Combination of oncolytic virus and CAR T cell therapy

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Combination of oncolytic virus and CAR T cell therapy

Combination of oncolytic virus and CAR T cell therapy.  Oncolytic viruses (OVs) have recently attracted great interest in the field of cancer treatment.

In particular, OVs can help CAR-T cells overcome certain immunosuppressive mechanisms in the tumor microenvironment (TME) through the intrinsic effects of OV or the delivery of immunostimulants. A large number of preclinical studies have shown that combining CAR-T cells with OVs can increase the transport of CART cells, anti-tumor activity and eliminate antigen-negative tumor cells.

Although the preclinical results are encouraging, there is only one ongoing clinical trial (NCT03740256) in which CAR T and OV combination therapies are studied, highlighting the challenges of applying this approach to the clinic.

Despite the overall success in targeting hematological malignancies, CAR-T therapy is still struggling against the background of solid tumors. Various factors in the tumor microenvironment (TME) can inhibit T cell function (Figure 1A) [6].

It is known that tumor cells can up-regulate PD-L1, thereby negatively regulating T cell function, and tumor cells can also induce tumor-associated macrophages (TAM) to polarize toward T cells. The immunosuppressive phenotype further hinders T cell function through a variety of surface receptors (such as CTLA-4) and cytokines (such as TGF-β).

In addition, tumors recruit bone marrow-derived suppressor cells (MDSC) to TME, where they prevent T cell infiltration and metabolize suppressor T cells. Long-term exposure to antigens in TME will promote the differentiation of regulatory T cells (Treg).

These Tregs promote the immunosuppressive environment by secreting IL-10 and TGF-β, metabolically destroying conventional T cells and lysing effector T cells [7] . TME also induces the up-regulation of PD-L1 and indoleamine 2,3 dioxygenase (IDO), which is characterized by an immunosuppressive phenotype, which hinders the immune response [6].

In addition, TME excludes T cells by disrupting T cell extravasation and trapping T cells in a dense matrix network [8]. Overcoming these physical and molecular mechanisms of immunosuppression may improve the efficacy of CAR T cell therapy in solid tumors.

Oncolytic viruses (OVs) are viruses that selectively infect and replicate in cancer tissues while retaining normal tissues [16,17]. Many clinical trials involving OVs are currently being conducted [18]. References [17,19,20] reviewed strategies for designing tumor-specific OV.

In short, the inherent defects of the innate antiviral pathway in tumor cells, such as the suppression of interferon response, can allow attenuated viruses to replicate in tumor tissues. Alternatively, the viral gene can be placed under a tumor-specific promoter (such as the human telomerase reverse transcriptase (hTERT) promoter) to prevent transcription of the viral gene in normal cells. A more targeted method to determine virus tropism is to add tissue-specific microRNAs to the viral genome to silence viral genes in tissues suffering from OV-related toxicity, thereby preventing side effects outside the tumor [21]. OV exerts its anti-tumor effect by directly lysing tumor cells [22]. OV-mediated tumor cell lysis results in the release of tumor-associated antigens (TAA), pathogen-associated molecular patterns (PAMP) and risk-related molecular patterns (DAMP) into TME.
These pro-inflammatory molecules reactivate the local immune response and provide activation signals for innate and adaptive immune T cells [25,26], helping to eliminate tumor cells (Figure 1B). Note that OVs can also induce immune responses against viral epitopes, thereby limiting the intratumoral spread of OV, although antiviral immunity has been observed to enhance the antitumor effect of OV therapy [22].

In addition to the intrinsic role of OV, many research groups have also designed OVs with transgenes [20] to help T cells overcome immunosuppressive TME (Figure 1C). The immunostimulatory properties of OVs and the potential to arm OVs with therapeutic transgenes enable them to enhance CAR T cells in the body.

Here, we summarize the current OV in clinical trials and discuss how OV can be combined with CART cells in preclinical models to improve tumor control. In addition, we highlighted promising combinations that have yet to be developed and discussed the transformational challenges faced by OV and CAR T combination therapies.

Clinical results of oncolytic virus monotherapy

At present, most OVs in clinical trials have been genetically modified to enhance their tumor-specific tropism. The oncolytic herpes simplex virus type 1 (oHSV-1) derivative is the first OV approved by the FDA for the treatment of melanoma [29]. oHSV-1 is produced by deleting neurovirulence genes to prevent pathogenicity and deleting virus-infected cellular proteins 47 (ICP47) can provide tumor specificity because ICP47 usually interferes with antigen loading and presentation [30]. Although many clinical trials have proved the safety of natural oHSV-1, it cannot effectively improve the prognosis of patients [31].

However, talimogene laherparepvec (TVEC), created by “arming” the granulocyte macrophage colony stimulating factor (GM-CSF) gene, is the key to obtain the clinical efficacy of anti-melanoma [30]. GM-CSF enhances T cell activation and dendritic cell maturation [32]. Tumor cells infected with TVEC release GM-CSF into TME after virus lysis, which significantly improves the prognosis of patients [33, 34].

A detailed review of OV treatment can be found in Reference 22. In short, OV monotherapy has several important lessons. A major limitation of OV therapy is the limitation of intratumoral spread due to antiviral immunity. The delivery method of OV and the modification of OV can delay the clearance of OV, but clinical studies have shown that antiviral immunity is closely related to anti-tumor immunity, and it is necessary to achieve a balance between these two immune responses [18]. Another key finding is that even though OVs directly lyse tumor cells, various studies have shown that T cells play a vital role in the anti-tumor effect of oncolytic viruses [36]. In addition, OVs increase the infiltration of CD8+ T cells into tumors, making tumors more susceptible to T cells [37]. Therefore, despite the limited success of monotherapy, OVs may still work synergistically with other immunotherapies (such as adoptive cell therapy and immune checkpoint inhibitors) [38, 39].

CAR T and Armed Oncolytic Virus Combination Therapy

Enhanced activation and delivery to solid tumors:

Many solid tumors destroy the expression of adhesion molecules on endothelial cells, reduce the production of chemokines and secrete extracellular matrix (ECM) components, physically exclude T cells from tumor cells, thereby interfering with immune cell infiltration [ 8].

Loaded with RANTES gene (a chemokine that promotes the migration of effector and memory T cells) and IL-15, showed the effect of enhancing CAR-T cell activity to oncolytic adenovirus (Ad5Δ24) [40]. In the neuroblastoma xenograft model, the local treatment of armed OV combined with the systemic infusion of GD2-specific CAR-T cells produces RANTES and IL-15, thereby improving tumor control and enhancing CAR T The transportation of cells to tumor cells. Tumors and the persistence of these cells in TME. A similar method is to use vaccinia OV with another chemokine CXCL11 [41]. CXCL11 is another chemokine that attracts effector T cells by interacting with CXCR3. It is known to be highly affected by effector T cells. expression. Therefore, the enrichment of chemokines matches the receptors expressed on effector T cells. Promote the recruitment of CAR T cells to tumors, thereby improving tumor control.

Adenovirus carrying tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) and binding to mesothelin-specific CAR T cells also showed improvement in tumor control in a pancreatic ductal adenocarcinoma model [42]. IL-2 does not directly attract T cells, they are known to induce the production of chemokines, and in their model, the authors reported an increase in CXCL10 and macrophage chemotactic protein 1 (MCP-1) in tumors. Both act as chemotactic agents for effector T [42]. These results indicate that even in the case of immunosuppressive solid tumors, OV equipped with pro-inflammatory cytokines and chemokines can support the infiltration and activation of CAR T cells.

Fight against immunosuppressive signals in TME:

Many molecular inhibitory pathways in TME can negatively regulate T cell function, including immune checkpoint receptor signaling from various cells (such as PD-1). Immune checkpoint inhibitors (ICIs) can rejuvenate T cells in TME. It has significant clinical activity [6], which promotes the development of OV equipped with immune checkpoint inhibitors (ICIs), with the goal of delivering ICI to TME more selectively, thereby avoiding the toxicity associated with their systemic delivery. Many solid tumors express PDL1 and IFN-γ produced by activated CAR-T cells to further promote PD-L1 expression [6,43]. The treatment of oncolytic adenovirus carrying PD-L1 mini antibody (HDPDL1) and human epidermal growth factor receptor 2. HER2-specific CART cells have enhanced anti-tumor efficacy in prostate cancer models, and delivery of ICI via OV vector is superior to systemic ICI [43]. Therefore, OV equipped with ICI can enhance CAR-T cell function by blocking the PD-1/PD-L1 inhibitory pathway within the tumor.

Prevent antigen-negative recurrence:

Like other antigen-specific therapies, CAR-T cell therapy can promote tumor immunoediting, leading to the emergence of antigen-negative tumor cells [3,4]. In solid tumors, antigen escape is caused by the high heterogeneity of tumor cell composition. OV directly dissolves tumor cells and causes a pro-inflammatory environment, increasing the priming effect of TAA. However, OVs can also be equipped with bispecific T cell adaptors (BiTE) to selectively target TAA, which may further reduce tumor immune escape through antigen loss. BiTE is composed of CD3scFv and scFv targeting specific TAA, so they transiently localize T cells on tumor cells and activate T cells, leading to tumor cell lysis [44]. The researchers proved that folate receptor-α-specific CAR T cells combined with adenoviruses with BiTE-targeted EGFR use CAR T cells to directly kill tumor cells and recruit non-CAR T through BiTE targeting the second antigen. Cells to enhance tumor control in colon cancer xenograft models [44].

Similar to BiTEs, membrane-integrated T cell adaptors (MiTEs) use CD3 scFv to locate T cells on the surface of tumor cells, leading to tumor cell lysis. However, MiTE remains on the tumor cell membrane, so only tumor cells infected with MiTE-armed OV will be targeted for lysis. Although this limits T cell activation to OV-infected cells, compared with BiTEs, this approach may limit targeting and non-tumor effects because activation depends on the targeting of the virus rather than the specific surface markers. Expression [19].

In summary, these studies suggest that OV equipped with MiTE or BiTE can resist antigen-negative recurrence by targeting multiple TAAs.

Current limitations and future directions

Clinical translation challenges:

Despite extensive preclinical studies and promising results, there is only one approved clinical trial evaluating the combination of oncolytic virus and CAR-T therapy for HER2-positive cancer (NCT03740256). Preclinical results are difficult to translate into clinical trials, because preclinical models often use immunodeficient (NSG) mice, and the experimental system cannot simulate the relationship between OV and CAR T cells in the context of a fully capable immune system. interaction.

In addition, the combination therapy of OVs and CAR-T cells raises questions about the safety of the combined use of two effective pro-inflammatory immunotherapies. Although CD19-directed CAR-T cells have been approved by the FDA, they still carry the risk of life-threatening adverse events, such as cytokine release syndrome and severe neurotoxicity [1]. Although the only OV (T-VEC) approved by the FDA is relatively well tolerated, drugs that arm OV to enhance T cell function may increase the incidence and/or severity of CAR-T cell side effects [31]. Therefore, it is essential to establish a safe dose of these drugs in combination with each other. Another important consideration is cost. The current strategy of generating CAR-T cells for treatment has a high cost per unit [45], and the cost of the entire T-VEC treatment process is estimated to be approximately US$65,000 [46]. The combined use of these therapeutic drugs must also be combined with their costs, which may become an important obstacle to widespread use.

Deliver:

One of the challenges of OV therapy is to successfully deliver the virus to the tumor site. Although many clinical trials are studying the method of intratumoral administration of OVs, systemic administration is more suitable for targeting metastatic cancer. 24 However, in the blood, OVs encounter neutralizing factors in the serum, are isolated in the liver and spleen, and impair the extravasation to the tumor. Systemic administration may also cause a cytokine storm response, leading to severe systemic inflammation and even death. 47 Although strategies such as carrier cells, polymer coating and targeting tumor vascular endothelium are used to circumvent these challenges, [24] OVs and CAR combined with T therapy may not need to infiltrate the metastatic site to promote its clearance. As Rosewell et al. found in their study, OV equipped with PD-L1 ICI could not infiltrate lymph node metastasis, but the HER2.CAR T cells of mice that received the OV still showed better control over metastasis. 48 Maybe the activity of CAR enhanced the T cells at the primary tumor site enough to improve the CAR T cell clearance rate of metastases, although other CAR constructs and tumor models should be used for further research. In order to overcome the barriers to OV transmission, one group used CAR T cells to deposit virus at the tumor site. 49 They successfully loaded OV in CAR T cells and delivered OV to tumor cells without impairing the function of CAR T, as well as the synergy between HER2.CAR T. Cellular and oncolytic vesicular stomatitis virus (VSV) have been observed to target breast cancer cell lines. Although this interesting system shows promise in vitro, its application in vivo may be limited to immunologically hot tumors, because in this case, if no cells infiltrate the tumor first to deposit OV, OV cannot enhance CAR T Of infiltration.

Expand the combination of armed OVs and CAR T cells:

Since CD19-specific CAR T cells show the strongest preclinical and clinical efficacy, the researchers produced an OV with human CD19 transgene to force CD19 to be expressed on solid tumor cells so that it can be targeted by CD19-specific CAR To-T cells. In the B16 melanoma model, OV equipped with murine CD19 caused CD19-specific CAR-T cells to slow down tumor growth.

Another group used a similar method to deliver CD19 to MC38 tumors [51]. They observed that CD19-specific CART can improve anti-tumor efficacy. Inject its cells in combination with truncated CD19 armed OV [52]. Even if this concept is conceptually attractive, its clinical potential is still questionable due to the unnecessary elimination of normal B lymphocytes and the limited biodistribution of viruses. The significant improvement of the infectivity of OVs may be necessary for this strategy to achieve clinical efficacy.

There are some transgenes that can work well with CAR-T cell therapy, but they have not been tested yet. In terms of improving transport to solid tumors, the combination of CAR-cells and OV targeting TME physical barriers remains to be explored. OV equipped with hyaluronidase [53] and matrix metalloproteinase 8 (MMP-8) [54] both showed increased intratumoral spread of OV in preclinical models, both of which reduced the dense ECM of solid tumors characteristic. Combining the OV with CAR-T cells can also improve the infiltration of CAR-T cells and thus increase their anti-tumor activity.

Monoclonal antibodies (mAbs) targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and monoclonal antibodies targeting PD-1 have recently been approved by the FDA for the treatment of melanoma, which indicates that the OV carrier Block CTLA-4 [55]. Engeland et al. describe an oncolytic measles virus (MV) with a full-length anti-CTLA-4 mAb. They demonstrated that the OV improved tumor control compared with the natural OV in a mouse model with immune function. However, the authors did report that MV-aCTLA-4 has a better survival rate than the combination of systemic aCTLA-4 treatment and natural MV [55]. However, it should be explored to use CAR T cells in combination with OV encoding a blocked CTLA-4 reagent.

Another potential method is to use OV to modify the metabolism of TME to make it more friendly to CAR-T cells. Recent studies have shown the importance of metabolism in T cell biology. For example, CD39 and CD73 hydrolyze extracellular ATP into immunosuppressive adenosine, which can accumulate in TME and impair T cell function. The CD39 blocking antibody can improve the efficacy of oxyplatin chemotherapy in immunologically functional mouse models, which prompted a clinical trial to study the efficacy of this antibody and PD-1 blocking antibody (NCT04261075) [56]. Arming OV with this antibody may represent a promising way to improve CAR T cell therapy by reducing adeninergic signaling, while stimulating T cells through the intrinsic effects of OV.

Even if combining CAR-T cells with OVs can overcome the challenges of TME, the combination therapy may also bring unique problems. Recent work describes a VSV encoding an IFN-β transgene, which interferes with CAR-T therapy by promoting the apoptosis of CART cells. However, removing the IFN-β transgene can reduce the loss of CAR-T cells [59]. Type I IFN, including IFN-α and IFN-β, stimulates the immune system, but chronic type I IFN signaling can cause immune dysfunction [60]. The duality of pro-inflammatory and anti-inflammatory is a common feature of immune molecules, which makes designing combination therapies tricky and needs to be balanced. The heterogeneity of the proteome among malignant tumors, and possibly even the heterogeneity between patients with the same malignant tumor, may further complicate the combination therapy. When used in combination with CAR-T cell therapy, careful consideration should be given to the potential immunosuppressive effects of the transgene used to arm the OV.

Conclusion

TME in solid tumors has the characteristics of multiple immunosuppressive mechanisms, which may require simultaneous suppression of different pathways to eradicate tumors.

The large capacity of OV can be used to express a variety of transgenes, including chemokines, cytokines, ICI and BiTE, and other molecules to form TME and promote the anti-tumor effect of CAR T cells [48].

In addition, the high flexibility of the engineering process, the combination of OVs and CAR-T cells can customize the most suitable gene combination and CAR-T cell costimulation for the specific characteristics of the target tumor and TME.

Although the vast majority of OV studies have focused on solid malignancies, the combination of armed OV and CAR T therapy to combat hematological malignancies can reduce disease recurrence.

(source:internet, reference only)

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