Category Archives: CAR T cells

Novel Immunotherapeutic Approaches to the Treatment of Cancer: Drug Development and Clinical Application

Our new immunotherapy book has been published by Springer:

http://www.springer.com/us/book/9783319298252

I want to take a moment to acknowledge the stunning group of authors who made the book a success. I’d also like to promote our fund raising effort in memory of Holbrook Kohrt, to whom the volume is dedicated – 5% of net sales will be donated by me, on behalf of all of our authors, the the Cancer Research Institute in New York. So please consider buying the book or just the chapters you want (they can be purchased individually through the link given above.

Now, the authors:

from Arlene Sharpe and her lab (Harvard Medical School, Boston):

Enhancing the Efficacy of Checkpoint Blockade Through Combination Therapies

from Taylor Schreiber (Pelican Therapeutics, Heat Biologics):

Parallel Costimulation of Effector and Regulatory T Cells by OX40, GITR, TNFRSF25, CD27, and CD137: Implications for Cancer Immunotherapy

from Russell Pachynski (Washington University St Louis) and Holbrook Kohrt (Stanford University Medical Center)

NK Cell Responses in Immunotherapy: Novel Targets and Applications

from Larry Kane and Greg Delgoffe (University of Pittsburgh School of Medicine):

Reversing T Cell Dysfunction for Tumor Immunotherapy

from Josh Brody and Linda Hammerich (Icahn School of Medicine, Mt Sinai, NYC)

Immunomodulation Within a Single Tumor Site to Induce Systemic Antitumor Immunity: In Situ Vaccination for Cancer

From Sheila Ranganath and AnhCo (Cokey) Nguyen (Enumeral Inc, Cambridge MA)

Novel Targets and Their Assessment for Cancer Treatment

From Thomas (TJ) Cradick, CRISPR Therapeutics, Cambridge MA):

Cellular Therapies: Gene Editing and Next-Gen CAR T Cells

From Chris Thanos (Halozyme Inc, San Diego) and myself:

The New Frontier of Antibody Drug Conjugates: Targets, Biology, Chemistry, Payload

and a second topic covered by Chris Thanos (Halozyme):

Targeting the Physicochemical, Cellular, and Immunosuppressive Properties of the Tumor Microenvironment by Depletion of Hyaluronan to Treat Cancer

and finally, my solo chapter (and representing Aleta Biotherapeutics, Natick MA and SugarCone Biotech, Holliston MA):

Novel Immunomodulatory Pathways in the Immunoglobulin Superfamily

Please spread the word that all sales benefit cancer research and more specifically, cancer clinical trial development and execution through the Cancer research Institute, and as I said, consider buying the book, or the chapters you want to read.

cheers-

Paul

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!)

The Tumor Ecosystem: some thoughts stirred up at the NY International Immunotherapy meeting

Ecosystems in tumor immunity

The buzzword ‘ecosystem’ has popped like a spring dandelion, and it is now used everywhere in biotech. I’m as guilty as anyone of rapid adoption: the term does capture essential elements of modern biomedical science. Complex and interlaced, with key control nodes at work at all levels – scientific, financial, clinical, commercial – and also dynamic, constantly driving adaptation, and, we hope, innovation. Scientifically the ecosystem connections are easily spotted. CRISPR technology appears in cellular therapies including TCRs and CAR-Ts as we simultaneously learn that the mechanisms of immune checkpoint suppression deployed by tumor cells can derail genetically engineered CAR T cells as readily as normal T cells. Further, those genetically engineered CAR T cells and TCRs owe their existence in large measure to our newly developed ability to sequence tumors at the individual level, with great sensitivity, to identify novel targets. The whole enterprise in turn requires ever faster, cheaper, smaller and more reliable equipment (RNA spin columns and PCR cyclers and cloning kits and desktop sequencers and on and on) and software to handle the data. Enterprises like these in turn drive discovery and innovation.

Within the tumor is another ecosystem – the tumor microenvironment or TME. While TME is a fine term it does blur the notion that this microenvironment is in nearly all cases part of a larger environment and not a walled-off terrarium (perhaps primary pancreatic cancer is an exception, within its fibrous fortress). The tumor ecosystem is a more encompassing term, allowing for the ebb and flow of vastly different elements: waves of immune cells attempting attack, dead zones of necrotic tissue being remodeled, tendrils of newly forming blood vessels, a fog of lactate, a drizzle of adenosine, energy, builders, destroyers, progenitors, phagocytes, parasites, predators. When viewed this way we might wonder how any single drug could treat a tumor, since it is not a singular thing that we attack with a drug, but an ever-changing world we are seeking to destroy.

So it’s hard to do.

Our understanding of the tumor as a complex entity was first informed by pathology, then microscopy, then histology and immunohistochemistry, myriad other techniques and of course genetics, the latter leading to the identification of tumor oncogenes, tumor epigenetics, tumor mutations (referred to above) etc, etc. This ecosystem – that of the cell and it’s mutational hardware and software (genome and exome, or genotype and phenotype) we can hardly claim to understand at all, not matter how many arrows we might draw on a figure for a paper or a review. A few recent examples: we think that tumor cells adapt to immune infiltration in part by engaging CTLA4 expressed on T cells, and when that fails they secrete IDO1, or express PD-L1 on their cell surface, or the tumor cells direct tumor associated cells to do the work for them – maybe monocytes, or macrophages, perhaps fibroblasts, perhaps the endothelium, i.e. the ecosystem. As we know from studying patterns of response to PD-1 and PD-L1 therapeutics, it is hardly so simple, as patients who don’t express the therapeutic target will respond to therapy and patients who express the therapeutic target sometimes, in fact often, will not respond. Which just says we don’t know what we don’t know, but we’ll learn, the hard way, in clinical trials.

The abundance of therapeutic targets and our lack of knowledge is best displayed, with some irony, when we try to show what we do know, as in this figure from our recent paper on immune therapeutic targets:

 Screen Shot 2015-10-07 at 4.29.43 PM

from http://www.nature.com/nrd/journal/v14/n8/full/nrd4591.html 

The picture is static, and fails to represent or visualize complexity (spatial, temporal, random, quantum), and we therefore cannot formulate meaningful hypotheses from the representation. Without meaningful hypotheses we just have observations. With observations we can only flail away hopefully, and be happy when we are right 15 or 20% of the time, as is the case with most PD-1 and PD-L1-directed immune therapeutics in most tumor indications, at least as monotherapies. Why focus so on the PD-1 pathway? Because at least for now, it is the singular benchmark immune therapeutic, stunning really in inducing anti-tumor immunity in subsets of cancer patients.

The success of the “PD-1″ franchise has created another ecosystem, clinical and commercial. The key approved drugs, and the 3 or 4 moving quickly toward approval, are held by some of the world’s largest drug companies (BMS, Merck, Astra Zeneca, Sanofi, Pfizer, Roche). Playing in that sandbox has proven very lucrative for some small companies, and very difficult for many others. There is competition for resources, for patients, for assets and ideas. This has created new niches in the commercial ecosystem, as companies try to differentiate from each other and carve out their own turf – Eli Lilly for example has focused on TME targets, distinguishing itself from other oncology pharmaceutical companies in choice of targets, followed closely of course by smaller contenders – Jounce, with a T cell program directed at ICOS but perhaps more buzz around their macrophage targeting programs, and Surface, whose targets are kept subterranean for now. Tesaro and others are betting on anti-PD-1 antibodies paired rationally with antibodies to second targets in bispecific format. Enumeral is focused on building rationale for specific combinations of immune therapeutics in specific indications, perhaps even for the right subset of patients within that indication. And so on.

It’s complicated.

Lets imagine you are right now pondering an interesting idea, have a small stake, and want to engage this landscape of shifting ecosystems. What might you do?

Lets start with a novel target. You’ve read some papers, woven together some interesting ideas, formulated some useful hypotheses. The protein has been around, maybe there are patents, but not in the immune oncology space, so you think you might have some freedom to operate. Good, best of both worlds. You dig around, find you can buy your target as purified protein, or find a cell line that expresses the target. Now what? Maybe you would hire an Adimab or Morphosys or X-Body to perform an antibody screen. Different companies, varied technologies, but all directed at antibody discovery. My favorite of this group was X-Body, who had an extraordinary platform to screen human antibody sequences and produce antibodies with really stunning activity and diversity. Juno bought them in early 2015, seeking the antibody platform and a TCR screening platform built with the same technology. I hadn’t seen anything quite so powerful until recently, with the introduction of a novel screening technology from Vaccinex. This platform is about as diverse as the X-Body platform (i.e. ~108 Vh sequences and up to 106 Vl sequences; that’s a lot of possible Vh-Vl pairs). What sets them apart is that the entire selection process happens as full length IgG in mammalian cells rather than surrogates like bacteria or yeast.  The net result is a reduction in risk associated with manufacturing.  They’ve used it to power their own clinical programs and have selection deals with some big names including Five Prime Therapeutics. Remarkably (I think) you can access their platform to screen targets for your own, i.e. external, use. Their website explains the platform further (http://www.vaccinex.com/activmab/) but here is one nice sample of their work on FZD4 (a nice target by the way):

 Screen Shot 2015-10-07 at 4.18.17 PM

So now via Vaccinex or someone else you’ve acquired a panel of antibodies that you are ready to test for immune modulatory activity in models that are relevant to immune oncology. You can build out a lab (expensive, time-consuming), find a collaborator with a lab, or find a skilled CRO. The immune checkpoint space was until recently devoid of really focused CRO activity, that is, having diverse modelling capability and careful benchmarking. However, Aquila BioMedical in Scotland, UK placed a solid bet on developing these capabilities around a year ago, and that effort is yielding a terrific suite of assays in both mouse and human cell systems, with multiple readouts, solid benchmarking (e.g. to nivolumab) and careful controls. I like this very much, rich in functional data in a way that a binding assay simply can’t reproduce. Aquila BioMedical seeks to become a driving force in this area, and I like their chances very much: see http://www.aquila-bm.com/research-development/immuno-oncology/ for more information on assays like this IFNgamma secretion assay:

 Screen Shot 2015-10-07 at 4.40.04 PM

Those are clean and robust data.

Now you come to the point of translation to actual use, that is, targeting an indication. How does one proceed? We can probe the TCGA and other databanks for clues, stare at the IHC data online (not recommended), try to cobble together enough samples to do our own analyses (highly recommended but difficult). The goal is to make some educated guesses about two distinct features of the tumor ecosystem: First, is your target expressed on a relevant cell within the ecosystem (tumor, TME, vasculature, draining lymph nodes, etc) in a specific indication or indications, and second, is that ecosystem likely to respond in a clinically meaningful way to manipulation of your target with your antibody?

That second question is a troubling one. What we are really asking is that we deconstruct the ecosystem and look for clues as to how the therapeutic might impact that ecosystem. What are we looking for during deconstruction? Several things, and they are assessed using diverse techniques, adding to the challenge. First, a highly mutated tumor is more likely to respond to immune therapy, and there are several aspects to these phenomena. One is to understand the underlying genomic changes underpinning the oncogenetics of the tumor: what is driving its ability to outcompete the natural surroundings – in our ecosystem analogy perhaps the tumor can be considered starting out life as an invasive species. Genomic sequencing can accurately identify the mutations that support the tumor, but also a potentially vast array of “passenger” mutations that accumulate when tumors turn off the usual mutation repair machinery. Various algorithms exists that can predict which mutated proteins may be immunogenic, that is, capable of stimulating an anti-tumor immune response. Another method designed to determine if an immune response has in fact be stimulated (and has stalled) is to sequence the mRNA expressed in the tumor: exome sequencing. This will reveal, among other things, what the TCR usage is within the tumor, and that in turn will inform you if there is a very narrow anti-tumor response and a broad one, based on the breadth of TCR clonality. That sounds complex, but really isn’t – suffice to say that a broader TCR response in suggestive of immune potential, leashed T cells awaiting clear orders to attack.

More complex is the nature of those orders, and counter-orders. Various methods are being developed to measure the “quality” of the immune response that confronts the tumor. Are key costimulatory molecules present on T cells that would allow stimulation? Are the T cells instead coated with immunosuppressive receptors? Are the tumor cells masked with inhibitory proteins, are they secreting immunosuppressive factors, have they hidden themselves from immune view by downregulating the proteins that T cells “see” (i.e. the MHC complex). What are the cells within the TME doing? Are they monocytes, macrophages, fibroblasts? Where are the T cells? Within the tumor, or shunted off to the side, at the margin between the tumor and normal tissue? Are NK cells present? And on and on it goes. It seems impossible to answer all these diverse questions.

You might try IHC, as mentioned, or targeted PCR for select genes, and Flow Cytometry to look at the distribution of proteins on various cells, or try deep sequencing. All of this is achievable with equipment, labs and people, or by assembling various collaborators, but all in all, quite a challenge. Very recently an interesting company called MedGenome came to my attention, offering a diverse range of services, starting with neo-epitope prioritization and immune response analyses. These offerings, plus some routine IHC, should give most researchers a comprehensive look into tumor ecosystems, informing indication selection, mechanism of action studies and patient profiling. They explain the technology at http://medgenome.com/oncomd/. This is a schematic they sent me showing their neoepitope prioritization scheme that enriches for peptides that trigger anti-tumor immunity, e.g. in a vaccine setting or perhaps in a cellular therapeutic format.

 Screen Shot 2015-10-07 at 4.22.16 PM

It’s a good start on democratizing a suite of assays typically available only to specialty academic labs and well-funded biotechs and pharma companies.

So now you’ve gotten your antibodies (Vaccinex), performed critical in vitro (and soon, in vivo) assays (Aquila Biomedical), and analyzed the tumor immune ecosystem for indication mapping (Medgenome).

You’ll have spent some money but moved quickly and confidently forward with your preclinical development program. Your seed stake is diminished though, and it’s time to raise real money. Now what? … now you face the financial/clinical/commercial ecosystem.

stay tuned.

How far can a CAR get you?

The publication of a paper from scientists at Cellectis (NASDAQ: CLLS) got me thinking. Here is a company with a very interesting idea – to engineer “universal” off-the-shelf CAR T cells by using gene-editing techniques to knock out the elements of an allogeneic T cell that would render it visible to the host immune system. The result – an immunologically “quiet” CAR T cell that you could give to any patient needing the treatment. Sounds good I think. Two things though:

FIRST, some definitions.

A CAR T cell is typically a cancer patient-derived T lymphocyte that is genetically engineered to express a hybrid molecule on its cell surface that can both recognize and then signal the destruction of a cancer cell. The T lymphocyte is most often a cytotoxic T cell (Greek: ‘cyto’ is cell; ‘toxic’ is poison) so this equals a T cell that kills other cells that it sees as foreign to the body with poisons. Cytotoxic T cells express CD8 and can be recognized due to this expression (more on this later).

Gene editing is the use of various technologies to edit (remove in this case) specific genetic elements within a cell (or an organism, a topic for another day). Techniques of interest include those using elements of TALEN, CRISPR or ZFN gene-editing systems.

Allogeneic (Greek: ‘allo’ is other, ‘geneic’ is race) literally means a foreigner, of another race, and biologically means: “denoting, relating to, or involving tissues or cells that are genetically dissimilar and hence immunologically incompatible, although from individuals of the same species”.

So now we understand that what Cellectis is proposing is to genetically alter allogeneic CAR T cells so that, although they are foreign to the patient, they will not be recognized and eliminated. So, “off-the-shelf”, universal, CAR T cells, ready to use. But…

SECOND, to quote a friend of mine: What Problem Are We Solving? In other words, while all of these layers of technology that Cellectus is implementing sound very impressive and appealing, of what utility will they be? Do they address a fundamental and intractable issue in the CAR T field? Should we be excited? Perhaps.

We can step back and ask of the CAR T field: what problems does it have? There are several and they are well known.

1) CAR T cells must be highly selective for the target cancer to avoid unwanted killing of other cells, tissues, organs

2) CAR T cells must proliferate and persist once injected into the patient (i.e. in vivo)

3) Since most CAR T technologies are based on a personalized medicine approach – your cancer attacked by your engineered T cells – there is a fair amount of cell culture to do between harvesting your T cells, altering them (via retroviral or other cell transduction technique), expanding those altered T cells so there are enough to “take” upon injection back into the patient. All of this is expensive, with a typical guess at the price tag of 500K USD

4) CAR T therapy is dangerous (although a bit like Formula One racing – very dangerous and just barely controlled). The danger comes from the potential for off-tumor cell killing but also from tumor lysis syndrome, which happens when large numbers of tumor cells are suddenly killed – all sorts of cellular signals get released and this causes an intense and systemic physiological breakdown – very dangerous, but controllable in an appropriate intensive care unit (so recovery care is also very expensive)

5) CAR T therapy to date has had limited success outside of refractory acute lymphocytic leukemia (ALL). Now, while refractory ALL is a poster child of an indication – intensely difficult to treat, with many pediatric patients – there are about 4000 such patients in the US each year. Commercially, this is limiting.

6) Cancer-specific targets suitable for CAR T technology are very rare.

OK, back to Cellectis, whose lead product targets … refractory ALL. So, what problem are they solving? According to company messaging – control over costs by eliminating the personalzed aspects of the therapy. But we’ve already noted that, right now, that is only one of the critical issues facing CAR T cell technology. That may be enough to grab a piece of the refractory ALL market (and some other indications), and drive valuation for a few years, but a sustainable business, hmmm.  And that we see here is true of all of the CAR T cells targeting the refractory ALL antigen, CD19. Refractory ALL is not a big enough pie for everyone, nor are the niche indications lumped under the non-Hodgkin Lymphoma label, like Diffuse Large B cell lymphoma and Follicular Lymphoma. CAR T companies will get a portion of these patients,  but that will not sustain an industry with a dozen big players. So Cellectis will need more. Of course Cellectis knows this and is looking well past this near term application.

What else happened last week? On the heals of it’s billion dollar 10 year deal with Celgene, JUNO announced the initiation of a CAR T clinical trial employing the impressive sounding “Armoured CAR”. While the term plays nicely to our adolescent/aggressive-minded car culture, what does it actually mean, and, again, what problem are they solving? The armoured CAR T cell is not so much armoured as it is accessorized, carrying a pro-inflammatory cytokine called IL-12 that it expresses as it circulates around the patient looking for tumor cells to kill. Once it finds the tumor, or tumor metastases, the CAR T cell does its usual work, secreting poisons (perforin, granzymes, cytokines, etc) but now, in addition, secreting IL-12, which can amplify the immune response to the tumor via its effects on nearby T and natural killer (NK) cells, including induction of IFN-gamma, enhancement of cell-mediated cytotoxicity and cell proliferation. This approach may work to unlock one of the biggest issues confronting CAR T cell companies – getting solid tumors (as opposed to the “liquid” leukemias and lymphomas) to respond to CAR T therapy at all. So far the results have been disappointing, possibly because the solid tumor microenvironment is so darn immunosuppressive. The JUNO trial is targeting the ovarian cancer antigen MUC16 and will be run at partner hospital, MSKCC. While MUC16 is strongly expressed in ovarian carcinoma (and also pancreatic cancer) the literature indicates normal expression on diverse epithelial cells, including in the lung, the lining of eye and elsewhere. For this reason, as well as the threat of tumor lysis syndrome, JUNO’s armoured CAR also has a off switch that can be activated in case of toxicity. So we are rolling the dice here. Why? Ovarian carcinoma is a large indication with enormous unmet medical need, and pancreatic equally so. Improving patient outcomes in these large and difficult indications would be very notable, and of course, very good business.

Lets look at some data on CAR antigens:

LIST OF SOME ANTIGENS FOR HEMATOLOGIC CANCERS

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THIS SHORT LIST IS REFLECTED IN ONGOING COMPANY-SPONSORED CLINICAL TRIALS

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ACADEMIC CENTERS ARE AHEAD OF THE CURVE, AS IS ALWAYS TRUE IN THIS FIELD

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BUT EVEN HERE, LEUKEMIA AND LYMPHOMA TARGETS DOMINATE (CD19, CD20, CD30, KAPPA Ig, BCMA, ETC)

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AS OF 2014, CD19 TRIALS DOMINATED CLINICAL WORK IN HEMATOLOGIC MALIGNANCIES

THE SOLID TUMOR ANTIGEN FIELD IS SIMILARLY CONSTRAINED

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AND ANOTHER PAGE BELOW

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ALTHOUGH THE DISTRIBUTION OF TRIALS/ANTIGEN IS MORE EVEN, THE NUMBER OF PROTOCOLS IS SMALL (AS OF DATE OF THE REFERENCED PUBLICATION)

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So my hope is that we can engineer CAR T cells with sufficient machinery to “rescue” CAR T technology from the reality of an antigen-poor landscape. The technology is stunning, but I wonder if in the face of such challenges one ought not to look around, and perhaps take another approach. As it turns out, nearly all cellular therapy companies that have taken on the CAR T field have begun to diversify - we’ve been asking what problems we are solving with these clever twists on the basic technology – and this is well worth pursuing. However in the face of a limited pool of targets, lets perhaps consider a technology with a much much larger target list: tumor neoantigens as recognized by T Cell Receptors (TCR). TCR and TIL technologies offer some interesting solutions, and their own unique challenges…

stay tuned.