CN111040039B - Positive polypeptide pool for tumor vaccine immune response detection and preparation method thereof - Google Patents
Positive polypeptide pool for tumor vaccine immune response detection and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6818—Sequencing of polypeptides
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Abstract
The invention discloses a positive polypeptide pool for detecting immune response of tumor vaccine and a preparation method thereof, wherein the positive polypeptide pool consists of a plurality of combined peptides prepared from the following polypeptide sequences; the polypeptide sequence includes: labeling an epitope peptide sequence which can be combined with MHC I type molecules, is efficiently presented and causes immune response as an A section sequence; labeling an epitope peptide sequence which can be combined with MHC II molecules, is efficiently presented and causes immune response as a B segment sequence; connecting the A section sequence and the B section sequence through a Linker sequence to form a complete polypeptide sequence; the positive polypeptide pool prepared by the invention can be used as a polypeptide pool for detecting a positive control stimulant in vitro by using the immune response of the polypeptide vaccine, so that the obtained universal positive polypeptide pool has an action mechanism similar to that of the tumor polypeptide vaccine for stimulating an immune system to generate response, the false negative condition is avoided, and the detection result is high in accuracy, stable and reliable.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a positive polypeptide pool for immune response detection of tumor vaccines and a preparation method thereof.
Background
In 2019, 1 month, the latest first-stage statistical data is released by the national cancer center, and in 2015, about 392.9 ten thousands of people suffer from malignant tumor and about 233.8 thousands of people die. On average, over 1 million people per day were diagnosed with cancer, and 7.5 people per minute were diagnosed with cancer. Although tumor targeting has been a goal pursued by researchers, there are significant difficulties. One of the important causes is the widespread tumor heterogeneity. Not only within the tumor tissue, but even tumors of the same type vary greatly from patient to patient. Rapidly growing and proliferating cancer cells have no time to repair coding errors generated in the DNA replication process, so that a new mutant protein, namely tumor neoantigen (neoantigen), appears. With the intensive research of scientists, two independent research results published in Nature journal in 2017 show that the individualized tumor neoantigen vaccine aiming at each patient is designed and prepared by screening and predicting the tumor neoantigen specifically expressed in the body of the tumor patient, the autoimmune system of the high-risk patient with late-stage recurrence in melanoma can be effectively activated, and the real individualized tumor precision treatment is initially realized. The types of neoantigen tumor vaccines that have been reported in the research today are mainly polypeptides, RNA, DNA and immune cells (e.g. dendritic cells and T cells). Therefore, based on the importance of tumor vaccine research, it is necessary to develop a technology or product capable of rapidly and accurately detecting the immune response effect of the vaccine.
In tumor immunotherapy, in vitro validation of polypeptide-stimulated immunocytokines and antibodies is an effective method for testing immunogenicity of polypeptides. Enzyme linked immunospot (ELISpot) is a well-established technique for detecting the immune response of polypeptide vaccines in vitro [3 ]. After the human immune system is stimulated by the stimulus, part of the stimulus can be directly recognized by T cells to generate immune response, and the other needs to process and present the antigen information of the stimulus to the T cells through APC (antigen presenting cells), then the T cells secrete specific cytokines, and finally the specific cytokines are expressed in the form of spots through ELISpot technology, wherein the strength and the number of the spots reflect the response capability of the cells to the stimulus and the capability of secreting the target cytokines. The ELISpot assay requires the provision of complex controls, including positive, negative and blank controls. Currently, the positive control stimulators commonly used in ELISpot experiments are CEF peptide pool (viral short peptide pool: consisting of several polypeptides of 9-15 amino acids in length), PHA (phytohemagglutinin), PMA (phorbol ester) and Ionomycin (Ionomycin). T cells cannot directly recognize natural protein antigen information, and can only recognize antigen peptide components that form complexes with MHC molecules, requiring the assistance of other immune cells. The APC of the antigen presenting cell can endocytose protein antigen, degrade into polypeptide and express on the cell surface to form MHC-antigen complex, and further be recognized by T cells and generate immune response. In the case of DC cells, the DC has a strong ability to take up and digest long polypeptides at the immature stage, but has a weak ability to present antigen information to T cells, whereas when the DC develops and matures, it forms MHC-antigen complexes and becomes more presented to T cells. Current positive controls, in which CEF can bind directly to MHC to form MHC-antigen complexes, require presentation by Antigen Presenting Cells (APC) (no uptake process), while PHA, PMA, Ionomycin are all direct T cell stimulations, and do not require APC involvement [4], so these positive controls do not fully mimic the complex process of protein antigen presentation to T cells via immature DCs to mature DCs.
However, the peptide vaccine used in the current tumor immunotherapy is generally a long peptide with a length of 20-35 amino acids, and after entering the human body, the peptide vaccine needs to be taken up, processed and presented to T cells by APC to generate specific immune response, and the individualized tumor neoantigen has strong complexity according to different individuals. If the CEF, PHA or PMA is also used as the positive control group of the tumor polypeptide vaccine, due to the difference of action mechanism between the CEF, PHA or PMA and the long peptide, some false negative results may not be excluded (for example, when the polypeptide vaccine group is negative, if both CEF and positive polypeptide pool are negative, the cell itself is abnormal in function or the experimental process is abnormal, if CEF is positive and positive polypeptide pool is negative, the APC cell is abnormal in function, the false negative condition can be determined, and if both CEF and positive polypeptide pool are positive, the polypeptide vaccine does not cause immune response or the immune response is very weak), thereby reducing the credibility of the experimental results. Therefore, in the experiment for detecting the immune response of the tumor polypeptide vaccine by using the ELISpot technology, a general positive control group stimulant which has a similar and effective action mechanism with the tumor polypeptide vaccine is used, and the possible false negative condition is eliminated as much as possible. The market needs a positive stimulant with a similar action mechanism to that of tumor polypeptide vaccine stimulating immune system to generate response, and the present invention solves the problem.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a positive polypeptide pool for detecting the immune response of a tumor vaccine and a preparation method thereof, which can be used as a polypeptide pool for detecting a positive control stimulant in vitro through the immune response of the polypeptide vaccine, so that the obtained universal positive polypeptide pool has an action mechanism similar to that of the tumor polypeptide vaccine for stimulating an immune system to generate response, the false negative condition is avoided, and the accuracy of detecting the immune response of the tumor polypeptide vaccine is further improved.
In order to achieve the above object, the present invention adopts the following technical solutions:
a positive polypeptide pool for detecting immune response of tumor vaccine comprises a plurality of combined peptides prepared from the following polypeptide sequences;
the polypeptide sequence includes:
labeling an epitope peptide sequence which can be combined with MHC I type molecules, is efficiently presented and causes immune response as an A section sequence;
labeling an epitope peptide sequence which can be combined with MHC II molecules, is efficiently presented and causes immune response as a B segment sequence;
connecting the A segment sequence and the B segment sequence into a complete polypeptide sequence.
The positive polypeptide pool for detecting the immune response of the tumor vaccine consists of a plurality of combined peptides prepared from the following polypeptide sequences;
the polypeptide sequence includes:
labeling an epitope peptide sequence which can be combined with MHC I type molecules, is efficiently presented and causes immune response as an A section sequence;
labeling an epitope peptide sequence which can be combined with MHC II molecules, is efficiently presented and causes immune response as a B segment sequence;
the A segment sequence and the B segment sequence are connected through a linker sequence connected with amino acids, and a polypeptide sequence with an A-L-B or B-L-A structure is designed.
The positive polypeptide pool for the immune response detection of the tumor vaccine comprises: respectively and independently connecting 23 CEF epitope peptides marked as A section sequences with a tetania Toxin 830-844 marked as B section sequences through a linker sequence and synthesizing combined peptides to enable the lengths to reach 20-35 amino acids; the antigen information of the Tetanus Toxin 830-844 is presented to CD4+ T cells by APC through MHC II, and the antigen information of the 23 CEF epitope peptides is presented to CD8+ T cells by APC through MHC I.
The positive polypeptide pool for detecting the immune response of the tumor vaccine consists of a plurality of combined peptides prepared from the following polypeptide sequences;
the polypeptide sequence includes:
PB1(591–599)VSDGGPNLY-KK-QYIKANSKFIGITEL；
Influenza A NP(44–52)CTELKLSDY-KK-QYIKANSKFIGITEL；
BMLF1(259–267)GLCTLVAML-KK-QYIKANSKFIGITEL；
Matrix 1(58–66)GILGFVFTL-KK-QYIKANSKFIGITEL；
pp65(495–503)NLVPMVATV-KK-QYIKANSKFIGITEL；
NP(265–273)ILRGSVAHK-K-QYIKANSKFIGITEL；
BRLF1(148–156)RVRAYTYSK-K-QYIKANSKFIGITEL；
EBNA3A(603–611)RLRAEAQVK-K-QYIKANSKFIGITEL；
EBNA3B(416–424)IVTDFSVIK-K-QYIKANSKFIGITEL；
BRLF1(134–143)ATIGTAMYK-K-QYIKANSKFIGITEL；
BRLF1(28–37)DYCNVLNKEF-KK-QYIKANSKFIGITEL；
NP(91–99)KTGGPIYKR-KK-QYIKANSKFIGITEL；
pp65(415–429)RKTPRVTGGGAMAGA-KK-QYIKANSKFIGITEL；
EBNA3A(379–387)RPPIFIRRL-KK-QYIKANSKFIGITEL；
EBNA3A(158–166)QAKWRLQTL-KK-QYIKANSKFIGITEL；
EBNA3A(325–333)FLRGRAYGL-KK-QYIKANSKFIGITEL；
BZLF1(190–197)RAKFKQLL-KK-QYIKANSKFIGITEL；
NP(380–388)ELRSRYWAI-KK-QYIKANSKFIGITEL；
EBNA3C(258–266)RRIYDLIEL-KK-QYIKANSKFIGITEL；
NP(383–391)SRYWAIRTR-KK-QYIKANSKFIGITEL；
EBNA3A(458–466)YPLHEQHGM-KK-QYIKANSKFIGITEL；
EBNA3C(281–290)EENLLDFVRF-KK-QYIKANSKFIGITEL；
pp65(511–525)QEFFWDANDIYRIFA-KK-QYIKANSKFIGITEL。
the positive polypeptide pool for detecting the immune response of the tumor vaccine consists of a plurality of combined peptides prepared from the following polypeptide sequences; the polypeptide sequence consists of the following three sequences:
Matrix 1(58–66)GILGFVFTL-KK-QYIKANSKFIGITEL；
pp65(495–503)NLVPMVATV-KK-QYIKANSKFIGITEL；
EBNA3B(416–424)IVTDFSVIK-K-QYIKANSKFIGITEL。
a preparation method of a positive polypeptide pool for tumor vaccine immune response detection comprises the following steps:
step one, selecting a plurality of CEFs, connecting the CEFs with a Tetanus Toxin 830-844, and adding two lysine Ks between the two sequences for connection to obtain a plurality of polypeptide sequences;
and step two, synthesizing the polypeptide sequence into polypeptide by a chemical synthesis method to obtain a polypeptide preparation with the purity of more than 95 percent, preparing the polypeptide freeze-dried powder, filling, sealing and storing in dark place at the storage temperature of-20 ℃, and dissolving the polypeptide freeze-dried powder into corresponding concentration by using normal saline before use.
In the preparation method of the positive polypeptide pool for immune response detection of tumor vaccine, the method for synthesizing the polypeptide sequence into the polypeptide comprises the following steps: chemical synthesis method or gene recombination technology.
The invention has the advantages that:
the action mechanism of the invention is similar to that of the polypeptide vaccine commonly used in the current tumor immunotherapy;
the invention can eliminate the false negative condition caused by the abnormal function of APC cell;
the matching of the A section sequence and the B section sequence adopted by the invention can further ensure that the detection result is stable and reliable, stable response can be kept among different batches, the result is clear and clear, and the number of spots is moderate;
the Linker sequence can increase the water solubility of the polypeptide and increase the restriction enzyme sites between the AB, thereby reducing the possibility of generating a new antigen in which the C terminal of the A is connected with the N terminal of the B;
the preparation method of the positive polypeptide pool is simple, and the polypeptide sequence in the polypeptide pool can be flexibly adjusted.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
A positive polypeptide pool for detecting immune response of tumor vaccine comprises a plurality of combined peptides prepared from the following polypeptide sequences; the polypeptide sequence includes: labeling an epitope peptide sequence (with the length ranging from 7 to 11 amino acids) which can be combined with MHC class I molecules, efficiently presented and cause immune response as an A segment sequence; an epitope peptide sequence (usually 15 amino acids in length) which can be combined with MHC II molecules and efficiently presented to cause immune response is marked as a B segment sequence; the A segment sequence and the B segment sequence are connected into a complete polypeptide sequence through a connected amino acid (linker, L) sequence (the sequence of A and B in the front and back of L can be interchanged: ALB and BLA). And preparing the polypeptide according to the complete polypeptide sequence to obtain a positive polypeptide pool for detecting the immune response of the polypeptide vaccine.
The positive polypeptide pool can be prepared without adding a linker, but the method has more advantages after adding the linker, and the main advantages are that the water solubility of the polypeptide is increased, the enzyme cutting sites between AB are increased, and the possibility of generating new antigens with the C terminal of A and the N terminal of B connected is reduced.
The A section sequence is an epitope peptide sequence which can be combined with MHC I type molecules, is efficiently presented and causes immune response; including but not limited to the sequences obtained by the following steps. And acquiring the polypeptide sequence information of a standard positive stimulant CEF mixed T cell epitope polypeptide database which is universal internationally. The CEF polypeptide library is prepared by mixing 32 polypeptides, which are respectively taken from Cytomegalovirus (CMV), Epstein-Barr Virus (EBV) and Influenza Virus (Influenza Virus). The 32 polypeptides are human T cell epitope peptides at the same time, and are limited by HLA alleles A1, A2, A0201, A68, A3, A11, A6801, A24, B7, B8, B18, B27, B35, B44 and B0702 respectively. Among them, 23 polypeptides are the most frequently used polypeptides in the polypeptide library to elicit immune responses. Since healthy people may be infected with CMV, EBV or influenza virus and generate specific cellular immunity, the CEF stimulation of PBMC of healthy people also generates specific ELISPOT spots, almost all people can generate immune response to CEF polypeptide, and therefore the polypeptide library can be used as a positive control stimulant for in vitro detection.
The information of the sequence of the 23 epitope peptides with the highest reaction frequency in the CEF polypeptide library is obtained as shown in the following table 1-polypeptide sequences and HLA restriction (Peptide sequences and HLArestrions):
Peptide sequences and HLA restriction
aThsse eptides were added to increase the representation of epitopes from 16to 23.
hThe bolded letters represent the optimal epitope.
TABLE 1
The B segment sequence is an epitope peptide sequence which can be combined with MHC II type molecules, can be efficiently presented and can cause immune response, and includes but is not limited to the following sequences, such as a partial sequence of tetanus toxin.
Tetanus toxin has the characteristics of broad spectrum and strong immunogenicity, the probability that the fragment is recognized by antigen presenting cells in a human body is high, and the integral recognition rate of a positive polypeptide pool can be effectively improved by using tetanus toxin as a constituting fragment of positive peptides. Obtaining 830-844 sequence information of Tetanus Toxin: QYIKANSKFIGITEL are provided.
Linker sequences generally provide better hydrophilicity (e.g., lysine K sequences), stability, and structural flexibility (e.g., glycine G sequences) while providing cleavage sites. Commonly used sequences include, but are not limited to: k, KK, G, GG sequence.
The polypeptide sequence with the structure of A-L-B or B-L-A is obtained by design.
The following exemplary method can be used to design and prepare a positive polypeptide pool:
respectively and independently connecting 23 CEF epitope peptides with a Tetanus Toxin 830-844 through linker sequences and synthesizing a combined peptide, wherein the length of the combined peptide reaches 20-35 amino acids; the antigen information of the Tetanus Toxin 830-844 is presented to CD4+ T cells by APC through MHC II, and the antigen information of the 23 CEF epitope peptides is presented to CD8+ T cells by APC through MHC I, so that 23 combined peptides can be connected.
It should be noted that: the linker sequence (such as basic amino acid lysine) is added between the two sequences, so that the water solubility can be increased, the synthesis difficulty can be reduced and/or the sequence stability can be improved, and the complete retention of antigen information after degradation can be ensured as much as possible.
The resulting 23 polypeptide sequences are shown below:
PB1(591–599)VSDGGPNLY-KK-QYIKANSKFIGITEL；
Influenza A NP(44–52)CTELKLSDY-KK-QYIKANSKFIGITEL；
BMLF1(259–267)GLCTLVAML-KK-QYIKANSKFIGITEL；
Matrix 1(58–66)GILGFVFTL-KK-QYIKANSKFIGITEL；
pp65(495–503)NLVPMVATV-KK-QYIKANSKFIGITEL；
NP(265–273)ILRGSVAHK-K-QYIKANSKFIGITEL；
BRLF1(148–156)RVRAYTYSK-K-QYIKANSKFIGITEL；
EBNA3A(603–611)RLRAEAQVK-K-QYIKANSKFIGITEL；
EBNA3B(416–424)IVTDFSVIK-K-QYIKANSKFIGITEL；
BRLF1(134–143)ATIGTAMYK-K-QYIKANSKFIGITEL；
BRLF1(28–37)DYCNVLNKEF-KK-QYIKANSKFIGITEL；
NP(91–99)KTGGPIYKR-KK-QYIKANSKFIGITEL；
pp65(415–429)RKTPRVTGGGAMAGA-KK-QYIKANSKFIGITEL；
EBNA3A(379–387)RPPIFIRRL-KK-QYIKANSKFIGITEL；
EBNA3A(158–166)QAKWRLQTL-KK-QYIKANSKFIGITEL；
EBNA3A(325–333)FLRGRAYGL-KK-QYIKANSKFIGITEL；
BZLF1(190–197)RAKFKQLL-KK-QYIKANSKFIGITEL；
NP(380–388)ELRSRYWAI-KK-QYIKANSKFIGITEL；
EBNA3C(258–266)RRIYDLIEL-KK-QYIKANSKFIGITEL；
NP(383–391)SRYWAIRTR-KK-QYIKANSKFIGITEL；
EBNA3A(458–466)YPLHEQHGM-KK-QYIKANSKFIGITEL；
EBNA3C(281–290)EENLLDFVRF-KK-QYIKANSKFIGITEL；
pp65(511–525)QEFFWDANDIYRIFA-KK-QYIKANSKFIGITEL。
it should be noted that: these sequences are not exhaustive and are but one example.
Any combined peptide is 20-35 amino acids in length, can be taken up and processed by APC, then presents and stimulates T cells, the APC processed product is consistent with the characteristics of each epitope peptide, the immune recognition information is not lost, and the combined peptide can be used as an in vitro polypeptide vaccine immune response ELISpot detection positive control substance; after the tumor vaccine is recognized by DC, some antigens enter a classical MHC II presentation pathway to activate CD4 < + > T cells; while the antigen, which mediates endocytosis via some receptors, enters the MHC class i molecule presentation pathway, (this presentation pathway is called cross-presentation), activates CD8+ T cells.
Compared with the existing positive control, the compound can simultaneously activate CD4+ T and CD8+ cells, so that whether the phagocytosis, restriction, transportation or presentation functions of APC in an experimental system are healthy or not can be verified, and the T cells of CD8+ and CD4+ can be simultaneously verified.
The combined peptide can be prepared by the following method: chemical synthesis method or gene recombination technology.
The effect of the invention is verified by experiments as follows:
firstly, preparing a sample for experimental verification, and specifically designing and preparing the method as follows:
as a preference, CEF was selected in which 3 strands were linked to a Tetanus Toxin 830-844 sequence designed to increase the stability of the sequence by adding lysine between the sequences.
The specific process is as follows:
firstly, matrix (58-66) is selected to be connected with the Tetanus Toxin 830-844, and two lysine K connections GILGFVFTLKKQYIKANSKFIGITEL are added between the two sequences;
secondly, pp65(495-503) is selected to be connected with Tetanus Toxin 830-844, and two lysine K connections NLVPMVATVKKQYIKANSKFIGITEL are added between the two sequences;
thirdly, EBNA3B (416-424) is selected to be connected with the Tetanus Toxin 830-844, and a lysine K connection IVTDFSVIKKQYIKANSKFIGITEL is added between the two sequences;
synthesizing 3 polypeptides by chemical synthesis method to obtain polypeptide preparation with purity higher than 95%. Preparing into lyophilized polypeptide powder, filling into penicillin bottle, sealing in dark place, storing at-20 deg.C, and dissolving with normal saline to corresponding concentration before use.
Experiment one, the false negative condition of the polypeptide vaccine is eliminated by using the positive polypeptide pool;
in an in vitro verification experiment of the curative effect of the polypeptide vaccine, a false negative condition caused by abnormal APC (APC) of a patient often occurs, and when only a CEF (cytokine induced plasma) polypeptide pool is used as a positive control, the false negative condition cannot be accurately eliminated, so that the actual effect of the polypeptide vaccine is misjudged. This example will illustrate that the positive polypeptide pool is used to eliminate the false negative condition of the polypeptide vaccine, so as to accurately determine the actual effect of the polypeptide vaccine, which is an advantage of the positive polypeptide pool compared to the CEF polypeptide pool.
The experiment was performed using clinical specimens obtained from the same patient P016, with the specimen numbers WJMI190311 and WJMI 190401. Clinically injecting polypeptide vaccine for immunotherapy, extracting blood sample, separating PBMC, performing in vitro verification of polypeptide vaccine curative effect, and detecting IFN-gamma secretion after polypeptide stimulation. The ELISpot method [5] was used with the polypeptide vaccine as the stimulus, the positive polypeptide pool of the invention as the positive control 1, the CEF polypeptide pool as the positive control 2, and the blank as the negative control.
The specific process of clinical polypeptide immunotherapy in vitro validation is as follows:
venous blood from patients undergoing polypeptide immunotherapy was drawn, fresh blood received at room temperature, and the blood was expressed using pre-warmed PBS as "blood: PBS 1: 1' and mixing uniformly for separation. Mononuclear cells were isolated using lymphocyte isolate Ficoll-Paque PLUS and washed 2 times with 37 ℃ pre-warmed PBS or 1640 medium. Cell mass and cell viability were determined by trypan blue counting. The number of experimental wells was determined from the amount of polypeptide using the human IFN-. gamma.ELISpot kit (Dayuu), and 100. mu.l of cell suspension was added per well. The corresponding polypeptide is added to a polypeptide concentration of 20. mu.g/ml. The ELISpot plate was incubated in a 37 ℃ carbon dioxide incubator for 48 h. And performing color development operation after the culture is finished. The specific color development step is described in the Specification of the Dayou human IFN-gamma ELISpot kit.
Observing the spot result, use
Spot counts were performed on the S6FluoroSpot Line instrument and ELISpot results for clinical sample WJMI190311 are shown in table 2 for sample number WJMI 190311.
| Experimental group | Positive control group 1 | Positive control group 2 | Negative control group |
| Polypeptide vaccine A | Positive polypeptide pool | CEF polypeptide pool | Blank group |
| Counting the number of spots: 0. 0 to | Number of spots 0, 1 | Counting the number of spots: 167, 149 | Counting the number of spots: 0,0 |
TABLE 2
From table 2, it can be seen that the results of the polypeptide vaccine a group and the positive polypeptide pool (positive control group 1) of the present invention are negative, while the result of the CEF group (positive control group 2) is positive. At this point it is not possible to tell whether the polypeptide vaccine is a negative result. Further experiments are needed to eliminate false negatives in patients with APC dysfunction, such as poor polypeptide uptake. Aiming at the clinical sample WJMI190401 of the same patient P016, the experimental method is improved, the incubation time of the polypeptide vaccine and the patient cells is prolonged, and the amount of the polypeptide taken by APC cells is increased, so that whether the polypeptide vaccine A is in a false negative condition in the batch experiment of the WJMI190311 sample is verified. The results are shown in Table 3-ELISpot test results for sample No. WJMI 190401.
| Experimental group | Positive control group 1 | Positive control group 2 | Negative control group |
| Polypeptide vaccine A | Positive polypeptide pool | CEF polypeptide pool | Blank group |
| Counting the number of spots: 67. 65 of the formula | Counting the number of spots: 53. 55 of a glass fiber | Counting the number of spots: 152. 151 of a gas turbine | Counting the number of spots: 0,0 |
TABLE 3
As can be seen from table 3, the CEF polypeptide pool (positive control 2) was still positive as in table 2; the results of the polypeptide vaccine A and the positive polypeptide pool (positive control group 1) of the present invention were changed from the negative results in Table 2 to the positive results.
The polypeptide vaccine A in the table 2 has no spot, the positive polypeptide pool has no spot, the CEF result is normal, and the polypeptide vaccine A is reminded of the false negative condition; by improving the experimental method, the results in table 3 confirm that the false negative condition caused by abnormal APC such as too weak polypeptide uptake capacity does appear in table 2, and thus the misjudgment of the effect of the polypeptide vaccine a is avoided. Therefore, compared with the CEF polypeptide pool only used as a positive control, the adoption of the positive polypeptide pool as a control group can effectively eliminate the false negative condition of the polypeptide vaccine.
Experiment II, verifying the effect stability of the positive polypeptide pool as a positive control of the polypeptide vaccine;
in order to further verify the stability and reliability of the positive polypeptide pool as the positive control group of the polypeptide vaccine, the same experimental method is adopted, and two ELISpot tests are carried out on immune cell samples of another patient P021, wherein the samples are numbered as LLM190109 and LLM 190218. The results are shown in Table 4-ELISpot test results of sample number LLM190109 and Table 5-ELISpot test results of sample number LLM 190218.
| Experimental group | Positive control group 1 | Positive control group 2 | Negative control group |
| Polypeptide vaccine B | Positive polypeptide pool | CEF polypeptide pool | Blank group |
| Counting the number of spots: 20. 25 of | Counting the number of spots: 105. 187 of the invention | Counting the number of spots: 334. 347 is prepared by | Counting the number of spots: 0,1 |
TABLE 4
| Experimental group | Positive control group 1 | Positive control group 2 | Negative control group |
| Polypeptide vaccine B | Positive polypeptide pool | CEF polypeptide pool | Blank group |
| Counting the number of spots: 19. 25 of | Counting the number of spots: 185. 173 the formula | Counting the number of spots: 286. 270 to k | Counting the number of spots: 0,0 |
TABLE 5
In the results in tables 4 and 5, the positive polypeptide pool (positive control group 1) and the polypeptide vaccine B of the present invention both have positive results, and the abnormal cases that the polypeptide vaccine results are positive and the positive control group 1 is negative do not occur. From the results, the positive polypeptide pool can keep stable response among different batches of different patients, the spot result is clear and clear, the quantity is moderate, the positive control effect is good, and the stability and the reliability of the positive polypeptide pool are verified.
Thirdly, verifying the necessity of the positive polypeptide pool as a positive control group of the polypeptide vaccine;
further analysis and comparison of the ELISpot test results of all patients in the existing clinical tests are performed, and as shown in table 6-positive control mode, the positive detection rate comparison of polypeptide vaccines (53 patients in total, 212 times in total for ELISpot test batches, and 701 in total for polypeptide vaccines) shows, different positive polypeptide vaccine detection rate results are obtained by respectively using the positive polypeptide pool and the CEF polypeptide pool of the present invention as the positive control group 1 and the positive control group 2.
TABLE 6
From the results in table 6, it can be seen that the detection rate (59.2%) of the positive polypeptide vaccine obtained by increasing the positive polypeptide pool of the present invention as the positive control group of the polypeptide vaccine is significantly higher than that of the experimental group (36.2%) using only the CEF polypeptide pool as the positive control group. The experimental results prove the necessity of the positive polypeptide pool as a positive control substance of the immune response result of the polypeptide vaccine.
Compared with stimulants such as CEF short peptide and the like, the positive polypeptide pool has the advantages that: 1. similar to the action mechanism of the polypeptide vaccine commonly used in the current tumor immunotherapy, the false negative condition caused by the abnormal function of the APC can be eliminated. 2. The control result generated by the positive polypeptide pool is stable and reliable, stable response can be kept among different batches, the result is clear and clear, and the number of spots is moderate. 3. The preparation method of the positive polypeptide pool for detecting the immune response of the polypeptide vaccine is simple, and the polypeptide sequence in the polypeptide pool can be flexibly adjusted.
The action mechanism of the invention is similar to that of the polypeptide vaccine commonly used in the current tumor immunotherapy; the invention can eliminate the false negative condition caused by the abnormal function of APC cell; the matching of the A section sequence and the B section sequence adopted by the invention can further ensure that the detection result is stable and reliable, stable response can be kept among different batches, the result is clear and clear, and the number of spots is moderate; the Linker sequence can increase the water solubility of the polypeptide and increase the restriction enzyme sites between the AB, thereby reducing the possibility of generating a new antigen in which the C terminal of the A is connected with the N terminal of the B; the preparation method of the positive polypeptide pool is simple, and the polypeptide sequence in the polypeptide pool can be flexibly adjusted.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Sequence listing
<110> Hangzhou Nianjin Biotechnology Co., Ltd
<120> positive polypeptide pool for tumor vaccine immune response detection and preparation method thereof
<141> 2019-12-30
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 1
Val Ser Asp Gly Gly Pro Asn Leu Tyr Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 2
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 2
Cys Thr Glu Leu Lys Leu Ser Asp Tyr Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 3
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 3
Gly Leu Cys Thr Leu Val Ala Met Leu Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 4
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 4
Gly Ile Leu Gly Phe Val Phe Thr Leu Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 5
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 5
Asn Leu Val Pro Met Val Ala Thr Val Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 6
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 6
Ile Leu Arg Gly Ser Val Ala His Lys Lys Gln Tyr Ile Lys Ala Asn
1 5 10 15
Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 7
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 7
Arg Val Arg Ala Tyr Thr Tyr Ser Lys Lys Gln Tyr Ile Lys Ala Asn
1 5 10 15
Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 8
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 8
Arg Leu Arg Ala Glu Ala Gln Val Lys Lys Gln Tyr Ile Lys Ala Asn
1 5 10 15
Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 9
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 9
Ile Val Thr Asp Phe Ser Val Ile Lys Lys Gln Tyr Ile Lys Ala Asn
1 5 10 15
Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 10
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 10
Ala Thr Ile Gly Thr Ala Met Tyr Lys Lys Gln Tyr Ile Lys Ala Asn
1 5 10 15
Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 11
<211> 27
<212> PRT
<213> Artificial Sequence
<400> 11
Asp Tyr Cys Asn Val Leu Asn Lys Glu Phe Lys Lys Gln Tyr Ile Lys
1 5 10 15
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 12
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 12
Lys Thr Gly Gly Pro Ile Tyr Lys Arg Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 13
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 13
Arg Lys Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Lys
1 5 10 15
Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25 30
<210> 14
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 14
Arg Pro Pro Ile Phe Ile Arg Arg Leu Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 15
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 15
Gln Ala Lys Trp Arg Leu Gln Thr Leu Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 16
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 16
Phe Leu Arg Gly Arg Ala Tyr Gly Leu Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 17
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 17
Arg Ala Lys Phe Lys Gln Leu Leu Lys Lys Gln Tyr Ile Lys Ala Asn
1 5 10 15
Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 18
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 18
Glu Leu Arg Ser Arg Tyr Trp Ala Ile Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 19
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 19
Arg Arg Ile Tyr Asp Leu Ile Glu Leu Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 20
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 20
Ser Arg Tyr Trp Ala Ile Arg Thr Arg Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 21
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 21
Tyr Pro Leu His Glu Gln His Gly Met Lys Lys Gln Tyr Ile Lys Ala
1 5 10 15
Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 22
<211> 27
<212> PRT
<213> Artificial Sequence
<400> 22
Glu Glu Asn Leu Leu Asp Phe Val Arg Phe Lys Lys Gln Tyr Ile Lys
1 5 10 15
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25
<210> 23
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 23
Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Lys
1 5 10 15
Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu
20 25 30
Claims (4)
1. A positive polypeptide pool for immune response detection of tumor vaccine is characterized by consisting of the following polypeptides;
the polypeptide is:
labeling an epitope peptide sequence which can be combined with MHC I type molecules, is efficiently presented and causes immune response as an A section sequence;
labeling an epitope peptide sequence which can be combined with MHC II molecules, is efficiently presented and causes immune response as a B segment sequence;
connecting the A section sequence and the B section sequence through a linker sequence connected with amino acids, and designing to obtain a polypeptide sequence with an A-L-B or B-L-A structure;
respectively and independently connecting 23 CEF epitope peptides marked as A section sequences with a tetania Toxin 830-844 marked as B section sequences through a linker sequence and synthesizing combined peptides to enable the lengths to reach 20-35 amino acids; the antigen information of the Tetanus Toxin 830-844 is presented to CD4+ T cells by APC through MHC II, and the antigen information of the 23 CEF epitope peptides is presented to CD8+ T cells by APC through MHC I;
the sequence of the polypeptide includes:
VSDGGPNLY-KK-QYIKANSKFIGITEL；
CTELKLSDY-KK-QYIKANSKFIGITEL；
GLCTLVAML-KK-QYIKANSKFIGITEL；
GILGFVFTL-KK-QYIKANSKFIGITEL；
NLVPMVATV-KK-QYIKANSKFIGITEL；
ILRGSVAHK-K-QYIKANSKFIGITEL；
RVRAYTYSK-K-QYIKANSKFIGITEL；
RLRAEAQVK-K-QYIKANSKFIGITEL；
IVTDFSVIK-K-QYIKANSKFIGITEL；
ATIGTAMYK-K-QYIKANSKFIGITEL；
DYCNVLNKEF-KK-QYIKANSKFIGITEL；
KTGGPIYKR-KK-QYIKANSKFIGITEL；
RKTPRVTGGGAMAGA-KK-QYIKANSKFIGITEL；
RPPIFIRRL-KK-QYIKANSKFIGITEL；
QAKWRLQTL-KK-QYIKANSKFIGITEL；
FLRGRAYGL-KK-QYIKANSKFIGITEL；
RAKFKQLL-KK-QYIKANSKFIGITEL；
ELRSRYWAI-KK-QYIKANSKFIGITEL；
RRIYDLIEL-KK-QYIKANSKFIGITEL；
SRYWAIRTR-KK-QYIKANSKFIGITEL；
YPLHEQHGM-KK-QYIKANSKFIGITEL；
EENLLDFVRF-KK-QYIKANSKFIGITEL；
QEFFWDANDIYRIFA-KK-QYIKANSKFIGITEL。
2. the positive polypeptide pool for the immune response test of tumor vaccine according to claim 1, which is composed of the following polypeptides; the sequence of the polypeptide is:
GILGFVFTL-KK-QYIKANSKFIGITEL；
NLVPMVATV-KK-QYIKANSKFIGITEL；
IVTDFSVIK-K-QYIKANSKFIGITEL。
3. the method for preparing the positive polypeptide pool for the immune response detection of the tumor vaccine according to claim 1, which comprises the following steps:
step one, selecting a plurality of CEFs, connecting the CEFs with a Tetanus Toxin 830-844, and adding two lysine Ks between the two sequences for connection to obtain a plurality of polypeptide sequences;
and step two, synthesizing the polypeptide sequence into polypeptide to obtain a polypeptide preparation with the purity of more than 95 percent, preparing the polypeptide preparation into polypeptide freeze-dried powder, filling, sealing and storing in dark at the temperature of-20 ℃, and dissolving the polypeptide freeze-dried powder into corresponding concentration by using physiological saline before use.
4. The method for preparing the positive polypeptide pool for the immune response detection of the tumor vaccine according to claim 3, wherein the method for synthesizing the polypeptide sequence into the polypeptide comprises the following steps: chemical synthesis method or gene recombination technology.
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| CN110343667A (en) * | 2019-07-17 | 2019-10-18 | 贝赛尔特(北京)生物技术有限公司 | Immunocyte of engineering and its preparation method and application |
| CN110499324A (en) * | 2019-09-02 | 2019-11-26 | 中生康元生物科技(北京)有限公司 | A method of for identifying the bacterial expression vector and screening and identification tumour neoantigen of tumour neoantigen |
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| WO2016198470A2 (en) * | 2015-06-08 | 2016-12-15 | Lophius Biosciences Gmbh | Composition for determination of cell-mediated immune responsiveness |
| CN110343667A (en) * | 2019-07-17 | 2019-10-18 | 贝赛尔特(北京)生物技术有限公司 | Immunocyte of engineering and its preparation method and application |
| CN110499324A (en) * | 2019-09-02 | 2019-11-26 | 中生康元生物科技(北京)有限公司 | A method of for identifying the bacterial expression vector and screening and identification tumour neoantigen of tumour neoantigen |
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