Approaches to Improve Clinical Efficacy of CD19-Redirected Chimeric Antigen Receptor (CD19 CAR) T Cell Immunotherapy of Non-Hodgkin’s Lymphoma

Clinical use of CD19-redirected chimeric antigen-receptor (CD19 CAR) T cell therapy has shown promise in the treatment of various B cell malignancies including acute lymphoblastic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL). However, its clinical use is limited due to inherent or acquired resistance of tumor cells to apoptotic death signals delivered by CAR transduced T cells as well as by the undesired side effects. Approaches to improve the clinical efficacy of CAR T cell therapy is urgently needed. In 2006, the histone deacetylase inhibitor vorinostat (SAHA) received Food and Drug Administration (FDA) approval for treatment of cutaneous T-cell lymphoma. It affects the acetylation of histones and centromeres, and non-histone proteins, and regulates gene expression. Similarly, the nonsteroidal anti-inflammatory drug (NSAID) Celecoxib (Celebrex®) received FDA approval in 1998 for treatment of osteoarthritis and rheumatoid arthritis; its use is extended to other cancers. NHL is highly dependent on inflammatory micro-environment for its growth, which is inhibited by celecoxib. Celecoxib and HAD Cihinder tumor growth via regulation of various apoptotic genes, such as cyclooxygenase-2 (Cox-2), proand anti-apoptotic Bcl-2 family members, Akt/PKB pathway, IAP family members, and other cell survival signaling pathways. Moreover, technological advances to incorporate various suicide gene systems in eukaryotic cells has limited the non-selective effects and reduced serious adverse effects (SAEs) of CD19 CAR T cell therapy. Considering their broad regulatory effects on apoptotic gene products, Celecoxib and HDACiare promising drugs to improve CD19CAR T cell therapy.CD19 CAR T cell immunotherapy of NHL can further be optimized through incorporation of suicide gene systems.

Engineering T cells to directly target tumor associated antigens (TAA) helps eliminate the requirement for MHCmediated peptide presentation as most tumors down-regulate surface MHC in an attempt to avoid immune detection and destruction Cheadle et al. [1]. Chimeric antigen receptor transduced human peripheral blood T lymphocytes (CAR T cells) meet this criterion. A CAR T cell commonly has an antigen-binding domain, which is encoded by single chain variable fragments (scFv), CD28 co-stimulatory domain, and CD3ζ signaling domain that triggers T cell activation Davila et al. [2], Kenderian et al. [3]. For CD19 CAR T cells, scFv is specific for the CD19 receptor, directing the CAR T cells selectively to CD19+ NHL cells Davila et al. [4]. CD19 CAR therapy has shown potential as an alternative cancer therapy for B cell malignancies. In a phase I/II trial, fortyone patients with NHL were infused with CD19 CAR T cells containing a defined ratio of CD8+ and CD4+ CAR T; an objective response rate (ORR) of 84% and a complete response rate (CR) of 47% were observed in those who received CD19 CAR T cells in addition to cyclophosphamide and fludarabine Turtle et al. [5]. Phase I trial involving patients with chemo-refractory NHLs underwent CAR T cell therapy following autologous hematopoietic stem cell transplantation (HSCT) showed modest results Wang et al. [6]. In NHL1 trial, out of eight patients who received HSCT and CD19 CAR T cells infusion, 3 (38%) had a CR and 2 (25%) had a partial response (PR); in NHL2 trial, out of 8 patients, 6 (75%) had a CR and 2 (25%) had a PR Wang et al. [6].
The modest to low response rates observed in clinical trials might be due to resistance mechanisms that NHLs have developed to avoid apoptosis induced by CD19 CAR T cells. CD19 antigen expression on tumor cells might become lost or down regulated after infusion of CD19 CAR T cells. Frame shift mutations that result in deletion of C-terminal tyrosine residues critical for signal transduction through CD19, were reported in patients with very low or undetectable levels of surface CD19 Van Zelm et al. [7]. A patient who had normal CD19 allele but have defect in CD81 gene was reported to also lack CD19 expression Van Zelm et al. [8]. Since CD81 works in conjunction with CD19/ CD21 complexes in B cell receptor (BCR) signaling, it is possible that defects in CD81 will affect the expression of CD19 Cherukuri et al. [9]. Thus, CD81 defective individuals are non-responsive to CD19-redirected CAR therapy.
NHLs could potentially undergo alternative differentiation and signaling pathways to avoid recognition by CD19 CAR T cells. The biopsy of a patient with plasmablastic lymphoma (PBL) contained tumor cells lacking CD19 and other markers of pre-plasmacytic B-cell differentiation, suggesting that PBL might have used alternative B-cell differentiation pathways to proliferate and avoid apoptosis Evans et al. [10]. Mutations in genes that code for key regulators in cell proliferation and survival signaling pathways might also play a role in apoptosis resistance in tumor cells. Mutated BRAF, NRAS, and p53 genes, all of which are involved in cell cycle regulation, have been reported in a wide range of cancers Davies et al. [11], Daya-Grosjean et al. [12], Chiappetta et al. [13]. Aberrant BCR signaling pathways, including those that involve the Src family kinases Lyn, Syk, PI3K/Akt/mTOR, Bruton tyrosine kinase (Btk), NF-κB, have been observed in chronic lymphocytic leukemia (CLL) and Non-Hodgkin's lymphoma (NHL) Woyach et al. [14], Arita et al. [15]. Abnormal levels of pro-and anti-apoptotic proteins expressed in tumor cells might also be responsible for apoptosis resistance. Whether the extrinsic and intrinsic apoptotic pathways are initiated or shut down depends on the balance between the expression levels of pro-and anti-apoptotic proteins A common trend found among various types of cancers is a decrease in Bax and Bak (pro-apoptotic members of Bcl-2 family) levels and an increase in Bcl-2, Mcl-1, and Bfl-1 (anti-apoptotic members of Bcl-2 family) Bentires-Alj et al. CAR T cell therapy can result in long-term toxicities because the CAR transduced T-cells can persist in the circulation even after cessation of treatment. One of these effects is the release of large quantities of cytokines from activated CAR T cells. Elevated cytokine levels can cause cytokine release syndrome or cytokine storm in more severe cases. During cytokine storm, the immune system creates a positive feedback loop of proinflammatory cytokine production. Cytokine storm is associated with hypotension and systematic inflammatory reaction syndrome where the entire body develops severe inflammation. These immune reactions are typically resolved using steroids or neutralizing antibodies, however, they can be fatal in some cases. Neurological toxic effects such as delirium, aphasia, hallucinations, and seizure-like activity have also been reported upon CAR T cell therapy Onea & Jazirehi [20].
Several strategies have been proposed to overcome the resistance mechanisms of NHL cells to CAR T cell therapy and to reduce the serious adverse effects (SAE). These approaches include the use of FDA-approved drugs with proven anti-cancer properties, such as histone deacetylase inhibitors (HDACi) and celecoxib, in combination with CD19 CAR T cell therapy. Incorporation of various suicide gene systems will further optimize and improve the clinical efficacy of CAR T cell therapy.

Use of Histone Deacetylase Inhibitors (HDACi) to Improve CAR T Cell Immunotherapy
Histone deacetylase inhibitors (HDACis) are promising anti-tumor agents. In eukaryotic cells, histone acetylation and deacetylation processes are central to regulation of transcription and gene expression Xu et al. [23]. The balance between these two processes is required for normal cell growth; improper activity of histone acetyltransferases (HAT) and deacetylases (HDAC)

Use of Celebrex (Celecoxib) to Improve CAR T Cell Immunotherapy
Celecoxib, also known as Celebrex, is a nonsteroidal antiinflammatory drug (NSAID) commonly used to treat rheumatic diseases. Due to its ability to inhibit cyclooxygenase-2 (Cox-2), in recent years Celecoxib has been used to treat several types of malignancies, including breast, colorectal, and prostate cancers, Gupta et al.

Incorporation of Suicide Genes to Improve CAR T Cell Immunotherapy
Clinical utilization of CAR T cell based immunotherapy has shown promising results in various tumor models, however, its clinical efficacy is somewhat hampered, in part, due to cytokine release syndrome (CRS) as well as on target/off tumor effects

Gene-Directed Enzyme Prodrug Therapy (GDEPT)
Using metabolic machineries, in genetically engineered CAR T cells a nontoxic drug is converted to a toxic compound Springer

Incorporation of Inducible Caspase-9 (iCasp9) to Improve CAR T Cell Immunotherapy
With the advance of genome-editing technology, the dimerization inducing mechanism of suicide gene using inducible caspase 9 (iCasp9) has become more efficacious. . In contrast to HSV-TK/GCV suicide system, iCasp9 is non-immunogenic and allows for rapid elimination of CAR T cells; however, the efficacy of iCasp9 system needs to be optimized Jones et al. [50].

Use of FDA Approved Therapeutic mAb to Improve CAR T Cell Immunotherapy
In addition to iCasp9, approaches using therapeutic mAbmediated mechanism can be utilized to circumvent immunogenic responses and improve CAR T cell therapy. The chimeric mouse anti-human CD20 mAb (Rituximab, Rituxan®) is most commonly used. In this approach, CAR T cells are engineered to express CD20 receptors, and after adoptive transfer of CAR T cells, anti-CD20 mAb is administered resulting in the elimination of unwanted or excess CAR T cells Introna et al. In addition to CD20 receptors, CAR T cells can be designed to express or not express certain receptors to enhance their potency. CAR T cells therapy has been reported to be unsuccessful partly due to tumor-induced immunosuppressive mechanisms, namely the production of adenosine Beavis et al. [62]. In tumor microenvironment, adenosine is being produced at immunosuppressive concentrations Ohta et al. [63]. Targeting the adenosine receptor A2AR (using A2AR antagonists or short hairpin RNA [shRNA]) in a murine model of HER2+ selfantigen tumors greatly improved the efficacy of CAR T cell-based immunotherapy. This effect was more pronounced when CART cell therapy was combined with other checkpoint inhibitors such as mAbs directed against program death (PD)-1 Beavis et al. [62].

Conclusion
Recent advances in using the immune system to treat NHL suggest a promising role of CD19 CAR T cell therapy in NHL clinical therapy. However, a subset of NHLs is either inherently resistant or develop resistance to CAR T cell-mediated immunotherapy upon long-term exposure to genetically engineered CAR T cells.
Outgrowth of apoptosis-resistant malignant B cells is a major hurdle in successful NHL clinical therapy. Aberrant activity of signaling pathways and distorted apoptotic machinery concludes in NHLs that develop cross-resistance to anti-cancer agents with different modes of action. Thus, it is practical to develop new means to regulate apoptotic machinery to circumvent resistance. Based on the broad apoptotic gene regulatory effects of Celecoxib and HDACi, we propose that combination of CD19CAR T cell therapy and these FDA approved anti-cancer drugs can potentially improve the outcome of CD19CAR T cellbased treatment of NHL patients. Another challenge with CAR T cells therapy is that genetically modified T cells can persist in patients long after infusion. These T cells can induce various AEs including delirium, aphasia, hallucinations, seizure-like activity, fever, hypotension, and Cancer Therapy & Oncology International Journal hypoxia. Alloreactive CD19 CAR T cells can be eliminated via incorporation of various suicide gene systems into genetically modified T cells, thus, reducing the undesired toxic side effects. Future studies are warranted to optimize the schedule and doses of CD19 CAR T cell therapy (alone or combined with other drugs) to evade development of NHL resistance to CD19 CAR T cells, high grade AEs, and progressive immunodeficiency (Figure 1).

CD19-redirected CAR T cells can induce apoptosis in CD19+
sensitive NHL B-cells via TRAIL (Apo2L), FasL, TNF-a, and perforin/granzyme apoptotic pathways. However, prolonged exposure of NHL B-cells to CD19 CAR T cells results in selective outgrowth and expansion of resistant cells. Resistant NHL cells have distorted apoptotic machinery and exhibit resistance to CD19CAR T cells despite adequate surface CD19 expression. The anti-inflammatory drug Celebrex® (Celecoxib), and the chromatin remodeling drug HDACi(SAHA and panobinostat[LBH 589]) regulate the expression profile of apoptotic genes, favoring the generation of a pro-apoptotic milieu; thus, CD19 CAR T cell-resistant NHL cells undergo apoptosis upon receiving death signals from CD19CAR T cells. Incorporation of suicide gene system in clinical CD19CAR T cell protocols will eliminate alloreactive CAR T cells, hence, reducing the undesired toxic side effects.