CN118240853A - SynNotch structure of target HIV antigen protein and application thereof - Google Patents
SynNotch structure of target HIV antigen protein and application thereof Download PDFInfo
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Abstract
The invention relates to a synNotch structure of a target HIV antigen protein and application thereof, relates to the technical field of biotechnology and cell preparation, and further relates to a preparation method of immune cells of a target secretion anti-HIV broad-spectrum neutralizing antibody and an HIV specific T cell re-targeting antibody prepared by utilizing the synNotch structure and application of the immune cells in AIDS treatment. The synNotch structure designed by the invention can regulate and control the expression of two downstream antibody genes after specifically responding to HIV Env antigen, and the two antibodies respectively mediate the clearance effect on free viruses and the killing capacity on infected cells, thereby having good clinical application prospect and potential important economic value and significance.
Description
Technical Field
The invention relates to the technical field of biology and cell preparation, in particular to a synNotch structure of a target HIV antigen protein. Further relates to immune cells which are prepared by utilizing the synNotch structure and are used for targeted secretion of anti-HIV broad-spectrum neutralizing antibodies and HIV specific T cell re-targeting antibodies and application thereof in the aspect of treating AIDS.
Background
AIDS is a global serious epidemic disease seriously jeopardizing human health caused by HIV (Human immunodeficiency virus) infection, and the combined antiretroviral therapy (Combined antiretroviral therapy, cART) used in the current clinical treatment cannot thoroughly remove latent viruses in infected persons, and patients are easy to have drug resistance and side effects after taking the medicines for a long time, so that development of better treatment strategies is urgently needed. Adoptive cell therapies, such as CAR-T cells (CHIMERIC ANTIGEN receptor-T cells), have the fundamental requirement of achieving functional cure of aids: long-term in vivo effectiveness and sustained control. However, it has not been observed to date in human clinical trials that HIV-specific CAR-T cells or TCR-T cells (T cell receptor-T cells) can continuously control viral replication in patients after termination of the art drug. Therefore, the search for optimal T cell engineering is a critical task to be solved in the current aids therapeutic field.
Broad-spectrum neutralizing antibody (Broadly neutralizing antibody, bnAb) treatment is used as one of the forefront hot spots, and can identify the area which is not easy to change in the structure of HIV surface antigen, thereby having the capability of capturing most variant strains and effectively reducing the viral load in a patient. The current treatment scheme of bnAb for AIDS patients has entered the clinical test stage, but the present treatment scheme still has the defects of short half-life in vivo, easy induction of anti-antibody reaction and the like. Splicing and combining the bnAb and Single-chain variable region (scfv) of other functional antibodies in different ways can also obtain the double-affinity re-targeting antibody with the functions of HIV broad-spectrum recognition and T cell targeting. Such re-targeting antibodies are capable of targeting the infected cells to the surface of T cells, thereby mediating direct killing of HIV-infected cells by T cells. However, immunotherapy with bnAb alone still fails to completely clear the latent viral reservoir in HIV-infected individuals, nor does HIV-specific T cells inhibit the reinfection of new host cells with extracellular free virus.
After the synNotch receptor regulation system is reported by Cell for the first time in 2016, the function customization potential of the user customization and a simple and independent signal regulation channel enable the system to have more flexible designability and wider application prospect, and the functions of T cells, such as secretion of cytokines, antibodies, functional small molecules and the like, are controlled more accurately by directly recognizing Cell surface specific antigens and regulating a transcription program freely customized at the downstream, and breakthrough progress is obtained in tumor treatment application research. However, synNotch-T cells have not been used for anti-AIDS therapy. If a synNotch system is used for designing an HIV specific T cell capable of continuously expressing therapeutic antibodies, the effect aging of the bnAb can be greatly prolonged, and the possibility can be provided for further clearing a virus latent reservoir and reconstructing the immune system function under the combined action of the T cell with the self-killing function. Therefore, the invention organically combines the bnAb and the T cell killing effect, designs a synNotch receptor regulation system structure capable of targeting HIV antigen protein, then carries out immunocyte transformation based on the structure, and completes the verification of the in-vitro treatment effect of the structure, thereby having very important significance for clinical treatment and functional cure of AIDS patients.
Disclosure of Invention
The present invention provides a novel synNotch receptor modulation system for the generation of anti-HIV broad-spectrum neutralizing antibodies and HIV-specific T cell re-targeting antibodies after recognition of HIV antigen proteins.
The technical scheme of the invention is as follows:
A synNotch structure consists of a receptor and a downstream gene regulated by the receptor, wherein the receptor part comprises an HIV antigen binding region, a Notch core region and a transcription factor regulation region; the receptor can regulate gene expression of downstream anti-HIV broad-spectrum neutralizing antibodies (such as VRC 01) and HIV-specific T cell re-targeting antibodies (such as N6-alpha CD 3) simultaneously.
The receptor portion of the SynNotch structure includes the CD4-17b extracellular region, the Notch core region, and the Gal4-VP64 transcription factor regulatory region. The extracellular region of CD4-17b is used for specifically recognizing HIV antigen protein Env, and has Myc tag protein for identifying receptor expression before the sequence; notch core region is used to anchor receptors to cell membranes; gal4-VP64 transcription factor regulatory region is used to initiate protein expression of downstream genes. The specific nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.
The downstream gene of the SynNotch structure comprises a tandem gene of VRC01 and N6- αCD3, the structure of which is VRC01 heavy chain-P2A-VRC 01 light chain-T2A-N6 scfv- αCD3 scfv, the sequence being preceded by a transcription factor binding region nucleic acid sequence for initiating transcription of the gene, the bound transcription factor being Gal4-VP64 in the acceptor moiety. The specific nucleotide sequence is shown in SEQ ID NO 3, and the amino acid sequence is shown in SEQ ID NO 4.
An expression vector comprising a gene encoding the synNotch construct.
The recombinant virus is characterized by comprising the coding gene of the synNotch structure or being prepared by using the expression vector.
In one embodiment, an expression vector containing the synNotch structural-encoding gene is used to prepare a recombinant virus.
An immune cell, characterized in that the immune cell contains the coding gene or is modified by the expression vector and recombinant virus.
In one embodiment, the expression vector or recombinant virus containing the synNotch structural coding gene is used to engineer an engineered immune cell, and the receptor moiety is expressed on the surface of the immune cell for initiating downstream gene expression after specifically recognizing HIV antigen protein Env; the regulated downstream gene expresses VRC01 and N6-alpha CD3 simultaneously after expression is started, and immune cells secrete VRC01 and N6-alpha CD3 to outside cells.
In one embodiment, the means for delivering synNotch structural coding genes when engineering the immune cells comprises one or more of recombinant lentiviruses, recombinant retroviruses, common plasmid expression vectors, nanodelivery systems, electrical transduction or transposons.
In one embodiment, a biological agent comprises a nucleic acid sequence of synNotch structure or an expression cassette of its encoded amino acid sequence, or a constructed expression vector, or a recombinant virus produced, or an engineered immune cell, etc., wherein the immune cell used for engineering is preferably a T lymphocyte.
Compared with the prior art, the invention has the following beneficial effects:
1. CD4-17b is taken as an extracellular recognition domain of a receptor part in synNotch structure, so that the risk of the engineering cell being infected by HIV can be effectively avoided while the high targeting of the engineering cell to HIV antigen is ensured; 2. the production of the neutralizing antibody and the re-targeting antibody is regulated and controlled through synNotch structures, so that the defect of short half life of the antibody can be effectively avoided and the generation of anti-antibody reaction can be reduced; 3. by combining broad-spectrum neutralizing antibody and T cell re-targeting antibody, humoral immunity and cellular immunity can be organically combined to form a novel controllable specific combined immune treatment strategy, and HIV free virus and infected cells can be removed.
The invention discloses a novel synNotch receptor regulation system applied to AIDS treatment, which is proved by experiments that the engineered synNotch-T cells modified by the invention can specifically recognize HIV antigen, activate the expression of downstream antibody genes, and generate the effects of resisting virus infection and eliminating infected cells. The synNotch structure designed by the invention expands the application range of the synNotch receptor regulation system, provides a new application prospect for the cell transformation technology for treating AIDS, and has potential important significance and economic value.
Drawings
FIG. 1 is a schematic diagram showing the structural design of HIV-specific antibody secretory synNotch receptor regulatory system (abbreviated as CD4-17 b-syn-Ab). Ab refers to a downstream antibody gene expressed under synNotch receptor modulation, where VN represents a tandem gene comprising VRC01 and N6- αCD3, expressed alone as a single functional control. The expression vector of the downstream gene contains red fluorescent protein DsRed expressed under the promotion of PGK promoter, and is used for flow sorting and identification of engineering cells.
FIG. 2 shows the positive rate of the modified cells detected by the flow method after CD4-17b-syn-Ab structure was used to modify the T lymphocyte line Jurkat. NC, a negative control group, refers to Jurkat cells that were not transduced with CD4-17b-syn-Ab structure.
FIG. 3 shows the relative expression levels of antibody mRNA detected by qRT-PCR after antigen stimulation of three Jurkat cells with CD4-17b-syn-Ab structure. The antigen-stimulated cells were provided as 293T-gp160 cells expressing the HIV antigen Env on the cell surface. Mock, a group of corresponding CD4-17b-syn-Ab Jurkat cells co-incubated with 293T cells without antigen expression.
FIG. 4 is a graph showing the expression of antibodies in CD4-17b-syn-Ab Jurkat cells secreting VRC01 antibodies after antigen stimulation. UTD, jurkat cells not transduced with CD4-17b-syn-Ab structure.
FIG. 5 is a graph showing the extent of infection of various HIV strains (NL 4-3, SF162, X1632, 246F3, CNE55 and CE 0217) by CD4-17b-syn-Ab Jurkat cells capable of secreting VRC01 antibodies. The viral infectivity in UTD group was 100%.
FIG. 6 is a flow assay for CD4-17b-syn-Ab Jurkat cell mediated cell killing of target cells 293T-gp 160-EGFP. The 293T-gp160-EGFP cell is prepared by transducing EGFP fluorescent protein by slow virus on the basis of the 293T-gp160 cell.
FIG. 7 shows the viral p24 replication profile detected during long-term incubation with HXB 2-infected H9T lymphocytes (abbreviated as H9/HXB 2) after using the CD4-17b-syn-Ab structure for engineering human CD8 + T cells.
Detailed Description
Embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. The following examples are further illustrative of the invention and are not intended to be limiting thereof. The experimental methods used in the following embodiments are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Taking an example of preparing immune cells with synNotch structure by using a T lymphocyte line Jurkat and CD8 + T cells in human peripheral blood, a preparation method and function verification of the immune cells are described in detail:
1. Structural design of antibody secretion type synNotch receptor regulation system;
2. engineering Jurkat T cells expressing the antibody secretory synNotch system;
3. in vitro functional verification of CD4-17b-syn-Ab Jurkat T cells;
4. In vitro functional validation of CD8 + T cells with antibody secreting synNotch system.
The specific implementation is as follows:
Example 1
1. Structural design of antibody secretion type synNotch
The structure consists of two parts: synNotch receptor molecule with CD4-17b as extracellular antigen recognition domain, its nucleotide sequence is SEQ ID NO 1, and its amino acid sequence is SEO ID NO 2; the nucleotide sequence of the downstream gene expressing VRC01 and N6-alpha CD3 after being regulated by synNotch receptor is SEQ ID NO 3, and the amino acid sequence is SEO ID NO 4.
The structural design is shown in figure 1, the N end of the synnotch receptor molecule is the serial sequence of the first two domains of the HIV natural receptor CD4 and the anti-HIV broad-spectrum neutralizing antibody 17b scFv, and can specifically bind to the viral envelope protein Env. synNotch receptor molecules were constructed to the pHR receptor expression vector (Addgene, plasmid # 85421). The anti-HIV broad-spectrum neutralizing antibody VRC01 and HIV specific T cell re-targeting antibody N6-alpha CD3 are selected as downstream expression proteins, wherein VRC01 can eliminate free virus particles, N6 alpha-CD 3 molecules can mediate the contact of CD3 + T cells with target cells expressing HIV antigen and activate T cells, the elimination of the target cells is enhanced, and downstream genes are respectively constructed to pHR downstream gene expression vectors (Addgene, plasmid# 79130). After the cells with the CD4-17b-syn-Ab molecular structure specifically recognize HIV envelope proteins, the synNotch receptor located intracellular transcription regulator Gal4-VP64 is sheared, and the transcription regulator enters the nucleus to initiate the expression of downstream genes.
Example 2
2. Engineering Jurkat T cells expressing antibody secreting synNotch system
HEK293T cells in the logarithmic growth phase were inoculated at a density of 6-8X 10 6/dish in 100mm dishes treated with 0.01% PLL, incubated at 5% CO 2 at 37℃for 24 hours until the cell density reached about 80%, 8. Mu.g shuttle Plasmid, 5. Mu.g lentiviral envelope Plasmid pMD2.G (Addgene, plasmid # 12259) and 10. Mu.g lentiviral packaging Plasmid psPAX2 (Addgene, plasmid # 12260) were added to the HEK293T cell dishes as described above, virus-containing culture supernatants were collected 48 hours and 72 hours, respectively, centrifuged at 3000rpm at 4℃for 10 minutes to remove cell debris, and filtered using a low protein adsorption 0.45 μm filter, and the virus supernatants were added to PEG8000-NaCl-PBS mixtures and allowed to stand overnight at 4 ℃.
The ratio of the virus supernatant to the PEG8000-NaCl-PBS mixed solution is as follows:
| Viral supernatant | 30mL |
| 50％PEG8000 | 8mL |
| 4MNaCl | 4mL |
| PBS | 6mL |
Centrifuging the mixed solution at 4deg.C, 7000 Xg for 10 min, removing supernatant, adding appropriate volume of RPMI 1640 fresh medium, suspending and precipitating to obtain lentivirus concentrate, packaging, freezing at-80deg.C, and taking small amount of virus titer.
We paired lentiviruses carrying the CD4-17b synNotch receptor (CD 4-17b SNR) gene with the single antibody group VRC01, N6-. Alpha.CD3 and the bifunctional group VN lentiviruses, respectively, co-transfecting the T lymphocyte line Jurkat. Proper amount of Jurkat cell suspension with good growth state is taken and placed into a centrifuge tube for centrifugation at 300 Xg for 5 minutes. The supernatant was discarded, a lentiviral concentrate expressing CD4-17b SNR was added at a ratio of 100. Mu.L/1X 10 6 cells, supplemented to 1mL with 1640 medium, and the final concentration of 8. Mu.g/mL of the transfection-facilitating reagent Polybrene was added and gently mixed by gentle pipetting. The cell suspension was transferred to a 24-well plate, centrifuged at 350 Xg for 2 hours, and returned to the incubator for continuous culture after centrifugation. After 20 hours or so of infection, the second virus infection was performed as described above, and after 20 hours of infection, the cells were resuspended in fresh medium by centrifugation. After continuous culture for 7 days after infection, the stably expressed cells were purified by flow sorting, and expanded culture, and after 15 days of culture, the modified cells were examined for CD4-17b SNR and expression of regulatory antibody genes by flow cytometry, and the results are shown in FIG. 2. The ordinate represents the expression efficiency of CD4-17b SNR, the abscissa represents the transduction efficiency of the regulated antibody gene, and the analysis result shows that the co-expression positive rate of the single antibody group VRC01, N6-alpha CD3 and the bifunctional group VN and CD4-17b SNR is more than 65% (74.7%, 71.4% and 65.9%, respectively), and the establishment of a regulation system is completed.
Example 3
3. In vitro functional verification of CD4-17b-syn-Ab Jurkat T cells
After the construction of the regulatory system was completed, three CD4-17b-syn-Ab Jurkat cells were tested for both neutralizing and cell killing ability of the antibodies secreted under antigen stimulation. The purified three cells of VRC01, N6-alpha CD3 and VN are respectively and uniformly mixed with target cell 293T-gp160 (surface expression HIV antigen) or control cell HEK293T in the amount of 2X 10 5：2×105 cells per group, and are placed in an incubator for culture after 400X g centrifugation for 1 min in a U-shaped 96-well plate. Total RNA was extracted from each group of cells after 24 hours, and the expression level of the corresponding antibody gene in each group of cells was detected by qRT-PCR method. Relative expression levels of mRNA of the corresponding antibodies after stimulation of the engineered cells with target cells 293T-gp160 were calculated using the group of engineered cells stimulated with antigen-free expressing cells (HEK 293T) as a control (FIG. 3). According to the qRT-PCR detection result, the expression level of the antibodies of the VRC01, N6-alpha CD3 and VN modified cells after target cell stimulation is obviously improved.
After detecting the expression level of antibody mRNA in the remodeled cells before and after stimulation, we also detected the secretion level of antibody in the supernatant. The same stimulation mode is adopted, namely three purified VRC01 and VN cells are respectively and uniformly mixed with target cell 293T-gp160 or control cell HEK293T according to the cell quantity of 2X 10 5：2×105 of each group, and are placed in an incubator for culture after 400X g centrifugation in a U-shaped 96-well plate for 1 minute. The culture supernatants of each group were taken at three time points of 12 hours, 24 hours and 48 hours after co-incubation, and after cell debris was removed, the expression of IgG antibodies in the culture supernatants was detected by ELISA, and IgG antibody expression curves were drawn according to the expression of antibodies at each time point. As can be seen from the expression curve of FIG. 4, the purified modified cells had significant antibody secretion in the supernatant after target cell stimulation, wherein VN group antibody secretion amounts up to 8ng/mL were obtained.
To verify the inhibitory effect of antibodies secreted by synNotch regulatory systems on viral infection, we incubated untransduced cells (UTD) and purified CD4-17B-syn-Ab cells with pseudoviruses MW965 (subtype C, tier 1), SF162 (subtype B, tier 1), X1632 (subtype G, tier 2), 246F3 (subtype AC, tier 2), CNE55 (subtype CRF01, tier 2) and CE0217 (subtype C, tier 2), respectively, of various strains of 20ng p24 for 24 hours, and the supernatant was taken to infect TZM-bl cells and the inhibition efficiency of the secreted antibodies on free viral infection in the system was detected by reaction with luciferase substrates 48 hours after infection. The relative infection efficiency of the viruses after co-incubation with CD4-17b-syn-VRC01 and CD4-17b-syn-VN cells, respectively, was calculated by selecting the non-transduced cells and the virus incubation group as a reference control group. As shown in the results of fig. 5, the two groups of CD4-17b-syn-VRC01 and CD4-17b-syn-VN showed significantly reduced infectivity compared to the control group, demonstrating that in the co-incubation system, the engineered cells were able to respond to multiple viral particle stimuli and secrete antibodies, and then effectively neutralize the infectious activity of the free virus, exhibiting flexible antigen response activity and broad spectrum neutralization activity against HIV virus.
After evaluating the broad-spectrum neutralizing capacity of bifunctional cells against different HIV viruses, we have again evaluated their mediated cell killing capacity. The killing capacity of the bifunctional T cells is evaluated by detecting the change degree of the proportion of the target cell population in the co-incubation system through flow cytometry. To facilitate flow assays, we constructed 293T-gp160-EGFP cells based on the target cell 293T-gp160 cell line by transduction of EGFP into 293T-gp160 cells and screening by puromycin. In testing effector cell-mediated killing using flow-through, we examined the extent of change in the proportion of target cell populations by co-culturing the bifunctional group CD4-17b-syn-VN, the single N6-. Alpha.CD3-expressing CD4-17 b-syn-N6-. Alpha.CD3 group, and the untransduced Jurkat cells with target cells 293T-gp160-EGFP, respectively, at 1X 10 5：1×105 per group, with 400 Xg centrifugation in U-96 well plates for 1 min, adding 1X 10 5 human CD8 + T cells to each well for 24 hours. N6-alpha CD3 generated by effector cells can directly mediate the killing of CD8 + T cells in a conductor system to target cells, and the lysis efficiency of the target cells can be calculated by taking the UTD group of untransduced cells as a reference group. From the cell lysis efficiency (FIG. 6), CD4-17 b-syn-N6-. Alpha.CD3 and CD4-17b-syn-VN cells significantly mediate lysis of target cells compared to the untransduced cell line, and the killing capacity mediated by the bifunctional cell line CD4-17b-syn-VN and the single functional control line CD4-17 b-syn-N6-. Alpha.CD3 was similar, enabling about 30% killing of target cells to be performed. The novel bifunctional T cells established based on synNotch regulation system can mediate virus neutralization and cell killing functions through a specific secreted broad-spectrum neutralizing antibody VRC01 and a bispecific antibody N6-alpha CD 3.
Example 4
4. In vitro functional validation of CD8 + T cells with antibody secreting synNotch system.
15ML of lymphocyte isolate Lymphoprep (Stemcell, # 07861) was added through the central well to the SepMate TM isolation tube according to SepMate TM isolation tube (Stemcell, # 85460) instructions, whole blood from healthy donors was diluted with an equal amount of PBS added to 2% fetal bovine serum, and diluted human blood samples were added to the tube along the wall and centrifuged at 1200 Xg for 10 minutes at room temperature. Part of the in-tube supernatant was discarded, and the supernatant containing the buffy coat was rapidly poured into a new sterile 50mL centrifuge tube, washed with PBS, and centrifuged at 300×g for 10 minutes to obtain peripheral blood mononuclear cells PBMC. CD8 + T cells were isolated using the CD8 + T cell magnetic bead isolation kit (Miltenyi Biotec, # 130-096-495).
CD8 + T cells were cultured using X-VIVO 15 medium (Lonza, # 04-418Q) containing 100IU/mL rhIL-2, 5ng/mL IL-7, and 5ng/mL IL-15, cell densities were adjusted to 1X 10 6/mL, CD8 + T cells were activated using 3 μg/mL anti-CD 3 monoclonal antibody (Biolegend, # 317347) and 3 μg/mL anti-CD 28 monoclonal antibody (Biolegend, # 302934) for 48 hours, and activated CD8 + T cells were infected with concentrated lentiviruses every 2-3 days.
Culturing was continued for 5 days after infection, and purified CD4-17b-syn-VN CD8 + T cells were obtained by sorting and expanded. T lymphocyte H9 was infected with HIV strain HXB2 and used as target cells to stimulate CD4-17b-syn-Ab CD8 + T cells. 2X 10 5 H9/HXB2 cells were incubated with 2X 10 5 CD4-17b-syn-VRC01, CD4-17b-syn-VN and UTD cells, respectively, and incubated in 12-well plates for 7 days in series, and HIV virus content in the supernatants of the co-culture systems was quantified using p24 ELISA quantification kit on days 0, 3,5 and 7, as shown in FIG. 7. After co-incubation of CD4-17b-syn-VRC01, CD4-17b-syn-VN with H9/HXB2 cells, the HIV virus content in the supernatant was significantly reduced compared to the untransduced cell group, wherein the inhibition effect of the bifunctional CD8 + T cell group secreting VRC01 and N6-alpha CD3 simultaneously was most significant, indicating that the bifunctional T cell can effectively inhibit HIV infection and replication in target cells.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and use various exemplary embodiments of the invention and to make various selections and modifications. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (6)
1. The synNotch structure is characterized in that after recognizing HIV antigen protein Env, the expression of downstream anti-HIV broad-spectrum neutralizing antibody and HIV specific T cell re-targeting antibody is regulated, and the sequence table SEQ ID NO is 1-4.
2. An expression vector comprising the synNotch construct of claim 1.
3. A recombinant virus comprising the synNotch construct of claim 1.
4. An immune cell comprising the synNotch construct of claim 1.
5. A biologic comprising the synNotch construct of claim 1 or the immune cell of claim 4.
6. Use of the immune cell of claim 4 or the biological agent of claim 5 in the preparation of a medicament for treating aids.
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