The Evolution of CAR-T Cell Therapy

The development of Chimeric Antigen Receptor (CAR) T cell therapy has been a decades-long road from conception in the 1980s to the FDA approval of the Novartis CAR-T cell therapy, tisagenlecleucel in 2017 that heralded the onset of this multibillion dollar healthcare industry. The initial concept for CAR-T therapy was developed with the construction of recombinant TcRs that replaced the TcR V regions with antigen-specific antibody V regions. The chimeric TcRs retained the normal extracellular C region, the transmembrane segment, and the cytoplasmic signaling domains, and therefore maintained the ability to induce T-cell proliferation, interleukin production and cell lysis. Furthermore, these chimeric TcRs were non-MHC-restricted and universal in the sense that a given chimeric construct could be transfected into T-cells from any individual.  T cells expressing CAR have been shown to recognize a wide range of surface antigens, including glycolipids, carbohydrate moieties and proteins (Morello et. al.  2016) and can attack malignant cells expressing these antigens.  

The initial CAR-T trials in cancer patients targeted advanced epithelial ovarian carcinoma using CAR-T cells programmed against the folate receptor (Lamers et. al. 2006), and metastatic renal cell carcinoma with CAR-T cells targeting carbonic anhydrase IX (CAIX) (Kershaw et al. 2006). Unfortunately, these trials were largely unsuccessful due to poor CAR-T cell persistence and the development of CAR-T auto-toxicity in some patients.  Fast forward to 2010, and the National Cancer Institute published the first case report of successful therapeutic intervention of a patient with non-Hodgkin’s lymphoma using a second generation of anti-CD19 CAR-T cells. This opened other clinical opportunities for similar clinical trials for other lymphomas including diffuse large B-cell lymphoma, refractory primary mediastinal large B-cell lymphoma and transformed follicular lymphoma. Most patients showed an objective response to the treatment, which resulted in the FDA granting the ‘breakthrough’ biological license application for anti-CD19 CAR-T (axicabtagene ciloleucel) on the 18^th of October 2017.  CAR-T is well suited for the treatment of B-cell malignancies they express B-cell lineage specific markers such as CD19, CD20 and CD22 that are not expressed on other tissues. As our understanding of CAR-T design and engineering has matured, the therapeutic applications for CAR-T cells in oncology have markedly expanded. 

Table 1. The Evolution of CAR-T Design and Mechanism of Action. Adapted from Advances on chimeric antigen receptor-modified T-cell therapy for oncotherapy.

CAR-T Targeting of Solid Tumors

Despite efforts to translate the principals of CAR-T cell therapy from hematological malignancies to solid tumors, there exists significant challenges. These include the lack of eligible and effective surface targets on solid tumors (the equivalent to CD19 that proved to be so effective for Acute Lymphoblastic Leukemia, ALL) to which CAR-T can be programmed to target. In addition, the tumor can be a highly heterogeneous with respect to cell types and the surface antigens presented, but more significantly, the tumor microenvironment (TME) can be a hostile environment for T-cells resulting in reduced effector functions. It has been proposed that tumor infiltrating CAR-T cell activity could be significantly inhibited by immunosuppressive cells such as Tregs and myeloid-derived suppressive cells (Balkwill et. al. 2012).

In spite of these challenges, there have been a number of small clinical studies that have demonstrated the potential effectiveness of this approach (summarized in Table 2) that may be influenced by the route of delivery into the patient (intrapleural and intracranial delivery versus IV). 

Table 2. Summary of clinical trial studies for CAR-T targeting solid tumors. These studies all demonstrated the potential of CAR-T therapy for the management of solid tumors. 

Overcoming the Risks of CAR-T Cell Therapy

As the CAR-T cells proliferate in the body, some patients have experienced severe side effects that are largely due to the release of high levels of cytokines. Side effects of this cytokine release syndrome (CRS) include high fevers and low blood pressure and in some cases neurotoxicity, all of which need to be managed for several days after treatment. 

CRS occurs through a series of interactions; as the CAR-T cells target the tumor cells, there is a release of IFN-γ and TNF-α that triggers the activation of monocytes and macrophages with enhanced tumoricidal capacity. These activated macrophages in turn secrete high levels of the proinflammatory cytokines IL-6, IL-1, IL-10 as well as induce iNOS to release nitric oxide; collectively these mediators can create a cascade reaction leading to the development of CRS. There are various grades of CRS ranging in severity of symptoms. Severe CRS is associated with high bone marrow tumor burden, lymphodepletion (following the administration of cyclophosphamide and fludarabine) and the administering of very high doses of CAR-T cells. CRS is largely managed through monitoring of predictive markers including C-Reactive Protein (CRP), Ferritin, and monocyte chemoattractant protein 1-a levels in serum, as well as the monitoring of vital signs, and the use of anti-IL-6 and steroid therapy. However, for critically affected patients, other inflammatory modulators including anti-TNF-α or IL-1R inhibitors have also been used, and in fact the prophylactic blockade of the IL-1 receptor and GM-CSF are now being studied for the prevention of CRS and neurotoxicity. 

Neurotoxicity from CAR-T cell therapy is thought be to associate with the impairment of the blood brain barrier function relating to TNF-α, IL-6 and IL-1 levels, as well as the balance between angiotensin 1 and 2, resulting in activation of microglia that can result in an inflammatory state and brain damage (Santomasso et al, 2018). CAR-T cell related encephalopathy syndrome (CRES) and immune effector cell- associated neurotoxicity syndrome (ICANS) are recognized conditions related to CAR-T cell therapy. 

Long term side effects of CAR-T cell therapy include cytopenias, cardiac toxicity that typically resolves within 6 months of treatment. Hypogammaglobulinemia may also be observed and therefore vaccination within several months of CAR-T therapy is not recommended.  

Tumor Escape from CAR-T Cell Therapy

Data arising from CAR-T trials in B-cell malignancies have highlighting a significant frequency of post-therapy relapse through acquired tumor resistance. The underlying mechanism of this so called ‘tumor escape’ appears to involve the downregulation or loss of expression of the targeted tumor antigen. For ALL, these relapse rates range from 30-60% of patients, with roughly 60% of these relapses associated with the loss of CD19 expression. Although we have only limited data on this process for CAR-T treatment of solid tumors, tumor escape is likely to be even more prevalent for these tumors, that display much higher heterogeneity in target-antigen expression. 

In the example of pediatric B-ALL, antigen loss has been shown through several distinct mechanisms: 

1. antigen escape-after remission in response to CD19-CAR-T, patients relapse with a phenotypically similar but distinct disease that lacks CD19 expression. This most likely occurs through the selection of cells expressing splice variants of CD19 that infers some level of resistance to CD19-CAR-T cells
2. lineage switch- patient relapses with a genetically related by phenotypically different malignancy, frequently this malignancy is acute myeloid leukemia. 
3. In the example of CD22-CAR-T therapeutic use, the diminished expression of CD22 was associated with downregulation of CD22 gene expression that was mediated at the post-transcriptional level (Fry et. al. 2018)

There are several strategies to combat tumor escape through antigen loss. The include equipping CAR-T cells with two CARs targeting two different tumor associated antigens. In this model, tumor escape would require the simultaneous mutation of both target antigen genes, and preliminary data has shown high levels of anti-tumor function in these bivalent or tandem CAR-T cells (Hegde et. al. 2013) (Fig. 2). However, identifying not just one, but two tumor associated antigens on a single tumor, that are expressed at low enough levels of other healthy cells to enable their safe and effective targeting with CAR-T is highly challenging for most malignancies.

Fig. 2. Engineering CAR-T cells to help mitigate tumor escape. CAR-T cells CARs can be engineering in several ways to provide more effective anti-tumor activity. A single vector can be designed to express two independent CARs on a single T-cell (Bicistronic CAR-Ts). Alternatively, a bivalent or tandem CAR that recognizes two different tumor associated antigens can be engineered, resulting in enhanced CAR-T function when both antigens are engaged. Beyond this concept, the generation of two species of CAR-T, targeting different antigens, that are co-administrated, has been proposed. Alternatively, T-cells can be modified by co-transduction with two separate vectors to achieve and random mixed population of CAR-T cells, targeting either one of both tumor-associated antigens.   

Other studies have indicated that combining CAR-T cell therapy with checkpoint blockade to overcome the unresponsiveness of tumor-specific T-cells and strengthen the immune response to the tumor. It is not yet fully understood how successful this strategy could be, but preliminary studies are very promising.

Another tactic to improve CAR-T anti-tumor activity involves the engineering of ‘armored’ CAR-T cell that have been modified to secrete immune stimulatory cytokines such as IL-12. IL-12 secretion appears to increase the anti-tumor activity of CAR-T cells directly, as well as through the modulation of the TME to reduce the suppressive action of Tregs and myeloid-derived suppressor cells on the armored CAR-T cells.  Armored CAR-T cells have also been designed to express co-stimulatory molecules such as CD40L, CD80 or 4-1BBL that work to enhance CAR-T function by maturing and activating dendritic cells, and increasing tumor immunogenicity through the modulation of the tumor phenotype to promote an effective endogenous antitumor response. 

What is in the future for CAR-T Cell Therapy?

The optimization of the basic CAR construct over the course of the years has included the addition of costimulatory domains that have markedly enhanced T-cell activation and survival, and in so doing have improved the outcomes of patients with B-cell malignancies significantly. 

Beyond this, an area of continued focus includes the treatment of malignancies for which such strong tumor associated antigens may not be available. There are several proposed strategies to address these hurdles including the identification of tumor-specific antigen pairings for CAR-T cell combinatorial antigen targeting (Fig. 2) as well as the development of transient CAR expression platforms that could potentially utilize suicide constructs to limit CAR-T activity and reduce potential damage to healthy tissue.  

The ability to engineer antigen targeting CAR-T cells, has resulted a wide array of early phase clinical CAR-T cell studies that target solid tumors.  However, overcoming the hostile TME for impactful CAR-T cell activity and identifying universally expressed tumor antigens has certainly impacted progress in this field. Radiation and epigenetic therapy are now being employed to increase the expression of tumor-associated antigens prior to CAR-T cell infusion with the aim of augmenting their anti-tumor activity. 

In parallel there have been innovations in the technical design of CARs to reduce toxicity and improve efficacy and survival of the T-cells. The co-administration or co-transduction of CAR-T cells, such as the combination of CAR-T cells targeting CD19 and CD22 appears to reduce antigen-negative relapse. The fourth generation ‘armored’ CARs utilize cytokine secretion (typically IL-2) by TRUCKSs on encountering the target antigen, thereby shifting the TME away from immunosuppression and towards immune-activation and tumor cell killing. Other concepts for CARs along these lines include the engineering of CAR-T cells with chemokine receptors to aid in trafficking or with elements to activate in the presence of hypoxia to help overcome the barriers associated with solid tumors.  

Final Thoughts 

CAR-T cell approaches have revolutionized the landscape of cancer therapeutics particularly against hematological malignancies and remains the most promising approach for the treatment of many types of cancers. The coupling of CAR-T cell with checkpoint blockade therapies holds the potential for extending the efficacy of anti-tumor activity, and the engineering of CARs to improve penetration of solid tumors and T-cell persistence holds great promise for the advancement of this area of medicine. This goal of targeted and more effective cancer therapeutic interventions justifies the unprecedented investment of effort and resources into CAR research over the last 20 plus years. It is now finally entering into mainstream medicine, thanks to a combination of innovative science, forward thinking engineering and most of all, persistence.

References

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Dr. Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Torrey Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists.