Category Archives: CLL

chronic lymphocytic leukemia

CAR T updates – tangled tales unwound

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:

Screen Shot 2016-03-20 at 9.55.18 AM

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:

Screen Shot 2016-03-20 at 10.00.38 AM

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:

Screen Shot 2016-03-20 at 10.05.07 AM

Again we can pull out some of the data:

Screen Shot 2016-03-20 at 10.07.43 AM

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/).

Screen Shot 2016-03-20 at 9.39.58 AM

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.

Screen Shot 2016-03-20 at 10.12.07 AM

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.

Screen Shot 2016-03-20 at 10.12.27 AM

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):

Screen Shot 2016-03-20 at 10.17.40 AM

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!)

Three high-altitude take aways from AACR14

The American Association for Cancer Research (AACR) 2014 meeting last week was high energy and high impact. We will dive into particular talks and specific pathways and indications in later posts, in the meantime I wanted to mention a few key themes.

1) Immunotherapy Versus The World.  That’s a deliberate overstatement of a subtle shift in emphasis from last year’s big meetings, where combinations of immunotherapy with just about anything else were the hot topic. This year there were several talks which emphasized the futility of chasing oncogenic pathways and all of their resistance mutations, one after the other, as opposed to letting the immune system do the work. However, it seems to me overly optimistic to believe that immune modulation can defeat a high percentage of patient  tumors on its own, as some speakers acknowledged. Combinations remain necessary although we will have to work past some notable failures in combo trials, such as the liver toxicity seen in the ipilimumab + vemurafenib combination phase 1, discussed briefly by Antonio Ribas               (see http://www.nejm.org/doi/full/10.1056/NEJMc1302338).

2) Immunotherapy Versus Itself.  In the ultimate battle of the titans, we see different immunotherapeutic modalities squaring off. This is a theme we’ve touched on before in this space, but the  competition is getting heated. In some indications, the leukemias, lymphomas, perhaps melanoma and some other solid tumors, there is an abundance of therapeutic choices, and the hard question of which therapy best suits which patient will ultimately need to be addressed outside of the context of clinical trial enrollment. Several talks really brought this message home. Roger Perlmutter of Merck (and before that, Amgen) envisions an important role for multiple immune therapies including bi-specific antibodies, chimeric antigen receptors (CARs), and immune checkpoint modulators like Merck’s anti-PD-1 antibody MK-3475.  For B cell lymphoma for example, there is blintumumab (Amgen), a potent bi-specific that redirects T cells to CD19+ tumor cells (and normal B cells), and there is CTL019, a CAR therapeutic which does much the same thing. The therapeutic profiles and toxicity differ, but the general idea is the same. One big difference is that while CTL019 drives T cell expansion and the development of long term anti-tumor memory, the bi-specific does not. Which is better? We don’t know yet. He did not mention that one might do well trying a course of BTK inhibition plus anti-CD20 antibody therapy, perhaps with restricted chemotherapy first e.g ibrutinib plus rituximab and chemo (R-BR or R-F). That choice comes down to efficacy, then toxicity, and eventually cost. Efficacy seems to be a home run with the CAR therapeutics, although these may run into trouble in the area of toxicity and cost calculation. Renier Brentjens discussed the CAR therapies being developed under the Juno Therapeutics umbrella. Acute lymphoid leukemia (ALL) can be treated with CAR 19-28z modified T cells to achieve a >80% complete response rate with >70% of patients showing no minimal residual disease, an outstanding result. However, 30% of treated patients end up in the ICU due to cytokine release syndrome and other toxicity, and recently patients in the ALL trials have died from unanticipated tox causes. Juno stopped 5 trials of their CAR technology last week due to toxicity. Apparently one patient died of cardiovascular complications and another of CNS complications (severe uncontrolled seizures) – it was hard to nail down as Dr Brentjens had gone off his prepared talk for these remarks which were off the cuff, so comment please if you have better info on this. Carl June discussed Dr Brentjens’ presentation, noting that the clinical results were really quite striking, and contrasting the CD28 motif-based CARs with the 4-1BB-based CARs (as designed by Dr June with U Penn and licensed to Novartis). He also stressed that in chronic lymphocytic leukemia (CLL) they have had patients who have failed up to 10 prior therapies, including rituximab and/or ibrutinib, and these patients have responded to CAR treatment. That’s very impressive data. The roadblocks to widespread use of CAR therapy however are large and include the toxicity, the “boutique” nature of the current protocols, the cost. Perhaps, Dr June suggested, CAR will end up as third line therapy, reserved for salvage therapy. I for one hope not.

Also in the immunotherapy space were hot new targets (e.g. CD47, OX40, GITR), advances on the vaccine front, and a few surprises. We’ll update soon.

3) The Medicinal Chemists Have Been Busy.  Not to be drowned out by the Immunotherapy tidal wave, small molecule therapies targeting specific oncogenic pathways continue to be developed and show promise. Most readers will be aware of the high stakes showdown (so billed) between Novartis, Pfizer and Lilly in the field of specific CDK4/6 inhibitors – in addition to bringing forward some really nice phase 2 data (we’ll discuss these another time) this “showdown” also illustrates that current portfolio strategy drives a lot of overlapping effort by different companies. As expected, much of the action is moving downstream in the signaling pathways, so we saw some data on MEK1 inhibitors and ERK1/2 inhibition. There were some new BTK inhibitors, nice advances in the epigenetics space, and some novel PI3K inhibitors. All grist for the mill.

stay tuned.

Hematological Malignancies – who will win the battle for patients? Part 2: BiTEs & CARTs targeting CD19

 We talked last time about the potential of Macrogenic’s DART bi-specific technology and we focused primarily on the T cell engaging bi-specifics, such as DART006, a CD3 x CD123 therapeutic. Lets just quickly state the hypothesis:

Bi-specific modalities will allow the targeting of the patients T-cell driven immune       system to a precise (tumor-expressed) antigen.

Other outcomes are possible. For example, the drugs might not work at all, or they might not be as specific as designed, or they act in ways we have not anticipated. In the context of the Macrogenics platform, we actually don’t know yet, as DART006 is very early in clinical development. BiTEs (Bi-specific T cell Engagers), Micromet’s version of a bi-specific technology, have been around a while and are further advanced. Acute Lymphocytic Leukemia (ALL) patients are now being recruited into Phase 3 clinical trials for blinatumomab, the anti-CD3 x anti-CD19 BiTE, with study completion due in July 2017. Micromet was acquired for 1.2BB dollars in January 2012 by Amgen. At the time Amgen R&D head Roger Perlmutter pointed to the Phase 2 clinical trial results in ALL as driving Amgen’s interest in the technology. Indeed, blinatumomab has produced some remarkable data in ALL. Historically, chemotherapy treated ALL patients had a complete response rate (CR) of about 38% and a median overall survival (OS) of 5 months. Rituximab (anti-CD20) didn’t perform much better than chemo. In the blinatumomab Phase 2 trial of adult relapsed/refractory (r/r) ALL, patients received a continuous IV infusion of blinatumomab for 28 days followed by 14-days off drug. Patients who responded could re-up for 3 more cycles of treatment or proceed to allogeneic stem cell transplantation (HCST). There was a very high rate CR of ~70% and the apparent absence of minimal residual disease (MRD) in many patients. Blinatumomab also impacted overall survival (OS) in ALL, as reported at the American Society of Hematology conference (ASH) in 2012 (Abstract #670). The CR was still 69% with most patients being MRD negative. The OS for responders was 14.1 months while the OS for non-responders was 6.6 months (so median OS = 9.8 months). Thirteen of the 36 patients enrolled were able to receive allogeneic HSCT.

The most common adverse events (AEs) were fever, headaches, tremors, and fatigue. Some patients experienced severe AEs (SAEs) such as cytokine release syndrome (CRS) and central nervous system events, including seizures and encephalopathy. One patient stopped treatment due to fungal infection leading to death. So, there is tox to consider.

A smaller study directed to salvaging patients with MRD despite prior treatments showed even more dramatic results: 16/21 patients became MRD negative and the probability for relapse-free survival was 78% at a median follow-up of 405 days. This is a remarkable result. An SAE led to one drug discontinuation.

Last year at ASH (Abstract #1811) we saw early results from an open label phase 2 study in r/r Non-Hodgkin’s Lymphoma (NHL), specifically, Diffuse Large B cell Lymphoma (DLBCL). Blinatumomab was administered by continuous IV for 8 weeks. Patients received either stepwise blinatumomab dosing of 9, 28, and 112 μg/d during weeks 1, 2, and thereafter, or received 112 μg/d throughout. All patients received prophylactic dexamethasone. So you can see some dose modifications here designed to reduce SAEs. After a 4-weeks off drug, patients who had responded could receive a 4-week consolidation cycle. 11 patients had been enrolled, 7 patients were evaluable for response. These patients had failed >2 prior therapies, including some patients who had relapsed after HSCT. The overall response rate (ORR) was 57% (14% CR plus 43% partial response (PR); 30% had progressive disease (all from the stepwise dose regimen). Note this is a very small sample size so every patient has a large impact on the response numbers. Ten of 11 patients had at least one grade ≥3 AE with 2 patients having grade 4 AEs (one patient with neutropenia and leucopenia; one with respiratory insufficiency). There were no drug related fatalities. Ten of 11 patients had central nervous system (CNS) AEs, mostly tremor, speech disorder and disorientation: in 5 patients these CNS toxicities were grade 3. The overall benefit/risk assessment suggested stepwise dosing (9, 28, 112 μg/d) to be the recommended dose.

Well first of all let’s point out here that blinatumomab has orphan drug status for ALL and NHL. That’s just to remind ourselves that these are pretty rare diseases with high unmet need. For ALL in particular this seems a good risk/benefit scenario. Within the diseases that make up NHL, DLBCL is not the most treatable (nor the least), and we note also that there is no attempt in the open-label phase 2 to characterize DLBCL into its subclasses – these have different oncogenic drivers and different outcomes for patients. Blinatumomab has also been in Phase in in other NHL classes, including Mantle Cell Lymphoma and Follicular lymphoma. Response rates were generally below current standard of care. Similarly, we can go back to look at rituximab, ofatumumab, and even ibrutinib, idelalisib and ABT-199 in NHL and likely find better treatment paradigms for r/rDLBCL than this, although maybe not as a monotherapy (see those earlier posts here: http://www.sugarconebiotech.com/?p=16).

Given the modality (CD3 x CD19 bi-specific) maybe the most interesting comparison is with Novartis’ CAR-T CD19 technology CTL019. CTL019 is the product of genetic engineering technology developed by Carl June’s group at U Penn, and is currently advancing in close to 20 clinical trials. The most advanced is a Phase 2 trial in r/r ALL, with a primary outcome completion due in July of 2015. As a quick reminder, CARs combine a single chain variable fragment (scFv) of an antibody (e.g. anti-CD19) with intracellular signaling domains from CD3 and 4-1BB into a single genetically engineered chimeric protein. The CD19-specific version of this technology is termed CTL019. Patient’s T cells are lentivirally transduced with a CAR, expanded ex vivo then infused back into the patient. Infusion of these cells results in 100 to 100,000x in vivo T cell proliferation, anti-tumor activity, and prolonged persistence in patients carrying CD19+ B cell tumors. Results from a pilot study in pediatric and adult r/r ALL were presented at ASH in 2013 (Abstract #67). Most patients received lymphocyte-depleting chemotherapy just a few days prior to infusion. This helps de-bulk the malignancy. In this small trial, 82% achieved a CR, 18% did not respond. Of the patients achieving CR, 20% subsequently relapsed. The rest of the patients are being followed and there has been no update. Responding patients all developed CRS, and about 30% of patients were treated with the IL6-receptor antagonist tocilizumab plus corticosteroids to control CRS symptoms.

We have a little more data on CTL019 from NHL studies, specifically r/r CLL. In December 2013, Phase 2 data were presented at ASH (Abstract #873).  Patients with r/r CLL received lymphocyte depleting chemotherapy and then one of several doses of transduced T cells (this is a dose study in that regard, although, cutting to the chase, no dose response was seen, so lets skip over that). Median follow-up for analysis was 3 months at which time the ORR = 40% (20% CR plus 20% PR, with clearance of CLL from the blood and bone marrow and at least a 50% reduction in lymphadenopathy. The toxicity profile was similar to that described above, dominated by treatable CRS. In a small Phase 1 study (Abstract #168), adult patients with r/r NHL including patients with chemotherapy-refractory primary mediastinal B cell lymphoma and DLBCL were enrolled. They received chemo to reduce disease burden and then an infusion of CTL019. 12 of 13 evaluable patients responded (ORR = 93%), the CR = 54% and PR = 38%. These are outstanding responses.

So let’s take a step back. It is a bit hard to compare these regimens head-to-head as they are in different stages of clinical development, the trails are generally small, and in the case of NHL, we have limited data on different types of lymphomas. At the same time we have to consider the larger landscape of therapies available, and ask ourselves how patients will best be served. In the case of the T cell engaging bispecific antibody landscape, it is very clear that robust anti-tumor responses are generated with very low concentrations of antibody. It seems to me very likely that there will be malignancies or subsets of malignancies where this technology will be very useful, including ALL, as we just saw. It will be important to either improve the antibody construction or alter the dose regimen sufficiently to reduce the toxicities associated with the BiTE therapeutic and competing modalities, including the DARTs. Now, people will claim that the tox is not so bad, and that it is only efficacy that matters, and that’s fine, but in the face of competition from CTL019 and other therapeutics, maybe this becomes a differentiating issue. This might also be different for the pediatric population (a critically important population in ALL) versus the adult population. When we look at the CAR T cell transduction technologies we need longer follow-up on the phase 2 studies but certainly anecdotal evidence from smaller trials suggests that some patients will experience long-lasting remissions. If this observational information holds up in the larger clinical trials than the technology will cement itself a place in ALL therapy, and perhaps in other diseases as well. We don’t know yet whether the BiTE therapeutic blinatumomab or the CAR therapeutic CTL019 will have a top-tier profile in NHL. This may change as more data become available, as some of the small studies are very encouraging. One of the interesting twists to the CAR technology is the question of how to make it widely available. In host-institutions (The U Penn system, MD Anderson, NCI) this is a centralized procedure, and in medical institutions world-wide, core patient cell facilities are commonplace. However it is rumored that Novartis at least wants to maintain the core facility model, as they picked up the Dendreon facility in Morris Plains New Jersey (at a bargain price) specifically to support CAR technology, and plan to duplicate those capabilities in Basel and in Singapore. Perhaps yesterday’s pickup of Israel’s Gamida Cell also plays into this centralized cell handling model. None of these complexities will bother the bi-specific therapeutics as these are injectable – that said, I’m not sure anyone will choose walking around with an IV pump for two months if they can avoid it.

So while these therapies and those like them are very potent, we will have to see how patients and providers ultimately use them.

Now, we’ve unfairly used blinatumomab and CTL019 to illustrate what are both pretty large areas of therapeutic development. We’ll come back to talk about the other players in the bispecific antibody and CAR spaces very soon.

stay tuned.

The Cancer Genomic Ecosystem

There have been several important recent advances in our understanding of tumor genomic ecosystems, and these advances have interesting implications for drug discovery in oncology.

The Journal Nature recently published a large data set on gene mutations in 21 distinct tumor types (http://www.nature.com/nature/journal/v505/n7484/full/nature12912.html). Much of the data came from the The Cancer Genome Atlas (TCGA) database, with additional data generated by the study authors. This study is sufficiently powered to uncover significance in several different ways. There is a cluster of mutations that are significant only in the combined tumor analyses, that is, when lumping different tumor types together. Conversely there a large cluster of mutations that are significant only in the analysis of individual tumor types, that is, the significance is lost if you look too broadly. Therefore these are genes that are important for specific tumor types. Finally there is a large cluster of gene mutations that are significant in both the combined analyses and in individual tumor analyses. This complexity of analysis is nicely shown in Figure 3 (http://www.nature.com/nature/journal/v505/n7484/full/nature12912.html#f3).

I spent a fair amount of time staring at this figure and going through the supplemental data (posted online and see also http://www.tumorportal.org/) and there are some results that I found interesting. First, the study confirmed many known cancer-related genes. The study also identified a fair number of new cancer-related genes mutated across or within tumor types, although these were found at the lower levels of significance. This is because they are mutated at a low rate, or the sample size for a particular tumor type was small, or both. The authors are transparent about this, and call for larger studies to increase sample size. This does beg the question as to the rate of gene mutation below which the knowledge is no longer actionable (because there will be so few patients), regardless the data will be critical to understanding tumor pathway biologies. Another interesting question is the extent to which new patterns of gene-mutation will emerge across tumor types, allowing binning (across tumor types) to complement subsetting (within a tumor type). Finally, the data might allow a different type of query, which is to ask which combinations of mutations are found within specific tumor types.

I want mention a few of the more common mutations, because these data held some surprises for me (although some readers know all this already, I’m sure). First, the best known cancer-related gene mutations cluster at the very highest levels of significance both across the 21 tumor types and within specific tumors. This makes sense, as these genes include those that contribute obligate cancer mutations: TP53; PTEN, PIK3CA and PIK3R1; KRAS, BRAF and NRAS; APC; EGFR, etc. There were a few genes in this category that surprised me, not so much because they made the list but because these at first glance appear more common than I had thought. GATA3 is a good example. Mutations in this gene are most commonly see in breast cancer but there are enough mutations in other tumor types to drive significance in the pooled tumor analysis, even though no tumor type other than breast is significantly associated with GATA3 mutations. Examination of the FTL3 data reveal a very similar pattern: mutations are significantly associated with acute myeloid leukemia (AML), as is well known, but also present in other tumor types, notably endometrial tumors and lung adenocarcinomas. When the mutational data across tumor type is pooled, significance is achieved. What are we do with such data? I think the answer perhaps is to simply know that these mutations can occur, and to look for them when typical mutations are missing in a given patient’s tumor. Such cataloging is of course the goal of personalized medicine. The other use of such data is to raise awareness of rare drug resistance mutations that may arise when targeting the major tumor oncogenic pathway in a particular tumor type. Many examples of this phenomena have been described (more on this below).

A different pattern emerges when we look at some other genes that are commonly mutated across tumor types but whose significant in these analyses is lower, due to a lower mutational rate. IDH1 is a good example here, having significant association with AML and glioblastoma multiforma, as is well known, but also with multiple myeloma (MM) and perhaps chronic lymphocytic leukemia (CLL). IDH2 is also most commonly associated with AML, but is present in colorectal cancer at “near significance” (love that fuzzy language). Notably, no other tumor metabolism genes appear in the analysis.

There are same gaps too I think. Looking at those genes that are significantly mutated only in a specific tumor type or types, we find some interesting genes. TGFBR2 has been described as a mutational driver in colorectal cancer, along with SMAD4. In the present analysis SMAD4 and SMAD2 are found to be significantly mutated in colorectal cancer, but the TGFBR2 mutation rate only reaches significance in Head and Neck cancer, although a few mutations do appear in the colorectal cancer data set used. Either the original studies are incorrect, which does not make sense biologically (TGFBR2 protein signals through the SMAD pathway), or this is an example of sampling error. Again, bigger data sets may be needed. Other tumor-type restricted patterns of gene mutation are very well known, such as EZH2 and CARD11 mutations in diffuse large B cell lymphoma (DLBCL). The CARD11 observation is interesting, as these mutations are associated with activation of MYD88, a gene known to be mutated in DLBCL and CLL.

There are lots of examples like these, and the data are easy to see and analyze: this is fun data to play with so have at it (see http://www.tumorportal.org/).

There is much discussion in the paper on new genes identified, and we’ll have to see how much of it is actionable at the drug development level.

That brings us to a different data set. If you go to the tumor portal you can sort by tumor type. Choosing melanoma, a highly mutated cancer, brings forth a whole spectrum of genes. Here’s a screengrab right from the tumor portal site (http://cancergenome.broadinstitute.org/index.php?ttype=MEL)

Screen Shot 2014-02-02 at 11.51.43 AM
In the table above, blue refers to known cancer-related genes, red indicates genes whose function is relevant to cancer biology, and black are novel genes. As many readers know, BRAF mutations are the canonical melanoma oncogenic driver, signaling through the MEK/ERK pathway to drive melanoma cell proliferation, migration and metastasis. Antagonists of the BRAF and MEK proteins have emerged as the best line of defense against melanoma, but its a complicated fight. BRAF inhibitors were developed several years ago, starting with vemurafenib (Roche). Although BRAF inhibition induced responses in many melanoma patients, BRAF resistance mutants and MEK1 escape mutations evolve quickly and patients relapse. Common BRAF resistance mutations include V600E and V600K mutations that confer protection against the first generation drugs. Second generation inhibitors that target the resistance mutations were developed, such as dabrafenib (GSK). In addition, the MEK inhibitor trametinib (GSK, Japan Tobacco) was approved last year for use in treating melanoma. Several weeks ago the combination of these two drugs was granted accelerated approval for the treatment of advanced (metastatic) or unresectable melanoma that is positive for either mutation (V600E or K). This is a great example of cancer genetics=driven drug development in action.

However, other mechanisms of resistance are independent of BRAF mutational status because of additional MEK resistance mutations. These additional mutational strategies were discussed in a series of papers published online on November 21, 2013, in Cancer Discovery. These studies used tumor samples from patients that had relapsed after either BRAF inhibitor of dual BRAF/MEK inhibitor therapy. Mutations were found in the MEK1, MEK2, ERK1, ERK2 pathway and the PI3K, AKT1, PTEN pathway (PTEN is a negative regulator of PI3K signaling to mTOR and AKT1). The papers were reviewed in the January 9th issue of SciBx.

What does this single example tell us about the mutational landscape and drug discovery. First let’s note that some of the resistance mechanisms for melanoma do not show up in the proposed melanoma mutational landscape chart above, that is, these did not appear in the tumor ecosystem until that ecosystem came under selective pressure via drug treatment. This has 2 implications: the first is that the TCGA type overview of tumor mutations is just one source of data and following patients longitudinally as they experience therapy is another source of data. The other implication is that the mutational landscape contains putative additional mechanisms of escape at least in some patients. So using our melanoma example, we see in the table evidence of other potential escape pathways (NRAS, several checkpoint genes, and KIT stand out to me). So how many drugs will any individual patient need to keep a rapidly evolving melanoma under control?

The good news is that drug developers have taken notice and ERK1/2. AKT and PI3K inhibitors of various specificity are under development. The bad news I guess is that this is just one example of how complicated cancer therapy is likely to become. One good question not addressed here is how the immune checkpoint drugs will overlay with targeted therapies, for melanoma and many other tumors. Thats a question for another day.

stay tuned.

ASH13 just around the corner – quick update of CAR-T technology

December 2, 2013.
by Paul D Rennert 

Part 7. Chimeric Antigen Receptor T cell technology (CAR-T) in the treatment of hematopoietic malignancies.

The American Society of Hematology Meeting will take place in New Orleans, December 7 – 10, 2013. The abstracts are available at http://www.hematology.org/Meetings/Annual-Meeting/Abstracts/5810.aspx
Having detoured briefly into myelofibrosis (see parts 6a and b), there are just a few more subjects to try to cover this week. With luck and time, I’ll get through this bit today and then maybe on to lymphoma genetics, we’ll see.
This is from the introduction to Carl June’s seminal 2011 NEJM case report:
“We designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with CD137 … (4-1BB) and CD3-zeta … signaling domains. A low dose (approximately 1.5×10^5 cells per kilogram of body weight) of autologous chimeric antigen receptor–modified T cells reinfused into a patient with refractory … CLL expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo, with delayed development of the tumor lysis syndrome and with complete remission. Apart from the tumor lysis syndrome, the only other grade 3/4 toxic effect related to chimeric antigen receptor T cells was lymphopenia.” (Porter et al. 2011. NEJM 365: 725-733). The therapy induced long term remission is a patient who had failed 4 rounds of rituximab+chemo, and then had failed alemtuzumab, anti-CD52, therapy. Pretty amazing.
The anti-CD19 CAR is essentially an antibody fragment containing a single chain Fv (antigen binding domain). The CD3-zeta chain induces T cell activation and the addition of the 4-1BB cytoplasmic domain ensures prolonged and robust response – 4-1BB is in immune checkpoint activator, and is gaining some favor in its own right in immunotherapy, through the development of agonist anti-4-1BB antibodies. The CAR-T components are introduced to the patient’s own T cells ex vivo via lentivirus transduction, then given back to the patient in hopes of inducing a T cell mediated immune response to the cancer (e.g. a CD19+ CLL). The original case reports were followed for up to 3 years, as reported in Abstract #4162. Of 14 patients treated in the pilot studies, the ORR = 57% (21% CR and 30% PR). 43% of patients did not respond. Of the PR cohort, 40% progressed within 4 months. So that’s about 1/3 of patients with a durable response.
Additional clinical trials have been funded via collaboration with Novartis, who has bought the technology and patents. A few of these are updated at ASH. The CLL and ALL data for patients treated with the anti-CD19 CAR T cells (CTL019) are summarized in Abstract #163. 24 rrCLL patients have been treated using 2 different protocols that vary by the number of CTL019 cells given back to the patient. The response rates were CR = 21%, PR = 29% (so ORR = 50%) and non-responders = 50%. In pediatric ALL (n=14) the CR = 57%; the rest of the patients (43%) either did not respond or progressed. In adult ALL, all 3 patients had a CR (=100%). CRs were always accompanied by in vivo expansion and persistence of CTL019 cells. Tumor cells were eliminated from circulation and also, importantly, from bone marrow. Molecular analyses showed that tumor cells were essentially eliminated in patients with CR – this is defined as minimal residual disease (i.e. not detectable). Additional data specific to these studies are reported in Abstract #873 (CLL) and Abstract #67 (ALL) – the latter study reports persistence up to 15 months. Another group at U Penn reported similarly high RRs in ALL. Lee et al (Abstract #68) report the use of an CD19-CD28-CD3zeta CAR construct to engineer t cells for use in ALL, with an initial CR (n=7) of 71.5%, with other 2 PR responders and 2 non-responders. These are impressive early data from multiple studies.
Steve Rosenberg’s group is also reporting use of anti-CD19 CAR-T cells, these made using a gamma-retrovirus construct to genetically modify the T cells. The technology differs also by use of the CD28 signaling domain instead of 4-1BB, along with CD3-zeta. Of 14 patients with rrCLL, rrDLBCL or primary mediastinal BCL, 36% achieved a CR and 43% a PR, the rest being non-responders or SD. All responders (PR + CR = 79%) were ongoing at the time of abstract submission. The study will be further updated at the meeting. The trial was funded under a CRADA-based collaboration between the NCI and Kite Pharma, a private biotech company.
Given the compelling response rates observed, it is unclear whether the ex vivo selection and expansion methods employed by the MD Anderson group will add benefit. Laurence Cooper and colleagues will present a CD19 CAR technique that utilizes artificial antigen-presenting cells to select the T cell population that is then given following hematopoietic stem cell transplantation in ALL and NHL patients (Abstracts #166 and #4208). Their very early results will be updated at the meeting. Additional efforts targeting CD19 include the trial in rrCLL patients who have received only 1 prior chemotherapy regimen; the idea is that these patients are earlier in the disease course and may have better response rates. It is not possible to tell from the Abstract (#874) if this effort is succeeding, but an update is promised at the meeting.
Turning from the CD19-directed technologies, Carl June’s group is presenting the first clinical data on the use of engineered T cells in multiple myeloma. The T cells are expanded using CD3/CD28 beads (a technology I worked on 20 years ago at Repligen, in the context of HIV therapy) and are engineered to express a modified TCR that recognizes the MM antigens NY-ESO-1/LAGE1. The recognition of this peptide complex is HLA-class restricted, so the patients are screened in advance for responsive HLA haplotypes. The T cells are infused followed depletion and stem cell transplantation, so CAR-T is used here in the context of adjunct therapy. Best response ORR = 100%, although some patients have since progressed. An update will be given at the meeting. Also of note is a trial in which this novel CAR-T therapy is used in a non-transplant setting (no data available yet). An interesting twist is the use of kappa-light chain of surface immunoglobulin expressed on malignant B cells (as opposed to lambda light chain expressed by most normal B cells? – I guess that’s right). A group from Baylor, funded by Celgene, will present Phase 1 data (Abstract #506). Another CAR antigen technology in preclinical development at U Penn targets CD123 for AML (Abstract #143). Preclinical data from the OSU group show that a different MM antigen can be used in the CAR-T setting. Abstract #14 shows that a CS-1 directed CAR works in a mouse xenograft model. I like the straightforward description of the technology: “We successfully generated a specific CS1-CAR construct with a lentiviral vector backbone, sequentially containing a signal peptide (SP), a heavy chain variable region (VH), a linker, a light chain variable region (VL), a hinge, CD28 and CD3epsilon.” Simple, right? Finally, Haso et al from the NIH compare CD22-targeting CAR constructs using different signaling chains (4-1BB v CD28) in preclinical mouse models of ALL, and report superior results using the 4-1BB construct (Abstract #1431). This is nice as they used a humanized mouse model, the NOD/SCID/Common gamma chain KO mouse (NSG), engrafted with a human ALL line. Love the humanized mouse technology, right up my alley.

A persistent theme in the evolving treatment of leukemias and lymphomas is the use of combination therapies. We see a similar trend developing with CAR-T technologies. Paolo Ghia et al combine a CAR directed to CD23 along with low dose lenalidomide treatment using the Rag2/Common gamma chain KO humanized mouse model and cell from CLL patients – nice work (Abstract #4171). A second study evaluated the use of mTOR modulation in the context of CAR-T therapy (Abstract #4488). As this technology continues to advance we can expect to see additional uses of targeted or other therapies in combination.