genetically modified DCs and CD8 ⁺ T cell co-culture.

Preferably, the acclimation method comprises DC to CD8 ⁺ T cell ratio of 1 (1-15), more preferably 1 (8-15), still more preferably 1:10.

Preferably, the culturing step includes half-volume replacement of the culture medium every 3 days.

Preferably, the culturing step comprises 1 round of stimulation every 7 days for a total of 2-3 rounds of stimulation.

In a seventh aspect of the invention, there is provided a domesticated CD8 ⁺ T cells, the acclimatized CD8 ⁺ T cells were obtained by the acclimatization method described above.

In an eighth aspect of the invention, there is provided a genetically modified DC or domesticated CD8 as described above ⁺ Use of T cells in the preparation of a medicament.

Preferably, the medicament is used for treating or preventing diseases such as tumors and the like.

More preferably, the tumor is derived from any malignancy, and more preferably, the malignancy includes a malignancy of the reproductive system, respiratory system, cardiovascular system, digestive system, urinary system, endocrine system, nervous system, blood system, skeletal muscle system, and the like. Further preferred, the malignancy includes malignancy of the reproductive system, digestive system, blood system, such as ovarian cancer, cervical cancer, liver cancer, acute leukemia, and the like.

In a ninth aspect, the invention provides a pharmaceutical composition comprising the genetically modified DC or domesticated CD8 as described above ⁺ T cells. Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Preferably, the pharmaceutical composition may be in any dosage form, such as injection and the like.

In a specific embodiment, the pharmaceutical composition comprises dendritic cells into which RNA encoding CA125, WT1, claudin 6 and/or NY-NSO-1 has been introduced;

In a specific embodiment, the pharmaceutical composition comprises dendritic cells into which RNA encoding AFP, CA125, MAGEA3 and/or HSP70 is introduced;

in a specific embodiment, the pharmaceutical composition comprises dendritic cells into which RNA encoding WT1, BAGE, PRTN3 and/or PRAME is introduced.

In a specific embodiment, the pharmaceutical composition comprises dendritic cells into which RNA encoding a tumor neoantigen is introduced.

Preferably, the introduction is a separate introduction.

In a tenth aspect of the present invention, there is provided a method of preparing a tumor-bearing non-human animal model, the method comprising implanting the genetically modified tumor cell line described above into a non-human animal.

Preferably, the non-human animal comprises an immunodeficient non-human animal, more preferably the method of preparation comprises humanizing the immunodeficient non-human animal with human PBMCs.

Preferably, the implantation means include subcutaneous injection, intraperitoneal injection, and the like.

More preferably, the genetically modified tumor cell line has the same HLA type as the candidate drug or vaccine.

Preferably, the non-human animal is selected from any non-human animal that can be studied, such as rodents, pigs, rabbits, monkeys, etc.

Preferably, the non-human mammal is a non-human mammal, more preferably, the non-human mammal is a rodent, even more preferably, the rodent is a rat or a mouse.

Preferably, the immunodeficient mouse is selected from NOG or NOD immunodeficient mice, e.g. NOD-Prkdc ^scid IL-2rγ ^nul Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG)、Rag 2 ^-/- -IL2rg ^-/- (RG), NOD/SCID, NOG-dKO, etc.

According to an eleventh aspect of the invention, a tumor-bearing non-human animal model obtained by the preparation method is provided.

In a twelfth aspect, the present invention provides an application of the above genetically modified tumor cell line or tumor-bearing non-human animal model, the application comprising:

1) The application in the product development of the immune process involving human cells, screening vaccines and medicaments;

2) Use as a disease model system for pharmacological, immunological, microbiological and/or other medical research;

3) The application in developing new diagnostic and/or therapeutic strategies.

Preferably, the vaccine or medicament comprises various exogenous antigens, autoantigens, tumor antigens, or their coding RNA or DNA sequences, more preferably, the vaccine or medicament is a DC carrying various exogenous antigens, autoantigens, tumor antigens, or their coding RNA or DNA sequences. Further preferred, the dendritic cells have the same HLA type as the tumor cell line implanted in the tumor-bearing non-human animal model.

Further preferably, the foreign antigen is derived from a virus, a bacterium, a fungus, or the like.

Preferably, the use is not a diagnostic or therapeutic method for a disease.

In a thirteenth aspect of the invention, there is provided a method of screening for a vaccine or drug, the method comprising using any of the tumor-bearing non-human animal models described above for screening for a candidate vaccine or drug.

Preferably, the method further comprises the step of acclimating CD8 ⁺ A step of implanting T cells into a tumor-bearing non-human animal model, more preferably, the acclimatized CD8 ⁺ T cells are acclimatized with a candidate vaccine or drug, further preferably, the acclimatized CD8 ⁺ T cells include CD8 which is acclimatized using any of the above dendritic cells ⁺ T cells.

In a fourteenth aspect of the present invention, there is provided a method for the prevention and/or treatment of a tumor, said method comprising administering the above pharmaceutical composition to the body.

Preferably, the vaccine and/or medicament is for the prevention or treatment of an infection, autoimmune disease, allergic disease, or malignancy as defined above.

More preferably, the infection is derived from an infection caused by a virus, bacteria, fungus, or the like. In a fifteenth aspect of the present invention there is provided the use of a acclimatized cd8+ T cell, the use comprising:

1) The application in preparing tumor-bearing non-human animal models;

2) The application in preparing medicines.

Preferably, the tumor-bearing non-human animal model is as defined above, CD8 will be acclimatized ⁺ T cells were implanted into tumor-bearing non-human animal models.

Preferably, the acclimated cd8+ T cells have the effects of secreting the cytotoxic factor IFN- γ and killing tumors.

The term "treatment (treating, treat or treatment)" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "preventing" refers to avoiding the occurrence of a disease, ameliorating or reducing the symptoms, pathological states or progression of a disease.

The invention has the beneficial effects that;

1. a genetically modified tumor cell line is constructed, and the tumor cell line can be used for effectively screening medicines in vitro, can better reproduce clinical characteristics of tumor diseases after being implanted into animals, and can effectively screen medicines in vivo, in particular to DC vaccine for treating tumors.

2. A genetically modified dendritic cell is constructed which can highly express exogenous antigens, with all antigens being expressed in greater than 50%, and some even in greater than 80%. The dendritic cells have the effect of treating related tumors, and can induce proliferation of antigen-specific T cells, produce effector factors and inhibit tumor growth.

3. Constructing a humanized animal model for effectively screening vaccine and medicine, wherein the animal model does not have GVHD reaction, the experimental window can be prolonged to 13 weeks, and the humanized animal model shows good CD4 for exogenous antigen ⁺ T cells and CD8 ⁺ Activation of T cells.

4. CD8 acclimatized with in vitro antigen ⁺ T cells are implanted into a tumor-bearing non-human animal model, candidate genetically modified dendritic cells are inoculated into the animal model, the candidate genetically modified dendritic cells can be used as a vaccine for tumor specific antigen immunotherapy or a good disease model for evaluating the drug effect of a drug, and the screened dendritic cell vaccine can effectively treat corresponding tumors.

The foregoing is merely illustrative of some aspects of the present invention and is not, nor should it be construed as limiting the invention in any respect.

All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. It will be appreciated by those skilled in the art that certain changes may be made thereto without departing from the spirit or scope of the invention.

The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

FIG. 1 shows the results of body weight of PBMC humanized NOG-dKO mice, wherein A is the result of ovarian cancer PBMC humanized NOG-dKO mice; b is the result of humanizing NOG-dKO mice by liver cancer PBMC; c is the result of the humanization of NOG-dKO mice by acute leukemia PBMC.

FIG. 2 shows the percentage of human CD45+ cells in the peripheral blood of mice from PBMC humanized NOG-dKO mice, wherein A is the result of ovarian cancer PBMC humanized NOG-dKO mice; b is the result of humanizing NOG-dKO mice by liver cancer PBMC; c is the result of the humanization of NOG-dKO mice by acute leukemia PBMC.

FIG. 3 shows the percentage of human CD 3T cells in the peripheral blood of mice from PBMC humanized NOG-dKO mice, wherein A is the result of ovarian cancer PBMC humanized NOG-dKO mice; b is the result of humanizing NOG-dKO mice by liver cancer PBMC; c is the result of the humanization of NOG-dKO mice by acute leukemia PBMC.

FIG. 4 shows human CD4 in the peripheral blood of mice from the PBMC humanized NOG-dKO mice ⁺ T cell percentage; wherein A is the result of humanizing NOG-dKO mice with ovarian cancer PBMC; b is the result of humanizing NOG-dKO mice by liver cancer PBMC; c is the result of the humanization of NOG-dKO mice by acute leukemia PBMC.

FIG. 5 shows human CD8 in the peripheral blood of mice from a PBMC humanized NOG-dKO mouse ⁺ T cell percentage; wherein A is the result of humanizing NOG-dKO mice with ovarian cancer PBMC; b is the result of humanizing NOG-dKO mice by liver cancer PBMC; c is the result of the humanization of NOG-dKO mice by acute leukemia PBMC.

FIG. 6 shows human peripheral blood of mice of PBMC humanized NOG-dKO mice CD4 ⁺ T cell percentage; wherein A is the result of humanizing NOG-dKO mice with ovarian cancer PBMC; b is the result of humanizing NOG-dKO mice by liver cancer PBMC; c is the result of the humanization of NOG-dKO mice by acute leukemia PBMC.

FIG. 7 shows human peripheral blood of mice of PBMC humanized NOG-dKO mice CD8 ⁺ T cell percentage; wherein A is the result of humanizing NOG-dKO mice with ovarian cancer PBMC; b is the result of humanizing NOG-dKO mice by liver cancer PBMC; c is the result of the humanization of NOG-dKO mice by acute leukemia PBMC.

FIG. 8 shows the results of animal model construction after ovarian cancer cell inoculation, wherein A is the in vivo growth characteristics of OVCAR 8; b is over-expression of WT1 and HLA-A 02:01 in OVCAR8 cell lines; c is the tumor volume of tumor cells with different cell numbers and subcutaneous tumor loading.

FIG. 9 shows the statistics of tumor volumes of tumor cells subcutaneously charged with different cell numbers of HepG 2.

FIG. 10 shows statistics of tumor volumes of tumor cells subcutaneously charged with tumors of different numbers of MV 4-11.

FIG. 11 shows the results of ovarian cancer tumor antigen expression.

FIG. 12 shows the results of expression of liver cancer tumor antigens.

FIG. 13 shows the results of acute leukemia tumor antigen expression.

FIG. 14 shows DC versus CD8 carrying ovarian cancer antigen ⁺ T activation, wherein A is the result of an A IFN-. Gamma.ELISPOT assay; b is the detection result of tumor killing experiment.

FIG. 15 shows DC versus CD8 carrying liver cancer antigen ⁺ T activation, wherein A is the result of an A IFN-. Gamma.ELISPOT assay; b is the detection result of tumor killing experiment.

FIG. 16 shows DC versus CD8 carrying an acute leukemia antigen ⁺ T activation, wherein A is the result of an A IFN-. Gamma.ELISPOT assay; b is the detection result of tumor killing experiment.

Fig. 17 shows the weight change of mice in tumor model, wherein fig. A, B, C shows the weight change of mice in animal model of ovarian cancer, liver cancer and acute leukemia, respectively.

Fig. 18 shows the percentage and total number of humanized cells of mice in tumor model, wherein fig. A, B, C shows the percentage and total number of humanized cells of mice in ovarian cancer, liver cancer, and acute leukemia, respectively.

FIG. 19 shows the results of IFN-r ELISpot detection of spleen cells of mice in tumor model, wherein FIG. A, B, C shows the results of IFN-r ELISpot detection of spleen cells of mice in ovarian cancer, liver cancer and acute leukemia, respectively.

Fig. 20 shows the tumor inhibition effect of DC, wherein fig. A, B, C shows the tumor inhibition results of ovarian cancer, liver cancer, and acute leukemia, respectively.

FIG. 21 shows the results of an ovarian cancer humanized mouse model to evaluate the efficacy of PD-1 or PD-L1 antibodies.

Fig. 22 shows the results of evaluating the efficacy of breast cancer DC vaccine in a humanized mouse model of breast cancer.

Figure 23 shows the results of evaluating the efficacy of sarcoma DC vaccine in a humanized mouse model of sarcoma.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Example 1: humanized mouse system establishment

PBMC humanized mice have short window period because the mouse cells elicit immune cell responses in humans, and lethal tissue rejection (GVHD) occurs. Humanized cells mainly survive as T cells and are mouse antigen activated or memory effector T cells, which are difficult to primary immunize against human tumor neoantigens. We used NOG-dKO mice knocked out mouse MHC I and MHC II molecules to create a humanized system, reduce GVHD response and increase the window period for drug efficacy assessment.

Female 5-7 week old NOG-dKO mice (purchased from Experimental animal technology Co., ltd., beijing Vitre.) were implanted with OVCAR8, hepG2 or MV4-11 to establish models of ovarian cancer, liver cancer and acute leukemia respectively, and were inoculated with 1.0 x 10 by tail vein ⁷ Human PBMC cells, followed by 2 times per week monitoring of mouse body weight and dayIn a normal state, the proportion of humanized cells in peripheral blood was detected by a flow cytometer 1 time per week, and the total observation was performed for 13 weeks.

The experimental results show that:

PBMC humanized NOG-dKO mice were continuously observed for 13W, and no significant weight loss was observed due to GVHD response (15% of the initial weight loss with mice body weight, see FIG. 1 for GVHD response). Meanwhile, humpback, depilation and hemolysis caused by GVHD reaction are not found in the period, and skin yellowing and the like are caused.

Human CD45 cells in the peripheral blood of mice tended to increase with time for humanization, peaking at 5-6 weeks with a consequent decrease (see figure 2).

Human CD45 cells in peripheral blood are almost 100% CD3 ⁺ T cells (see FIG. 3), and CD4 therein ⁺ T cells and CD8 ⁺ T cell ratios were close to 1:1 (see FIGS. 4, 5).

Initial CD4 increases with time for humanization ⁺ T cells were able to maintain a proportion of at least 10% (see figure 6), but initially CD8 ⁺ T cells decreased from around 60% to around 0% from the first four weeks, and were not subsequently increased (see fig. 7).

From the experimental results, it can be seen that the PBMC humanized NOG-dKO mice were observed continuously at 13W, no significant weight loss caused by GVHD reaction was observed (15% of the initial weight loss was observed in mice to produce GVHD reaction), and no humpback, depilation and hemolysis caused by GVHD reaction were observed during this period. Based on this result, we found that NOG-dKO mice were able to reduce GVHD response, increasing the potency window period to 13W.

Human CD45 cells in the peripheral blood of the mice tend to increase with the humanization time, peak at 5-6 weeks, and decrease. Human CD45 cells in peripheral blood are almost 100% CD 3T cells, whereas the ratio of CD 4T cells to CD 8T cells approaches 1:1, with the initial CD 4T cells maintaining a 10% ratio as the time of humanization increases, but the initial CD 8T cells decrease from around 60% to around 0% of the initial.

Based on this result, we subsequently selected CD8 cells that were acclimated in vitro to antigen loaded DC cells for human immune system establishment.

Example 2: establishment of tumor cell lines

The WT1 gene is constructed into a lentiviral packaging plasmid, then the corresponding gene is transduced into an ovarian cancer tumor cell line by using lentivirus, and the stable expression is carried out after the screening of corresponding antibiotics; and infecting the tumor cell line by using HLA-A 0201 slow virus, and allowing the corresponding tumor cell line to stably express the HLA-A 0201, thereby obtaining the HLA0201 over-expressed WT 1+ovarian cancer tumor antigen cell line.

Corresponding tumor antigens are selected according to different tumor cell lines and introduced into the tumor cell lines, and the sequence structures are shown in table 1. Other specific tumor cell lines, e.g. HLA-A 02:01AFP ⁺ HepG2、HLA-A*02:01WT1 ⁺ The construction method of MV4-11 is similar and will not be described in detail herein.

Example 3: human ovarian cancer disease model establishment

Xenografts (CDX) and patient-derived xenografts (PDX) of human cell lines are both preclinical tools for assessing immunotherapy. How to select CDX or PDX, various criteria related to tumor molecular characteristics and experimental design should be considered.

Ovarian cancer is a heterogeneous tumor that includes at least five tissue types: clear cells, endometrium-like, mucinous, low grade serous and high grade serous tumors. High Grade Serous Ovarian Cancer (HGSOC) is the most common and deadly type of the disease, considered the "prototype" of epithelial ovarian cancer, characterized by significant structural genomic changes and widespread TP53 gene mutations. More than 50% of HGSOCs have defects in the homologous recombination dependent repair (HR) pathway, mainly related to genetic and epigenetic alterations of HR pathway genes, such as BRCA1, BRCA2 and PTEN. HR deficient HGSOC responds well to platinum chemotherapies and PARP inhibitors. About 20% of HGSOCs show an increase in cyclin E1 (CCNE 1) gene, an intact HR pathway, adverse effects on platinum-based chemotherapy and poor clinical outcome. The association of genomic and molecular signatures with useful laboratory model systems is therefore critical to the development of new therapies. Thus the in vivo growth characteristics of the HGSOC cell model determine its application.

To assess which HGSOC cell lines reproduced clinical features of OC in vivo, we performed the assessment of tumor formation in OVCAR8 cell line peritoneal female NOG-dKO mice and observations after 30 days. As a result, it was found that tumor-bearing in the abdominal cavity was represented by metastasis spread over the abdominal cavity, colonized in the peritoneum, diaphragm, omentum, spleen, gastrointestinal tract, liver and reproductive organs, indicating that this cell model was able to reproduce clinical features, localized in specific sites in vivo, and was also suitable for studying interactions between tumor cells and microenvironment (fig. 8A). Thus, OVCAR8, as a line that can grow rapidly in vivo and is aggressive, can well reproduce clinical features, helps reduce time and cost of xenograft studies, and is suitable as an ovarian cancer disease model for subsequent evaluation of dendritic vaccine treatment.

WT1 plays an important role in invasion and metastasis of ovarian cancer and is a marker affecting the therapeutic effect and prognosis of the disease, so we constructed over-expressed WT1 ⁺ OVCAR8 cell line (fig. 8B). Further for the purpose of binding to dendritic cells and CD8 in healthy subjects ⁺ HLA type matching of T cells we constructed HLA-A 02:01WT1 ⁺ OVCAR8 cell line in the hope of assessing in vivo efficacy of DC products in HLA-A 02:01 background. To evaluate the ovarian cancer cell line HLA-A 02:01

WT1 ⁺ OVCAR8 growth in vivo we injected subcutaneously cells of different cell numbers into female NOG-dKO mice and observed after 30 days to assess tumor formation. As a result, HLA-A of different orders of magnitude was found to be 02:01WT1 ⁺ OVCAR8 cells were able to grow subcutaneously with tumors all growing in size with increasing cell number. The results are shown in FIG. 8C.

Example 4: liver cancer disease model establishment

For evaluation of liver cancer cell lines HLA-A 02:01afp ⁺ HepG2 growth in vivo, with reference to the procedure of example 3, we injected subcutaneously cells of different cell numbers into female NOG-dKO mice and assessed tumor formation by observation after 30 days. As a result, HLA-A 02:01AFP of different orders of magnitude was found ⁺ HepG2 cells can grow subcutaneously with tumors and tumor size increases with cell number. The results are shown in FIG. 9.

Example 5: acute leukemia disease model establishment

For evaluation of acute leukemia cell line HLA-A 02:01wt1 ⁺ MV4-11 in vivo growth, referring to the method of example 3, we subcutaneously injected cells of different cell numbers into female NOG-dKO mice and assessed for tumor formation by observation after 30 days. As a result, HLA-A of different orders of magnitude was found to be 02:01WT1 ⁺ MV4-11 cell subcutaneous tumor can grow, and the tumor size increases with the cell number. The results are shown in FIG. 10.

Example 6: DC cell carrying tumor-associated antigen

In order to more truly simulate and objectively evaluate the therapeutic effect of the dendritic vaccine in human body, the dendritic vaccine is prepared according to the production flow. HLA-A 02:01 healthy subjects derived from monocytes differentiated into dendritic cells, and then respectively introduced into RNA sequences encoding 4 tumor-associated antigens of ovarian cancer (CA 125, WT1, claudin 6, NY-NSO-1), liver cancer (AFP, CA125, MAGEA3, HSP 70) or acute leukemia (WT 1, BAGE, PRTN3, PRAME), recombinant autologous DC therapeutic products were prepared, all of which were expressed by more than 50%, and some even more than 80% (see FIGS. 11, 12, 13, respectively), demonstrating that the DC cells prepared according to the present invention were able to efficiently express the antigen of interest.

Wherein the amino acid sequence of the tumor-associated antigen and the RNA sequence encoding the tumor-associated antigen are shown in Table 1.

Example 7: DC cell in vitro effect assessment

To evaluate the in vitro efficacy of the 12 DCs products prepared in example 6, we evaluated the immune effects of the DC vaccine using IFN- γ ELISPOT and tumor killing function index. The specific method comprises the following steps:

1. cd8+ T cell acclimation:

co-culturing antigen-assembled DCs of example 6 with CD8+ T cells at a ratio of 1:10, half-cell-changing every 3 days, re-adding DC cells every 7 days, and stimulating for 3 rounds of stimulation, and harvesting CD8 after 17 days ⁺ T cells are used for IFN-. Gamma.ELISPOT and tumor killing function detection.

2. IFN-gamma ELISPOT detection

IFN-. Gamma.ELISPOT detection was performed according to standard methods with reference to kit instructions.

3. Tumor killing test

Tumor killing experiments, tumor cells were plated overnight first, following day according to CD8 ⁺ T cell and tumor cell 5:1 ratio add-on photo CD8 ⁺ T cells were incubated for 20h and then tumor cell numbers were counted by flow cytometry.

The results of ovarian cancer, liver cancer and acute leukemia are shown in FIGS. 14-16, respectively, and the secretion of the cytotoxic factor IFN-gamma and tumor killing by CD8+ T cells induced by DC stimulated groups compared with blank control groups are statistically significant.

Example 8: evaluation of therapeutic effect of DC cell vaccine on tumor by in vivo immunization of humanized tumor-bearing mice

In the above humanized mouse system, we explored the finding that NOG-dKO mice were able to delay GVHD, more suitable for subsequent in vivo evaluation, but still were unable to alter the original CD8 ⁺ T cell loss, which makes it difficult for human immune cells to respond to primary immunization with neoantigen, thus, promoting in vitro domestication of dendritic cells used by us to tumor antigen-specific CD8 ⁺ T cells to establish the human immune system to maintain CD8 ⁺ T cells mount an immune response to tumor antigens.

Specific CD8 ⁺ The T cell acclimation method is described in example 7.

1. Experimental method

Female NOG-dKO mice are divided into 2 groups, a group 1 tumor-bearing mouse model and humanized tumor antigen DC in vitro domesticated CD8 ⁺ T cells and frozen stock (vehicle control), group 2 tumor-bearing mouse model, humanized tumor antigen DC in vitro domesticated CD8 ⁺ T cells and tumor antigens are recombined into autologous dendritic cell injection, wherein the tumor-bearing mouse model refers to the tumor-bearing mouse models of ovarian cancer, liver cancer and acute leukemia prepared in examples 3-5 respectively; the tumor antigen recombinant autologous dendritic cell injection is three groups of dendritic cells prepared in example 6, wherein the first group comprises the dendritic cells carrying CA125 and WT respectively1. Dendritic cells of Claudin 6 and NY-NSO-1, the second group comprises dendritic cells carrying AFP, CA125, MAGEA3 and HSP70 respectively, the third group comprises dendritic cells carrying WT1, BAGE, PRTN3 and PRAME respectively, the dendritic cells are obtained by dissolving in physiological saline, and the effect of the three groups of dendritic cells is evaluated by injecting the three groups of dendritic cells into tumor-bearing mice models of ovarian cancer, liver cancer and acute leukemia respectively.

The frozen stock used in the experiments was derived from CryoStor CS10 frozen stock from Stem cell company.

Tumor cells were subcutaneously injected and administered 3 days later, and CD8 was acclimatized in vitro by intravenous injection one hour prior to administration ⁺ T cells, then vehicle or tumor antigen recombinant autologous dendritic cells were given by intravenous injection, once every 7 days, 5 times until 2 weeks after 5 times of administration, all mice were euthanized and dissected. The detection indexes of animals during the test include body weight, human immune cell change in mice, antigen-related T cell secretion effector and tumor inhibition rate.

2. The results were as follows:

1) Weight of body

During the trial, no abnormal changes in body weight gain were seen in all groups of animals, nor was a dramatic decrease in body weight due to GVHD observed (ovarian cancer, liver cancer and acute leukemia groups are shown in figures 17a, b, c, respectively).

2) Activating tumor antigen specific T cell proliferation

The significant increase in both the percentage of human cells and the total number of cells in the spleens of the animals in the DC-injected group compared to the control group indicated that the DC cell injection caused proliferation of human T cells (ovarian cancer, liver cancer and acute leukemia groups, respectively, see fig. 18A, B, C).

3) Induction of tumor antigen specific T cell production of effector

Spleen cells of each group of mice are isolated, and the tumor antigen specific T cells in the spleen cells are re-stimulated to secrete cytotoxic effector IFN-r, IFN-r ELISpot kit for detection. The results showed a significant increase in IFN-r cytokines in the DC treated group compared to the vehicle group, indicating human antigen-specific CD8 ⁺ T cells respond to dendritic cell vaccine presentation antigens and produce cytotoxic effectors (ovarian cancer, liver cancer and acute leukemia groups, respectively, see figure 19A, B, C).

4) Dendritic cell vaccine for tumor inhibition

After 2 weeks of observation, the subcutaneous tumor size of mice was measured using a small animal living imager, and the results showed a significant decrease in tumor volume in the DC treated group compared to the vehicle-irradiated group (ovarian cancer, liver cancer and acute leukemia groups are shown in fig. 20, A, B, C, respectively).

Because of the heterogeneity of tumors in tumor patients, a single antigen cannot represent all tumor cells. In order to better simulate the clinical situation and evaluate the drug effect, we reform the corresponding tumor cell line, express the corresponding antigen, treat with the DC vaccine loaded with the mixed antigen, avoid the failure of treatment caused by the omission of the antigen. In the in vivo evaluation experiment of the invention, T cell tumor inhibition rate induced by DC cells of the mixed antigen is higher.

In conclusion, the NOG-dKO mice are loaded with the modified human tumor cells subcutaneously, then the humanized antigen DC domesticates the CD8 cells in vitro, and then the tumor antigen recombinant autologous dendritic cell injection is repeatedly injected intravenously to cause immune reaction and effectively inhibit tumor growth.

Example 9: ovarian cancer humanized mouse model for assessing PD-1 or PD-L1 antibody efficacy

Referring to the methods of examples 3 and 8, female NOG-dKO mice, subcutaneously bearing tumor HLA-A 02:01mart+ovcart 8 cells (constructed similarly to the method in example 7), were acclimatized in vitro by intravenous injection after 3 days, and then divided into 3 groups, group 1 being a control group to which an equal volume of PBS (control group) was simultaneously administered, group 2 being a PD-1 antibody treatment group to which a PD-1 antibody treatment (PD-1 antibody treatment group), 5mg/kg, intraperitoneally injected, 2 times per week for 4 weeks; group 3 PD-L1 antibody treatment group was given PD-L1 antibody treatment (PD-L1 antibody treatment group), 5mg/kg, i.p. injection, 2 times per week for 4 weeks. Tumor size was monitored by in vivo imaging of small animals, observed for 1 week 4 weeks after dosing.

The results are shown in FIG. 21The tumor fluorescence signal of the control group was shown to be approximately 1.5X10 ¹⁰ The tumor cell after the gene modification can successfully construct a tumor animal model, and after the PD-1 antibody and the PD-L1 antibody which are known to have the tumor inhibiting effect are treated, the tumor inhibiting rate respectively reaches 36.1% and 32.1%, so that the animal model constructed by the method can be used for screening other vaccines, medicines, researches and other purposes for preventing or treating tumors.

Example 10: breast cancer humanized mouse model for evaluating efficacy of breast cancer DC vaccine

Female NOG-dKO mice, fourth inguinal mammary fat pad in situ tumor-bearing HLA-A 02:01MDA-MB-453 cells, after 3 days, in vitro acclimation of CD8+ T cells by intravenous injection, then dividing into 3 groups, wherein group 1 is tumor-bearing control group, and simultaneously, an equal volume of frozen stock solution (tumor-bearing control group) is given, group 2 is no-load DC control group, and simultaneously, the same number of no-load DC treatments (no-load DC control group) are given; group 3 is a DC vaccine treatment group to which tumor neoantigens were administered recombinant autologous dendritic cell therapy (DC vaccine treatment group), once every 7 days, 5 times, and 2 weeks after the end of administration, and the tumor size of animals was continuously tracked during the test.

The results are shown in figure 22, where the DC vaccine treated group showed significant differences in tumor size (P < 0.001) from the other two groups, demonstrating that the DCs of the present invention were effective in treating tumors. The invention also shows that the animal models with different tumor types constructed by the invention can be used for screening vaccine, medicine, research and other purposes for preventing or treating tumor.

Example 11: method for obtaining tumor neoantigen (TSA analysis method)

Example 12: sarcoma humanized mouse model for evaluating efficacy of sarcoma DC vaccine

Female NOG-dKO mice, subcutaneous tumor-bearing HLA-A 02:01WT1+MNNG/HOS cells, were acclimatized in vitro by intravenous injection after 3 days, and then divided into 3 groups, wherein group 1 is tumor-bearing control group and simultaneously given an equal volume of cryopreserved solution, and group 2 is empty DC control group and simultaneously given the same number of empty DC treatments; group 3 is DC vaccine treatment group given tumor neoantigen recombinant autologous dendritic cell treatment, once every 7 days, 5 times, 2 weeks after the end of administration, and animal tumor size is continuously tracked during the test.

The results are shown in figure 23, where the DC vaccine treated group showed significant differences in tumor size (P < 0.02) from the other two groups, demonstrating that the DCs of the present invention were effective in treating tumors. The invention also shows that the animal models with different tumor types constructed by the invention can be used for screening vaccine, medicine, research and other purposes for preventing or treating tumor.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims

1. A genetically modified dendritic cell, wherein the genetic modification comprises introducing into the dendritic cell an RNA sequence encoding a tumor antigen comprising an ovarian, liver, breast, sarcoma and/or acute leukemia antigen.

2. The dendritic cell of claim 1, wherein said tumor antigens comprise tumor-associated antigens and tumor neoantigens;

wherein the tumor associated antigen comprises CA125, claudin 6, NY-NSO-1, AFP, MAGEA3, HSP70, BAGE, PRTN3, PRAME and/or WT1 proteins;

tumor neogenesis antigen is obtained from tumor tissues of patients through biological information analysis.

3. The dendritic cell of claim 1 or 2, wherein said genetic modification further comprises introducing an RNA sequence encoding an immunoadjuvant into the dendritic cell, preferably wherein said dendritic cell comprises an HLA gene comprising an HLA-I gene, an HLA-II gene, or an HLA-III gene.

4. A method of constructing a dendritic cell according to any of the claims 1-3, characterized in that the method of constructing comprises introducing RNA encoding a tumor antigen into a dendritic cell, preferably the method of constructing further comprises introducing RNA sequences encoding an immunoadjuvant into a dendritic cell.

5. Use of a dendritic cell according to any of claims 1-3 for the preparation of a pharmaceutical composition.

6. A pharmaceutical composition comprising the dendritic cell of any one of claims 1-3.

7. CD8 ⁺ A method for the domestication of T cells, which comprises combining the genetically modified DC and CD8 according to any one of claims 1 to 3 ⁺ T cell co-culture.

8. Domesticated CD8 ⁺ T cells, the acclimatized CD8 ⁺ T cells obtained by the acclimation method according to claim 7.

9. The domesticated CD8 of claim 8 ⁺ Use of T cells, comprising:

1) The application in preparing tumor-bearing non-human animal models;

2) The application in preparing medicines.

10. The use of claim 9, wherein the non-human animal comprises an immunodeficient non-human animal.