Thoughts on Bluebird's Striking BCMA CAR-T Data

Bluebird bio just released early data from their initial first-in-human trial of a BCMA CAR-T product for multiple myeloma that will be presented tomorrow at the #ENA2016 meeting. This is a 2nd generation 4-1BB CAR targeted to BCMA with a cyclophosphamide + fludarabine based conditioning regimen. The early results are quite impressive, with a 78% response rate in all 9 patients evaluable for response, and 100% in the 6 patients treated at the two highest cell doses. Even more impressive, and surprising, has been the lack of grade 3 or higher cytokine release syndrome or neurotoxicity that is frequently observed in CD19 CAR trials, as well as other BCMA CAR trials. While this data is still very early, with only 11 patients treated, dose escalation ongoing, and less than 1 year follow-up, the results compare very favorably to other BCMA-directed CARs, which I'll compare below.

Here is the data from Bluebird's press release:

The time to response seems a bit slow (2 & 4 months) so it's possible that some of the PRs in the highest dose cohort will improve with further follow-up. They also mention that all patients in the two highest dose cohorts that had detectable MM in bone marrow had no detectable MM in bone marrow by day 14 and beyond. They do not mention if any of the patients have relapsed, although the wording regarding the sCRs suggests that they have not relapsed at 4 and 6 months follow-up.

These data compare favorably to a 4-1BB BCMA CAR being developed by UPenn/Novartis that had some preliminary data on 6 patients in their recent ASH abstract. In this first cohort, they did not lymphodeplete with conditioning chemotherapy prior to CAR-T infusion. Even without lymphodepletion, they were able to get significant expansion of CAR-T cells in two of the six patients. These two patients also had significant responses - a VGPR and an sCR, with the sCR ongoing at 7 months, but the patient with the VGPR relapsing after 5 months. They administered a similar number of cells as Bluebird, 1.8-5 x 10^8 cells. Importantly, both patients who responded, and had significant expansion of CAR-T cells, also had grade 3 cytokine release syndrome, and one of the two had grade 4 reversible neurotoxicity. CAR-T cells were found in the CSF of this patient. This data is summarized in the table below:

These results were fairly similar, but perhaps more variable, than results published from a clinical trial on an earlier CD28 BCMA CAR at the NCI. In this study, they dose escalated up to 9x10^6 cells/kg and were given conditioning chemotherapy of cyclophosphamide and fludarabine. They treated 12 total patients, with both patients treated at the highest dose having responses of greater than 8 weeks (sCR of 17 weeks and VGPR ongoing at 26 weeks). Patients treated with lower doses of CAR-T cells had either no response, or responses of shorter duration.

From Ali et al., 2016

Importantly, similar to the UPenn study, it was the patients (10 & 11) that had the most dramatic expansion of CAR-T cells that had significant clinical responses:

From Ali et al., 2016

It was also the two patients with these dramatic expansions of CAR-T cells that had CRS and neurotoxicity (reversible).

Thus what is surprising is that the patients in Bluebird's trial achieved significant responses without these significant toxicities. This is quite different from both the UPenn trial and original NCI trials, where activity came with grade 3 and higher CRS and/or neurotoxicity. I'll discuss this difference further below.

These trials also began to characterize relapses after initial responses. In CD19 CAR trials, relapses frequently fall into two categories - loss of the target antigen (CD19) or (/and) loss of persistence of the CAR-T cells. Loss of antigen expression/availability is a concern for all CAR-T therapies. In UPenn's trial, one patient relapsed with loss of BCMA expression and concomitant loss of CAR-T persistence. The patient with an ongoing response still had detectable CAR-T levels. A patient in the NCI CD28 BCMA CAR trial who had a transient response also was found to have lost BCMA expression on their MM cells, but a number of patients who relapsed were not evaluable for BCMA expression, so this may be an underestimation of antigen loss frequencies. Importantly, in this trial, the CAR-T persistence was fairly poor, with loss of CAR-T persistence by 3 months, which could also partially explain the transient responses. The general thinking seems to be that 4-1BB CAR-T cells have better persistence, and CD28 CAR-T cells have greater expansion (and perhaps greater toxicity).

Bluebird will present their data tomorrow, so we should get some additional details.

Questions I'm thinking about going into their presentation:

What was the disease burden in their treated patients? Lower disease burden may cause less CAR-T cell expansion and toxicity. In the NCI trial, both responding patients had bone marrow disease burdens of >80%, and in the UPenn trial, both responding patients had bone marrow disease burdens of >70%.

What were the kinetics of tumor response? What was the speed of decreases in tumor biomarkers and soluble BCMA levels - these decreases were fairly rapid (on the order of weeks) in the NCI trial.

What were the kinetics and peak of CAR-T cell expansion? Did the cells expand to less dramatic levels, or less rapidly, than in previous BCMA CAR-T trials, but were still able to induce responses?

How well do their CAR-T cells persist over time?

What was the state of their CAR-T cells at peak levels? %CD8+ T cells and levels of activation markers.

How are the conditions different (if different at all) for how Bluebird manufactures their T cells compared to others? What is the composition of their final product in terms of T cell markers? At last year's ASH they had presented their finding that adding a PI3K inhibitor during manufacturing led to better efficacy preclinically.

How durable have the responses been? Bluebird started treating patients in February, and the data cutoff for the abstract was November 18th, so I am not expecting a lot of data to answer this question.

Have there been any relapses, and have these been associated with antigen loss or loss of CAR-T persistence (perhaps at the lower dose levels)?

*Update 12/1
Bluebird presented their data in a conference call this morning. Slides of their presentation are available here.

Of the above questions, Bluebird mainly presented data on the initial kinetics of CAR-T cell expansion and tumor marker decrease, as well as cytokine levels. They did not, however, want to speculate on differences between their trial and other BCMA CAR-T trials, and did not appear to provide any additional information on the initial tumor burden levels in these patients. They also did not characterize the T cell product for CD4:CD8 ratios or other T cell markers either prior to infusion or during treatment. They did not want to speculate on manufacturing differences between their product and others, but mentioned they plan to bring along their next generation BCMA-CAR program, which will use the PI3K inhibitor during manufacturing over the next year.

Below are some of the key additional data they presented. While they did not want to compare data too closely to other BCMA CAR-T trials, I will make some very preliminary, premature, any other diminishing adjective you want, comparisons just to start getting a sense of some of the early similarities and differences. Of course, this is all subject to change with further follow-up and additional patients, so CAUTION is warranted when trying to draw any conclusions.

Response timing and durability:

Above, you can see the time to first response and time to best response in their treated patients, as well as time to disease progression. In the first three patients treated at the lowest dose level, only one achieved a PR, and then subsequently relapsed after >12 weeks, but they did not disclose any data on the nature of this relapse (although persistence of CAR-T cells was shorter for this dose level, as shown below). Another thing to note is the conversion of initial responses into better responses over time in the cohort treated at the second dose level. Thus, it is still possible that the PRs with shorter follow-up in the highest dose cohort will continue to improve.

We can see some of the kinetics of response over time below:

The serum M-protein levels appear to continue to be trending down in the highest treated dose-cohort, so again, it is possible that the responses will continue to improve. The rapidity of the response appears to be roughly in line with what was seen previously in the NCI trial, but it's too few responses to really be able to have a good sense of this yet.

Bluebird also presented some initial CAR-T expansion and persistence data:

The CAR-T cells expanded for all dose cohorts, but appeared to expand to a greater extent for the two higher dose levels. The peak levels of the CAR-T cells - 10^5-10^6 vector copies per ug genomic DNA, appears to be similar to the peak expansion of 1.74 x 10^5 and 2.20 x 10^5 vc/ug genomic DNA in the two patients who had significant responses in the UPenn trial (table above). So it does not appear that the, as of yet, unobserved grade 3 or higher CRS is solely due to poor peak expansion of Bluebird's CAR-T cells.

The persistence also looks potentially improved versus the CD28 CAR in the NCI trial, which had minimal levels of CAR detectable by 3 months in any of their patients. While it is still very early in follow-up and patient numbers, Bluebird's 4-1BB CAR-T cells seem to maintain detectable levels in the majority of patients treated with the two highest doses through 8 weeks. Preliminary data from UPenn's 4-1BB BCMA CAR trial, as mentioned above, also had increased persistence, with CAR detectable for up to 7+ months in patients.

Decreases in serum BCMA levels correlated with response. For one patient with the longest follow-up in this figure (through 24 weeks), the CAR-T persistence appeared to wane by 8 weeks, and serum BCMA levels have begun to rise from 16-24 weeks, with re-emergence of CAR detection by 24 weeks. Further follow-up will be necessary to determine if this preludes a relapse.

Bluebird also presented a more detailed list of treatment emergent adverse events:

The grade 3 & higher toxicities were attributable to the lymphodepleting regimen. Again the headline has been the lack of observed grade 3 or higher CRS or neurotoxicity to date, even in patients who have had clinical responses.

They also gave additional details about the peak cytokine levels in their patients:

They show that their peak levels of cytokines in blood were all orders of magnitude below the very high levels observed in the NCI trial, in the two patients who had responses but also CRS (shown below). This is somewhat surprising that even with apparently significant CAR-T expansion they did not see very high levels of cytokines produced.

From Ali et al., 2016

Lastly, they said they plan on having a flexible trial design, potentially with different approaches for patients with higher disease burden versus low (using 50% bone marrow MM cells as a threshold). They also stated that patients enrolled so far were required to have BCMA detectable on >50% of their MM cells, but were considering loosening this criterion due to the impressive responses seen to date, hoping that their CAR will be able to detect even very low levels of antigen present on cells.

Summary

The data released by Bluebird on their still very early BCMA CAR-T program are certainly quite encouraging. They appear to compare favorably in terms of response rate to other early anti-BCMA CAR-T trials, although we are comparing small numbers here, and dose escalation in Bluebird's trial (and others) is still ongoing. More surprisingly, and impressively, unlike in previous trials, the activity of Bluebird's CAR-T cells has not been accompanied by grade 3 or higher CRS or neurotoxicity. One question that Bluebird has still not given further clarity on is what the disease burden was in these patients and we will have to see how disease burden will correlate with responses and toxicity going forward. The CAR-T cells did appear to expand significantly, but this has surprisingly not come with severe increases in cytokine levels and subsequent toxicity. These are definitely exciting results, but seeing how durable these responses are, as well as if these preliminary levels of activity and safety hold up with greater patient numbers will be of critical importance. As with CD19, loss of the target antigen, BCMA, will most likely be a difficulty going forward. If the safety and efficacy are, in fact, differentiated from other BCMA CAR-T programs, understanding why will be essential as well.



Disclosure: I own shares of Bluebird Bio

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.