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Delivery of CAR target virus to solid tumors can achieve effective cell therapy
Delivery of CAR target virus to solid tumors can achieve effective cell therapy. Chimeric Antigen Receptor (CAR) T cell therapy has limited efficacy against solid tumors, largely due to the lack of selective and highly expressed surface antigens. In order to avoid relying on the endogenous antigens of the tumor, we describe here a method for tumor-selective delivery of surface antigens using oncolytic viruses to achieve a wide range of CAR-T cell therapy.
Solid tumors pose several challenges to effective CAR T cell therapy, and so far these challenges have limited the success of the effort. First, solid tumors are usually full of suppressor cells of myeloid origin, which can produce an immunosuppressive microenvironment that can inhibit the function and proliferation of T cells . Secondly, solid tumor CAR targets that are uniformly and selectively expressed on malignant cells can be identified to prevent “non-tumor target” toxicity , depending on the antigen density, which indicates that even solid tumors that express tumor-related targets have low expression The level of antigen may also not respond to treatment [5,6,7].
So far, efforts to overcome these challenges have mainly focused on the potency and durability of CAR T cells. In order to avoid the immunosuppressive effects of the tumor microenvironment, reverse T cell exhaustion and prevent antigen escape, a combination involving radiotherapy, oncolytic virus , oncolytic virus  and checkpoint blockade [10,11] has been proposed. therapy. Genetic engineering has also been used to destroy T cell activation with cytokines that maintain activation  or “armor” T cell negative regulators .
However, despite new methods to improve CAR T cell function, there are few efforts to engineer tumors for effective adoptive cell therapy, and the lack of targetable surface antigens selectively expressed on malignant cells is still a major problem. Solve the challenge. This restriction must also design a new CAR for each newly proposed antigen. CAR T cells targeting putative solid tumor antigens, including ganglioside G2 (GD2), mesothelin, B cell maturation antibody (BCMA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor variants III (EGFRvIII), mucin 1 (MUC1) and esophageal squamous cell carcinoma 1 (NY-ESO-1) are all under active investigation .
In order to overcome the challenge of relying on endogenous solid tumor antigens, here we describe a method using oncolytic viruses to selectively deliver ectopic CAR target antigens to malignant cells, so that a potential general method can be used for solid tumor CAR T cell therapy.
Oncolytic viruses are ideal partners in this regard for the following reasons: (1) They can selectively infect and/or replicate in malignant cells to minimize the delivery of CAR targets to healthy tissues ; (2) ) It has been shown that they can reprogram the microenvironment of immunosuppressive solid tumors into a microenvironment that is more conducive to T cell activity [15,16]; (3) They have the ability to selectively lyse infected cells and recruit endogenous cells. The ability of antiviral immune response  and (4) They have demonstrated the effectiveness and effectiveness of safety in clinical trials, which has been approved by the U.S. Food and Drug Administration (FDA) (talimogene laherparepvec, 2015) .
In the proof of concept, we designed a thymidine kinase destroyed (TK-) oncolytic vaccinia virus (VV) to selectively induce CD19 expression on malignant cells, and then treat tumors with CART cells targeting CD19. This method represents an important conceptual advancement in tumor-centric CAR T cell therapy, and may make possible a general method of CAR T cell therapy for solid tumors, which has nothing to do with the natural surface expression profile of tumors.
mCD19 CAR T cells show activity against mCD19-positive melanoma cells
In order to characterize the activity of primary mouse CD19 (mCD19) CAR T cells against solid tumors uniformly expressing mCD19, we first stably expressed mCD19 and TurboRFP/Renilla luciferase (TR) fusion in the B16 mouse melanoma cell line Protein (Figure S1).
The second-generation murine mCD19 CAR T cells containing CD3ζ and CD28 costimulatory domain 19 showed effective cytotoxicity to the engineered B16-TR-mCD19 cell line in the co-culture test, the ratio of effector to tumor (E:T) 0.5 or higher, by bioluminescence imaging (p<0.0001; n=3 in each case) (Figure 1A). Even if the E:T ratio was 4:1, the vitality of mCD19-negative B16 was not affected. Mock T cells that were similarly activated by interleukin 2 (IL-2), IL-7 and anti-CD3/CD28 activated beads in culture but were not transduced by mCD19 CAR also lacked activity against mCD19-positive or mCD19-negative B16 cells .
CD19 CAR T cytotoxicity also depends on antigen density. Compared with the B16-mCD19 high cell line, the B16-mCD19 low cell line showed a weakened response (p=0.0116; n=5 in each case) (Figure 1B). Only under appropriately matched B16-mCD19+ mCD19 CAR T cell conditions, the cytotoxicity of antigen-specific T cells was confirmed by the up-regulation of the early T cell activation marker CD69 on CD4 and CD8 T cells (Figure 1C).
In order to evaluate the solid tumor activity of mCD19 CAR T cells in vivo, we established an orthotopic syngeneic model of B16 and B16-mCD19 melanoma, and used intratumoral mCD19 CAR T cells or simulated T cells immediately after sublethal lymphectomy. Treatment of tumors The previously demonstrated 5 Gy total body irradiation (TBI) is required for CD19 CAR activity in the immune competence model. 19 Similar to the results of the in vitro study, the antigen-matched treatment group showed delayed tumor growth in all mice, and completely eliminated tumor% of the mice in 33 mice (p <0.0001; B16+ CAR: n=5, B16-mCD19+ simulation; n=6, B16-mCD19+ CAR: n=6) (Figure 1D). Even for antigen-positive tumors, a single intravenous injection of CAR T cells is not an effective treatment (Figure S2).
In summary, the data indicate that mCD19 CAR T cells can exhibit strong activity in solid tumors engineered to express ectopic mCD19.
Recombinant VV can deliver mCD19 to malignant cells
In order to selectively express the ectopic surface protein to malignant cells, we generated a recombinant VV with a transgene inserted into the viral TK locus. VV destroyed by TK relies on cellular TK for replication, and because of its higher nucleotide turnover rate, it can selectively reproduce in tumor cells . We designed a control (Ctrl) oncolytic VV (Ctrl VV) protein (YFP) expressing firefly luciferase (Fluc) and yellow fluorescence  and a version that also encodes mCD19 (mCD19 VV) (Figure 2A). The effective VV replication in B16 cells was confirmed by the time and dose-dependent expression of Fluc, YFP and mCD19 (Figures 2B and 2C). After 48 hours of virus culture, as many as 75% of the cells expressed mCD19, and the multiple of the virus It is 1 for infection (MOI).
Although transgene expression can be detected, the oncolytic virus did not induce significant cell death at MOI of 0.01 or 0.1, highlighting the limitations of oncolytic virus therapy as a single drug (Figure 2B).
Infection of mCD19 VV can achieve antigen-specific mCD19 CAR T cell activity
Next, we aimed to demonstrate that the ectopic CAR target expression driven by oncolytic viruses can be selectively killed by antigen-matched CAR T cells (Figure 3A). Before adding mCD19 CAR T cells or mock T cells, the cultured B16 cells were infected with Ctrl or mCD19 VV with MOI of 0.2 for 48 h. The toxicity profile of each type of VV or T cell as a monotherapy was also evaluated. In the B16 co-culture, the combination of mCD19 VV and mCD19CAR T cells showed the highest toxicity at 24 and 48 h after the addition of T cells (Figure 3B).
This effect was also observed in the SB28 murine glioma cell line, indicating that the method can be used for various tumor types. VV-mediated mCD19 delivery also enhances CAR T cell activity against B16-mCD19 low cell lines, highlighting the potential of this method to “raise” tumor-associated surface antigen levels before treatment or as a method to overcome low antigen resistance use. The selective upregulation of CD69 on CD4 and CD8 T cells in the antigen-matched combination reflects enhanced cytotoxicity (Figure 3C). 24 or 48 h after adding T cells, the flow cytometry of the remaining B16 cells further confirmed the killing effect mediated by T cells (Figure 3D).
In the absence of T cells, both Ctrl and mCD19 VV can be expected to induce the expression of mCD19 and/or YFP. The addition of simulated T cells does not affect these profiles, and similarly, YFP expression from Ctrl VV still persists 48 hours after the addition of CART cells. It is worth noting that after 48 hours of co-cultivation with mCD19VV-infected cells, CAR T cells eliminated all mCD19+ and YFP+ cells.
Tumor selective delivery of mCD19 enhances the lethality of mCD19 CAR T cells in vivo
To assess our ability to force mCD19 tumor expression in vivo, we injected three doses of 108 plaque forming units (PFU) of mCD19 VV into orthotopic B16-TR tumors (days 1, 3, and 5). The dosing interval was 48 hours, and the YFP and mCD19 expressions on TR + cells from the resected tumor were measured on day 1 (day 6) after the final virus dose was administered. Without any lymphadenectomy protocol, we observed an average of 24% mCD19+ and 14% YFP+ cells in the TR+ population (Figure 4A). It can be expected that the infection rate of T cells in the tumor is significantly reduced (T cell ratio B16-TR% CD19+, 0 Gy p = 0.0042, 5 Gy p = 0.0014), highlighting the tumor selectivity of TK-deficient VV and minimizing the reduction This raises the potential concern about CAR T cell killing agents.
As a single drug, oncolytic virus therapy can either directly lyse infected tumor cells or recruit endogenous antiviral responses. Although beneficial, this endogenous immune response can also impair the spread of the virus . Since patients receiving CAR T cell therapy will receive chemotherapy or radiation lymphocastration therapy to expand the adoptively transferred cells, we speculate that this lymphopenia may also have the following benefits: enhanced oncolytic virus Spread to the entire tumor. Consistent with this hypothesis, we observed that lymphatic clearance with 5 Gy TBI immediately before the first viral dose (n=2) significantly increased mCD19+ (62%; p = 0.0144) and YFP+ (34%; p = 0.008) ratio. Compared with the TBI-free group, the cells in the TR+ population (n=3). In the same model, we did not observe significant differences in anti-tumor efficacy in vivo through the two-tailed unpaired t-test between Ctrl (n=5) and mCD19 (n=4) VV, and both achieved moderate Delay (day 11: relative to TBI only group (n=6), tumor volume in tumor growth Ctrl VV and TBI only, p=0.004; mCD19 VV and TBI only, p =0.0246) (Figure S3).
Due to the lack of solid tumor surface antigens that can be effectively targeted by CAR T cells, here we describe a method for selective delivery of CAR target tumors using oncolytic viruses to make a potential general approach to adoptive cell therapy. Using CD19 as a model antigen, we have shown both in vivo and in vitro that TK-VV can selectively deliver surface antigens, which can then be targeted by CAR T cells with homology with high specificity. We further proved in vitro that this method can be extended to two different cell lines and is effective for two tumors that do not express ectopic targets or express ectopic targets at low levels.
Many functions of our method are modular and require further study. Although we have demonstrated the packaging ability of using TK to delete VV (genome size is about 190kb), easy genetic manipulation, high immunogenicity  and clinical translatability  alternative oncolytic viruses (especially with higher replication In addition, FDA-approved drugs such as herpes virus or polio/measles virus that can cross the blood-brain barrier can also be used as antigen delivery vehicles . Considering the potential variability of tumor susceptibility, this is an important consideration.
Finally, although we succeeded in infecting about 60% of tumor cells in the body with de-lymphocyte hosts, it is also possible to use multiple oncolytic viruses with multiple tumor selective mechanisms to increase tumor coverage. For example, although VV has shown impressive infectivity among multiple tumor types [27, 28, 29], the use of multiple oncolytic viruses may be particularly useful, which can account for the variability of tumor entry receptor expression. Provide adequate coverage.
Delivery of therapeutic agents to solid tumors also remains a limitation of many immunotherapies, and our approach is also hindered in this regard. Consistent with previous studies on the efficacy of CAR T cells against B16 melanoma , we have shown that even when paired with TBI and exogenous cytokines, CAR T cells matched to the intravenous delivery of antigens can only achieve moderate anti-tumor effects. Efficacy, therefore, the number of CAR T cells that must be delivered intratumorally. Although this delivery route is the most effective in clinical trials of oncolytic viruses [18,24] and CAR T cells , further work is still needed to achieve effective intravenous delivery of the two drugs to treat transmission Disease. Indeed, oncolytic viruses have been shown  to reshape the tumor microenvironment and promote the recruitment of adoptively transferred T cells, so they may be ideal partners in this regard.
There is also evidence that CAR T cells can induce the spread of antigenic determinants and trigger an endogenous anti-tumor response against non-immunogenic tumor neoantigens [13,33]. Future work will investigate whether the strategy described here can generalize this effect and trigger an anti-tumor response on untreated tumor foci. In theory, this would alleviate the need for viral infection of every malignant cell and delivery of CAR T cells to all neoplastic lesions. It is believed that CAR T cell therapy has unique equipment that can induce antigenic determinants by secreting large amounts of interferon gamma (IFN gamma) and expressing high levels of CD40 ligand (CD40L) , both of which recruit and activate antigen presentation Key cells. In this way, the combination therapy can achieve a certain degree of epitope diffusion that cannot be achieved by using viral monotherapy alone.
Finally, despite considering that CD19 CAR is the most advanced clinically, although we use CD19 in the proof of concept, future iterations will use surface antigens and homologous CARs that are not expressed on healthy tissues to avoid unnecessary B cells Dysplasia. We are optimistic about this. Tumor engineering will become a complementary method of immune engineering in adoptive cell therapy.
Delivery of CAR target virus to solid tumors can achieve effective cell therapy.
Delivery of CAR target virus to solid tumors can achieve effective cell therapy.
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