CAR-T safety: Traffic laws needed

by Robert Kruse

The Juno CAR-T cell clinical trial halt brought shockwaves throughout the immuno-oncology world. While the previous toxicities for CD19 CAR-T's were well known, the death of several patients from them during later phase trials that were going to enabling for FDA approval was alarming. Is this therapy really ready for clinical approval and launch if the toxicities are not currently manageable?

The addition of fludarabine to the conditioning regiment was quickly noted by Juno to be the cause of the toxicity issue, since the patient deaths only occurred when fludarabine was added to cyclophosphamide for the conditioning regimen. Perhaps even more surprisingly, the FDA quickly agreed with them, and the trial halt only lasted 48 hours. The scare to the CAR-T sector was over, and the stocks could rise again.

The question for scientists, clinicians, and investors is if the correct culprit was really identified, or if fludarabine was a scapegoat for larger issues for the CD19 CAR-T strategy. Such issues will affect adoption and patient outcomes, even if the FDA approves the therapy. We wanted to delve into the issue more at the blog, in order to consider the opposing sides on this still controversial finding.

Why is lymphodepletion a part of CAR-T regimens?
As a starting point, those unfamiliar to the CAR-T space might ask why lymphodepletion (using chemotherapy to kill the patient's lymphocytes) is even needed. Why can't one just infuse CAR-T cells into the patient, after which they will home to the tumor and destroy it. The reason is that CAR-T cells, so far, have not been noted to remarkably persist under such conditions. Instead, helping them by depleting the host of its own T cells allows the infused CAR-T cells to expand and fill this niche. Growing into larger numbers and feeding on host cytokines like IL-7 facilitates their long-term engraftment. Furthermore, the expanded numbers help facilitate the clearance of the tumor, and the cells don't solely have to depend on target antigen driven expansion, but also utilizing homeostatic expansion mechanisms.

Drugs to lymphodeplete patients are many, having regular use in bone marrow transplants and now being adopted into CAR-T preparation regimens. Previously, cytokine release syndrome was a regular occurrence with CD19 CAR-T trial, and was in fact associated with good outcomes and complete tumor response. The lymphodepletion helped foment the cytokine release syndrome and clinical response. Cytokine release syndrome has been known to cause some neurotoxicity, thought to be in part from the systemic inflammation. Indeed, in clinical trials before the most recent death, the neurotoxicity could resolve with the administration of anti-IL-6 antibodies, suggesting the link between the inflammation and the neurological findings. It has been found that IL-6 could be transported across the blood brain barrier, forming a connection to the observed clinical findings. So before this recent adverse event then, the paradigm had been established for lymphodepletion and then toxicity management with anti-IL-6 antibodies.

Is cyclophosphamide + fludarabine combination truly the issue?
The FDA seems to be satisfied that the added fludarabine was the culprit, with the trial now already resumed. However, it is interesting that other academic investigators and companies have used the "cy/flu" combo without any CNS death attributable to the CAR-T cells. For example, Juno's competitor, Kite Pharma, is currently using it in their trials right now. What is the difference then? While I can only speculate, the differences might have to do with the potency of the CAR-T cells themselves. Differences in signaling domains between CD28 and 41BB can alter the persistence and cytokine release of the CAR-T cells, however, both Kite and Juno's CAR used here use CD28. Novartis employs the 41BB endodomain by contrast. Less reported and focused on are differences between the preparation of the cells, the cytokines they are cultured in, how long they were cultured in vitro before infusion, and donor to donor differences. All of these parameters could influence the peak CAR-T potency post infusion. Other trials using cy/flu might lack toxicity simply because the CAR-T cells are not as potent leading to a lack of profound cytokine release initially.

Is cyclophosphamide & fludarabine integral to the therapy?
Another question is if the function of cy/flu is needed for therapeutic efficacy. As discussed above, conditioning is important for establishing a niche for CAR-T cells to expand in, replacing the normal T cells. However, more than that, the conditioning might be needed to suppress the host immune system against immune responses against the CAR molecule. Most CARs in trials represent early generations that use mouse-derived single chain variable fragments to bind to CD19 antigen. Humans can develop human anti-mouse antibodies (HAMA) that would lead to elimination of CAR-T cells. This would, of course, would also prevent the elimination of tumor cells and the desired therapeutic effect. Previous studies offer clues that fludarabine might be needed for treatment efficacy. I will list the quotes in full from the paper below:

"Although a high rate of relapse was observed among adults treated at FHCRC, this may reflect inadequate lymphodepletion in patients receiving Cy monotherapy, with rejection of the murine component of the scFv; early data suggest Flu may help to overcome this limitation."

"CAR T-cell persistence was short in most of the 12 patients treated with Cy-based lymphodepleting chemotherapy, and similar to FHCRC’s experience in adults with ALL, a cytotoxic T lymphocyte–mediated response to the murine component of the CAR transgene was observed."

Given that some data indicates conditioning with both agents might be required in the current form of CAR-T cells for optimal therapeutic effects, the reduced conditioning could diminish the high level of complete remissions that clinicians, payers, and investors are looking for. This potential concern will be worth monitoring as Juno generates data with the Cy only regimen. The JCI paper reporting Juno trial data reiterates these findings that responses were much better in the cy/flu group, likely due to a lack of anti-CAR immune response, similar to the Blood paper above. In the long run, though, the immune response issue will likely be solved as almost all new generation CARs employ human scFv's. In the short run, this might prove problematic for initial product launches.

Another aspect on this point is whether cy/flu and conditioning is required for CAR-T efficacy, or if there will be future potential to do without it. Notably, the NCI had a study with allogeneic CAR-T cells post transplant where there was still anti-tumor activity with no lymphodepletion. However, since the patient was already bone marrow transplanted, forms of ablation had already occurred before the adjuvant CAR-T cell infusion period making this an uncertain comparison. We do know that early CD19 CAR-T cell trials failed without the lymphodepletion, as mentioned above. 

Other explanations for the adverse events
The other possibility that has received some attention is that the CD19 antigen might exist in the central nervous system in some form, which then leads to an on-target response in the brain that causes intense inflammation and in this case, too much brain swelling and and death.

There are two different possibilities for this. One is that the brain tissue expresses low levels of CD19 protein that have evaded detection on histological stains. Given that CAR-T cells are exquisitely sensitive to very low amounts of antigen, this possibility can not necessarily be ruled out. However, it would be unexpected given that our current understanding is that CD19 is a B cell restricted antigen. Similarly toxicities with blinatumomab might suggest that it is restricted to CD19, but it could still be driven by cytokine release syndrome.

The second is that the CD19 tumor has infiltrated the CNS, so it is an on-target on-tumor effect as intended, but that the location of the vigorous immune response leads to these adverse events. It should be possible to look for tumor by imaging, but this could also be missed on MRI or CT imaging. Perhaps more vigorous screening is needed before CAR-T infusion. On the flip side, early reports have shown that even with CNS leukemia involvement, that it wasn't necessarily predictive of toxicity development. Clearly more research is needed in order to robustly predict what will happen when patients are treated with CD19 CAR-T cells. In that respect, the autopsies of these brave patients will be crucial in educating the entire CAR-T field on the safety of these therapies, and their sacrifice should hopefully afford many more patients in the future to be successfully treated of cancer.

Importance of a safety switch
The other question from this result is the necessity of a safety switch going forward. The first question to ask is if the Juno CD19 CAR-T cells had the inducible caspase-9 safety switch from Bellicum in them, would this neurotoxicity death have been prevented. Importantly, the caspase-9 mechanism triggers an apoptotic event within the T cells that does not produce any further inflammation. This compares to the other suicide methods that leverage therapeutic antibodies to target CAR-T cells for their elimination. These approaches could cause more inflammation, and even if they didn't, it's uncertain that antibodies could fully penetrate CNS and cross the blood brain barrier to eliminate CAR-T cell that have trafficked there.

Neurotoxicity can occur concurrently or after cytokine release syndrome, and is generally less responsive to corticosteroids or anti-IL-6 antibodies as reported here. Development of severe neurotoxicity did correlate with early cytokine levels in patients, but it may be difficult to determine when to activate the safety switch to allow sufficient activity, but prevent the delayed neurotoxicity. Additionally, if CAR-T cells in the CNS are playing a role in the neurotoxicity, the dimerizer drug would also have to reach the cells there. This has not necessarily been tested in any clinical trials that I have seen for Bellicum, which focus on T cells in their periphery. In all, while Bellicum's safety switch may turn out to be the most effective way to reduce CAR-T levels safely, there may be difficulty in determining when and in which patients to titrate their CAR-T levels to prevent neurotoxicity. However, this may be able to be determined effectively with experience in the clinic. It may, in fact, be easier for the safety switch to deal with acute cytokine release syndrome, which seems more immediately responsive to interventions.

Implications for other approaches
Currently, many efforts are being made to make CAR-T cells even more powerful, in order to solve the problem of lack of efficacy in solid tumors. One push has been to use CRISPR/Cas9 gene editing to make the T cells resistant to immune checkpoints and suppressive signaling, which is commonly co-opted by tumors as a defensive mechanism. Indeed, the clinical trials to edit PD-1 in CAR-T cells for therapy will begin shortly. In light of the recent CD19 CAR data, however, the question arises if this is indeed safe, and if the FDA should reconsider. If we are getting unanticipated side effects for a well described antigen target, what about for less validated tumor antigen targets, which may occur in healthy tissue and now suddenly have permanent mutations rendering the hosts ability to combat them limited. Perhaps regulators need to take a closer look at these consequences before approving them. The same will be true for TIL and TCR trials where the antigens are more defined, but the off target toxicity could be unknown. Notably, cytokine release syndrome has been observed in a trial with virus specific T cells against EBV, so this is not just a phenomenon with CAR-T cells, and we must consider the safety for all cell therapy approaches.

Conclusion
As exciting as the triumphs of CD19 CAR-T cells have been, the recent adverse events show that the therapy still has an inherent identity crisis, trying to become a scalable standardized therapeutic moving beyond its days as a boutique therapy, where every patient was almost its own self-contained experiment. CAR-T cells in their current form represent a series of several procedures, therapies, and extended patient management, with only one step of that process being cell infusion. CAR-T cell efficacy is still dependent on these other factors, such as donor influences, cell collection, lymphodepletion, and post-infusion toxicity management. Unless the problems of toxicity can truly be standardized and contained within rigorous protocols, the therapy may never take off among clinicians. However, there will be a strong desire to achieve this, due to the impressive efficacy of these therapies. Of course, more robust safeguards like safety switches might help for managing toxicities. Furthermore, it will be crucial going forward to know if this is just a CD19 antigen issue that is less likely to occur in other CAR-T therapies. However, until these other targeted therapies start having success, we won't know about their respective efficacy/safety profiles. I'm still bullish and hopeful about this field, but events like this should be considered more going forward. Every patient and antigen is truly novel, and the tumor xenograft mouse models often utilized aren't informative for these toxic events. On the hopeful side, though, we've come a long way from the early days of CAR-T therapy where no efficacy was seen, so if the problem now is having efficacy but needing to manage toxicity, progress has been made offering hope for the future that potency and safety will be maximized together.