CAR-T cells and oncolytic viruses jointly treat solid tumors

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CAR-T cells and oncolytic viruses jointly treat solid tumors

CAR-T cells and oncolytic viruses jointly treat solid tumors.  The adoptive transfer of chimeric antigen receptor (CAR) modified T cells has led to long-term and unprecedented success in patients with leukemia and lymphoma.

However, CAR-T cells have limited therapeutic effects on solid cancer. The new method will need to simultaneously overcome the multiple challenges encountered by CAR-T cells in solid tumors. The main reasons include the immunosuppressive tumor microenvironment and the heterogeneity of antigen expression. Oncolytic viruses are anticancer drugs with solubility and immunogenicity.

They have the potential to act synergistically with CAR-T cells and can be used to treat solid tumors. In addition, the virus can be further modified to selectively deliver therapeutic transgenes to the tumor microenvironment, which can enhance the effector function of tumor-specific T cells. This review summarizes the main limitations of CAR-T cells in solid tumors and discusses the potential role of oncolytic viruses as partners of CAR-T cells in the fight against cancer.

CAR-T cells in solid tumors: challenges and limitations

Although most of the early CAR-T cell trials for solid tumors have led to poor treatment results, some case reports have shown that they have controllable treatment-related toxicity and good clinical responses provide clear reasons for optimistic prospects ( 13, 14).

The latest reports on second-generation CAR-T cells indicate that CAR-T cells can be transported, sustained and proliferated in tumors (9, 10). In addition, evidence of transient anti-tumor activity has been observed in patients with refractory tumors, such as glioblastoma (13), neuroblastoma (14), pancreatic cancer (12), and sarcoma.

Here, we summarize the lessons learned from these clinical trials and discuss the obstacles that must be overcome for effective CAR-T cell therapy, focusing on the challenges that OV may help to solve.

Delivery, diffusion and persistence

The ability of tumor-specific T cells to deliver, proliferate and last to tumors is considered essential to achieve an effective anti-tumor response (14, 29, 30). Although T cells can be actively transported to the disease site, tumors are usually inflamed to a low degree and lack the chemokines required for migration. 

Similarly, physical disorders, such as abnormal vasculature, increased matrix stiffness and higher tissue pressure, may impair T cell infiltration. Once in the tumor, CAR-T cells must proliferate effectively and continue until the tumor is completely eliminated.

However, due to the internal (T cell adaptability) or external factors (tumor microenvironment) of T cells, the proliferation and persistence of T cells are often hindered. If local delivery and redo of CAR-T cells is a treatment option, the requirements for proliferation and persistence can be relaxed in some cases (31).

For example, in a recent clinical trial, for patients with glioblastoma, multiple intracranial injections of CAR-T targeting IL13Rα2 mediated a transient complete response (13). In this patient, two intracranial CAR-T cell delivery routes were tested: intracavity and intracerebroventricular.

Although intracavitary therapy can only control the growth of local tumors, intraventricular therapy has led to a significant reduction in the size of all intracranial and spinal tumors. These results highlight the importance of transportation and administration routes to achieve optimal tumor response.

Developing strategies to enhance delivery and durability to increase the input of therapeutic CAR-T cells in tumors will represent a direct development in this field.

Tumor immunosuppression

After reaching the tumor, CAR-T cells will encounter an immunosuppressive environment, which prevents T cells from exerting their full therapeutic potential. The main obstacles that CAR-T cells need to overcome once in a tumor include:

1. Inhibition of immune regulatory cells, including myeloid-derived suppressor cells (MDSC), tumor-related macrophages and neutrophils, and regulatory T cells;
2. There are a series of immunosuppressive molecules, such as IL-10, TGF-β, PD-L1, IDO and arginase-1;
3. Microenvironmental factors such as low oxygen, low pH and nutrient depletion. These conditions, coupled with long-term antigen exposure, can cause T cells to enter different stages of dysfunction (32-34). In addition, the stromal microenvironment can actively exclude T cells from the vicinity of cancer cells (35).

Finally, a recent clinical report indicated that after the activation of CAR-T cells in the tumor, the tumor microenvironment may become more immunosuppressive, which may be caused by the initial production of IFN-γ (10). Finding ways to prevent or reverse T cell dysfunction by reversing tumor immunosuppression will be the key to improving treatment.

Tumor escape due to lack of antigen expression or heterogeneity

One of the main limitations of using CAR-T cells to treat solid tumors is the lack of cancer-restricted antigens that are uniformly expressed in tumor cells and do not exist in basic organs. Tumor escape due to heterogeneity or lack of antigen expression is an emerging threat to CAR-T cells because it may lead to overgrowth of target-deficient tumor cells that are invisible to CAR-T cell therapy (36-38).

Preclinical studies have shown that CAR-T cells can preferentially eliminate tumor cells that express high levels of targeted antigens, while tumor cells that express the lowest levels can survive (39-41).

Reduced expression of targeted antigens after CAR-T cell therapy has been observed in some clinical trials, including those against Her2 (9), EGFRviii (10), IL13Rα2 (11) and mesothelin (12). These results demonstrate the potential of CAR-T cells to eliminate antigen-positive tumor cells, but they also highlight the importance of designing new strategies that target different antigens at the same time.

Several groups are designing new CAR constructs that can target more than one antigen at the same time (39, 42, 43). While reducing the risk of escape, these strategies may also lead to increased extra-tumor reactivity outside the target, because most targeted antigens can be expressed at low levels in healthy tissues (17-19).

Another method is to find a strategy to activate the endogenous immune response, which can be combined with CAR-T cells to completely eliminate tumors. Some reports indicate that CAR-T cell-mediated tumor destruction may lead to the release of other tumor antigens, which are cross-presented in a process called epitope spread (44, 45). This observation requires further research, but it can explain the complete elimination of tumor lesions even when tumors cannot uniformly express their targets (13).

Oncolytic viruses: lessons learned in clinical trials

To date, there are three viruses that can be used to treat cancer: T-VEC approved in the United States, H101 approved in China and Rigvir approved in Latvia, Georgia and Armenia. Several other viruses are also in clinical trials and may eventually be added to this short list of listed viruses (46).

Some of the lessons learned from clinical trials will drive the design of future treatments, including:

(A) OV can produce therapeutic benefits for cancer patients without serious adverse reactions, including complete remission (47-50). Interestingly, some of these complete responses can be achieved after virus elimination, which suggests that complete tumor elimination may depend on the activation of immune-mediated anti-tumor responses (48). Based on this observation, a recent clinical trial reported that patients treated with chimeric poliovirus can maintain an overall survival rate of 21% for a few months after 1 year of treatment (51). This plateau in long-term survival is similar to the plateau observed in Kaplan-Meier curves for cancer patients treated with other cancer immunotherapies, and highlights the role of the immune system in the emergence of long-term survivors (52).

(B) Antiviral immunity is an obstacle to OV because it isolates or neutralizes virus particles before they reach the target. A major problem is how to effectively deliver the virus to the tumor.

(C) A few days after treatment, virus replication was detected in the tumor biopsy. However, antiviral T cells limit the ability of OV to survive and spread in tumors (47, 48, 53).

(D) OV-treated tumors usually show increased immune cell infiltration, including activated macrophages and cytotoxic T cells, as well as pro-inflammatory cytokines (47, 48, 53). Tumor-specific T cells have been detected after treatment with OV (53, 54). Although the ability of OV to expand neoantigen-specific T cells deserves further study, the potential of OV in combination with immunotherapies such as immune checkpoint inhibitors has been widely recognized (28, 55-58). Several clinical trials are currently testing the combination of OV and immune checkpoint therapy, and preliminary reports have shown encouraging results (23, 24).

Oncolytic virus: an ideal ally for CAR-T cells?

It is possible that OV can work synergistically with CAR-T cells by helping CAR-T cells to overcome multiple obstacles found in solid tumors at the same time.

First, the virus provides a danger signal that can reverse the immunosuppressive effect of tumors, thereby promoting the transport, proliferation and persistence of CAR-T cells in the tumor microenvironment.

Second, the direct lysis of cancer cells by OV leads to tumor lysis and the release of tumor-associated antigens (TAA), which can induce anti-tumor adaptive responses, which may alleviate tumor escape caused by antigen loss.

Third, OV can be equipped with therapeutic transgenes, which can further enhance the effector function of T cells. Here, we provide an overview of the biological characteristics of OV that can be considered when selecting a viral platform to combine with CAR-T cells, and we summarize the recent preclinical strategies that have been explored to combine CAR-T cells and OV.

Oncolytic viruses as immunotherapeutics

The immune system is well-equipped to carry out innate inflammatory response to the virus, which will eventually induce the infiltration of effector T cells. In particular, OV has pathogen-associated molecular patterns (PAMPS) detected by pattern recognition receptors (PRR) on tumors and epithelial cells, as well as macrophages and dendritic cells (59).

These PRRs can induce a dangerously related molecular pattern (DAMP) that is characteristic of immunogenic cell death (60, 61). PRRs also use NF-kB signaling to induce the expression of cytokines such as TNF-α and IL6, and induce type I interferon through IFN regulatory factors (IRF) and activate mature IL-1β caspase 1 (62) .

This pre-immune cytokine environment can promote the maturation and function of DCs, macrophages and epithelial cells, leading to the recruitment of neutrophils and natural killer (NK) cells, monocytes and memory T cells to the site of infection (63 -65).

Tumor cells that die due to the lytic activity of OV can release TAA. The activated DC of MHC with virus and/or tumor epitope can flow to the draining lymph node, combine with specific T cells and stimulate their proliferation and circulation into the blood.

The chemokines of the infected tumor can induce the expression of integrins on these T cells and the expression of selectins on endothelial cells, thereby allowing them to penetrate. Under these conditions, T cells can be effectively recruited to infected tumors, and as mentioned above, increased T cell infiltration is usually detected in the tumors of patients treated with OV therapy.

Interestingly, viral infections have been shown to induce neo-antigen-directed T cell responses (53, 54), which can act synergistically with CAR-T cells and virus-specific T cells to eliminate tumors. The limitation of studying the influence of OV on the immunomodulatory effect of CAR-T cell therapy is the lack of good animal models.

However, it can be assumed that after a more immunogenic tumor environment is established, the target cells may be killed more effectively due to the cooperation between effector T cells. The ability of OV to induce an anti-tumor immune response is now considered to be a key mechanism of action for obtaining a long-term anti-tumor response. Therefore, most of the current efforts aimed at improving the therapeutic potential of OV have focused on improving its ability to induce systemic anti-tumor responses.

Oncolytic virus arsenal

Many types of viruses are used in the treatment of cancer viruses, each of which has its own unique characteristics (Table 1) (66). Here, we discussed some of the different factors that should be considered when choosing an OV that binds to CAR-T cells. Generally speaking, viruses that replicate in the cytoplasm (RNA viruses) kill tumor cells faster than nuclear cells (DNA viruses) because they do not need to reach the nucleus of the infected cell. But for the same reason, they provide fewer opportunities for selective tumor control.

Most oncolytic RNA viruses (such as reovirus, picornavirus (Coxsackie virus, Riga virus), rhabdovirus (vascular stomatitis virus [VSV], Malaba virus) and paramixed virus ( Measles virus, Newcastle disease virus [NDV]) tumor-selective replication depends on the defect of the interferon pathway in tumor cells. Since IFN induction is the central pathway of virus innate response and can enhance adaptive T cell response, it is expected that these viruses The inflammatory response will be lower.

DNA viruses (such as adenovirus) have a slower replication cycle, but tumor-selective promoters can be used to control the nucleus of infected cells. The presence of the envelope also determines the oncolytic properties of the virus. Enveloped viruses (for example, measles virus, NDV, VSV, herpes simplex virus, and vaccinia virus) germinate from cells and are less capable of “decomposing” than naked viruses. Envelope also contributes to the main clearance mechanism in the blood, and complement plays a major role in enveloped and non-enveloped viruses. Size is also an important parameter of OV properties. The smaller the virus, the easier it is for the virus to penetrate and spread throughout the tumor. But larger viruses with larger genomes can insert non-viral transgenes. Arming OV with therapeutic transgenes can provide opportunities to supplement OV in a variety of ways.

Among RNA viruses, in contrast to parvovirus and reovirus, VSV, measles virus, and NDV can accept transgenes, while for DNA viruses, adenovirus, herpes simplex virus, and vaccinia virus can be equipped with transgenes, compared with parvovirus.

The list of potential genes contained in OV that may be used in combination with CAR-T cells is long and has recently been reviewed (67). It includes: (a) inducers of immunogenic cell death (68), (b) transgenes that directly regulate the immune system, such as cytokines (22, 69–71), chemokines (72, 73), co-stimulation Proteins (74–77), bispecific T cell adaptors (BiTE) (78, 79) and immune checkpoint blockers (80–83), and (c) matrix-degrading proteins that may promote the spread of OV and T cells Inside the tumor (84, 85).

Considering the limitations of preclinical immunological mouse models, comparing viruses and transgenes is a very challenging task. In this model, many human viruses have replication defects, and tumors cannot be as slow as humans. And gradually edit the immune system.

Combining CAR-T cells and oncolytic virus to treat solid tumors

At the pre-clinical level, several groups have begun to combine CAR-T cells to test different transgenic arms OV (Figure 1). Most of these studies have evaluated the anti-tumor effects of these therapies on NOD scidγ (NSG) mice, which are completely lacking in adaptive immunity and severely lacking innate immunity (86). NSG mice allow the implantation and persistence of adoptively transferred CAR-T cells, while human tumor xenotransplantation allows virus replication and transgene delivery.

Therefore, these studies provide important information in the anti-tumor effects of combining CAR-T cells with oncolysis and transgene delivery. An important limitation is that these tumor xenografts cannot be used to assess the ability of OV to induce anti-tumor immunity.

It has been shown that modified oncolytic adenoviruses that can express IL-15 and RANTES (87) or IL-2 and TNF-α (88) can increase the accumulation and survival of CAR-T cells in the tumor microenvironment. Similarly, for the purpose of enhancing the internal transport of tumors, vaccinia virus expressing CXCL11 (a CXCR3 ligand) is used to attract effector cells after metastasis (89).

Another report showed that the expression of BiTE targeting the second tumor antigen by oncolytic adenovirus can resolve the heterogeneity of antigen expression (40). In the absence of CAR-targeting antigen or CAR expression (ie, untransduced T cell population), the CAR-T cell preparation is combined with OV-BiTE-induced T cell activation. In a slightly different way, the oncolytic adenovirus is combined with a helper-dependent adenovirus expressing PD-L1 blocking mini-antibody to reverse T cell dysfunction by preventing the PD1:PDL1 interaction (90). The co-expression of IL12p70 and PD-L1 further enhanced the therapeutic efficacy of the combination (91).

As expected, all these combinations of CAR-T cells and equipped OV can enhance tumor control and prolong survival compared to each drug as a monotherapy. The interesting discovery of Watanabe et al. is that CAR-T cells as a monotherapy cannot control the growth of primary tumors, while OV can inhibit the progression of primary tumors, but the mice die of metastatic disease. The combination of CAR-T cells and OV loaded with IL-2 and TNF-α can control primary tumors and tumor metastasis (88).

Finally, in a completely different and very preliminary approach, CAR-T cells have been used to deliver OV to tumors (92). Loading OV onto tumor-specific T cells (by adhering to the surface of T cells) can protect the virus from neutralizing antibodies while still maintaining anti-tumor efficacy after being released in the tumor microenvironment (96). OV tumor delivery of CAR-T cells can enhance virus delivery to tumors, and subsequent oncolysis can attract more CAR-T cells and establish a positive feedback loop.

Existing problems and future directions

For so many kinds of oncolytic viruses, it is difficult to know which one is most suitable for binding to CAR-T cells. In fact, it is difficult to imagine a virus that is commercially developed only for binding to CAR-T cells. Therefore, it is expected that the virus on the market or the virus undergoing clinical research will be the first virus to be combined with CAR-T cells for clinical use.

Although the general value of the virus in attracting T cells into tumors has been widely accepted (53, 97), practical issues regarding the optimal delivery route and dosing regimen are more difficult to predict. Intratumoral administration of OV can provide a large amount of virus in the injected tumor, but it is technically challenging for visceral tumors or metastatic tumors, and it is unlikely that any virus will change the immunosuppressive microenvironment if the tumor is not injected. Systemic intravenous administration is easier and may effectively reach all metastases, but it effectively neutralizes the virus in the blood, especially when high titers of neutralizing antibodies are produced after the first virus administration, which will be repeated Delivery set obstacles. If the virus is injected intratumorally or systemically, the immune response to the virus may also be very different. Usually immunization is performed subcutaneously or intramuscularly, because the immune system does not respond positively to systemic pathogens, partly because the inflammatory response of the cells is lower than that of the dendritic cells in the tissue, and the liver is tolerogenic (98).

Therefore, when the system detects the virus, it may tame or regulate the immune response caused by OV replication in the tumor. The timing of the virus and CAR-T cells will also affect the results. In principle, the virus should first change the immunosuppressive tumor microenvironment, induce direct lysis of tumor cells, and create a more suitable environment to attract CAR-T cells. Pretreatment of the patient should also be considered before treatment. Although the immune-stimulating environment generated by the virus may bypass the need for lymph node dissection to promote the expansion of CAR-T cells, lymph node dissection can still be a good method to promote virus replication and persistence in tumors, and at the same time is a common use. CAR provides advantages-T cells (4, 99, 100).

Oncolytic viruses provide a powerful inflammatory self-amplifying oncolytic mechanism of action, and can also lead to the release of TAA. However, the ability of OV to induce anti-tumor immune responses is not well understood. Given that there are a large number of viral non-self peptides after treatment with OV, the immune response to viral epitopes is likely to dominate the mixture with tumor neoantigens (101-103). New strategies are needed to increase the immunogenicity of tumor epitopes and reduce the immunodominance of viral antigens to promote the spread of epitopes (104).

Finally, T cells can also be manipulated to make them better partners for oncolytic viruses. Virus-specific T cells have been used as a platform for CAR expression (105). Virus-specific CAR-T cells retain the ability to recognize viral infection targets and tumor targets through their natural receptors and chimeric receptors, respectively. Therefore, these T cells may be ideal for combination therapy with OV, because the presence of the virus can promote the expansion of CAR-T cells in tumors. The disadvantage of this method is that it will produce a faster OV gap.

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