Category Archives: cancer

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

“Combination Cancer Immunotherapy and New Immunomodulatory Targets” published in Nature Reviews Drug Discovery

Part of the Article Series from Nature Reviews Drug Discovery, our paper hit the press today

Combination cancer immunotherapy and new immunomodulatory targets. Nature Reviews Drug Discovery 14, 561–584. 2015.  doi:10.1038/nrd4591

by Kathleen Mahoney, Paul Rennert, Gordon Freeman.

a prepublication version is available here: nrd4591 (1)

Some Adjacencies in Immuno-oncology

Some thoughts to fill the space between AACR and ASCO (and the attendant frenzied biopharma/biotech IO deals).

Classical immune responses are composed of both innate and adaptive arms that coordinate to drive productive immunity, immunological expansion, persistence and resolution, and in some cases, immunological memory. The differences depend on the “quality” of the immune response, in the sense that the immunity is influenced by different cell types, cytokines, growth factors and other mediators, all of which utilize diverse intracellular signaling cascades to (usually) coordinate and control the immune response. Examples of dysregulated immune responses include autoimmunity, chronic inflammation, and ineffective immunity. The latter underlies the failure of the immune system to identify and destroy tumor cells.

Let’s look at an immune response as seen by an immunologist, in this case to a viral infection:

 immune viral

Of note are the wide variety of cell types involved, a requirement for MHC class I and II responses, the presence of antibodies, the potential role of the complement cascade, direct lysis by NK cells, and the potentially complex roles played by macrophages and other myeloid cells.

In the immune checkpoint field we have seen the impact of very specific signals on the ability of the T cell immune response to remain productive. Thus, the protein CTLA4 serves to blunt de novo responses to (in this case) tumor antigens, while the protein PD-1 serves to halt ongoing immune responses by restricting B cell expansion in the secondary lymphoid organs (spleen, lymph nodes and Peyer’s Patches) and by restricting T cell activity at the site of the immune response, thus, in the tumor itself. Approved and late stage drugs in the immune checkpoint space are those that target the CTLA4 and PD-1 pathways, as has been reviewed ad nauseum. Since CTLA4 and PD-1 block T cell-mediated immune responses at different stages it is not surprising that they have additive or synergistic activity when both are targeted. Immune checkpoint combinations have been extensively reviewed as well.

We’ll not review those subjects again today.

If we step back from those approved drugs and look at other pathways, it is helpful to look for hints that we can reset a productive immune response by reengaging the innate and adaptive immune systems, perhaps by targeting the diverse cell types and/or pathways alluded to above.

One source of productive intelligence comes from the immune checkpoint field itself, and its’ never-ending quest to uncover new pathways that control immune responses. Indeed, entire companies are built on the promise of yet to be appreciated signals that modify immunity: Compugen may be the best known of these. It is fair to say however that we remain unclear how best to use the portfolio of checkpoint modulators we already have in hand, so perhaps we can look for hints there to start.

New targets to sift through include the activating TNF receptor (TNFR) family proteins, notably 4-1BB, OX40, and GITR; also CD40, CD27, TNFRSF25, HVEM and others. As discussed in earlier posts this is a tricky field, and antibodies to these receptors have to be made just so, otherwise they will have the capacity to signal aberrantly either because the bind to the wrong epitope, or they mediate inappropriate Fc-receptor engagement (more on FcRs later). At Biogen we showed many years ago that “fiddling” with the properties of anti-TNFR antibodies can profoundly alter their activity, and using simplistic screens of “agonist” activity often led to drug development disaster. Other groups (Immunex, Amgen, Zymogenetics, etc) made very similar findings. Careful work is now being done in the labs of companies who have taken the time to learn such lessons, including Amgen and Roche/Genentech, but also BioNovion in Amsterdam (the step-child of Organon, the company the originally created pembrolizumab), Enumeral in Cambridge US, Pelican Therapeutics, and perhaps Celldex and GITR Inc (I’ve not studied their signaling data). Of note, GITR Inc has been quietly advancing it’s agonist anti-GITR antibody in Phase 1, having recently completed their 8th dose cohort without any signs of toxicity. Of course this won’t mean much unless they see efficacy, but that will come in the expansion cohort and in Phase 2 trials. GITR is a popular target, with a new program out of Wayne Marasco’s lab at the Dana Farber Cancer Institute licensed to Coronado and Tg Therapeutics. There are many more programs remaining in stealth for now.

More worrisome are some of the legacy antibodies that made it into the clinic at pharma companies, as the mechanisms of action of some of these agonist antibodies are perhaps less well understood. But lets for the sake of argument assume that a correctly made anti-TNFR agonist antibody panel is at hand, where would we start, and why? One obvious issue we confront is that the functions of many of these receptors overlap, while the kinetics of their expression may differ. So I’d start by creating a product profile, and work backward from there.

An ideal TNFR target would complement the immune checkpoint inhibitors, an anti-CTLA4 antibody or a PD-1 pathway antagonist, and also broaden the immune response, because, as stated above, the immune system has multiple arms and systems, and we want the most productive response to the tumor that we can generate. While cogent arguments can be made for all of the targets mentioned, at the moment 4-1BB stands as a clear frontrunner for our attention.

4-1BB is an activating receptor for not only T cells but also NK cells, and in this regard the target provides us with an opportunity to recruit NK cells to the immune response. Of note, it has been demonstrated by Ron Levy and Holbrook Khort at Stanford that engagement of activating Fc receptors on NK cells upregulates 4-1BB expression on those cells. This gives us a hint of how to productively combine antibody therapy with anti-4-1BB agonism. Stanford is already conducting such trials. Furthermore we can look to the adjacent field of CAR T therapeutics and find that CAR T constructs containing 4-1BB signaling motifs (that will engage the relevant signaling pathway) confer upon those CAR T cells persistence, longevity and T cell memory – that jewel in the crown of anti-tumor immunity that can promise a cure. 4-1BB-containing CAR T constructs developed at the University of Pennsylvania by Carl June and colleagues are the backbone of the Novartis CAR T platform. It is a stretch to claim that the artificial CAR T construct will predict similar activity for an appropriately engineered anti-4-1BB agonist antibody, but it is suggestive enough to give us some hope that we may see the innate immune system (via NK cells) and an adaptive memory immune response (via activated T cells) both engaged in controlling a tumor. Pfizer and Bristol Myers Squibb have the most advanced anti-4-1BB agonist antibody programs; we’ll see if these are indeed best-in-class therapeutics as other programs advance.

Agonism of OX40, GITR, CD27, TNFRSF25 and HVEM will also activate T cells, and some careful work has been done by Taylor Schreiber at Pelican to rank order the impact of these receptors of CD8+ T cell memory (the kind we want to attack tumors). In these studies TNFRSF25 clearly is critical to support CD8 T cell recall responses, and may provide yet another means of inducing immune memory in the tumor setting. Similar claims have been made for OX40 and CD27. Jedd Wolchok and colleagues recently reviewed the field for Clinical Cancer Research if you wish to read further.

Looking again beyond T cells another very intriguing candidate TNFR is CD40. This activating receptor is expressed on B cells, dendritic cells, macrophages and other cell types involved in immune responses – it’s ligand (CD40L) is normally expressed on activated T cells. Roche/Genentech and Pfizer have clinical stage agonist anti-CD40 programs in their immuno-oncology portfolios. Agonist anti-CD40 antibodies would be expected to activated macrophages and dendritic cells, thus increasing the expression of MHC molecules, costimulatory proteins (e.g. B7-1 and B7-2) and adhesion proteins like VCAM-1 and ICAM-1 that facilitate cell:cell interactions and promote robust immune responses.

I mentioned above that interaction of antibodies with Fc receptors modulates immune cell activity. In the case of anti-CD40 antibodies, Pfizer and Roche have made IgG2 isotype antibodies, meaning they will have only weak interaction with FcRs and will not activate the complement cascade. Thus all of the activity of the antibody should be mediated by it’s binding to CD40. Two other agonist anti-CD40 antibodies in development are weaker agonists, although it is unclear why this is so; much remains to be learned regarding the ideal epitope(s) to target and the best possible FcR engagement on human cells. Robert Vonderheide and Martin Glennie tackled this subject in a nice review in Clinical Cancer Research in 2013 and Ross Stewart from Medimmune did likewise for the Journal of ImmunoTherapy of Cancer, so I won’t go on about it here except to say that it has been hypothesized that crosslinking via FcgRIIb mediates agonist activity (in the mouse). Vonderheide has also shown that anti-CD40 antibodies can synergize with chemotherapy, likely due to the stimulation of macrophages and dendritic cells in the presence of tumor antigens. Synergy with anti-CTLA4 has been demonstrated in preclinical models.

One of the more interesting CD40 agonist antibodies recently developed comes from Alligator Biosciences of Lund, Sweden. This antibody, ADC-1013, is beautifully characterized in their published work and various posters, including selection for picomolar affinity and activity at the low pH characteristic of the tumor microenvironment (see work by Thomas Tötterman, Peter Ellmark and colleagues). In conversation the Alligator scientists have stated that the antibody signals canonically, i.e. through the expected NF-kB signaling cascade. That would be a physiologic signal and a good sign indeed that the antibody was selected appropriately. Not surprisingly, this company is in discussion with biopharma/biotech companies about partnering the program.

Given the impact of various antibody/FcR engagement on the activity of antibodies, it is worth a quick mention that Roghanian et al have just published a paper in Cancer Cell showing that antibodies designed to block the inhibitory FcR, FcgRIIB, enhance the activity of depleting antibodies such as rituximab. Thus we again highlight the importance of this sometimes overlooked feature of antibody activity. Here is their graphical abstract:

 graphical abstract

The idea is that engagement of the inhibitory FcR reduces the effectiveness of the (in this case) depleting antibody.

Ok, moving on.

Not all signaling has to be canonical to be effective, and in the case of CD40 we see this when we again turn to CAR T cells. Just to be clear, T cells do not normally express CD40, and so it is somewhat unusual to see a CAR T construct containing CD3 (that’s normal) but also CD40. We might guess that there is a novel patent strategy at work here by Bellicum, the company that is developing the CAR construct. The stated goal of having a CD40 intracellular domain is precisely to recruit NF-kB, as we just discussed for 4-1BB. Furthermore, the Bellicum CAR T construct contains a signaling domain from MYD88, and signaling molecule downstream of innate immune receptors such as the TLRs that signal via IRAK1 and IRAK4 to trigger downstream signaling via NF-kB and other pathways.

Here is Bellicum’s cartoon:

 cidecar

If we look through Bellicum’s presentations (see their website) we see that they claim increased T cell proliferation, cytokine secretion, persistence, and the development of long-term memory T cells. That’s a long detour around 4-1BB but appears very effective.

The impact of innate immune signaling via typical TLR-triggered cascades brings us to the world of pattern-recognition receptors, and an area of research explored extensively by use of TLR agonists in tumor therapy. Perhaps the most notable recent entrant in this field is the protein STING. This pathway of innate immune response led to adaptive T cell responses in a manner dependent on type I interferons, which are innate immune system cytokines. STING signals through IRF3 and TBK1, not MYD88, so it is a parallel innate response pathway. Much of the work has come out of a multi-lab effort at the University of Chicago and has stimulated great interest in a therapeutic that might be induce T cell priming and also engage innate immunity. STING agonists have been identified by the University of Chicago, Aduro Biotech, Tekmira and others; the Aduro program is already partnered with Novartis. They published very interesting data on a STING agonist formulated as a vaccine in Science Translational Medicine on April 15th (2 weeks ago). Let’s remember however that we spent several decades waiting for TLR agonists to become useful, so integration of these novel pathways may take a bit of time.

This emerging mass of data suggest that the best combinations will not necessarily be those that combine T cell immune checkpoints (anti-CTLA4 + anti-PD-1 + anti-XYZ) but rather those that combine modulators of distinct arms of the immune system. Recent moves by biopharma to secure various mediators of innate immunity (see Innate Pharma’s recent deals) and mediators of the immunosuppressive tumor microenvironment (see the IDO deals and the interest in Halozyme’s enzymatic approach) suggest that biopharma and biotech strategists are thinking along the same lines.

Hif, Hif, Hif, Hike!

Football season. Except is was 85 degrees here in Massachusetts today and felt more like mid-July. Thankfully there is “fallball” (fall softball season) so we got to enjoy that instead.

We got a good look at the convergence of immune and pathogenic pathways in this week’s issues of Science and Nature. Two papers in Science identify metabolic adjustments made by monocytes and macrophages that may support innate immune memory. The same pathway is hijacked by some tumors to redirect macrophage activity, as described in a very nice Nature paper.

Cheng et al from the Netea lab in The Netherlands used a b-glycan derived from the pathogenic fungus Candida albicans to “educate” monocytes, mimicking an infection event (Cheng et al). C. albicans b-glycan, a carbohydrate moiety, binds the dectin-1 receptor on monocytes, macrophages and other innate immune cells and induces cell activation. This activation response included changes in the epigenetic profile of the cells. The epigenetic signature suggests that monocytes “trained” by exposure to b-glycan alter their metabolic status, in particular by elevating aerobic glycolysis with increased glucose consumption. Key glycolysis enzymes such as hexokinase and pyruvate kinase were epigenetically upregulated, supporting the shift to glycolysis. Aerobic glycolysis produces lactic acid and increased lactate production was also observed: these b-glycan activated monocytes have really committed to this metabolic state.

This metabolic shift was mediated by signaling from dectin-1 to AKT and mTOR. This signaling pathway is responsible for many cellular responses, including induction of HIF-1α (hypoxia-inducible factor–1α). In turn, HIF-1α-dependent signals turn on many genes needed to adapt to the metabolic shift. This is a common tactic in hypoxic conditions for example. Blockade of any steps in the pathway abrogated the metabolic shift and prevented “trained immunity”. The role of epigenetic components in induction of the metabolic shift in monocytes was demonstrated using the epigenetic inhibitors methylthioadenosine, a methyltransferase inhibitor, and givinostat, a class I/II histone deacetylase (HDAC) inhibitor.

A second paper from the same group dives deeper into the monocyte to macrophage differentiation program (Saeed et al). Short-term culture of monocytes with LPS (a TLR4 agonist) or b-glycan yielded distinct macrophage populations. Serum culture (mimicking the homeostatic state) yielded yet a 3rd type. This paper is a technical grind so have at it if you want all the complex details. I was interested in the conclusions. As in the b-glycan study referenced above, LPS and serum culture induced distinct epigenetic signatures. Genome-wide mapping of histone modifications identified epigenetically marked clusters – that is, reactive regions of the genome. Within these clusters we would expect to find transcription regulatory regions, and indeed four such clusters were differentially modulated when monocytes were exposed to LPS or b-glucan. Targets within these clusters include G protein–coupled receptors, protein kinases, and additional epigenetic enzymes. The authors therefore affirm the “trained immunity” state identified in the first paper and now elucidate a macrophage “exhaustion” phenotype induced by short term exposure to LPS. By my reading of the paper it appears both of these induced phenotypes are extensions of the M-CSF/serum induced homeostatic differentiation profile. This makes sense, as monocytes are recruited from circulation so they can differentiate into macrophages at sites of inflammation, a process that optimally requires M-CSF.

In the first paper the production of lactic acid and lactate was noted as a consequence of differentiation to the “trained”, glycolysis-driven phenotype. Turning now to a paper in Nature from Medzhitov and colleagues at Yale, we find ourselves confronting a chicken and egg story (Colegio et al). In this study the crosstalk of tumor-resident macrophages and “client” tumor cells was examined. The premise is that tumor-associated macrophages (TAMs) perform key homeostatic functions that support tumor growth and survival. In this case it appears that the tumor microenvironment subverts macrophage function via production of lactic acid. There are important differences in the study designs – the papers published in Science use short-term culture techniques while the Nature paper relies on in vivo tumor/macrophage development in syngeneic mouse models – but with this caveat in mind the convergence of pathway data is striking. TAMs sorted from implanted lung (LLC) or melanoma (B16-F1) tumors expressed high levels of VEGF and arginase 1 (Arg1) mRNA, accounting for nearly all of the expression of these proteins in tumor samples. Strikingly, tumors induced macrophage expression of VEGF via stabilization of HIF1a in a manner that did not require hypoxia. This is interesting as it identifies a pathway by which tumor cells can stimulate angiogenesis (blood vessel formation) via VEGF and Arg1 prior to a hypoxic challenge. The soluble tumor cell effector capable of turning on this pathway was identified as … lactate. Here it is worth quoting from the paper:

“Warburg observed that cancer cells preferentially perform aerobic glycolysis: that is, they convert most glucose molecules into lactate regardless of the amount of oxygen present. Furthermore, the eponymous Warburg effect is also observed in most cells undergoing rapid proliferation. It has been hypothesized that aerobic glycolysis is conducive to cell proliferation because, despite the consequent reduction in ATP production, aerobic glycolysis produces metabolic precursors, such as lactate, for biosynthetic pathways, and these precursors may be the limiting factor during rapid cell proliferation”

The suggestion here is that tumor cells are going a step further in order to ensure that their supportive microenvironment, which includes TAMs, step in line. Lactate is taken up by TAMs via specific cell surface receptors (the monocarboxylate transporters) and the effect is potentiated by acidic pH (from all the lactic acid) and perhaps requires other mediators such as M-CSF. Once all is said and done the TAMs are surviving and thriving using the same machinery as the tumor cells.

From the drug development perspective it is probably worth asking whether AKT and mTOR inhibitors impact TAM activity in the tumor microenvironment (perhaps someone already has). Conversely, one might speculate on the impact of such inhibitors of macrophage responses to infection. More selectively, I suspect there is a clever way of targeting the epigenetic responses to derail the TAM phenotype and disrupt the tumor-supportive microenvironment while either simultaneously targeting the tumor, as in a combination therapy setting with a therapeutic that targets tumor biology directly. Also, in the era of immune checkpoint therapeutics I wonder if there isn’t some signal to “wake-up” these “trained” macrophages and have them turn on their clients – the tumor cells.

A few other questions:

How is the macrophage glycolysis pathway maintained once initiated by exposure to tumor derived lactate? There must be a feedback mechanism, perhaps similar to the one used by “trained” macrophages?

Do the HIF2-dependent tumors (some renal cell carcinomas for example) also hijack resident TAMs in the same manner, or different?

The tumor microenvironment includes tumor-associated fibroblasts – are these also impacted by exposure to lactic acid?

If there is intimate cross-talk between the macrophage and it’s client (a tumor cell) then disabling that conversation at the level of the macrophage (and other stromal cells) should be therapeutic – or will the tumor (in this case) simply adapt? Remember that in this setting the epigenetic changes are not necessarily addictive (oncogenic).

interesting stuff to consider in this new era of combination therapies….

stay tuned

Immune Checkpoint Therapeutics – Part 3: a) Innate immunity targets and b) IDO

Lets quickly set the stage. In part 1 we reviewed the CTLA4 and PD-1 pathways and therapeutics targeting these pathways. In part 2 we brought in a few more targets within the immunoglobulin superfamily: LAG-3, TIM-3, B7-H3, B7-H4, and very briefly TIGIT and VISTA. Then we reviewed therapeutics being developed to target proteins in the TNF receptor (OX40, CD40, 4-1BB, CD27, GITR) and ligand (CD70) superfamilies.

 While some of these pathways play a role in the innate immune system, they are all more closely aligned with the adaptive immune system. The innate immune system is hard-wired, triggering a rapid immune response, while the adaptive immune system relies on the orchestrated interaction of antigen presenting cells (dendritic cells, macrophages, etc) with T cells and B cells, leading to a robust immune response and, importantly, immunologic memory, i.e. memory of “that which” induced the immune response in the first place. Memory underlies immunity, as in “I am immune to…”, and is the basis for vaccination. In the context of oncology, memory allows sustained immune response to cancer cells over time.

In most immune responses to pathogens, both the innate and adaptive arms of the immune system are critical for efficient and sustained protection. We are learning from work with innate immune checkpoint therapeutics that the same may hold true for anti-tumor immunity.

One of the critical cells in the innate immune response is the natural killer (NK) cell. The name tells their story, as these cells are primed to disgorge toxins onto pathogens and pathogen-infected cells or tissue. Recently, an adaptive immune role for NK cells has been described, a finding that only increases the importance of this cell type. The activity of NK cells is controlled to a great degree by the killer inhibitory receptors.

Killer inhibitory receptors come in 2 flavors: killer cell immunoglobulin-like receptors (KIRs) and C‑type lectin transmembrane receptors. There are many different proteins within these groups, with various functions. KIRs are normally kept quiescent through interaction with cell surface HLA proteins. Both HLA and KIR have variable genotypes, and not all are compatible. Further complicating the picture is the existence of multiple KIR family proteins. We are just beginning to understand the expression and regulation of these proteins in the context of tumor biology, and choosing which of the 20 or more receptors to target remains an open question. However, some progress has been made.

Innate Pharmaceuticals (OTC: IPHYF) is taking the first steps in exploring NK cell therapeutics. The company’s lead drug is lirilumab, a first-in-class anti-KIR antibody that specifically recognizes the KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The antibody increases NK cell–mediated killing of HLA-C–expressing tumor cells. A phase II study of lirilumab in 150 patients with acute myeloid leukemia is in progress. Lirilumab has been licensed by Bristol-Myers Squibb. BMY is sponsoring Phase 1 trials of lirilumab in combination with ipilimumab (anti-CTLA4) and nivolumab (anti-PD-1) in patients with solid tumors. These early lirilumab trials will start to read out over the next 2 years and the data will generate considerable interest.

Innate Pharma’s expertise in NK cell biology has produced several other programs. KIR3DL2 is another KIR family protein that is highly expressed in aggressive forms of cutaneous T cell lymphoma. Innate has developed an anti-KIR3DL2 antibody that has cytotoxic activity against cutaneous T cell lymphoma in vivo (mouse models) and ex vivo (primary patient cells). They gave an update at the T cell lymphoma forum in January:                                                  (http://www.innate-pharma.com/sites/default/files/tclforum2014_iph41_1.pdf). The company will file an IND this year. Innate Pharma also has several interesting earlier stage programs.

One of the reasons this is an exciting pathway is reflected in the combination therapy approaches mentioned, in which a boost in T cell activity is combined with a boost in NK cell activity. Other combinations worth considering include KIR inhibition with 4-1BB agonist activation. Ron Levy (Stanford) has described the transient expression of 4-1BB on NK cells that are exposed to tumor cells coated with antibody (e.g. lymphoma cells coated with rituximab or breast cancer cells coated with herceptin). This suggests that the presence of activating antibody induces 4-1BB expression on NK cells. If so, and if one could get the timing right, very potent combinations can be considered. One might also consider such mechanisms in the development of bispecific therapeutics. Obviously there are critical considerations here – one is toxicity (will the combination be safe) and second is timing, if the antibodies are administered separately.

Another critical cell in the innate immune response is the macrophage, that has an ancient and fundamental role in the clearance of dead, dying and infected cells from the body. Galectin-3 is an anti-apoptotic protein that is widely expressed, and may regulate apoptosis of tumor cells and tumor-associated macrophages. There are also reports that galectin-3 can regulate macrophage/T cell interaction, although the mechanism of action is unclear.

I honestly don’t know what to make of therapeutics targeting galectin-3 as this is a very promiscuous protein. That does not mean such therapeutics won’t be useful, it is just a point of risk assessment. Galectins bind to sugar moieties that are hanging off of proteins or bound to extracellular matrix (ECM). In this sense galectins are “sticky”, capable of binding distinct targets. There are about 15 different human galectins, and to add to the fun, some of these can oligomerize with each other. 

Specificity is imposed by the preference of galectins for sugars having a terminal galactose. Further specificity is imposed by the preference of different galectins for different sugars adjacent to the terminal galactose in the oligosaccharide chain. Oligomerization allows galectins to support cell-cell and cell-matrix interactions, either of which can induce cell signaling. Galectins are most highly expressed in macrophages but are pretty ubiquitous. Galectins, including galectin-3, are proposed to play a role in diverse diseases, including asthma, fibrosis, cardiovascular disease, inflammatory disease and oncology. Well, that gives me pause, as a lot of biology is involved here. Already some big bets on galectin-3 have failed, such as BG Medicine’s cardiovascular disease program.

Galectin Therapeutics Inc (NASDAQ: GALT) has several galectin-targeting programs in development for liver fibrosis (notably, non-alcoholic steatohepatitis aka NASH) and oncology. GR-MD-02 and is a polysaccharide polymer that binds to galectin-3 and galectin-1, with higher affinity for galectin-3.

Mouse models have demonstrated that GR-MD-02 plus ipilimumab enhances T-cell function and anti-tumor responses greater than either agent alone. The data suggest a role of GR-MD02 in promoting the CD8+ T cell response to tumor antigens. GR-MD-02 is in Phase 1 testing to establish dose and tolerability. Providence Portland Medical Center has filed an IND to test GR-MD-02 plus ipilimumab in a Phase 1B study, enrolling patients with metastatic melanoma. Galecto Biotech has also developed galectin-3 inhibitors, these are preclinical stage programs.

The mechanism of action remains unclear. This remains the biggest issue with galectin-3 drug development – we really have no idea how it the system works. Galectin Therapeutics has built mechanism of action studies into the oncology clinical trials, which is a good step forward. To be clear, this is not to suggest that galectin-3 is not a good target, but if it is it will be nice to know more about the mechanism of action.

Phosphatidylserine (PS) can be classified as a pattern recognition target, making its expression a component of innate immunity regulation. This may or may not have anything to do with the mechanism of action of Peregrine Pharmaceuticals (NASDAQ: PPHM) anti-PS antibody bavituximab. Bavituximab is being tested in multiple solid tumor settings. It’s an oversimplification, but let’s define PS as an immunosuppressive molecule. PS is an inner membrane protein that is “flipped” to the cell membrane surface in cells undergoing apoptosis. PS is also a component of ECM, and is found on the surface of activated cells, such as activated T cells, although at much lower levels than on apoptotic cells. Tumor cells can express a lot of PS on their cell surface, and this is thought to provide protection from immune cells because the immune system will often ignore cells undergoing normal (i.e. programmed) cell death, which always occurs by apoptosis. Bavituximab may act to block PS and therefore allow an immune response to cancer cells.

Somewhat amazingly, a Phase 2 trial of bavituximab in advanced NSCLC yielded positive results on PFS and more importantly, improved OS, from less than 6 months to 12 months. This was a second line study in patients who had failed chemotherapy. FDA granted fast track designation for bavituximab for second line NCSLC even though the drug had failed as a first-line therapy in Phase 2. In other words, there is some disconnect in the results. Peregrine is currently testing bavituximab in phase 3 trials of advanced NSCLC as second-line therapy.

Again what we have here is a drug with a poorly defined mechanism of action. This will not matter if the Phase 3 results are positive, but it will complicate rationale design of co-therapies. One wonders how this drug might be paired with another immunotherapeutic, or even with a targeted therapy like ramucirumab (anti-VEGFR2 from Eli Lilly), which just reported out positive Phase 3 data in a very similar patient population.

The SIRPalpha/CD47 system is another fundamental component of blocking innate immune responses, in this case by macrophages. There are a few companies trying to utilize antibodies to CD47 or to SIRPalpha (CD172a). Expression of CD47 on tumors provides a shutdown signal to macrophages via CD127a, that basically prevents phagocytosis. Of note, cancer stem cells also utilize CD47 to escape the attention of macrophages. The approach is effective in models of human tumors in mouse, and there is a report of synergy with rituximab in a non-hodgkin’s lymphoma model. Irving Weissman and colleagues at Stanford and Oxford Universities will initiate Phase 1 testing of an anti-CD47 antibody this year. Several very small biotechs have begun working on antibodies to CD47 and CD127a.

There are other targets in this class although most are even earlier in development. TGFbeta is a good example of a target around which there is a lot of early activity in oncology (among other things). There are also, scattered in the literature, hints as to the next wave of immune checkpoint targets emerging.

OK, on to IDO.

Indoleamine 2,3-dioxygenase (IDO1) is an IFN-inducible enzyme that catabolizes the essential amino acid tryptophan from the cellular microenvironment. IDO1 is induced by interferon gamma, and is therefore elevated in settings of innate or adaptive immune responses. Elevated tryptophan degradation stops T cell activation and induces T-cell apoptosis. Furthermore, generation of biologically active tryptophan metabolites are associated with the induction of immune tolerance. Therefore IDO expression in APCs or the tumor cells is a potential mechanism by which the immune tolerance to tumor antigens is induced.

Incyte Corporation (NASDAQ: INCY) has developed a clinical stage oral IDO1 inhibitor. INCB24360 is currently in Phase 1 and 2 for metastatic melanoma in combination with ipilimumab and as monotherapy for ovarian cancer. Incyte presented PK/PD and tolerability data from a phase 1 trial at ASCO in 2012, and monotherapy data in advanced disease was presented at ASCO in 2013. In general that data showed only modest efficacy, with some patients being able to maintain stable disease, and these were late-stage patients. Results from the ipilimumab combo trial should be available this year, probably at ASCO.

Earlier this month Incyte entered into a collaboration with Merck to evaluate INCB24360, in combination with Merck’s anti-PD-1 antibody MK-3475. The first trial is a Phase 1/2 study in advanced or metastatic cancers including melanoma and NSCLC. The trial is designed to set appropriate doses and then randomize to MK-3475 with or without INCB24360. NewLink Genetics (NASDAQ: NLNK) is also in phase 2 with indoximod, a tryptophan analogue inhibitor of IDO1. The mechanism of action of this drug is not well understood, as it does not appear to influence free tryptophan levels. NewLink presented Phase 1 data at ASCO last year, showing good tolerability and some early signals of clinical activity.

Several companies have preclinical inhibitors of IDO and related enzymes. Privately held iTeos Therapeutics has preclinical drug discovery programs on IDO1 and Tryptophan 2,3-dioxygenase (TDO2), a second  key enzyme in tryptophan catabolism. ToleroTech is developing an siRNA approach to targeting IDO. Other programs are no doubt underway in pharma and biotech.

Recall that in Part 1 we identified five or six distinct areas of immunotherapeutic development, and immune checkpoint inhibition was only one of these. We’ve stretched the definition a little bit to allow coverage of some of the targets in this last bit, Part 3. We’ve also deliberately skipped over the Toll-Like Receptor (TLR) field as these agents may best be viewed in the context of tumor vaccines and adjuvants.

There are compelling questions to consider.

1- how the heck will “healthcare” fold all these therapeutics together in a way that makes the best sense for individual patients?

2- how are companies coping with the overload of targets and modalities? How do you build a credible pipeline?

3- what modality is best suited for which tumor types? plus we’ll need biomarkers to sort out responses to these new therapeutics and combinations- what are they?

4- can we identify gaps that can be filled by new targets, perhaps new companies?

5- can we find hidden drug development gems already out there waiting to be licensed or bought?

6- what are the pivotal data coming up that will move companies and their stock prices?

7- can we foresee changes in clinical practice that will support some therapeutic modalities, and doom others?

8- where are the transformative therapies that will change the clinical landscape

We have spent a lot of time working on this competitive landscape, and have arrived at some very interesting answers (and lots more questions).

If we can help you or your company work through some of these issues feel free to connect with us via the Contact page.

As always you may leave a comment or reach us at rennertp@sugarconebiotech.com. Please follow us @PDRennert.

What will we cover next? Honestly, I don’t know yet.

stay tuned.

Immunotherapy: Companies Chasing Immune Checkpoint Therapeutics

Excitement continues to build in the Immunotherapeutic drug development space following a recent flurry of deals. In the most recent, we saw Novartis acquire Costim Pharma(http://www.fiercebiotech.com/story/novartis-beefs-its-cancer-immunotherapy-pipeline-biotech-buyout/2014-02-17).

The deal making begs the question as to what, and who, is next. The immunotherapeutic space is very large and diverse so it’s important to focus. Lets start by defining the space broadly, using the following categories:

1) Immune checkpoint modulators. These are therapeutics specifically designed to alter the way the immune system interacts with a tumor. This field is exemplified by the anti-CTLA4 antibody ipilimumab (Vervoytm), from Bristol Myers Squibb and the anti-PD-1 antibody MK-3475, from Merck.

2) Tumor depleting antibodies. These are antibodies with inherent or engineered cell-killing (cytotoxic) activity. The first generation of cytotoxic antibodies is best illustrated by the anti-CD20 antibody rituximab (Rituxantm) from Roche. Engineered antibodies have increased cytotoxic activity (ofatumumab from GlaxoSmithKline being an important example). Other formats include bispecific antibodies that recognize 2 different tumor proteins (antigens) simultaneously. All of these antibodies act by recruiting the immune system to kill cells that they have bound. The antibodies do this by activating cell killing NK and CD8+ T cells and by activating the complement cascade.

3) Bispecific antibodies and fusion proteins that recruit T cells, NK cells or dendritic cells and bind tumor antigen, simultaneously. These molecules function similarly to tumor depleting antibodies, but have the added activity of specifically engaging relevant immune cell types.

4) Modified T cells. Made famous by the CAR-T (CAR-19) technology developed by Carl June at U Penn, this technology uses genetic engineering to take a patients T cells and repurpose them for high impact tumor cell recognition and killing.

5) Cancer vaccines. Exemplified by Provengetm from Dendrion, these are techniques designed to induce an immune response to the tumor by immunizing with tumor antigens along with immune stimulants. There are ex vivo approaches (like Provenge) and in vivo approaches.

Note that we have left out the antibody-drug conjugates (ADC) and radiolabeled antibodies since they theoretically do not require the immune system to attack the tumors. In this class the cytotoxic drug or radioactive payload is brought to the tumor by the antibody.

Today we will only discuss novel and next generation therapeutics in the first class: immune checkpoint modulators.

The field has been dominated by discussion of the clinical stage drugs being developed to target the CTLA4 and PD-1 pathways. Blocking CTLA4 shuts down this T cell inhibitory pathway by preventing interaction of CTLA4 with it’s ligands, called CD80 and CD86, which are expressed on B cells, dendritic cells, macrophages and related cell types. This then allows these ligands to productively interact with the stimulatory receptor CD28, also expressed on T cells, thereby promoting T cell activation. In the case of the PD-1 pathway, blocking PD-1 or its ligand (PD-1L) prevents another inhibitory pathway on T cells, although in this case the ligand is often found overexpressed on tumor cells, that is, this is an active pathway for immune evasion.

Just for review, these are the key late stage clinical therapeutics:

drug

target

phase

company

 
     ipilimumab      CTLA4      approved      Bristol Myers Squibb
     nivolumab      PD-1      3      Bristol Myers Squibb
     MK-3475      PD-1      3      Merck
     MPDL3280A      PD-L1      3 (not yet recruiting)      Roche/Genentech

These are all monoclonal antibodies (mAbs). The approval and phase 3 designations refer to advanced metastatic melanoma however all of these drugs are in multiple clinical trials for many tumor types. Of equal interest are the ipilimumab/nivolumab co-therapy trials also underway.

So these are very advanced drugs. Earlier clinical trials with agents targeting the CTLA4 and PD-1 pathways are shown here:

drug

target

phase

company

       
     tremelimumab    CTLA4   1 and 2, in various solid     tumors      Astra Zeneca          (AZN)/Medimmune
     MEDI4736    PD-L1   1 and 1/2 in various solid   tumors      AZN/Medimmune
     pidilizumab    PD-1   2: hematological cancers,   solid tumors      CureTech Ltd
     BMS-9365569    PD-L1   1: multiple cancers      Bristol Myers Squibb
     AMP-224    PD-1   1: advanced cancers      Amplimmune/AZN
     AMP-514    PD-1   1: advanced cancers      Amplimmune/AZN

Again these earlier stage drugs are all mAbs, except AMP-224, a Fc-PD-L2 fusion protein that serves as a soluble inhibitor of PD-1. Pidilizumab had been partnered with Teva, but was returned last year. According to Nature Reviews Drug Discovery (NRDD), CureTech is seeking a partner for this drug to advance its’ development (http://www.nature.com/nrd/journal/v12/n7/full/nrd4066.html). The NRDD report is free to read and download.

There are other immune checkpoint modulators in the clinic, and we’ll get to those in a bit. What has been really shocking is how aggressive large pharma and biotech have been in acquiring very early stage assets in the immune checkpoint area. The CoStim acquisition by Novartis is an excellent example. CoStim had no clinical assets, and probably not even any IND-enabled assets, and yet was scooped up. Why? And importantly, who is next?

“Why” is a pretty interesting question, and translates into “What did they own?” The answer in the case of CoStim was that they owned patents on novel antibody inhibitors of PD-1 and PD-L1/PD-L2. Possibly of greater importance, they owned intellectual property (IP) portfolios covering new checkpoint pathways, notably the LAG-3 pathway and the TIM-3 pathway. We have no clinical data yet on either of these pathways, but preclinical tumor models, and the expression profile of these pathways, suggests very strongly that they will be critical for the prosecution of specific tumor types. Therein lies the value of buying early into the science. Bruce Booth writing on the role of Atlas Venture in the CoStim deal, has a great take on this on the LifeSci VC blog (http://lifescivc.com/2014/02/immuno-oncology-startup-costim-pharmaceuticals-acquired-by-novartis/).

So are there other CoStim Pharmas just waiting to be scooped up? The question is critical for biopharma portfolio gurus trying to peer into the future, and for stock investors wondering who to bet on. That second category, stock investors, will be looking for public companies or venture owned companies about to go public. The recent surge in biotech IPOs has helped bring plenty of candidates into public view.

Lets have a look around, but as an organizing principal, we’ll let the biology of tumor immune evasion and response lead the way.

We briefly mentioned the ligands for CTLA4 (CD80 and CD86) and for PD-1 (PD-L1 and PD-L2). These proteins are all related by protein sequence, and are members of the B7 protein family. The receptors for these ligands are also related and can be considered members of the CD28 protein family. Lets start with these, and line them up:

Screen Shot 2014-02-23 at 4.27.58 PM

This image is from Drew Pardoll’s excellent review in Nature Reviews Cancer. This paper is free to read and download, and can be found here:                       http://www.nature.com/nrc/journal/v12/n4/full/nrc3239.html. At the top you see the PD-1 and CTLA4 pathways and corresponding ligands – note here that an activating receptor for PD-L1 and PD-L2 is proposed, although none has been found yet. At the bottom we see some newer members of the B7 family, B7RP-1 (ICOS-L), B7-H3 and B7-H4. There are both stimulatory and inhibitory pathways proposed. Not surprisingly, there have been a number of development deals across this spectrum of targets.

Novartis. We’ve already mentioned the CoStim/Novartis deal, which purportedly includes PD-1 and PD-L1/2 assets and IP.

Merck. Merck took the biopharma world by surprise a few weeks ago by announcing a suite of partnerships for MK-3475 anti-PD-1 mAb. The stance is bold and aggressive and shows that Merck recognizes the importance of anticipating combination therapy clinical practice and developing MK-3475 accordingly. The company is capitalizing on the momentum behind MK-3475 that has accelerated with FDA breakthrough therapy designation (for advanced melanoma) in April of last year and an aggressive rolling submission drug application, which should be completed by mid-year.

Merck plans to run clinical studies of MK-3475 in combination with axitinib, Pfizer’s small molecule kinase inhibitor for renal cell carcinoma. This deal is similar to the one that Merck did with GlaxoSmithKline (GSK) in December 2013, to pair MK-3475 with GSK’s kinase inhibitor, pazopanib, also in advanced renal cell carcinoma.

In a combination immunotherapy effort, MK-3475 will be paired with PF-05082566, Pfizer’s agonist mAb to the 4-1BB receptor. We’ll discuss 4-1-BB and related pathways later, as this is an interesting area. The combo therapy will be tested in multiple cancer types. In a similar effort, Merck will partner with Incyte to pair MK-3475 with INCB24360, an indoleamine 2, 3-dioxygenase (IDO) inhibitor, in patients with advanced or metastatic cancers. IDO inhibitors are a very hot subject, which we will tackle below. Finally, in collaboration with Amgen, Merck will combine MK-3475 treatment with Amgen’s investigational oncolytic immunotherapy talimogene laherparepvec, in patients with previously untreated advanced melanoma.

Merck also signed on with Ablynx in a very interesting deal to develop nanobody therapeutics to immune checkpoint targets. Nanobodies are derived from camelid (camels, llamas, etc) antibodies and have some nice intrinsic properties (small size, good pharmacodynamics). Of interest, the Merck deal specifies bi- and tri-specific nanobodies targeting different proteins.

Servier. In another very recent deal (February 13, 2014), French pharmaceutical firm Pierre Fabre licensed a peptide therapeutic directed to PD-1 from Biotech company Aurigene. This new therapeutic is IND-enabled, but clinical development has not begun. Servier also acquired rights to Macrogenic’s anti-B7-H3 mAb MGA271 in December 2011. B7-H3 is overexpressed by a variety of solid tumors (prostate, pancreatic, melanoma, renal cell, ovarian, colorectal, gastric, bladder, and NSCLC). It has been hypothesized that B7-H3 expression by tunors is a mechanism of immune evasion, however, since the receptor in unknown this remains a hypothesis. So, although an anti-B7-H3 antibody may have biological impact on the tumor, Macrogenics is taking no chances, and has engineered MGA271 for optimized interaction with cytotoxic immune cells, including NK cells, macrophages and CD8+ T cells. MGA271 is currently in phase 1, in patients with B7-H3+, refractory neoplasms.

Astra Zeneca. In October of 2013, AZN/Medimmune announced that it had acquired Amplimmune, a privately held company developing immune checkpoint modulators for oncology. This preclinical company’s assets included AMP-224, the Fc-PD-L2 fusion protein mentioned earlier, and AMP-514, an anti-PD1 mAb. In December of 2013, Amplimmune registered its first clinical trial for AMP-514, a phase 1 in patients with advanced solid tumors. As discussed in a column by FierceBiotech’s John Carroll “the widely acknowledged area for differentiation will be combinations … mAbs (anti-CTLA4 tremelimumab, anti-PD1 AMP514,  OX40 agonist MEDI6469) and … targeted therapies … AZN is gearing up for combination trials with Iressa & tremelimumab … AZN’s purchase of Amplimmune gained it access to other … targets … likely including another attractive checkpoint antibody to B7-H4″. You can see the article here:                     http://www.fiercebiotech.com/story/can-astrazeneca-catch-leaders-cancer-immunotherapy/2013-10-03

Amplimmune’s discovery portfolio covers many B7 family members and their patent portfolio includes both agonist and antagonist assets and IP. Within the database-visible patents there are claims to fusion proteins and antibodies targeting PD-1, PD-L1/2. B7-H3, B7-H4, “B7-H5″, ICOS and ICOS-L.

Bayer Healthcare. Late to the party is Bayer, who to date has not made a big play in immune modulatory drugs. The company took a step forward perhaps in a deal with Compugen (NASDAQ: CGEN), paying 10MM USD upfront in a collaboration/licensing agreement. The goal is to develop novel antibody based immune checkpoint regulators discovered by Compugen. While the company is secretive as to the specific targets, one may be TIGIT, a relatively new member immune regulatory protein with some very exciting preclinical biology.

Early stage assets like Compugen’s are hard to judge without the benefit of full due diligence. We can list some of the asset players however, and some are pretty easy to score just based on the prior reputation of the company:

-  Earlier this month Five Prime Therapeutics went on record as having novel ligands for B7-H3 and B7-H4 (http://www.biotech-now.org/business-and-investments/2014/02/bio-ceo-five-prime-therapeutics-company-snapshot#) among other targets. Five Prime has an antibody discovery and development deal with Adimab. As far as I can tell, none of these are visible in the patent databases to date. Five Prime recently went public (NASDAQ: FPRX).

-  Kadmon LLC, backed by the former head of Imclone, lists anti-PD-1 and anti-PD-L1 mAbs on its pipeline chart. However this company seems focused on other areas.

-  Locally, Third Rock funded Jounce Therapeutics is developing antibodies and proteins to undisclosed immune checkpoint targets. Jounce and Adimab have announced a collaboration to drive the antibody technology. It will of great interest to see if Jounce will take the IPO route over the next few years, or instead will be acquired while still private.

-  VISTA is another relatively new immune regulator being developed by privately held ImmuNext, in partnership with Johnson & Johnson.

-  In January of this year Johnson & Johnson’s Janssen unit agreed with BiocerOX Products to develop a new mAb to an immune checkpoint protein. The target was not released but is rumored to be PD-1.

-  By the way, J&J/Janssen really does seem to be taking a multi-pronged approach to get into this space. In late January J&J Innovation partnered with MD Anderson Cancer Center, as part of its “Moon Shots” oncology effort. The joint program will evaluate new combination therapies and identifying useful biomarkers for eight critical cancers. MD Anderson has a very similar agreement with Pfizer.

-  AnaptysBio, Inc has publicized a portfolio that includes an anti-PD-1 antibody, ANB011, and novel antibodies against other immune checkpoint receptors, including TIM-3 and LAG-3.

I’m going to assume that there are other CTLA4, PD-1, PD-L1 and PD-L2 assets out there in the hands of companies large and small. We’ll track the progress of these as they pop up, whether in the poster hall at AACR, or in press releases! Also, we will discuss companies targeting TIM-3 and LAG-3, along with 4-1BB, OX40, GITR, IDO, and various other interesting targets, next post.

 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.