Juno Halts JCAR015 Trial Again

Juno announced its lead CD19 targeted CAR-T product, JCAR015, in its phase II ROCKET study in adult ALL, was placed on clinical hold (again) after two patients experienced severe neurotoxicity, leading to death. The fate of this program is currently undecided, whether or not it will be modified or shuttered completely. This is after an earlier clinical hold in the same trial after similarly fatal neurotoxicity events. They resolved that hold very quickly by removing fludarabine from the conditioning regimen, which they thought would prevent, or at least reduce, severe neurotoxicity. Unfortunately, this did not appear to be sufficient to prevent this toxicity. Robert wrote his thoughts on the initial hold, and a lot of those concepts are again important after this hold. Juno held a conference call to discuss the hold, which is available here. The following is a quick summary of what they said, and my thoughts on this toxicity and things to consider with CAR-T development going forward.

While Juno still believes that fludarabine was a contributing factor to neurotoxicity, they stated that they always considered it to have multifactorial causes, but hoped removal of fludarabine was a significant contributing factor. They feel that ALL (compared to NHL and CLL) is particularly prone to CAR-T toxicity, both CRS and neurotoxicity, due to the high levels of accessible antigen, which allows for rapid expansion of CAR-T cells, and may lead to toxicity. Other factors are potentially the costimulatory domain used (CD28 vs 4-1BB), dose of cells, and the conditioning regimen, which all can impact the expansion of CAR-T cells. The rapid cell expansion in patients and an early high fever (2-3 days after CAR-T administration) appear to be fairly closely correlated with later development of neurotoxicity, which typically appears approximately 1 week after CAR-T administration and progresses rapidly. Going forward, Juno highlighted the lower levels of toxicity in both ALL and other B-cell malignancies using their 4-1BB CAR-T products with defined CD4:CD8 composition (JCAR017, and non-commercial product JCAR014). The CD28 costimulatory domain used in JCAR015 may lead to more rapid expansion of T cells, and thus potentially more toxicity, although I would not say this has been definitively proven yet.

In terms of how to deal with this toxicity, Juno mentioned that both patients were given an intervention, as per protocol, either with anti-IL-6 antibodies and/or corticosteroids, at the time of their high fevers. Dealing with the subsequent neurotoxicity they speculated would be difficult because it progresses so rapidly that makes it tough to use an intervention, such as a safety switch, to intervene in time while maintaining sufficient T cell activity. They also said this would make it difficult to treat prophylactically with steroids or anti-IL-6, due to concerns it would dampen CAR-T engraftment and activity across all patients.

The question arose whether this toxicity may be due to either on-target off-tumor activity against low levels of CD19 somewhere in the CNS, or on-target on-tumor toxicity from tumor burden in the CNS. Juno did not believe it was due to targeting CD19, either tumor burden in the CNS or off-tumor activity, as they have successfully been able to treat a patient with active CNS lymphoma with JCAR017 without neurotoxicity, although importantly, this is a different CAR product. However, there is some evidence that other therapies that activate T cells against CD19 also cause neurotoxicity. In particular, the CD19-CD3 BiTE, blinatumomab, which redirects T cells to CD19+ cells, has also shown signs of neurotoxicity. In particular, the CNS toxicity has been partially attributed to activating T cells against CD19 positive cells in the CNS causing cytokine release and disruption of the blood-brain-barrier, although I have not seen the primary data indicating this is the definitive mechanism.

From Ribera et al., 2015

Some evidence that neurotoxicity is not due to CAR activity against CNS disease comes from studies of CARs against other targets that can also induce neurotoxicity as an adverse event. In particular, two early studies of BCMA-targeted CARs in multiple myeloma have found that both a CD28 CAR as well as a 4-1BB CAR could cause neurotoxicity after large expansions of CAR-T cells. Strikingly, the 4-1BB study did not use any pre-conditioning chemotherapy, demonstrating that at least for this CAR, you can have significant CAR-T cell expansion and toxicity in the absence of conditioning. Surprisingly, a new 4-1BB anti-BCMA CAR from Bluebird Bio has recently shown responses without grade 3 or higher CRS or neurotoxicity in early initial clinical data, which I write about more in a post here. While very promising, it is too early to say if this construct will have a better safety profile than others, and what are the reasons for lower toxicity. Multiple myeloma only very rarely enters the CNS, so it is unlikely that the neurotoxicity is due to BCMA+ myeloma cells in the CNS. However, soluble BCMA can exist in the CSF. Soluble antigen, in general, is not thought to significantly interfere with, or activate, CAR-T signaling, such as in a mesothelin CAR:

From Lanitis et al., 2012

This has been found to be true for other CAR-T targets as well that can exist as soluble antigen, with the thinking being that a multimer of the antigen is required for stimulation of the CAR.

My thoughts on future CAR-T development:
Other groups (such as Kite/NCI) using CD28 CARs appear to have not had as significant troubles with neurotoxicity, even though they also use cyclophosphamide and fludarabine for conditioning. As Robert had previously discussed, an additional factor to consider, although we have no sense of whether it is playing a role, is the manufacturing process of the CAR-T cells leading to additional differences between the different products.

Expansion of CAR-T cells appears to be a double edged sword: while Juno's trials may suggest that rapid expansion of CAR-T cells can potentially lead to dangerous toxicity, a lack of robust expansion if not enough antigen is available immediately is correlated with worse activity. This has been shown preclinically with a mesothelin CAR where local administration of CAR-T cells near antigen-positive cells improved expansion and activity. Additionally, a recent clinical study by Kochenderfer using CD19 CAR-T cells from donor post-allogeneic transplant showed that low B cells (containing target antigen) correlated with poor expansion and activity of the CAR-T cells. Importantly, in another CD19 CAR trial in ALL by Juno and its collaborators, the addition of fludarabine to the conditioning regimen allowed significantly better expansion of CAR-T cells and durability of responses.

From Turtle et al., 2016

Going forward, synthetic biology tools or CAR constructs that would allow for a consistent, but less rapid, increase in CAR-T expansion may be useful to get sufficient activity and persistence but reduce excessive toxicity.

Another area that may be worth exploring is to try to identify ways to better separate the activity of CAR-T cells from the causes of cytokine release syndrome and neurotoxicity. For instance, IL-6 may be dispensable for CAR-T activity, but contributing to CRS, so anti-IL-6 antibodies might be able to be used more aggressively. However, the spike in IL-6 during CAR-T expansion may not be coming from the CAR-T cells, so knockout of IL-6 in CAR-T cells may not make for a safer CAR-T product. Continuing to look for ways to separate activity from toxicity in CAR-T cells to either engineer safer T cell therapies or allow better prophylactic toxicity management will probably be important in the continued development of these therapies.

In summary, I think it is still not extremely clear why Juno has, particularly recently, been running into such severe neurotoxicity in their JCAR015 trial. I thought removing fludarabine would lead to poorer activity and durability of responses, but would, at least substantially, reduce the levels of neurotoxicity, as fludarabine appeared to allow for significantly increased CAR-T expansion. However, there are clearly other factors that are important for modulating rapid CAR-T expansion, and whether or not rapid CAR-T expansion is even the primary factor leading to neurotoxicity is still unknown. The potentially better safety profile of the JCAR017 program as well as CD19 programs developed by others suggests that neurotoxicity may be particularly problematic for JCAR015, but I would say it's too early to draw strong conclusions. Even these other programs have severe CRS and neurotoxicity, so there is still a lot of work to be done in continuing to improve the safety of CAR-T therapies while maintaining their efficacy.

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.

Evolving Therapeutics Part I: Immunotherapy

I agree with fellow science guy, Bill Nye, when he said "Evolution is the fundamental idea in all of life science - in all of biology". One can view anything in biology through the lens of evolution. Thinking about how biology is shaped by the forces of evolution is a lot like thinking about how human behavior in economics can be shaped by the forces of incentives.

With that in mind, I wanted to take a look at a number of topics, relevant to therapeutics and disease, wearing evolution-colored glasses. I think this approach gives an interesting perspective on both the challenges evolutionary forces create in treating certain diseases, like cancer, as well as the ways in which evolution can be harnessed for our own benefit through things like selection screens or directed evolution.

Considering evolutionary forces can be critical for determining the best therapeutic approaches to take in a given disease, particularly cancer, which is essentially a disease of evolution and selection. So for my first post in this series, I would like to focus on the dynamic relationship between the immune system and cancer, two systems with great diversity and proliferative capacity - the fuel for an evolutionary arms race. In particular, I will be highlighting three recent papers that add to the growing amount of research using human immunotherapy clinical trial data to further our understanding of this dynamic battle between cancer and the immune system.


A Tumor-Immune System Arms Race - T Cells Try to Keep Up with Mutating Tumor Targets

One of the most appealing aspects of using the immune system to target cancer is both its ability to evolve along with the tumor, as well as its ability to potentially recognize multiple targets simultaneously in a tumor. These factors may be key for why some patients have been able to achieve very durable responses to immunotherapies.

In particular, a recent paper by Verdegaal et al. has formally demonstrated this evolving arms race between cancer and the immune system, and shows the ability of the immune system to adapt in response to tumor evolution (and vice versa).

The authors studied two melanoma patients treated with an adoptive T cell therapy. The patients had tumors removed and used to generate T cells reactive to that tumor, and also had those tumors sequenced to identify the potential mutations the T cells were reacting against. The T cells were not generated from TILs, as has been done by Steve Rosenberg's group, instead peripheral blood cells from the patient were stimulated repeatedly for reactivity against their own tumor-derived cell lines. The paper follows the clinical course of two patients, BO, who has an ongoing complete response after receiving the adoptive cell therapy, and patient AB, who unfortunately died of the disease.

For brevity, I will just go through the example of patient AB, as this case shows both the tumor evolving to evade the T cell response, as well as the T cell response evolving to react to additional neoantigens (novel peptides presented to the immune system due to tumor-specific mutations) over time.

Patient AB had a metastasis resected in 2004 (MEL04.01) that was used to produce the T cell product, which reacted against 3 neoantigens present in that tumor. The patient was treated with the T cell product, but progressed, and had further resections of metastases in 2008 and 2012. In the relapsed metastases, it was found that for one of the targeted neoantigens there was a loss of the mutation, essentially removing the target those T cells were reacting against. Another mutated gene producing one of the neoantigens was significantly reduced in its expression in the relapse, also potentially reducing the ability of T cells to react against that target.

While the immune system appeared to have applied a selective pressure that led to the loss of specific targets, surprisingly, the authors found that T cells found in the tumors of the later metastases were now reacting against additional neoantigen targets. Specifically, they found that T cells were reacting against a neoantigen that, while the mutation was present in the initial tumor in 2004, was expressed at much higher levels upon relapse. This correlated with the presence of T cells reactive against that mutation in that metastasis, whereas no T cells reacting against this target were found in earlier tumors. This shows that the immune system is also dynamic, and has the ability to change its reactivity toward a tumor depending on the presence of targets it can recognize. While this may not be surprising, this is the first time that immunoediting, the loss of specific antigens in response to pressure from the immune system, has been demonstrated in human tumors, and further establishes the neoantigen-reactive T cell as a mediator of anti-tumor activity.

Considering that the vast majority of mutations found in a tumor are probably not contributing significantly to its growth, and are so-called passenger mutations, it is not surprising that the tumor selects for loss of these mutations. The ability of TIL therapy to generate potential cures in a percentage of melanoma patients is most likely then due to the simultaneous targeting of multiple neoantigens by diverse T cell clones. In fact, the Rosenberg lab recently charaterized a TIL product, and found the T cells were able to react against 10 distinct neoantigens.

In all, this work shows that the relationship between the tumor and the immune system is not static, with both the tumor and the immune system able to change in response to each other. This has implications for targeting cancers with immunotherapies. Specifically, that you will need to generate a response against a multitude of neoantigens to get the potential durable responses desired, whether that is through the use of broad immunomodulation with checkpoint inhibitors, or designing adoptive cell or vaccine products with reactivities against neoantigens.


Are All Neoantigens Created Equal? - Clonality Matters

Next, a recent paper from Charles Swanton's group asked the question if all of these potential neoantigen targets created equal. This paper was also discussed nicely on this blog specifically focused on the concept of cancer and evolution. There is significant evidence that the neoantigen-reactive T cell is a/the critical mediator of checkpoint antibody efficacy, and the number of predicted neoantigens correlates with efficacy. However, the authors of this paper ask the question whether its just the total number of neoantigens that matters, or if also the clonality of those neoantigens matter. This is something I have wondered about as well. Specifically, does it matter if the neoantigens are present in almost every cell in the tumor (clonal), or only present in a subfraction of the tumor cells (subclonal). This is illustrated in a theoretical tumor below: shown is a clonal "trunk" mutation A, and subclonal "branch" mutations B-F.

From Translational Implications of Tumor Heterogeneity

The authors measured the clonality of the total of a tumor's predicted neoantigens and looked to see if that influenced survival and response to checkpoint blockade. Starting in lung cancer, they found that patients with lung adenocarcinomas with high neoantigen load had longer overall survival, not taking into account treatment, and that this stratification improved further when taking into account the clonality of the the neoantigens. Specifically, the subset of patients with high neoantigen load and low neoantigen heterogeneity had the best survival. Interestingly, they found that lung squamous cell carcinomas did not have this same association, and the authors present some evidence that these tumors might have reduced processing/presentation of antigens compared to adenocarcinomas.

The authors were able to identify neoantigen-reactive T cells in patients with tumors with both high and low intratumoral heterogeneity, but they could only find neoantigen-reactive T cells to clonal neoantigens and not subclonal neoantigens. This suggests that subclonal neoantigens may not induce a robust T cell response. Unsurprisingly, neoantigen-reactive T cells tended to express more inhibitory markers, such as PD-1 and LAG-3, which has also been seen by the Rosenberg group.

Next, they went on to see if the intratumoral heterogeneity of neoantigens, in addition to the total number of neoantigens, could effect the response to checkpoint blockade. Using sequencing data from recent PD-1 studies in melanoma, they stratified patients who had a durable clinical response to PD-1 blockade versus those who did not. They found that for a given threshold of high neoantigen burden and low intratumoral heterogeneity, 12 out of 13 patients had durable clinical benefit, compared to only 2 of 18 patients who had either high intratumoral heterogeneity or low neoantigen burden (shown below).

Further, they found that applying a threshold for the amount of intratumoral heterogeneity of neoantigens helped to better predict the outcomes of patients treated with PD-1 blockade in NSCLC (Rizvi cohort) or CTLA-4 blockade in Melanoma (Snyder cohort) than neoantigen burden alone.

These data raise the question - does inducing non-clonal neoantigens actually weaken the response a patient has to checkpoint blockade? This could have implications for treating patients who are heavily pretreated with radiation/chemotherapy which may have induced non-clonal neoantigens. The authors specifically looked at data from heavily pretreated melanoma patients, and found that treatment led to high levels of non-clonal neoantigens. In this population, the predictive power of neoantigens alone to predict response is lost, but if you combine this with intratumoral heterogeneity of neoantigens, this now almost able to reach statistical significance to predict response. The authors stated there were too few patients to sufficiently power this analysis, so this is still speculation.

A potential drawback of this study is that it is correlational, and low intratumoral heterogeneity tends to correlate with things already known to be positive predictors of response to therapy, such as higher PD-L1 expression. Also, surprisingly to me, high neoantigen burden, another positive predictor, correlates with those neoantigens being more clonal: 

I would have expected a higher neoantigen burden would be indicative of greater intratumoral heterogeneity, not less. This also makes me curious what this analysis would look like for for mismatch-repair deficient cancers, since their tumors might have higher ongoing genomic instability, instead of just accumulated mutations caused by previous carcinogens, like UV or smoking. Tumors with this deficiency have been found to be sensitive to checkpoint blockade, presumably due to their high number of mutations increasing the "tumor foreignness" for the immune system to recognize. Specifically, it would be interesting to see if mismatch-repair deficient tumors have high intratumoral heterogeneity, and if patients can have positive outcomes with checkpoint blockade despite their high levels of heterogeneity.

While mouse models have drawbacks, especially in the study of tumor immunology, it will be interesting to see if any groups try to experimentally test whether increasing or decreasing the heterogeneity of tumor antigens effects the immune response to them.

In addition, this work suggests that the strategy of mutagenizing a tumor to increase the number of neoantigens may not lead to a more productive immune response, and could potentially hamper it. The etiology of these mutations would be different from the mutations initially present in a tumor, which accumulated under the stringent selective pressures of initial tumor development. A way to recreate this accumulation of mutations but with higher clonality might be to first mutagenize the tumor (with chemo/radiation or disruption of DNA repair) and then try to create a new evolutionary bottleneck with a targeted therapy with a high response rate that could apply a selective pressure to increase the clonality of the tumor. You might need a really effective second therapy to reduce the heterogeneity sufficiently, though. It would be interesting to see how different therapies effect the clonality of a tumor, and Swanton's group is performing a longitudinal study, called TRACERx, looking at how clonality changes during the course of treatment, which may shed light on therapies that increase and decrease tumor heterogeneity.

While like other studies retrospectively looking for biomarkers of response, this study is correlational and further validation is needed, intratumoral heterogeneity is now on the map, and should be considered as a potentially predictive biomarker of response to checkpoint blockade, which are nicely summarized in this review. Better biomarkers may allow doctors to better stratify patients, and determine which are most likely to benefit from these therapies. This study also emphasizes the importance of targeting the clonal "trunk" neoantigens for cell or vaccine based therapies directly targeting neoantigens. It also suggests the potential importance of developing strategies to reduce intratumoral heterogeneity or targeting the causes of heterogeneity.


Tumors Go Silent - Loss of Antigen Presentation and Interferon Responsiveness

While the ability of T cell populations to recognize multiple neoantigens in a tumor may reduce the risk of relapse due to loss of single neoantigens, there is the potential for escape mutations that globally make the tumor less sensitive to T cell recognition or attack. These mutations could potentially be the most pernicious, as they could essentially make the tumor unresponsive to any therapy dependent on T cell recognition of the tumor. Unfortunately, evidence of these types of mutations has been observed in a recent paper looking for the causes of relapse after an initial response to checkpoint blockade.

The authors examined the cases of 4 patients with melanoma who had this clinical course of a relapse after a relatively prolonged period of response to therapy. By sequencing sequential biopsies of tumors pre-therapy and post-relapse, the authors were able to look for new mutations present at relapse that could explain their loss of responsiveness. The vast majority of mutations were conserved between the tumors at both time points, but they found homozygous loss-of-function mutations in JAK1 in one patient and JAK2 in a second patient.

Looking at the biopsies of from these tumors at relapse, they found that both of these patients lost PD-L1 expression at the interface of the tumor and stromal cells, despite the continued presence of CD8+ T cells at this interface, and presence of PD-L1 expression in adjacent stromal cells (shown below).

The authors wondered if these JAK1/2 mutations could be causing a loss of responsiveness in the tumor to interferon-gamma, which normally induces PD-L1 expression. They found that T cells could still recognize tumor cells, however, tumor cells from both patients lost responsiveness to the growth-inhibitory effects of IFN-gamma, and to all interferons for the JAK1 mutation (below). They subsequently showed these mutations were sufficient to cause this loss of responsiveness.

They went on to look for relapse-specific mutations in a 3rd patient, and found B2M mutations, which have previously been shown to cause loss of MHC class I surface expression, and inability of tumor cells to be recognized by T cells. Immunohistochemistry from this patient showed this loss of surface localization for MHC class I.

IHC stain for melanoma marker (left) and MHC class I expression (right)

Interestingly, B2M mutations have been observed in about 30% of colorectal cancers with microsatellite instability (mismatch-repair deficient). Surprisingly, these mutations were not a negative prognostic marker, and if anything, trended toward being a positive prognostic biomarker. This raises the question if there are negative consequences for the tumor for mutating B2M. Another question this raises is in mismatch-repair deficient CRC, which as a class are quite responsive to checkpoint blockade, if patients that already have baseline B2M mutations are more resistant to checkpoint blockade. This could be used as a predictive biomarker if found to be the case.

The results from this paper also may have implications for combining PD-1 + JAK inhibitors such as in this trial. The results in this paper would suggest sustained inhibition of JAK signaling could reduce tumor responsiveness to T cells.

In summary, this paper demonstrates how the tumor can develop adaptive resistance to checkpoint blockade by either reducing the ability of T cells to react to the tumor or the tumor to react to T cells. Considering the ability of cancer to evolve resistance to basically any therapy we throw at it, it is almost surprising that these mutations are not essentially inevitable, and that it is possible to get these extremely durable responses with current therapies.This paper, however, highlights the need for future strategies to deal with these types of mutations. For B2M mutations perhaps by engaging other elements of the immune system, such as NK cells, that might be able to recognize this "missing self", and react to the loss of MHC surface expression. And for JAK1/2 mutations, perhaps using strategies that are not reliant on interferon for efficacy. Additionally, it is possible these B2M or JAK1/2 mutations produce compensatory vulnerabilities that can be exploited therapeutically. It will be worthwhile to further study the mechanisms of adaptive resistance to checkpoint blockade to develop the best strategies for dealing with it.


Conclusions - Designing the Next Generation of Immunotherapies

These three papers highlight the ability for both the immune system to adapt to the evolving tumor, as well as the ability for tumor evolution to produce problems for the immune system. It will be critical to understand, for each individual patient, what is the status of this tumor-immune system interaction, and how the tumor is avoiding immune surveillance. While we may not currently have therapeutics to intervene in patients for all the different mechanisms of immune evasion, further understanding the mechanisms critical for tumor survival will help identify new targets and rationally combine therapeutics.

This thinking is summarized nicely in a review called "The Cancer Immunogram", which attempts to think about treating cancer in terms of defining the different aspects of tumor-immune interactions critical for an immune response in a patient's tumor. The authors describe seven categories critical for effective recognition of cancer by the immune system, they are (shown below): tumor foreignness, general immune status, immune cell infiltration, absence of checkpoints, absence of soluble inhibitors, absence of inhibitory tumor metabolism, and tumor sensitivity to immune effectors.

In summary, here are some of the implications of the above three papers for the design and implementation of immunotherapeutic approaches in the future:

- It will probably be necessary to target multiple mutations simultaneously to reduce the likelihood of immunoediting of antigens and tumor escape.

- It is important to understand the intratumoral heterogeneity of a potential target neoantigen, and, if possible, target clonal mutations.

- Intratumoral heterogeneity may become a useful predictive biomarker for response to checkpoint therapies, and become part of how we define tumor foreignness in the cancer immunogram.

- Intratumoral heterogeneity is a problem, and it will be important to develop strategies that either reduce heterogeneity or target the mechanisms that lead to heterogeneity directly.

- Mutagenizing a tumor, by itself, may not generate productive tumor-foreignness and immune recognition. Perhaps if we can provide a new evolutionary bottleneck for the tumor, we can increase the clonality of these induced mutations.

- Unfortunately, there are mutations that make tumors less sensitive to T cell attack generally, and it will be important to develop strategies that target tumors that evolve this response.

Xencor & Novartis Announce Bispecific Partnership

by Robert Kruse


Xencor and Novartis reached a huge partnership deal Tuesday, sending the stock of Xencor skyrocketing. We wanted to share our analysis on Biotechr on the deal and what it might mean for both Xencor ($XNCR), Novartis ($NVS), and their competitors. I will focus on the CD20 antigen target as a paradigm for contrasting the differences between current drugs and how Xencor could differentiate themselves.

CD20 is an antigen expressed on B cells that has gained much success in being targeted by antibody-based drugs, such as rituximab, to treat B cell malignancies. However, as rituximab took off, some limitations in its efficacy were seen. Importantly, it was determined that antibody-dependent cell cytotoxicity (ADCC) drove much of the mechanism of action of rituximab. ADCC engages the Fc domain on rituximab to an Fc receptor on an effector cell such as natural killer cells or macrophages. While potent, ADCC is somewhat limited in absolute toxicity. In order to boost efficacy, scientists found that engaging T cells through the CD3 receptor could cause potent T cell activation and cytotoxicity against a targeted cell antigen.

This same strategy of engaging T cells in a bispecific format has, of course, been utilized by many different companies to date. Prominently, blinatumomab is an approved bispecific antibody that connects CD19 and CD3 binding components. Owned by Amgen, the format links two different single chain variable fragments in tandem, such that that they will correctly pair heavy and light chains of the respective scFv's. The small protein forms a short synapse that afforded potent activation of T cells, compared to previous whole antibody versions. However, without the Fc domain, the half-life of the protein is very short, requiring constant infusion in order to obtain the therapeutic window necessary for efficacy and cross-linking of tumor and T cells.

Xencor Bispecific Ab (image from their site)

This leads us back to the Xencor program. On a simple level, Xencor uses an Fc domain in its construct, thereby affording greater half-life and thus reduced dosing. Their design resembles the "quadroma" format listed below, with differences in antigen binding domains. It is well known that Fc receptor binding must be eliminated, however, in order to avoid systemic toxicity from T cell activation and cytokine release syndrome, so it can be assumed this is built into the Xencor approach. Xencor also has mutations in its platform technologies to increase FcRn binding in order to afford even greater half-life. It's unknown, however, how potent Xencor's molecules are in activating T cells head to head versus a smaller BiTE or DART format (shown in the figure below), which were previously published to be more effective in cross-linking T cells and lack toxicity. Will the benefit of longer half-life outweigh potency reduction? The jury is out, and it's possible Xencor has something up its sleeve to solve this previously, including possibly using an scFv against a more effective CD3 epitope.

From http://www.mdpi.com/2073-4468/1/2/172/htm

The current portfolio against B cell tumors is wide, so forecasting where this fits into the market is uncertain. The traditional antibody drugs are only getting better, whether it is new targets or enhanced Fc domains or antibody-drug conjugates. It will be costly to onboard yet another factory to make a unique biologic, and it has been speculated that most of the cash will go toward production costs. Certainly, this strategy represents a more robust off-the-shelf product compared to the CAR-T platform Novartis has heavily invested in. Comparing the two, CAR-T cells can detect antigens at lower thresholds versus bispecific antibodies, have the benefit of proliferating in the patient, and the potential to be a long-lasting single dose therapy. Head to head, a CAR-T cell is more potent than a cross-linked antibody based molecule. However, scaling and ease of use is much more in the favor of the bispecific antibody format here, and safety will be advantageous as well. Rather than using CRISPR to enhance cell therapy as has garnered recent headlines, simple combination with checkpoint inhibitors should be sufficient for increased efficacy. I like the terminology "backup plan" used by Jacob Plieth to describe the deal, although I would suspect large pharmaceutical players view it as owning all technology platforms and then letting the winners develop from there, rather than deciding technology first. In time, one could imagine rituximab generic being used as first line therapy, followed by a bispecific or ADC targeting CD20, and followed lastly by a third line CAR-T therapy if those patients fail. In this scenario, a pay for performance scheme would have to reign, as it is hard to imagine payers paying hundreds of thousands of dollars for three separate lines of therapy, but rather a single sum for the entire management of the disease.

For investors trading in other antibody companies with bispecific platforms, the Xencor deal is enticing to see if these companies too might be targets for new partnerships and/or acquisitions. Most of the intellectual property in bispecific antibodies revolves around mutated sequences and production/isolation techniques, meaning the general strategy and shape of the proteins is largely the same and perhaps equivalent among the formats. A single success for the class could raise the value of others, although the challenges of commercializing me-too candidates in immuno-oncology is increasingly scrutinized, as covered by Bruce Booth in a recent post in Forbes. I think what might be more important going forward are companies distinguishing themselves by what antigens they are targeting, since the CD20 and CD123 antigens in the Xencor-Novartis deal are already heavily covered by all biotech companies using different modalities, meaning the fight is just over marginal differences in potency. In light of that, it makes sense why we still see antibody discovery companies make huge partnership deals today even after the technology has long matured, as the race for new and better candidates continues.

Gene Editing for HIV Therapies

by Robert Kruse

Human Immunodeficiency Virus (HIV) has been a truly intractable disease for researchers to eradicate since it's discovery over 35 years ago. HIV is a lentivirus that integrates into host T cells, along with monocytes and possibly other cell types causing lifelong infection. The advent of HAART (or highly active antiretroviral therapy) was able to suppress the production of newly formed virus preventing new cells from being infected. The drugs target multiple different processes important for the viral life cycle, including reverse transcription, protease activity, and more recently, integrase activity. This causes a plummet in viral loads to undetectable levels in the serum, representing a functional cure for the patient. Unfortunately, with the withdrawal of anti-retroviral drugs, the viral levels quickly rebound, being derived from stably integrated virus in T cells. Initially, there was hope that T cells with integrated virus could be waited out, such that they would all die out after a couple years, and then treatment could be stopped. Those hopes were dashed with the realization that HIV integrated into long lived memory T cells and possibly other cell types and represents a long term reservoir with a half-life of several years. Thus, for the foreseeable future, antiretroviral drugs must be maintained.

This best describes the hurdles and challenges of the current state of HIV medications. Into this frontier, many people are now hyping up using gene editing as a tool to treat HIV. The gene editing strategies can basically be broken down into two camps. The first targets host genes, in order to facilitate an advantage against HIV. The second targets the virus itself, in order to purge it from host cells. Both have received attention from academia and biotech alike.

1)  Gene Editing of Host Genome in order to Protect against HIV

The furthest along gene editing strategy against HIV targets removal of host CCR5 expression in T cells in order to protect those cells from HIV entry. HIV enters cells by first attaching to CD4 through gp120, and subsequently binding to CCR5 in order to induce a conformational change resulting in membrane fusion. In the human population, individuals with a deletion in CCR5 have been found to be resistant from HIV infection, through public health studies. This observation was then turned into a therapy with the treatment of the "Berlin patient" with a bone marrow transplant from a donor with CCR5 del32 genotype. The patient had concomitant leukemia, and necessitated undergoing a bone marrow transplant anyways. His physician hypothesized that infusing a new immune system with the CCR5 deletion would prevent residual HIV from infecting the new cells leading toward eradication. Remarkably, the strategy worked and the patient has been hailed as a paradigm toward the cure for HIV.

The excitement over the result is potentially dampened by a few questions. It's uncertain how much contribution the chemotherapy the patient was administered to treat his cancer and in turn wipe out all of his immune cells, actually helped eliminate his HIV via eliminating the reservoir cells, versus the cure happening because the new immune cells were resistant to infection; GVHD could have also helped eliminate HIV infected cells. Given that the patient took HAART throughout this clinical course, before withdrawing medication, it makes it difficult to determine. This fact matters a lot when contemplating how to translate this strategy into the clinic. Bone marrow transplants with fully ablative regimens are too dangerous to use on an otherwise healthy individuals. Instead the clinical application requires modifying ex vivo a small number of hematopoietic stem cells or T cells. These cells will initially represent a small proportion of the patient's immune system, and then could grow through a selective advantage of not being eliminated by HIV, in theory preventing loss of CD4 T cell levels. Another possibility is that the few protected, uninfected cells could restore enough of the patient's immune system to reverse course and clear HIV permanently. Both of these hypotheses remain to be born out in human patients, however. One concern is that the majority of T cell loss occurs without those T cells ever becoming infected, so how could a small minority exert any effect on this process? If any company will find out, it will be Sangamo Biosciences ($SGMO), with its zinc finger nuclease technology already has this strategy of gene editing knockout targeting CCR5 in Phase II clinical trials, and with several other trials planned or ongoing that consist of optimized versions of the approach. The Phase I trial results showed that the cells could be safely administered with a modest survival advantage.

2) Gene editing to Excise HIV from the Host genome

As discussed above, the main hurdle toward eradicating HIV is that it integrates into long-lived immune cells, such as memory helper T cells, which don't actively express all HIV genes, with the HIV genome remaining relatively latent inside the cell. HIV gene expression is tied to the activation state of the T cell, affording the possibility of latency periods in T cell lineages. There has also been some speculation of HIV integrations causing increased T cell divisions in an oncogenic effect to increase persistence.

An elegant solution to all these problems would be to simply cut the HIV genome out of the host cell, whereafter it forms episomal DNA that is lost with cell division. With the rise of CRISPR, one solution has been to use Cas9 to cut HIV out of the genome, as demonstrated here and here. A similar strategy has used TALENs to edit HIV from the genome. Just a couple weeks ago, a paper was published online showed in vivo administration of an AAV expressing Cas9 and gRNA could remove HIV sequences in a transgenic HIV mouse model where the target was in every chromosome, in a fraction of the lymphocytes and cells of other organs. However, the strategy has a number of limitations. A simple one is that the HIV sequence is heterogeneous, so a small fraction of circulating virus will not be cut. Alternatively, Cas9 cleavage itself is mutagenic, meaning that it can induce mutations that cause the resultant HIV genome to be resistant to further Cas9 cleavage. In essence, the therapy is aiding and abetting the escape of the criminal.

A strategy that accomplishes genome editing in a unique way that might be less resistant to mutational changes is using evolved recombinases that can recognize the HIV long terminal repeats (LTRs). The ends of the HIV genome are called LTRs, formed during the process of reverse transcription and harboring the promoter and polyadenylation elements respectively. They also harbor palindromic sequences, which are similar to the LoxP sites targeted by Cre recombinase. Researchers have been able to adapt Cre recombinase via evolutionary selection in bacteria to instead target HIV LTRs. The end result is efficient excision of the entire genome into an episome, very similar to what many science researchers use today with Cre excision of different exons or stop cassettes in transgenic mouse models.

The real question with both of these HIV targeted strategies are whether they are even practical and worth pursuing. HIV infected cells are a needle in a haystack. During active infection, levels might only be 1% of CD4 T cells, and the percentage is much lower for the targeted latently infected T cell types that are the real goal for this type of approach. These cells look functionally similar to non-infected cells, so no specific targeting approach is likely possible. One would need to introduce CRISPR into billions of your T cells, hoping some of them harbor HIV. Even withdrawing blood in an apheresis setting, the total would only be a fraction of the T cells in your body. A vector that could target memory T cell markers and cell types would also be useful, but again, if you only hit a portion, and the virus could grow back anyways that you didn't hit, what was the point exactly. Similar challenges present themselves for HSV and HBV applications, but those viruses at least operate under different replication kinetics than HIV. Either way, the HIV focused strategy has significant delivery challenges that might preclude it from ever seeing the clinic.

Conclusions

Sangamo's trials targeting CCR5 offer great insight into the immunology of HIV. I would hesitate that it's still too early to gauge success. HIV leads to disrupted lymph node architectures and dysregulation of the entire immune system, meaning the gene edited cells, even if protected, might not stand much of a chance of long term survival in a hostile environment. That said, it does offer some hope for the community. CRISPR strategies targeting CCR5 have already been published and might soon move into the clinic as well. On the other hand, the direct HIV targeting strategy has significant delivery challenges that might preclude it from ever seeing the clinic. The best hope would be a procedure to lower the latent provirus enough in patients where the remaining infected cells limited half-life could lead to a sterilizing cure under HAART without off-treatment rebound. Either way, by expanding our tool kit with genome editing, the ability for HIV to hide is continuing to diminish, and new treatments may be heading toward patients.

Treating TTR Amyloidosis: Introducing CRISPR

by Robert Kruse

Among the many different programs announced by Intellia Therapeutics ($NTLA) during their disclosures to announce their IPO, one particularly caught our eye at Biotechr. It is a favorite of the biotech world, being targeted by many different companies looking for orphan disease rapid approval and high reimbursement rates with little to no competition. Will the introduction of a CRISPR-Cas9 player lead to the demise of these competitors? Let us delve into the disease and the technologies to find out.

The rare disease of interest is Transthyretin-related (TTR) hereditary amyloidosis, also called Familial amyloid polyneuropathy, which is an autosomal dominant disease characterized by a mutation in the TTR gene (most common being V30M). This mutation leads to the formation of protein aggregates (hence amyloidosis), which are deposited in various organs across the body leading to various toxicities (pain, paresthesia, muscular weakness and autonomic dysfunction) and ultimately death from cardiac and kidney toxicities.

The TTR protein is mostly produced in the liver, and the current standard of care is a liver transplant, which can cure the disease by removing the source of the protein being produced. The downside is the morbidity risk from the liver transplant procedure, the limited availability of livers for transplants, and the need for immunosuppression. Notably, is it the complete transplant of the liver that is necessary, since any remaining sub-fraction of mutated cells can continue producing the TTR proteins causing more aggregates to be formed. That is why it's an autosomal dominant disease after all, since one gene and one mutation causes the effect over the presence of the normal gene. One can imagine that only replacing a portion of the liver will lead to a slower disease course, the extent to which biomedical science simply does not know right now.

This last fact is particularly important when considering the current therapeutic approaches in clinical trials right now. Several RNA knockdown companies have candidates in clinical trials to treat TTR amyloidosis. The players include Alnylam ($ALNY) and Ionis Pharma ($IONS). Alnylam is using the LNP delivery technology developed by Arbutus (formerly Tekmira) ($ABUS), with some royalties from the success of the drug flowing the Arbutus. Alnylam has reported efficacy in clinical trials of knocking down TTR up to 86.8% in patients, with the caveat of a significant number of patients developing infusion-related reactions. Ionis Pharma is exploring a similar strategy, but by using antisense oligonucleotides, that bind to mRNA and cause degradation by RNase H mechanisms (DNA-RNA binding in cytoplasm). The product can be delivered subcutaneously as a benefit, and optimized delivery can occur through conjugation to a GalNAc sugar to the oligonucleotide. Ionis is currently in Phase III trials with an unconjugated ASO, with the trial projecting to be concluded in 2017. Previous studies showed a maximum knockdown of 92% in trials. FDA approval seems likely, but there is the unknown of the long term benefit of the current knockdown levels. Alnylam is also exploring the use of GalNAc conjugates to siRNA and subcutaneous delivery to improve liver uptake for its TTR cardiomyopathy indication.

Into this picture enters Intellia and CRISPR technology. CRISPR allows the permanent cleaving of DNA to inactivate the gene, stopping protein production. In this manner, Intellia could pursue an approach where after a single injection, permanent knockdown of TTR mutants could be achieved, versus the lifelong dosing regimens of the RNA knockdown therapies. This represents a significant advantage that can not be underestimated from the perspective of patient ease and use. There have been no academic papers published on the topic, so the discussion here is all speculative. We don't have data on mouse models to know how much CRISPR could truly alleviate the pathology of the disease. That said, we can already imagine the clinical translation of the approach fairly easily. Given the success of LNP technology in targeting the liver for siRNA applications, the tech can also be applied for the delivery of Cas9 mRNA and gRNA, as has been previously published for Type I Tyrosinemia. Indeed, Intellia's SEC documents reveal a plan to use LNP technology for liver delivery. The transient nature of mRNA and gRNA delivery will alleviate concerns about continued off target editing by Cas9, and any potential immune reactions against Cas9 as well. What we don't know is how efficiently can Cas9 cleave the TTR locus within that 2-3 day window of time, and how much serum knockdown will this lead to eventually. Certainly, with efficient delivery to almost every hepatocyte combined with efficient editing, one could achieve a correction of almost every hepatocyte, replicating the liver transplant therapy in efficacy and representing a real cure.

We don't know if that efficiency will be achieved, but at least the upside of Intellia's approach is there. Autosomal dominant disorders appear to be a great fit for CRISPR therapy, given that the toxic gene must be removed in order for a cure to happen. A concern about the CRISPR approach though, is how they will design the guide RNA's to target TTR. The easiest design would remove both the normal WT allele and the mutant TTR allele, but the normal TTR protein serves an important function in carrying thyroid hormone, thyroxine (T4) around the body, so it might not be able to be removed. It was noteworthy that the current RNA knockdown approaches do target the WT transcript as well. Added complexity could be to cut out the mutant sequence with two gRNA's and try to have homologous recombination with the WT allele, although this process would seemingly cut the normal allele as well. These questions still need to be addressed, likely in academic papers to be published in the near future.

Looking at realistic timelines, Intellia hasn't even announced when a prospective TTR trial will begin. It is likely that they are only going through mouse experiments right now. Their competitor Editas Medicine ($EDIT) could certainly also get into the TTR game as well. If approval is 5-6 years away, in the absolute best case scenario, for Intellia in TTR, that should give Ionis and Alnylam some time with approved drugs on the market to recoup money for investors. Longer term data from Ionis and Alnylam will also provide valuable clinical insights into how TTR knockdown can alleviate symptoms in these patients, setting the benchmarks for future efficacy goals for CRISPR modalities that appear to have potential to replace the RNA knockdown technologies.

Interesting #AACR16 Abstracts and Themes

There were a number of AACR abstracts that I found interesting, and many more that just had provocative titles. Many of the symposiums and plenary sessions just had the speakers and titles of their talks, so I am sure there will be much more presented at AACR than what I can glean from the listed abstracts and titles. However, I decided to give my thoughts on a number of them, below. I kind of went down the rabbit hole, as I normally do, and made this blog post longer than I intended, so feel free to pick and choose the parts that seem most interesting. The AACR meeting starts April 16th, and while I unfortunately will not be able to go, I will absolutely be following the presentations and the coverage of the new research.

CARs that act like TCRs
Eureka Therapeutics: ET1402L1-CART, a T cell therapy targeting the intracellular tumor antigen AFP, demonstrates potent antitumor activity in hepatocellular carcinoma models (ADAP)
Eureka is a company that uses phage display to screen for antibodies that can bind to specific peptide-MHC complexes, so they can have the specificity of a T cell receptor (TCR), but in the modality of an antibody. This allows antibodies to "target" intracellular proteins. They are also developing an antibody against WT1 (called ESK1). This antibody was developed with the Scheinberg lab at MSKCC, where they made the original monoclonal antibody (described here), and more recently turned it into a CD3-bispecific antibody here. Not surprisingly, Eureka is also considering using these antibodies to make the scFv portion of a chimeric antigen receptor (CAR), and appears to have done so here, with their lead antibody ET1402, which apparently is able to target AFP. Interestingly, Adaptimmune also has an AFP program entering clinical trials, although they are using the more traditional engineered TCR approach to target it. I don't think it's clear whether it's better to target peptide-MHC complexes with normal TCRs, or with TCR-like antibody CARs (with costimulatory domains), so it will be interesting to see how it plays out. Eureka's pipeline (although very little is disclosed) is listed here.

CAR Design - new safety switches
UPenn: Reversible regulation of chimeric antigen receptor surface expression (NVS, BLCM, CLLS, JUNO, KITE)
Engineered T cells using CARs or TCRs have the potential for serious side effects, both on-target/on-tumor (CRS), and on target/off tumor (HER2 CAR lung tox), in addition to straight off-target (Titin cross-reactive MAGE-A3 TCR). There are numerous safety switch technologies different groups have developed to either remove the engineered T cells or, more recently, control CAR expression/function. The safety switch designs currently being used in the clinic generally rely on inducible removal of the engineered T cells, such as Bellicum's inducible caspase 9 approach, and Juno's truncated EGFR, although the latter has never been used as a safety switch in the clinic. In UPenn's abstract here, they wanted to design a CAR that could be inducibly down-regulated, to avoid irreversibly ablating the engineered T cells, which occurs with the previously mentioned strategies. UPenn's approach is to attach an inducible degradation component to the CAR construct. I believe the one they are using is this LID degradation system from Stanford published here. Basically, similar to Bellicum's approach, they use rapamycin or a rapamycin-related molecule to specifically bind to a protein, and in this case, reveal a degradation motif that targets the CAR to be degraded by the cell's machinery. From the abstract so far they have only tested it in culture, not in animal models, and they were able to achieve up to 80% reduction in the CAR levels by 24 hours, which they state was able to reduce the CAR-T cell's activity back down to near background levels.

There are a number of other approaches that try to tune CAR-T activity with destroying the engineered cells. One is Bellicum's inducible activation GoCAR-T approach, which uses their inducible dimerization construct to activate costimulatory signaling, as opposed to their CaspaCIDe construct, which activates cell death. Wendell Lim's lab previously published a system where they split the intracellular components of the CAR into two pieces that could be reconstituted into the full CAR after addition of a small molecule dimerizer (using a rapamycin-based molecule) to induce CAR activation. Cellectis also published a recent paper showing they could control that they could control the surface expression of CARs using novel extracellular architecture that could be regulated by a small molecule (also using rapamycin-based molecules).

These approaches are further distinguished from those that try to integrate multiple inputs to make smarter CARs that may be able to expand the potential pool of CAR-T surface targets. A recent example is a strategy from Wendell Lim's lab that was published in back-to-back cell papers which used a synthetic Notch receptor to control expression of a chimeric antigen receptor. Michel Sadelain's lab has previously published both systems that activate in response to the simultaneous presence of two antigens, or activate in response to cells that have one antigen but specifically lack the expression of a second antigen

There will also be a symposium at AACR specifically on next generation CAR approaches, which I expect will, at least in part, focus on the costimulatory domains, highlighted in these recent papers: 4-1BB vs CD28 (June), CD28 CAR + 4-1BBL surface expression (Sadelain).

TRK inhibitors face off (LOXO, RXDX)
Loxo and Ignyta are both presenting their early data for their TRK inhibitors in precision medicine clinical plenary session. While neither of the abstracts for their clinical data are available, both have published/presented case reports of clinical efficacy, as well as resistance (Loxo here & here; Ignyta here. Interestingly, there is a new abstract from Ignyta saying that the resistance mutations Loxo identified to Loxo-101 in the above presentation did not affect sensitivity to Ignyta's entrectinib (as well as other gatekeeper mutations in ALK or ROS1). This is a bit surprising, since one of those mutations was a G667 mutation, which was one of the mutated positions in the patient who became resistant to entrectinib in the paper above. It will definitely be interesting to see how the safety, efficacy, and durability of response shake out between Loxo and Ignyta for what appears to be a good target in the small % (although wide variety) of NTRK-rearranged tumors.

New MAGE-A3 TCR from NCI (KITE)
The NCI (in a CRADA with KITE) will be presenting data from its new MAGE-A3 TCR program in solid tumors in one of the immuno-oncology clinical trial plenary sessions. MAGE-A3 has been a difficult target for TCRs, with off-target neurological toxicity & cardiac toxicity in previous TCRs. This new TCR had some data presented previously showing a PR in 3/12 patients, and importantly no off target toxicity. MAGE-A3 is a cancer/testis antigen that are a class of antigens that appear to be good targets for engineered TCRs, with TCRs targeting another cancer/testis antigen, NY-ESO-1, already having some evidence of efficacy in the clinic in synovial sarcoma, melanoma (KITE), and multiple myeloma (ADAP). MAGE-A3 may also be expressed in a higher percentage of tumors than NY-ESO-1, and thus may have a greater addressable population.

Also, most likely just preclinical, but there will be a poster on an NCI/KITE KRAS TCR as well.

Immuno-oncology early trial updates (Genentech, KITE)
In the two plenary sessions for immuno-oncology trials, it appears there will be new clinical data for PD-1 (&CTLA-4) antibodies, but also some data from new targets in the second plenary session.

One presentation will be on an OX40 agonist from Genentech. OX40 is one of numerous additional T cell surface targets investigators are looking to target in combination with PD-1 or CTLA-4 antibodies. In contrast to those inhibitory receptors, where antibodies are used to block signaling, OX40 is a stimulatory receptor, and the therapeutic antibodies have been designed to activate its signaling

The other presentation will be on pegylated-IL10 from ARMO Biosciences. While there is no data in their abstract, I believe investigators have recently presented data from this program at the ITOC3 conference here (search pegylated), where they saw a 25% PR rate in RCC.

Kite will also be presenting updated CD19 CAR data in NHL, which I am sure will be closely watched.

Targeting Neoantigens (BioNTech, ZIOP, KITE)
Neoantigens are potentially personalized medicine to its most extreme for cancer therapy - directly targeting the presence of specific mutations unique to that patient's tumor. Neoantigens are thought to be a major target source for the endogenous immune response - reviewed here. Amplifying this response specifically, with vaccines or t cells (TILs or engineered TCRs), is a hot area of focus, with a sudden swelling of startups focusing on this approach. As far as I can tell, the only neoantigen vaccine presentations that will be at AACR are from BioNTech, which will present a number of neoantigen vaccines programs in Melanoma, GBM (collaboration with Immatics), and TNBC. While neoantigen vaccines are an interesting approach (and potentially easier to manufacture than neoantigen-TCR T cells), vaccines have had difficulty in producing robust efficacy in cancer, whereas neoantigen T cells (in the form of TILs) have had sometimes impressive efficacy (reviewed here). So I am reserving judgement on the potency of neoantigen vaccines until we start to see more clinical proof of concept.

While it appears Steve Rosenberg will only be presenting the MAGE-A3 TCR data, there are also two recent papers from his group showing they could prospectively identify neoantigen TCRs in peripheral blood by using PD-1 as a marker, and in a collaboration with Ziopharm, use a transposase-based system to make neoantigen-TCR T cells. Kite is also working with Rosenberg to develop neoantigen-specific TCRs. The key will be the feasibility of identifying, cloning, and expressing the neoantigen-specific TCRs, and I will be very interested in seeing how this approach is developed by the numerous groups pursuing it.

The best T cell composition for CAR-T products (KITE, JUNO)
There is also debate in what is the best T cell (or composition of T cells) for a CAR-T product. Kite Pharma will be presenting data saying that in NHL, neither the CD4:CD8 composition nor the differentiation state clearly correlated with response, while the final CAR-T product tends to be less differentiated than the initial T cell population. Paul Rennert reviewed this area in a very nice series of blog posts here. Alexey Bersenev also had a great post on this here.
 
Novartis EGFRvIII CAR-T in GBM : Phase I study of T cells redirected to EGFRvIII with a chimeric antigen receptor in patients with EGFRvIII+ glioblastoma (NVS)
While EGFRvIII seems like a great target for a CAR as it is surface protein and the target is exclusively found on the tumor, I do not believe there have been any significant responses reported for CARs targeting it. I will be interested in seeing if there have been any better responses, as well as the expansion, persistence, and trafficking to the brain (if data is available) for the CAR-T cells.

Targeting Ras
Ras has been a difficult, often called undruggable, but much sought after target in cancer due to its role in a large percentage of human tumors. Recently, there appears to be renewed emphasis on approaches to target Ras directly or downstream, and I will be interested in following what appears to be real progress in approaches to drug Ras.
There are two sessions on targeting Ras mutated tumors: Progress in Treating KRas Mutated Tumors session, and From Chemistry to the Clinic: Inhibition of K-Ras
While most of the abstracts have not been published yet, there are a number of approaches that are interesting:
Covalent inhibitors of Ras - one is described in an abstract here.
Trapping mutant KRas G12C in its inactive state - abstract (title only), paper.
Allosteric inhibition of Ras - abstract (title only), paper.

BET-bromodomain Inhibitors in NUT-midline carcinoma
There seem to be a ton of BET-bromodomain inhibitors in development, starting from JQ1, and now there seem to be multiple beginning to generate clinical data, primarily in NUT-midline carcinoma, where tumors appear to be especially dependent due to frequent NUT-BRD4 fusion events.
In a clinical trials minisymposium at AACR there will be data from: GSK525762, a selective bromodomain (BRD) and extra terminal protein (BET) inhibitor: results from part 1 of a phase I/II open-label single-agent study in patients with NUT midline carcinoma (NMC) and other cancers 

Previously there has been clinical data presented on a compound from Tensha: Clinically efficacy of the BET bromodomain inhibitor TEN-010 in an open-label substudy with patients with documented NUT-midline carcinoma (NMC)

There was also just recently a publication on a Merck-licensed compound that also had activity in NUT-midline carcinoma - here in Cancer Discovery.

Dissecting the complex ecosystem of malignant tumors with single cell RNA-Seq
While there was no abstract for this title, this is an approach that seems like it could significantly improve the resolution with which we can look at all the different cell types simultaneously and separately in a tumor. One of my favorite papers from last year used a cytolytic gene expression signature from large genomic data sets to see what tumor properties correlated with what appeared to be an immune response. I would imagine this analysis could be even more powerful in identifying ways in which the tumor and the immune system co-evolve if we had resolution of all the different cell populations instead of looking at a single tumor signature for both the tumor and all the different stromal cells. Update: The paper for the above presentation appears to just have been published in Science, here.
  
Single-cell TIL profiling (JUNO)
Juno does not appear to be presenting much work on CARs at this AACR, but I actually found this non-CAR abstract very interesting. This approach from Juno seems like it could be very powerful for identifying what is going on in individual TILs as at the single cell level, they claim to be able to sequence the TCR, while also getting a snapshot of that T cell's transcriptome and proteome. I could imagine this level of resolution could lead to the identification of additional T cell pathways of activation or repression in different T cell populations in the tumor, such as PD-1+ TILs or neoantigen-reactive TILs.

Identifying novel immunotherapy targets from human tumor data from PD-1 & CTLA-4 antibody trials:
There are so many potential immunotherapy targets for either monotherapy or combination with checkpoint blockade that it can be overwhelming. An approach I like that may help sort out which are the critical targets is starting with data sets generated from human tumors, particularly the differences in responders versus non-responders to checkpoint blockade. In this way, we can try to let what's going on in human tumors tell us what is important to escape immune surveillance. One can see this approach in a number of abstracts, such as:
The tumor immunity continuum as a framework for rational combinations or Immunogenomics and precision cancer medicine among others.

There have also been a number of recent papers published using approaches like this:
Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints

Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma

Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity.

There are many more interesting projects and themes in oncology research that I'm sure will be presented at AACR, but I will somewhat arbitrarily stop here. Any discussion is welcome here or on twitter - @Festivus159.