CN113493765A - BsAb in vitro loaded T cell - Google Patents
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Abstract
The invention belongs to the field of biomedicine and the technical field of cell therapy, and particularly relates to a BsAb in-vitro loaded T cell. The method comprises the following steps: the structure design and optimization of BsAb; preparing BsAb; analyzing the stability of BsAb; affinity analysis of BsAb; culturing immune cells; BsAb loads T cells in vitro; killing of in vitro tumor cells by BsAb loaded T cells; secretion of cytokines; expression of proto-oncogenes and cancer suppressor genes; killing of tumor cells in vivo by BsAb loaded T cells; the TaqMan chip screens the changes of immune related genes and tumor markers in tumor tissues; immunohistochemistry was performed to determine the CD3+ T cell content in tumor tissues. The invention provides a BsAb in-vitro loaded T cell which is prepared by preparing a BsAb through chemical crosslinking at an early stage, is intended to construct and synthesize a specific BsAb, increases the affinity of the BsAb to a tumor cell related antigen, establishes a high-efficiency and simple method for loading immune cells in vitro by the BsAb, and establishes an immune cell in-vitro amplification method which is simple and convenient to operate, safe, free of pollution and strong in repeatability.
Description
Technical Field
The invention belongs to the field of biomedicine and the technical field of cell therapy, and particularly relates to a BsAb in-vitro loaded T cell.
Background
Based on the technology of establishing chemical crosslinking preparation of BsAb in the early stage, a series of BsAb prepared based on a genetic engineering method is to be developed, and the tumor ultra-early treatment technology of loading autoimmune cells in vitro is used. The technology has the characteristics of safety, reliability, low toxic and side effect, strong specificity and the like, can effectively eliminate tiny focuses, prevent tumor recurrence, and provides an important intervention means for the ultra-early cancer treatment.
Autoimmune cell therapy has become a new tumor therapy model as the latest tumor immunotherapy technology, and despite the rapid development in recent years, the overall effect is still poor. The reason is mainly related to the immune escape of tumors through various mechanisms. For example, tumor cells express the same antigen as normal tissue cells to escape the recognition of the immune system of the body; inhibiting the function of immunocompetent cells by secreting immunosuppressive factors such as TGF-beta and IL-10; anti-tumor effects of cytokines and Cytotoxic T Lymphocytes (CTL) and the like are resisted by down-regulating TNF-alpha receptors, Fas receptors and the like. In order to further enhance the killing of tumor cells by immune cells, the targeting of immune cells needs to be increased.
The advent of bispecific antibodies (BsAb) provides a new approach for solving the targeting problem of autoimmune cell therapy. Because the BsAb contains two Fab sections which can recognize different specific antigens, the contact time of the tumor cells and the effector cells is prolonged by combining the surface antigens of the effector cells and the tumor cells, the escape of the tumor cells can be effectively prevented, the tumor killing capability of the effector cells is enhanced, and the effect of selectively killing tumors is achieved.
There are three main methods for preparing BsAb, namely chemical cross-linking method, hybridoma cell method and genetic engineering method.
As a new approach to immunotherapy, BsAb was originally called "antigenic cross", and in the early 90 s BsAb was constructed by chemical cross-linking two antibodies that specifically recognized different antigenic determinants on the surface of known tumor cells. The BsAb contains two monoclonal antibodies which can be specifically combined with different antigenic determinants, can be simultaneously combined with tumor cell-associated antigens CD19, HER-2 and the like and receptors of blood circulation immune effector cells such as Fc gamma RI (CD64), Fc gamma RII (CD32) and the like, so that the BsAb can selectively recognize tumor cells, can also introduce the immune effector cells (such as monocyte, macrophage, polymorphonuclear granulocyte, natural killer cell, activated T cell and the like) in the circulation blood to tumor tissues and can induce the immune effector cells to generate antitumor activity. The chemical crosslinking method is developed to present day and is a mature BsAb preparation method, but the cost is high, so that the market demand is difficult to meet.
In recent years, with the development of molecular techniques and genetic engineering techniques, the research on BsAb has been rapidly developed. The preparation of BsAb has also been followed by more advanced methods, such as hybrid hybridoma cell method and genetic engineering method. The hybridoma cell method is gradually eliminated by researchers because the screening of the hybridoma cells is complicated and the seed preservation is difficult. The genetic engineering method has become the main development direction of the BsAb preparation method because the structure can be designed, optimized and adjusted, and the large-scale industrial production can be realized.
In recent years, with the development of molecular techniques and genetic engineering techniques, the research on BsAb has been rapidly developed. The preparation of BsAb has also been followed by more advanced methods, such as hybrid hybridoma cell method and genetic engineering method. The hybridoma cell method is gradually eliminated by researchers because the screening of the hybridoma cells is complicated and the seed preservation is difficult. The genetic engineering method has become the main development direction of the BsAb preparation method and is also a developed technology because the structure can be designed, optimized and adjusted and the large-scale industrial production can be realized.
In conclusion, the technologies of efficiently amplifying NK cells in vitro and transfecting NKG2D to activate NK cells, the DC vaccine and DC-CTL technologies of cell-penetrating peptide mediated tumor antigen polypeptide sensitized and transfecting CD40L and the early intervention technology of BsAb in vitro modified T cell tumors are to be developed. The technical system has an upgraded NK technology for a broad-spectrum killing technology for pathological changes or tumor cells, and also has a DC-CTL and BsAb in-vitro modified immune cell technology which is safe, reliable, low in toxic and side effects and strong in specificity, and a proper immune technology can be selected according to the detection result of a high risk group of tumors, so that cells with tiny focuses or early pathological changes and cancer cells can be effectively eliminated, and the incidence rate of cancers is reduced. The provided intervention means which is important for treating the ultra-early cancer has important social significance and economic value.
The key problem to be solved by BsAb in-vitro loading T cell technology (1) anti-mouse antibody reaction, we intend to prepare BsAb by adopting a genetic engineering method, and realize effective in-vivo distribution of BsAb by using T cells in a way of activating T cells by BsAb in-vitro loading, so that the using amount of BsAb is reduced, and anti-mouse antibody reaction is reduced; (2) the selective killing of tumor cells is realized, and we intend to utilize the specificity of BsAb on tumor-associated antigens to realize the selective killing of T cells on tumor cells and further reduce toxic and side effects; (3) the design of a new target of the double-antibody to the tumor and an evaluation system of safety and effectiveness.
Disclosure of Invention
The invention provides a BsAb in-vitro loaded T cell which is prepared by preparing a BsAb through chemical crosslinking at an early stage, is intended to construct and synthesize a specific BsAb, increases the affinity of the BsAb to a tumor cell related antigen, establishes a high-efficiency and simple method for loading immune cells in vitro by the BsAb, and establishes an immune cell in-vitro amplification method which is simple and convenient to operate, safe, free of pollution and strong in repeatability.
The technical scheme adopted by the invention is as follows: BsAb loaded T cells in vitro, characterized in that: the method comprises the following steps:
s1: the structure design and optimization of BsAb; designing single-chain bispecific antibodies with different configurations, and optimizing the sequence and the structure;
s2: preparing BsAb; the preparation method of the BsAb comprises a hybrid hybridoma technology, a chemical crosslinking method and a genetic engineering method;
s3: analyzing the stability of BsAb; 10ug of double antibody was allowed to stand at 4 ℃ for 7 days, and the size of the double antibody fragment was then detected by non-reducing SDS-PAGE gel;
s4: affinity analysis of BsAb;
s5: culturing immune cells; t cells are the most main immune cells in the body, and are selected as effector cells;
s6: BsAb loads T cells in vitro; incubating BsAb and T cells for 1h, taking about 106 cells, incubating with corresponding fluorescence labeled antigen for 2h, washing free antigen after the BsAb loaded T cells are combined with the corresponding antigen, adding serum for sealing, washing excessive serum after incubation and combination, and counting by using a flow cytometer;
s7: killing of in vitro tumor cells by BsAb loaded T cells;
s8: secretion of cytokines;
s9: expression of proto-oncogenes and cancer suppressor genes;
s10: killing of tumor cells in vivo by BsAb loaded T cells;
s11: the TaqMan chip screens the changes of immune related genes and tumor markers in tumor tissues;
s12: immunohistochemistry was performed to determine the CD3+ T cell content in tumor tissues.
The affinity analysis of the BsAb is specifically as follows:
A) incubating the double antibody and 1X 106T cells for 2h, wherein the final concentration of the double antibody is 10nM concentration and 50nM concentration;
B) adding the cell suspension after double antibody incubation into a 2ml round-bottom centrifuge tube, centrifuging at 1500rpm for 5min, and removing the supernatant;
C) fixing with 1% paraformaldehyde-containing PBS at room temperature for 15min, and washing with PBS for 1 time;
D) incubating the goat anti-mouse or goat anti-human IgG, F (ab') 2 fragment specific antibody labeled with biotin at 4 ℃ for 2h, and washing the antibody with PBS for 1 time;
E) adding 200ul of PE-labeled streptavidin, mixing uniformly by blowing, incubating for 1h at 4 ℃, and keeping out of the sun;
F) washing with PBS 1ml by centrifugation for 2 times;
G) resuspending the cells in 500ul PBS, mixing, placing in a flow tube, keeping out of the sun, and detecting on a machine;
H) the affinity of the diabodies to the target cells was determined in the same manner.
The T cell in-vitro amplification culture method comprises the following steps:
A) coating: adding coating factor into 10ml of D-PBS, keeping out of the sun at 4 ℃, and coating 2T 175 culture bottles overnight; or placing in 37 deg.C incubator, and coating for at least 2 hr;
B) cell preparation:
a: drawing 100ml of anticoagulated peripheral blood, 800g, and centrifuging at room temperature for 15min (without breaking);
b: preparation of autologous plasma: collecting supernatant plasma, and placing in water bath at 56 deg.C for 30 min; then standing for 10min at-20 ℃; finally, centrifuging for 15min at 4 ℃ and 1100g, and storing at 4 ℃ for later use;
c: taking the centrifuged lower cell component, adding D-PBS to 50ml, mixing, adding into 4 50ml centrifuge tubes containing 20ml medical grade lymphocyte separation liquid, centrifuging at room temperature for 15min (without breaking) at 800 g;
d: taking the cell layer, adding the culture medium to 50ml, 600g for 10min, and removing the supernatant;
e: the separated PBMC was added to 80mlT cell culture solution to prepare a cell suspension, 5ml of plasma was added to 2T 175 flasks from which the coating solution was discarded, and the flask was simultaneously charged with the induction factor and kept at a saturated humidity of 37 ℃ and 5 ℃. Culturing in 0% CO2 incubator;
C) cell expansion: the concentration of various induction factors in the T cell culture solution is adjusted, the amplification speed of the T cells is accelerated, and the activity of the T cells is increased.
Killing of the BsAb-loaded T cells on in-vitro tumor cells takes the BsAb-loaded T cells as effector cells and cancer cell lines as target cells; setting a pure T cell control group and a target cell control group; t-test was used for each experimental group with P < 0. 05 is that the difference is statistically significant;
the killing rate (%) [1- (experimental OD value-T cell control OD value)/target cell control OD value ] × 100% experiment.
The secretion of the cell factors is realized by paving a cancer cell line in a logarithmic growth phase in a 12-hole plate by 105 cells/hole, adding T cells or BsAb loaded T cells in the next day, co-culturing for 4 hours at the concentration of an effective target ratio of 5:1, collecting supernatant, and detecting the secretion levels of IFN-gamma and TNF-alpha of the cells by using ELISA Kit Human IFN-gamma and ELISA Kit Human TNF-alpha.
The expression of the protooncogene and the cancer suppressor gene is that tumor cells are planted into a 6-well plate by 2 x106 cells per well, 2 x 107-effect T cells and BsAb-loaded T cells (effect target ratio concentration is 5: 1) are respectively added in the next day, untreated tumor cells are used as blank control, the blank control is co-cultured for 24 hours, supernate is discarded, PBS is used for rinsing the cells twice, TRIZOL1ml is added, cell lysate is collected, total RNA is extracted by using a total RNA kit, and the total RNA is reversely transcribed into cDNA; and (3) detecting the mRNA expression of the related gene by using the cDNA as a template through qPCR.
Killing of tumor cells in vivo by BsAb-loaded T cells 30 nude mice of 6 weeks of age, half of each nude mouse, and each nude mouse is injected with 1 × 107 positive tumor cell lines subcutaneously in the right axilla, and the nude mice are randomly divided into 3 groups: saline control group, T cell group and BsAb-loaded T cell group; BsAb-loaded T cells and T cells were used as effector cells, and injected into caudal vein at a rate of 1X 107 cells/time in a total volume of 200ul 2 times per week. The control group was replaced with the same volume of saline. Before each treatment, the long diameter and the short diameter of the tumor are measured by a vernier caliper, and the tumor volume is calculated (the length is a mm, the width is b mm, and the calculation formula of the tumor volume is 1/2ab 2); and counting the death number per day, and drawing a survival rate curve of the transplanted tumor model mouse.
The TaqMan chip screens the changes of immune related genes and tumor markers in tumor tissues, and after a tumor model mouse is transplanted for one month, cervical dislocation is killed and the tumor tissues are collected. Grinding tumor tissue to extract RNA, carrying out reverse transcription to obtain cDNA, and sending the cDNA as a template to a professional biological company for TaqMan chip method detection of expression changes of immune related genes and tumor markers.
The immunohistochemical detection of the content of CD3+ T cells in tumor tissues allows transplanted tumor model mice to be killed by dislocation of cervical vertebrae one month after treatment and tumor tissues to be collected. The tumor tissue was sectioned into tissue pieces with a thickness of 4 μm and then immunohistochemically stained. Formalin-fixed paraffin-embedded tumor tissue was sequentially soaked in alcohol and xylene at different concentration gradients, placed in citrate buffer PH10 and heated for 25 minutes for antigen retrieval, 3% hydrogen peroxide solution was reacted at room temperature for 5 minutes, 1: 50 diluted monoclonal CD3 antibody was reacted at room temperature for 60 minutes, the secondary antibody was reacted at room temperature for 30 minutes, reacted with DAB solution at room temperature for 5 minutes, stained with hematoxylin solution for 5 minutes, and the sections were placed on an Olympus microscope after being coverslipped for observation and evaluation.
The invention has the beneficial effects that:
aiming at the problem of BsAb affinity, the affinity of BsAb with T cells and related antigens is increased by using a codon optimization method.
Secondly, BsAb is completely designed and synthesized by humanized antibody sequence and a mammalian cell protein expression system, thereby reducing the immunogenicity of the antibody.
And thirdly, the BsAb loads T cells in vitro, so that the possible immunogenic reaction caused by the antibody is further reduced.
And fourthly, the autologous T cells are used as effector cells for early intervention of the tumor, so that the method is safe and reliable and has no toxic or side effect.
The tumor cells can be cracked under the condition of a low effective target ratio, compared with the traditional method of singly using the antibody or singly using a cell culture system, the using amount of the BsAb is greatly reduced, the cell culture system is reduced, and the early intervention cost of the tumor is greatly reduced; .
And sixthly, the BsAb is used as a medium to connect immune effector cells and tumor cells by simultaneously recognizing two targets, so that the distance between the immune effector cells and the tumor cells is shortened, the number of effector cells near the tumor cells is increased, and the killing efficiency of the tumor cells is enhanced.
And seventhly, the release of factors such as IFN-gamma and TNF-alpha secreted by the T cells can be greatly enhanced, a complement system and an Fc receptor related effector system can be effectively activated, and the tumor killing capability of the T cells is enhanced.
Drawings
FIG. 1 is a technical scheme of the BsAb loading T cells in vitro.
Detailed Description
BsAb loaded T cells in vitro, characterized in that: the method comprises the following steps:
s1: the structure design and optimization of BsAb; designing single-chain bispecific antibodies with different configurations, and optimizing the sequence and the structure;
s2: preparing BsAb; the preparation method of the BsAb comprises a hybrid hybridoma technology, a chemical crosslinking method and a genetic engineering method; because the hybrid hybridoma technology has low yield, consumes time and labor, is not easy to screen, and the chemical cross-linking method is easy to cause protein denaturation and can not activate antibodies, a genetic engineering method is adopted. To ensure the function of BsAb, we propose to use eukaryotic expression system for expression.
S3: analyzing the stability of BsAb; 10ug of double antibody was allowed to stand at 4 ℃ for 7 days, and the size of the double antibody fragment was then detected by non-reducing SDS-PAGE gel;
s4: affinity analysis of BsAb;
s5: culturing immune cells; t cells are the most main immune cells in the body, and are selected as effector cells;
s6: BsAb loads T cells in vitro; incubating BsAb and T cells for 1h, taking about 106 cells, incubating with corresponding fluorescence labeled antigen for 2h, washing free antigen after the BsAb loaded T cells are combined with the corresponding antigen, adding serum for sealing, washing excessive serum after incubation and combination, and counting by using a flow cytometer;
s7: killing of in vitro tumor cells by BsAb loaded T cells;
s8: secretion of cytokines;
s9: expression of proto-oncogenes and cancer suppressor genes;
s10: killing of tumor cells in vivo by BsAb loaded T cells;
s11: the TaqMan chip screens the changes of immune related genes and tumor markers in tumor tissues;
s12: immunohistochemistry was performed to determine the CD3+ T cell content in tumor tissues.
The affinity analysis of the BsAb is specifically as follows:
A) incubating the double antibody and 1X 106T cells for 2h, wherein the final concentration of the double antibody is 10nM concentration and 50nM concentration;
B) adding the cell suspension after double antibody incubation into a 2ml round-bottom centrifuge tube, centrifuging at 1500rpm for 5min, and removing the supernatant;
C) fixing with 1% paraformaldehyde-containing PBS at room temperature for 15min, and washing with PBS for 1 time;
D) incubating the goat anti-mouse or goat anti-human IgG, F (ab') 2 fragment specific antibody labeled with biotin at 4 ℃ for 2h, and washing the antibody with PBS for 1 time;
E) adding 200ul of PE-labeled streptavidin, mixing uniformly by blowing, incubating for 1h at 4 ℃, and keeping out of the sun;
F) washing with PBS 1ml by centrifugation for 2 times;
G) resuspending the cells in 500ul PBS, mixing, placing in a flow tube, keeping out of the sun, and detecting on a machine;
H) the affinity of the diabodies to the target cells was determined in the same manner.
The T cell in-vitro amplification culture method comprises the following steps:
A) coating: adding coating factor into 10ml of D-PBS, keeping out of the sun at 4 ℃, and coating 2T 175 culture bottles overnight; or placing in 37 deg.C incubator, and coating for at least 2 hr;
B) cell preparation:
a: drawing 100ml of anticoagulated peripheral blood, 800g, and centrifuging at room temperature for 15min (without breaking);
b: preparation of autologous plasma: collecting supernatant plasma, and placing in water bath at 56 deg.C for 30 min; then standing for 10min at-20 ℃; finally, centrifuging for 15min at 4 ℃ and 1100g, and storing at 4 ℃ for later use;
c: taking the centrifuged lower cell component, adding D-PBS to 50ml, mixing, adding into 4 50ml centrifuge tubes containing 20ml medical grade lymphocyte separation liquid, centrifuging at room temperature for 15min (without breaking) at 800 g;
d: taking the cell layer, adding the culture medium to 50ml, 600g for 10min, and removing the supernatant;
e: the separated PBMC was added to 80mlT cell culture solution to prepare a cell suspension, 5ml of plasma was added to 2T 175 flasks from which the coating solution was discarded, and the flask was simultaneously charged with the induction factor and kept at a saturated humidity of 37 ℃ and 5 ℃. Culturing in 0% CO2 incubator;
C) cell expansion: the concentration of various induction factors in the T cell culture solution is adjusted, the amplification speed of the T cells is accelerated, and the activity of the T cells is increased.
Killing of the BsAb-loaded T cells on in-vitro tumor cells takes the BsAb-loaded T cells as effector cells and cancer cell lines as target cells; setting a pure T cell control group and a target cell control group; t-test was used for each experimental group with P < 0. 05 is that the difference is statistically significant;
the killing rate (%) [1- (experimental OD value-T cell control OD value)/target cell control OD value ] × 100% experiment.
The secretion of the cell factors is realized by paving a cancer cell line in a logarithmic growth phase in a 12-hole plate by 105 cells/hole, adding T cells or BsAb loaded T cells in the next day, co-culturing for 4 hours at the concentration of an effective target ratio of 5:1, collecting supernatant, and detecting the secretion levels of IFN-gamma and TNF-alpha of the cells by using ELISA Kit Human IFN-gamma and ELISA Kit Human TNF-alpha.
The expression of the protooncogene and the cancer suppressor gene is that tumor cells are planted into a 6-well plate by 2 x106 cells per well, 2 x 107-effect T cells and BsAb-loaded T cells (effect target ratio concentration is 5: 1) are respectively added in the next day, untreated tumor cells are used as blank control, the blank control is co-cultured for 24 hours, supernate is discarded, PBS is used for rinsing the cells twice, TRIZOL1ml is added, cell lysate is collected, total RNA is extracted by using a total RNA kit, and the total RNA is reversely transcribed into cDNA; and (3) detecting the mRNA expression of the related gene by using the cDNA as a template through qPCR.
Killing of tumor cells in vivo by BsAb-loaded T cells 30 nude mice of 6 weeks of age, half of each nude mouse, and each nude mouse is injected with 1 × 107 positive tumor cell lines subcutaneously in the right axilla, and the nude mice are randomly divided into 3 groups: saline control group, T cell group and BsAb-loaded T cell group; BsAb-loaded T cells and T cells were used as effector cells, and injected into caudal vein at a rate of 1X 107 cells/time in a total volume of 200ul 2 times per week. The control group was replaced with the same volume of saline. Before each treatment, the long diameter and the short diameter of the tumor are measured by a vernier caliper, and the tumor volume is calculated (the length is a mm, the width is b mm, and the calculation formula of the tumor volume is 1/2ab 2); and counting the death number per day, and drawing a survival rate curve of the transplanted tumor model mouse.
The TaqMan chip screens the changes of immune related genes and tumor markers in tumor tissues, and after a tumor model mouse is transplanted for one month, cervical dislocation is killed and the tumor tissues are collected. Grinding tumor tissue to extract RNA, carrying out reverse transcription to obtain cDNA, and sending the cDNA as a template to a professional biological company for TaqMan chip method detection of expression changes of immune related genes and tumor markers.
The immunohistochemical detection of the content of CD3+ T cells in tumor tissues allows transplanted tumor model mice to be killed by dislocation of cervical vertebrae one month after treatment and tumor tissues to be collected. The tumor tissue was sectioned into tissue pieces with a thickness of 4 μm and then immunohistochemically stained. Formalin-fixed paraffin-embedded tumor tissue was sequentially soaked in alcohol and xylene at different concentration gradients, placed in citrate buffer PH10 and heated for 25 minutes for antigen retrieval, 3% hydrogen peroxide solution was reacted at room temperature for 5 minutes, 1: 50 diluted monoclonal CD3 antibody was reacted at room temperature for 60 minutes, the secondary antibody was reacted at room temperature for 30 minutes, reacted with DAB solution at room temperature for 5 minutes, stained with hematoxylin solution for 5 minutes, and the sections were placed on an Olympus microscope after being coverslipped for observation and evaluation.
Aiming at the problem of BsAb affinity, the affinity of BsAb with T cells and related antigens is increased by using a codon optimization method.
Secondly, BsAb is completely designed and synthesized by humanized antibody sequence and a mammalian cell protein expression system, thereby reducing the immunogenicity of the antibody.
And thirdly, the BsAb loads T cells in vitro, so that the possible immunogenic reaction caused by the antibody is further reduced.
And fourthly, the autologous T cells are used as effector cells for early intervention of the tumor, so that the method is safe and reliable and has no toxic or side effect.
The tumor cells can be cracked under the condition of a low effective target ratio, compared with the traditional method of singly using the antibody or singly using a cell culture system, the using amount of the BsAb is greatly reduced, the cell culture system is reduced, and the early intervention cost of the tumor is greatly reduced; .
And sixthly, the BsAb is used as a medium to connect immune effector cells and tumor cells by simultaneously recognizing two targets, so that the distance between the immune effector cells and the tumor cells is shortened, the number of effector cells near the tumor cells is increased, and the killing efficiency of the tumor cells is enhanced.
And seventhly, the release of factors such as IFN-gamma and TNF-alpha secreted by the T cells can be greatly enhanced, a complement system and an Fc receptor related effector system can be effectively activated, and the tumor killing capability of the T cells is enhanced.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
- BsAb loaded T cells in vitro, characterized in that: the method comprises the following steps:s1: the structure design and optimization of BsAb; designing single-chain bispecific antibodies with different configurations, and optimizing the sequence and the structure;s2: preparing BsAb; the preparation method of the BsAb comprises a hybrid hybridoma technology, a chemical crosslinking method and a genetic engineering method;s3: analyzing the stability of BsAb; 10ug of double antibody was allowed to stand at 4 ℃ for 7 days, and the size of the double antibody fragment was then detected by non-reducing SDS-PAGE gel;s4: affinity analysis of BsAb;s5: culturing immune cells; t cells are the most main immune cells in the body, and are selected as effector cells;s6: BsAb loads T cells in vitro; incubating BsAb and T cells for 1h, taking about 106 cells, incubating with corresponding fluorescence labeled antigen for 2h, washing free antigen after the BsAb loaded T cells are combined with the corresponding antigen, adding serum for sealing, washing excessive serum after incubation and combination, and counting by using a flow cytometer;s7: killing of in vitro tumor cells by BsAb loaded T cells;s8: secretion of cytokines;s9: expression of proto-oncogenes and cancer suppressor genes;s10: killing of tumor cells in vivo by BsAb loaded T cells;s11: the TaqMan chip screens the changes of immune related genes and tumor markers in tumor tissues;s12: immunohistochemistry was performed to determine the CD3+ T cell content in tumor tissues.
- 2. The BsAb in vitro loaded T cells of claim 1, wherein: the affinity analysis of the BsAb is specifically as follows:incubating a double antibody and 1X 106T cells for 2h, wherein the final concentration of the double antibody is 10nM concentration and 50nM concentration;b, adding the cell suspension after double antibody incubation into a 2ml round-bottom centrifuge tube, centrifuging at 1500rpm for 5min, and removing the supernatant;c, fixing the mixture for 15min at room temperature by using PBS containing 1% paraformaldehyde, and washing the mixture for 1 time by using PBS;d, incubating the goat anti-mouse or goat anti-human IgG and Fab' 2 fragment specific antibody labeled by biotin at 4 ℃ for 2h, and washing the antibody with PBS for 1 time;e, adding 200ul of PE-labeled streptavidin, mixing uniformly by blowing, incubating for 1h at 4 ℃, and keeping out of the sun;f, washing with 1ml PBS for 2 times by centrifugation;g, resuspending the cells in 500ul PBS, mixing uniformly, placing in a flow tube, keeping out of the sun, and detecting on a machine;the affinity of the H double antibody and the target cell is detected according to the same method.
- 3. The BsAb in vitro loaded T cells of claim 1, wherein: the T cell in-vitro amplification culture method comprises the following steps:coating A: adding coating factor into 10ml of D-PBS, keeping out of the sun at 4 ℃, and coating 2T 175 culture bottles overnight; or placing in 37 deg.C incubator, and coating for at least 2 hr;b cell preparation:a: drawing 100ml of anticoagulated peripheral blood, 800g, and centrifuging at room temperature for 15min with without breaking;b: preparation of autologous plasma: collecting supernatant plasma, and placing in water bath at 56 deg.C for 30 min; then standing for 10min at-20 ℃; finally, centrifuging for 15min at 4 ℃ and 1100g, and storing at 4 ℃ for later use;c: taking the centrifuged lower cell component, adding D-PBS to 50ml, mixing uniformly, adding into 4 50ml centrifuge tubes filled with 20ml medical grade lymphocyte separation liquid, centrifuging at room temperature for 15min at 800g, and centrifuging with out breaking;d: taking the cell layer, adding the culture medium to 50ml, 600g for 10min, and removing the supernatant;e: adding 80mlT cell culture solution into the separated PBMC to obtain cell suspension, adding 5ml of blood plasma, adding into 2T 175 culture bottles without coating solution, adding induction factor into each culture bottle, and standing at saturated humidity, 37 deg.C and 5 deg.C; culturing in 0% CO2 incubator;c, cell expansion: the concentration of various induction factors in the T cell culture solution is adjusted, the amplification speed of the T cells is accelerated, and the activity of the T cells is increased.
- 4. The BsAb in vitro loaded T cells of claim 1, wherein: killing of the BsAb-loaded T cells on in-vitro tumor cells takes the BsAb-loaded T cells as effector cells and cancer cell lines as target cells; setting a pure T cell control group and a target cell control group; using t test for each experimental group, and using P < 0; 05 is that the difference is statistically significant; percent killing [% OD value-T cell control OD value/target cell control OD value ] x 100% experiment.
- 5. The BsAb in vitro loaded T cells of claim 1, wherein: the secretion of the cell factors is realized by paving a cancer cell line in a logarithmic growth phase in a 12-hole plate by 105 cells/hole, adding T cells or BsAb loaded T cells in the next day, co-culturing for 4 hours at the concentration of an effective target ratio of 5:1, collecting supernatant, and detecting the secretion levels of IFN-gamma and TNF-alpha of the cells by using ELISA Kit Human IFN-gamma and ELISA Kit Human TNF-alpha.
- 6. The BsAb in vitro loaded T cells of claim 1, wherein: the expression of the protooncogene and the cancer suppressor gene is realized by that tumor cells are planted into a 6-well plate by 2 x106 cells per well, 2 x 107-effect T cells and BsAb-loaded T cell-effect target specific concentration 5 are respectively added in the next day: 1, carrying out co-culture on untreated tumor cells for 24 hours by taking a blank control, removing supernatant, rinsing the cells twice by PBS, adding TRIZOL1ml, collecting cell lysate, extracting total RNA by using a total RNA kit, and carrying out reverse transcription to obtain cDNA; and (3) detecting the mRNA expression of the related gene by using the cDNA as a template through qPCR.
- 7. The BsAb in vitro loaded T cells of claim 1, wherein: killing of tumor cells in vivo by BsAb-loaded T cells 30 nude mice of 6 weeks of age, half of each nude mouse, and each nude mouse is injected with 1 × 107 positive tumor cell lines subcutaneously in the right axilla, and the nude mice are randomly divided into 3 groups: saline control group, T cell group and BsAb-loaded T cell group; taking BsAb loaded T cells and T cells as effector cells, injecting 1 × 107 cells/time in tail vein, wherein the total injection volume is 200ul, and 2 times per week; the control group was replaced with the same volume of saline; before each treatment, the long diameter and the short diameter of the tumor are measured by a vernier caliper, the length a mm and the width b mm of the tumor volume are calculated, and the tumor volume calculation formula is 1/2ab 2; and counting the death number per day, and drawing a survival rate curve of the transplanted tumor model mouse.
- 8. The BsAb in vitro loaded T cells of claim 1, wherein: screening the changes of immune related genes and tumor markers in tumor tissues by the TaqMan chip, and killing the transplanted tumor model mouse by dislocation of cervical vertebrae and collecting the tumor tissues after the transplanted tumor model mouse is treated for one month; grinding tumor tissue to extract RNA, carrying out reverse transcription to obtain cDNA, and sending the cDNA as a template to a professional biological company for TaqMan chip method detection of expression changes of immune related genes and tumor markers.
- 9. The BsAb in vitro loaded T cells of claim 1, wherein: the immunohistochemical detection of the content of CD3+ T cells in tumor tissues comprises the steps of treating a transplanted tumor model mouse for one month, dislocating cervical vertebrae, killing and collecting tumor tissues; slicing the tumor tissue into tissue slices with the thickness of 4 mu m, and then carrying out immunohistochemical staining; formalin-fixed paraffin-embedded tumor tissue was sequentially soaked in alcohol and xylene at different concentration gradients, placed in citrate buffer PH10 and heated for 25 minutes for antigen retrieval, 3% hydrogen peroxide solution was reacted at room temperature for 5 minutes, 1: 50 diluted monoclonal CD3 antibody was reacted at room temperature for 60 minutes, the secondary antibody was reacted at room temperature for 30 minutes, reacted with DAB solution at room temperature for 5 minutes, stained with hematoxylin solution for 5 minutes, and the sections were placed on an Olympus microscope after being coverslipped for observation and evaluation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630146A (en) * | 2015-01-24 | 2015-05-20 | 马飞 | Preparation method and application of tumour cell specific polyclonal T cells |
| CN107854490A (en) * | 2017-09-26 | 2018-03-30 | 首都医科大学附属北京世纪坛医院 | A kind of T cell and its application through modification |
| CN109513003A (en) * | 2012-08-14 | 2019-03-26 | Ibc药品公司 | T- cell for treating disease redirects bispecific antibody |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109513003A (en) * | 2012-08-14 | 2019-03-26 | Ibc药品公司 | T- cell for treating disease redirects bispecific antibody |
| CN104630146A (en) * | 2015-01-24 | 2015-05-20 | 马飞 | Preparation method and application of tumour cell specific polyclonal T cells |
| CN107854490A (en) * | 2017-09-26 | 2018-03-30 | 首都医科大学附属北京世纪坛医院 | A kind of T cell and its application through modification |
Non-Patent Citations (1)
| Title |
|---|
| HOSSEINI SS等: "Bispecific monoclonal antibodies for targeted immunotherapy of solid tumors: Recent advances and clinical trials", 《INT J BIOL MACROMOL.》 * |
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