Last month we saw a biomedical media campaign go a bit off the rails. A press release from the American Association for the Advancement of Science (AAAS: see for example https://www.sciencenews.org/article/memory-cells-enhance-strategy-fighting-blood-cancers) and the Fred Hutchinson Cancer Center, was picked up by multiple media outlets who quickly spun the story of CAR-T-cell mediated rapid and complete clearance of B cell leukemias and some lymphomas from very ill patients and turned it into the “cancer cured” sort of headlines that serve as great click-bait but don’t do much to really educate the reader.

But what first caught my eye was an odd distortion of the data as presented in the session entitled “Fighting Cancer and Chronic Infections with T Cell Therapy: Promise and Progress” (see https://aaas.confex.com/aaas/2016/webprogram/Session12231.html). Several credible sources were telling very different stories about the progress presented. To take one example, BioWorld Today told the story of the clear benefit of using naive T cells as the recipient for cellular therapy, while FierceBiotech (and many other outlets) focused on the benefit of using memory T cells instead (see http://bit.ly/1UdLqDs). Indeed the claim was made that even a single memory T cell could affect a cure – which was not really the point, or an important conclusion of the presented works.

It follows that the pressers were used to talk up CAR T cell company stocks, which have been languishing along with the rest of biotech.

All of this came across as garbled and confusing. I found it all very frustrating.

So now I’ve gone through the abstracts presented at AAAS and some of the primary literature, and I’ve a Cliff Notes version of what data were actually presented and what the data mean and don’t mean. I seems clear that the confusion regarding the results arose from the oversimplified weaving of two talks (by Dirk Busch and by Steven Riddell) into one tangled “story”. Lets untangle the knot and follow the threads.

Riddell’s work is closely followed in the CAR T field – not surprising as Dr. Riddell, from the Fred Hutchinson Cancer Center in Seattle, is a technology leader and a cofounder of Juno Inc. The story presented at the AAAS symposium is interesting but perhaps more controversial than one might have gathered from the press reports. Some of the work was recently published (http://www.nature.com/leu/journal/v30/n2/full/leu2015247a.html). They start with the observation that in all reported CAR-19 clinical trials, patients have received back a random assortment of their (now CAR-transduced) T cells, meaning that the cell population is a collection of naive T cells, effector T cells and memory T cells representing both the CD4 and CD8 T cell lineages. This introduces a variable into therapy, as different patients are likely to have different percentages of these various T cell subsets. Indeed there is quite a list of variables that may impact the efficacy of CAR T cell treatment including baseline immune competence, prior treatments and antigen load. With this in mind Riddell and colleagues are trying to control the one variable that they can, which is the composition of the transduced T cells going into the patient. By analyzing CAR cell subsets for tumor cell killing function they arrive at the “most potent” combination of CD4+ T cells and CD8+ T cells and conclude that the findings will be important for the formulation of CAR T cells therapeutics for use in patients.

The data in the paper are derived from normal donor and cancer patient PBMC samples that are tested in vitro using cell culture assays and in vivo using humanized mice (NOD/SCID/yc-deficient mice; NSG) reconstituted with T cells and tumor target cells (Raji) that express CD19. The CAR T construct is a “generation 3” CAR having CD28, 41BB and CD3 signaling domains downstream of the well-studied FMC63-derived anti-CD19 scFv.

Some results:

– substantial differences were seen in the T cell populations between normal donors and cancer patients, with most patients having a higher percentage of CD8+ than CD4+ T cells.

– patient samples also contained more memory T cells than did normal donor samples. A further refinement to the memory T cell definition allows one to identify effector memory and central memory T cells. The latter are a long-sustained population of antigen-educated T cells that contribute to immunological memory, such as one retains after a vaccination against a virus for example.

– both CD4+ and CD8+ T cells were readily transduced with the CAR-19 construct, and when presented with target cells in vitro both cell types responded. CD8 T cells mediated target cell lysis more effectively than CD4+ T cells, but the latter proliferated more vigorously and produced more pro-inflammatory cytokines such as IFNy and IL-2.

– among the CD4+ subset, naive T cells (those not previously antigen-activated) produced more cytokines than the memory cell subsets. In vivo, naive T cells were more potent in controlling tumor growth than central memory T cells which were in turn more potent than effector memory cells.

– similar analyses of CD8+ cells revealed that, of the three subsets, central memory CD8+ T cells were the most potent in vivo, a result that was most closely associated with the enhanced proliferation and expansion of this subset.

– the activity of CD8+ central memory T cells was further enhanced by the addition of CD4+ T cells, notably those of the naive subset. This effect was seen using cells from normal donors and cells from B cell lymphoma patients (specifically, Non-Hodgkin Lymphoma (NHL) patients). The improved in vivo activity was due to enhanced proliferation and expansion of T cells in the NSG mouse model, specifically an increase in the peak of CD8+ cell expansion, in line with clinical results (see below). I’ll note as a reminder that all of the available clinical results are from CAR T cell populations that had not been sorted into naive and memory subsets. Also, many researchers in the field believe that naive T cells (CD4 and CD8) have the best proliferative capacity and potency.

Regardless, the Riddell work suggests a straightforward improvement in the ability to create more potent CAR T cell preparations for use in the clinical setting. There are some caveats however. In the in vitro and in vivo models used, antigen (CD19) is abundant, even in the NSG mouse, due to robust expression of rapidly dividing CD19+ Raji cells. As noted earlier, antigen availability may be an important limiting feature for some patients, and may be more important than the composition of the T cell subset tested. Fortunately the relative importance of these variables could easily be examined in vivo by using sub-optimal Raji cell numbers, or using transfected cells with different levels of CD19 expression, to vary the antigen load.

The Busch study at the same symposium was notable for dispensing with CD4+ T cells altogether and using just CD8+ central memory T cells to control CMV infection (that can occur following allogeneic hematopoietic stem cell transplantation). Nearly all of this work has been performed in mouse models, with a small number of patients treated under compassionate use protocols (see e.g. http://www.bloodjournal.org/content/124/4/628). In the mouse models very small numbers of antigen-specific memory T cells can expand to control viral infection, and this has been taken as evidence (in the popular press mainly) that similar technology could be applied in the CAR T setting. However, numerous studies have shown conclusively that very large-scale expansion is required to achieve optimal potency, to a degree that would seem beyond the capacity of a small number of cells or a single cell. Further, studies in acute lymphocytic leukemia patients presented by Carl June last fall at the Inaugural International Immunotherapy meeting in NY showed that clonal selection and perhaps competition was a component of successful therapy for some patients, a process that would be eliminated or reduced by using a limited cell number in preparing the CAR T cells. The Busch study makes the further argument that central memory CD8+ T cells themselves possess “stem-ness”, that is, they can give rise to functionally diverse CD8+ T cell lineages and as such should have no limit to their proliferative capabilities. While this was demonstrated convincingly in mouse models it would seem a difficult finding to translate to the CAR T setting, although the work may find utility in the adoptive cell transfer setting (e.g. of selected but not transduced T cells, such as tumor infiltrating T cells).

The “stem-ness” concept reminded me of older literature that aimed to dissect the basis for long-lived CD8+ T cell memory in the context of viral immunity (see here for a recent review: http://journal.frontiersin.org/article/10.3389/fimmu.2012.00357/abstract). There were at one time two broad classes of thought – first, that such memory required a consistent supply of antigen, for example, a depot that periodically re-stimulated the antigen-specific T cell population. The second school of thought, more reminiscent of the Busch finding, was that memory CD8+ T cells were self-renewing, and therefore did not require life-long antigen stimulus. The “big bang” hypothesis of T cell memory development, a hypothesis that the work of Dr. Busch and colleagues has definitively supported (see: http://www.bloodjournal.org/content/124/4/476?sso-checked=true) holds that once stem-like T cell memory is created, these cells can be used just like stem cells, i.e. to reconstitute cellular function, in this case, the ability to control viral infection.

Let’s get back to CAR T cells. Recent work has demonstrated clearly that the establishment of persistence in cellular therapy requires a robust response to abundant antigen. Only then can CD8+ T cell memory develop and from that point on be maintained. This observation informs the next set of studies, presented at the Clinical Application of CAR T Cells conference (#CART16 – https://www.mskcc.org/event/car-t-cell) held at the Memorial Sloan Kettering Cancer Center, the Adoptive T-Cell Therapy Congress held in London (http://tcellcongress.com/resource-center/) and the Advanced Cell Therapy Symposium (https://www.immunology.org/document.doc?id=1807) held at Guy’s and St Thomas’ NHS Foundation Trust and King’s College, also in London. Much of the work presented highlighted at these meetings addressed attempts to move CAR T cells into solid tumors.

Here I am a little hamstrung, as I’m relying on information presented on slides (as shared on Twitter by @JacobPlieth @VikramKhanna and others). Let’s try to define some themes here regardless.

Jacob has reviewed some #CART16 data: http://epvantage.com/Universal/View.aspx?type=Story&id=627150&isEPVantage=yes. Please see that link for his viewpoints.

First, to stick with CAR19 therapeutics, we have some posted Novartis data on responses in Non-Hodgkin Lymphoma (NHL). NHL is comprised of diverse B cell lymphomas, some of which are highly refractory to treatment. Examples of the refractory class include diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) among others. Here we see some rather impressive results treating these lymphomas:

The data are hard to read, but let’s pull out some numbers from the table. Note that essentially all patients got the optimal dose of 5 x 10e8 cells (1 exception) and that the peak cellularity is defined as %CD3+/CAR19+ cells in peripheral blood. We can therefore look at expansion, time to peak cellularity and outcome:

There seems no correlation between day to peak and outcome, unless it is very short – day 1 or 2 – and even then that is likely due to abortive expansion. If we arbitrarily set 10% as an exploratory setting with which to parse the %CD3+/CAR19+ data we quickly see that above 10% (black line), half of the patients responded, which below 10% only a third of patients responded.  So expansion is important, as we already knew. With respect to the earlier discussion, we do not know the critical variable at work here, be it CAR T cell persistence (likely), CAR T cellular composition (per Riddell), patient variability, antigen density, or something else.

The FL data are a little bit more confusing:

Again we can pull out some of the data:

And now we are really hard-pressed to see any correlation between outcome and peak cellularity, no matter where we might draw the arbitrary line for analysis. What data is missing? I suspect it is a measure CAR T cell persistence over time, as this is most often associated with positive response. We should note that CD19 is an unusual target antigen in that it is expressed on the cancer cells (B cell leukemia or lymphoma) and on normal B cells that we can deplete without undue harm to the patient.

Other B cell antigen targets are under development as CARs, including CD22 and BCMA. BCMA is expressed on plasma cells (relatively uncommon B cells that secrete antibodies) and on essentially all multiple myeloma cells. Early promising results generated using a BCMA CAR to treat multiple myeloma were presented at ASH (http://www.ascopost.com/issues/march-10-2016/car-t-cell-therapy-may-have-role-in-treating-multiple-myeloma/).

Updated results are a little less encouraging as the complete response patient (#10) has since relapsed as was reported at #CART16. It is unclear if advanced multiple myeloma is simply more refractory to CAR treatment, if the lower cell number infused led to poor persistence, if the CAR were different, or if antigen load was too low. Thus we are again faced with multiple variables to assess.

So now we can ask what happens when there is little or no persistence, which is the case with most CARs directed to solid tumors. This is data from Nabil Ahmed and Stephen Gottschalk from Baylor College of Medicine. This group is collaborating with Celgene on cellular therapeutics. Here we see the results of treatment of HER2+ advanced solid tumors with CAR-HER2 T cells. The response is minimal.

This is very likely due to the very short persistence of the CAR-HER2 T cells, in most cases gone in a week or so.

Interestingly, analysis of resected or biopsied tumor after treatment revealed that the CAR T cells had migrated preferentially into the tumor, but had not proliferated extensively. Novartis presented nearly identical data on an EGFRvIII targeting CAR T cell study at the Boston PBSS Immuno-Oncology Workshop (http://www.pbss.org/aspx/homeBoston.aspx), and similar data has been presented on a host of solid tumor targets.

To return briefly to CD19+ tumors, it was reported recently that the response to CAR19 therapy in chronic lymphocytic leukemia was about 25%, but that all of those responders were durable complete responders (i.e. potential cures). Why the seemingly digital nature of response here? Again this is most likely due to CAR T cell persistence which itself is most likely a reflection of antigen load (among other variables). With this in mind I was struck by a slide from Seattles Childrens Hospital (I’m not sure from which meeting):

In point #3 the presenter is basically suggesting injecting an artificial antigen presenting cell expressing CD19, i.e. increasing the antigen load.

We can conclude by saying that there is a fundamental issue with CAR T cell antigens – those that are tumor specific are either not abundantly expressed and/or have been removed during the course of therapy. This is an issue that may not be solved by adding 4-1BB or IL-12 or anti-PD-1 antibody or whatever other immunological “help” one might envision. This issue impacts the entire field, which is why we now see analysts who once talked of the emergence of dominant CAR T platform companies now wondering who will win the CAR19 race to the finish line. That is still a noble race to run, but the patient numbers cannot justify the number of companies competing for the prize. Yet change will come and progress will be made…

What to do?

stay tuned.

ps. thanks again @JacobPlieth @VikramKhanna and others for kindly sharing slides (and getting great seats at conferences!)