Cancer treatment has historically relied on what are called the three pillars of oncology: operation, chemotherapy, and radiotherapy. With recent advances in cancer immunotherapy, where the body’s immune system to used to treat cancer, it is being touted as the fourth pillar of oncology. The US FDA’s (Food and Drug Administration) approval of Kymriah™―developed by Novartis©―in August 2017 has cemented this idea. Kymriah™ is the first chimeric antigen receptor (CAR) T Cell Therapy approved to treat disease in the United States. This CD-19 targeted CAR-T therapy is for patients under the age of 25 who have refractory or relapsed (r/r) B-cell precursor acute lymphoblastic leukemia (ALL). “We’re entering a new frontier in medical innovation with the ability to reprogram a patient’s own cells to attack a deadly cancer,” said FDA Commissioner Scott Gottlieb.
In brief, CAR-T takes immune cells called T cells from a patient, genetically engineers them to express chimeric antigen receptors (CARs) that recognize the patient’s cancer cells, and re-infuses them back to the patient (this process is called adoptive cell transfer, or ACT). These engineered cells circulate in the bloodstream, becoming “living drugs” that target and kill the antigen-expressing cancer cells. With many early successes in clinical trials using CAR-T, there is great hope that it can be used to treat a wide variety of blood and solid tumor cancers.
What are T cells?
T cells are lymphocytes or white blood cells that play an important role in cell-mediated immunity. They can be distinguished from other lymphocytes by the presence of a T cell receptor (TCR). Foreign antigen fragments presented by specific major histocompatibility complex (in humans, called human leukocyte antigens or HLAs) molecules are essentially ligands for the TCR. Once bound, an intracellular signaling cascade is initiated resulting in T cell-induced apoptosis in the target cell.
The ability of T cells to recognize and eradicate cancer cells expressing foreign tumor-associated antigens (TAAs) has been an area of interest for cancer immunotherapy for the past few decades. Tumor-reactive T cells isolated from tumor-infiltrating lymphocytes (TILs) from patients with metastatic cancer can be expanded ex vivo and re-infused back into patients to enhance tumoricidal effects. The use of TILs to treat melanomas was pioneered by Dr. Steven Rosenberg’s group at the National Cancer Institute in the 1990’s (Topalian, 1987). Taken this idea a step further, other groups genetically engineered TCRs to recognize specific TAAs. TCRs engineered to recognize MART-1 antigen, which is highly expressed on malignant melanoma cells, resulted in T cells with high avidity for malignant cells. Adoptive transfer of these engineered cells led to tumor regression in a number of patients (Morgan, 2006).
While it is possible to engineer TCRs to recognize many different tumor antigens, the mechanism of T cell activation is a limiting factor for their use for ACT. The activation of T cells occurs in an MHC-dependent manner and is crucial for the immune system to differentiate ‘self’ from ‘non-self’. However, because the HLA genes are highly ‘polymorphic’ (containing many different alleles) across individuals T cells with engineered TCRs can only be used on histocompatible patients. The development of chimeric antigen receptors (CARs) provided a more universal approach to cancer immunotherapy since CARs can recognize cell surface antigens in an MHC-independent manner. The CAR is a fusion antigen receptor: part antibody, part TCR, made up of an extracellular antigen-binding, transmembrane and intracellular signaling domain(s).
Evolution of CARs
The first CAR T cells were developed at the Weizmann Institute of Science in Israel in the late 1980s by chemist and immunologist Zelig Eshhar (Gross et al., 1989). Chimeric antigen receptors (CARs) are composed of three components:
- Extracellular antigen-binding domain derived from a tumor-specific monoclonal antibody single chain variable fragment (scFv)
- A transmembrane domain anchoring the CAR to the T cell-derived from CD3, CD4, CD8 or CD28
- An intracellular T cell activation domain of CD3ζ with or without costimulatory molecules
The transmembrane domain connects the scFv, which specifies the T cell binding to a tumor antigen to the intracellular CD3ζ domain, responsible for T cell activation. The most commonly used method to stably insert DNA constructs encoding CARs into T cell genomes is viral vector transduction with either Ɣ-retroviral vector or lentiviral vector (Wei, 2017). When the CAR binds to a tumor antigen on the surface of a target cell, the CAR T cell will induce apoptosis using the same mechanism as normal T cells. Figure 1 summarizes the evolution CARs design.
Most of the clinical data we have is from 1st and 2nd generation CAR-T designs. Kymriah™ is a 2nd-generation CAR-T that combines an extracellular anti-CD19 antibody fragment with costimulatory intracellular signaling domains, CD3ζ and CD137 (4-1BB).
In 2012, Novartis and the University of Pennsylvania entered into a global collaboration develop and commercialize CAR-T cell therapies, including Kymriah™ (formerly known as CART-19 or CTL019), for the treatment of various malignancies. Kymriah™ targets the CD-19 antigen, which is uniquely expressed on most B cells and B lymphoid progenitors. CD-19 is also expressed on all tumor cells and its expression is retained during tumor cell progression (Essand, 2013), making it an attractive therapeutic target. The FDA approval was based on the results of their Phase II ELIANA trial where children and young adults were enrolled in 25 treatment centers in the US, EU, Canada, Australia and Japan. Of the 63 evaluable patients, 52 responded (83%), with 40 patients (63%) achieved complete remission (CR), and 12 (19%) achieving CR with incomplete blood count recovery (CRi) within three months of infusion with no evidence of minimum residual disease (MRD)–a blood marker that indicates potential relapse (Novartis, 2017).
The one-time treatment has a high price tag of $475,000 USD for patients who are responsive. Initially, Kymriah™ will be available at 32 hospitals and clinics that have been specially trained in administering the therapy and handle any side effects.
While not fatal in most cases, cytokine release syndrome (CRS) is the most common side effect of CAR-T resulting from the activation of a large number of T cells causing the release of inflammatory cytokines, primarily IFN-γ, IL-6, IL-10, TNF-α and IL-2, several hundred times above baseline. This flood of cytokines can cause systemic inflammatory responses, hypotension, fever, and neurological changes. Ironically, CRS is an “on-target”/”off-tumor” effect of the therapy. The cytokine release indicates the treatment is working; there are active CAR T cells in the body. The drug tocilizumab (Actemra®), which blocks IL-6 activity, used to treat juvenile arthritis has been found to mitigate the effects of CRS in patients.
CAR T-Cell Therapy Concerns
Improvements in molecular biology and our understanding of immunology over the last two decades have led to unprecedented successes with CAR-T while also highlighting their shortcomings. There is no more glaring illustration that we need to proceed with caution, despite stellar clinical results, than what happened in 2016 to Seattle-based Juno Therapeutics (NASDAQ: JUNO), one of the early leaders in the CAR-T field. Their phase II ROCKET trial examined JCAR015 (also a CD-19 targeted therapy) for patients with r/r B cell ALL. Sadly, 5 of 38 adults treated with JCAR015 died unexpectedly from severe neurotoxicity-triggered cerebral edema (brain swelling). This sent shockwaves through the scientific community and eventually caused Juno Therapeutics to abandon its JCAR015 program. It was a sobering reminder that, despite the utmost safety measures, these therapies are experimental and not without flaws. Unpredictable outcomes are always a risk when dealing with complex biological systems and patients that have diverse genetic backgrounds.
Are the risks worth the reward? Researchers are already exploring ways to make CAR-T therapy safer.
Future of CAR T-Cell Therapy
Currently, all the leading CAR-T companies (Novartis, Juno Therapeutics, Kite Pharma/Gilead) have focused their efforts on autologous treatments targeting the CD19 antigen to treat B-cell malignancies, which include several forms of lymphoma and leukemia. However, as with other cell therapies, cost is leading other companies to explore allogeneic, so-called “off the shelf”, approaches. Notably, Pfizer (NYSE: PFE) and Servier are in partnership with Cellectis (NASDAQ: CLLS) to develop allogeneic UCART19 product, which also targets CD-19, for treatment of B cell ALL.
Addressing Safety Concerns
Harnessing one’s own immune system to attack cancer cells is undoubtedly a powerful tool, but left unchecked, can lead to some deleterious results. Researchers are examining new iterations of CAR T cells that express additional accessory molecules to serve as safety switches, which allow for the elimination of CAR T cells from circulation, in the event of life threatening T-cell-mediated toxicity―essentially an “off” switch (Zhang, 2017). For example, Cellectis’ UCART19 product is engineering with a switch control system where the CAR T cell activation can only occur in the presence of rapamycin. Bellicum Pharmaceuticals (NASDAQ: BLCM) is testing a similar technology called GoCAR-T where T cells can only be activated in the presence of rimiducid. Other groups are working with an inducible caspase-9 (iCasp9) system. Inactive, monomeric caspase9 subunits expressed in CAR T cells can form a lethal dimer, causing the rapid clearance of CAR-T cells, when exposed to dimerizing agent (Abate-Daga, 2016). Synthetic control switches such as the iCasp9 system are likely to play a central role in the management of adoptive immunotherapy, either for the removal of toxic/incompatible CAR T cells or to allow for more sophisticated control over CAR-T cell activity.
Other CAR-T Approaches
Adapting the CAR technology to other cancers beyond B-cell malignancies has yet to deliver anywhere near the same level of clinical response. One main hurdle is identifying a suitable target antigen targeted by the scFv portion of the CAR that isn’t present cells in healthy tissues. Severe toxicities could develop if the target is expressed on an important healthy cell, like those of the heart or lungs. Companies like Celyad (Euronext Brussels and Paris, and NASDAQ: CYAD) are taking a different approach to CAR design. Their NKR-T platform aims to combine the rapid response of the natural killer (NK) cell with the immunological power of the T cell (Cyelad, 2017). Instead of inserting a gene that codes for an antibody in the T cell (like in traditional CAR design), the gene encoding the natural killer (NK) cell surface receptor―Natural Killer Group 2D (NKG2D) ―is used. The NKG2D protein is fused to the CD3ζ cytoplasmic domain of the T-cell receptor to form a novel CAR product called CYAD-01. The NKG2D receptor can bind NKG2D ligands, which are minimally expressed on normal cells, but are over-expressed by cancer cells in 80% of hematological and solid malignancies. Additionally, co-stimulatory molecules are also used to increase the potency but instead of being integrated into the CAR construct, NKG2D recruits DAP10 which is naturally expressed by T cells, making the CAR-T NKG2D construct much simpler than traditional CARs. Together, NKG2D-CAR and DAP10 create a new receptor complex which enables T cells to recognize and kill tumor cells (Celyad, 2017).
In March 2017, the U.S. FDA gave Celyad the go ahead for their second clinical trial: THINK (THerapeutic Immunotherapy with NKG2D) is a Phase I study of CYAD-01 in seven refractory cancers, including five solid tumors (colorectal, ovarian, bladder, triple-negative breast and pancreatic cancers) and two hematological tumors (acute myeloid leukemia and multiple myeloma). Recently (October 3, 2017), Cyelad reported the first complete response in an r/r acute myeloid leukemia (AML) patient in the THINK trial.
CAR-T for Solid Tumors
Targeting solid tumors is the biggest challenge in immuno-oncology. Until now, most of the reported clinical trials utilizing CAR T cells to treat solid tumors (i.e. lung, breast, and colon cancers) have had limited effectiveness. Low T-cell infiltration and an immunosuppressive environment prevent the immune system from effectively attacking solid tumors.
Currently, the latest approach in ACT for solid tumors is the fourth generation of CARs (see Figure 1). These T cell redirected for universal cytokine killing or TRUCK T cells are genetically modified to express CARs along with an inducible cytokine gene cassette driven by an NFAT(nuclear factor of activated T cells)-responsive promoter. Specifically, there is interest in the interleukin (IL)-12 cytokine which directly enhances T-cell activity and may help undo the immunosuppressive tumor microenvironment by triggering the apoptosis of inhibitory macrophages (Abate-Daga, 2016). However, since IL-12 cannot be administered systemically due severe toxicities, other approaches are required. With TRUCKs, IL-2 is only secreted when antigen binding activates the CAR T cell. Using this type of transcriptional control, Chmielewski et al. (2011) showed that IL-12 secretion by anti-carcinoembryonic antigen CAR T cells resulted in elimination of carcinoembryonic antigenexpressing tumor cells.
Such TRUCK T cells are envisioned to be applied in fields beyond cancer therapy including the therapy of virus infections, auto-immune diseases or metabolic disorders (Chmielewski, 2015).
Without question, the CAR-T field is flourishing. The approval of Kymriah™ by the FDA is only the beginning of what is to come, with no less than 76 CAR-T therapies under evaluation by the FDA. Studies are under way to look at ways to improve the CAR T-cell designs; to identify additional targets and receptors and to decrease the side effects of CAR T-Cell Therapy. While the current technology is not perfect, it does offers great hope for patients who have exhausted all traditional therapies. This is encouragement enough to continue to push the development of immunotherapies like CAR-T as treatment options for patients in the fight against cancer.