Category Archives: Pathological Cells and Processes

Radical optimism: considering the future of immunotherapy

I wrote recently about the sense of angst taking hold in the next-generation class of immuno-therapeutics – those targets that have come after the anti-CTLA4 and anti-PD-(L)-1 classes, and raised the hope that combination immunotherapy would broadly raise response rates and durability of response across cancer indications.

There are diverse next-generation immuno-therapeutics including those that target T cells, myeloid cells, the tumor stromal cells, innate immune cells and so on. A few examples are given here (and note that only a few programs are listed for each target):

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There are of course many other therapeutic targets – OX40/CD134, Glutaminase, ICOS, TIM-3, LAG-3, TIGIT, RIG-1, the TLRs, various cytokines, NK cell targets, etc.

In the last year – since SITC 2017 – there has been a constant stream of negative results in the next generation immuno-therapy space, with few exceptions. Indeed, each program listed in the table has stumbled in the clinic, with either limited efficacy or no efficacy in the monotherapy setting or the combination therapy setting, typically with an anti-PD-(L)-1 (ie. an anti-PD-1 or an anti-PD-L-1  antibody). This is puzzling since preclinical modeling data (in mouse models and with human cell assays) and in some cases, translation medicine data (eg. target association with incidence, mortality, or clinical response to therapy), suggest that all of these targets should add value to cancer treatment, especially in the combination setting. I’ve discussed the limitations of these types of data sets here, nonetheless the lack of success to date has been startling.

With SITC 2018 coming up in a few days (link) I think it is a good time to step back and ask: “what are we missing?”

One interesting answer comes from the rapidly emerging and evolving view of tumor microenvironments (TME), and the complexity of those microenvironments across cancer indications, within cancer indications and even within individual patient tumors. TME complexity has many layers, starting with the underlying oncogenic drivers of specific tumor types, and the impact of those drivers on tumor immunosuppression. Examples include activation of the Wnt-beta catenin pathway and MYC gain of function mutations, which mediate one form of immune exclusion from the tumor (see below), and T cell immunosuppression, respectively (review). In indications where both pathways can be operative (either together or independently, eg. colorectal cancer, melanoma and many others) it is reasonable to hypothesize that different strategies would be needed for combination immuno-therapy to succeed, thereby producing clinical responses above anti-CTLA4 or anti-PD-(L)-1 antibody treatment alone.

A second and perhaps independent layer of complexity is TME geography, which has been roughly captured by the terms immune infiltrated, immune excluded, and immune desert (review). These TME types are illustrated simply here:

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The different states would appear to be distinct and self-explanatory: there are immune cells in the tumor (infiltrated), or they are pushed to the periphery (excluded), or they are absent (desert). The latter two states are often referred to as “cold” as opposed to the “hot” infiltrated state. It is common now to propose as a therapeutic strategy “turning cold tumors hot”. The problem is that these illustrated states are necessary over-simplifications. Thus, immune infiltration might suggest responsiveness to immune checkpoint therapy with anti-PD-(L)-1 antibodies, and indeed, one biomarker of tumor responsiveness is the presence of CD8+ T cells in the tumor. But in reality, many tumors are infiltrated with T cells that fail to respond to immune checkpoint therapy at all. The immune excluded phenotype, alluded to above with reference to the Wnt-beta catenin pathway, can be driven instead by TGF beta signaling, or other pathways. The immune desert may exist because of active immune exclusion, lack of immune stimulation (eg. MHC-negative tumors) or because of physical barriers to immune infiltration. Therefore, all three states represent diverse biologies within and across tumor types. Further, individual tumors have different immune states in different parts of the tumor, and different tumors within the patient can also have diverse phenotypes.

There are yet other layers of complexity: in the way tumors respond to immune checkpoint therapy (the “resistance” pathways, see below), the degree to which immune cells responding to the tumor cells are “hardwired” (via epigenetic modification), the metabolic composition of the TME, and so on. Simply put, our understanding remains limited. The effect of this limited understanding is evident: if we challenge tumors with a large enough immune attack we can measure a clinical impact – this is what has been achieved, for example, with the anti-PD-(L)-1 class of therapeutics. With a lesser immune attack we can see immune correlates of response (so something happened in the patient that we can measure as a biomarker) but the clinical impact is less. This is what has happened with nearly all next-generation immuno-therapeutics. As a side note, unless biomarker driven strategies are wedded to a deep understanding of specific tumor responsiveness to the therapeutic they can be red herrings - one example may be ICOS expression, although more work is needed there. Understanding specific tumor responsiveness is critical regardless of biomarker use, due to the layered complexity of each indication, and even each patient’s tumors within a given indication.

So why should we be optimistic?

I propose that some of the next generation immuno-therapeutics will have their day, and soon, due to several key drivers: first, for some of these classes, improved drugs are moving through preclinical and early clinical pipelines (eg. A2AR, STING). Second, the massive amount of effort being directed toward understanding the immune status of diverse tumors ought to allow more specific targeting of next generation immuno-therapeutics to more responsive tumor types. The TGF beta signature presents a particularly interesting example. Genentech researchers recently published signatures of response and resistance to atezolizumab (anti-PD-L1) in bladder cancer (link). In bladder cancer about 50% of tumors have an excluded phenotype, and about 25% each have an immune infiltrated or immune desert phenotype. The response rate to treatment with atezolizumab was 23% with a complete response rate of 9% (note that responses did not correlate with PD-L1 expression but did correlate with both tumor mutational burden and a CD8+ T cell signature). Non-responding patients were analyzed for putative resistance pathways. One clear signature of resistance emerged – the TGF beta pathway, but only in those patients whose tumor showed the immune excluded phenotype. The pathway signature was associated with fibroblasts, but not myeloid cells, in multiple tumor types. The T cells were trapped by collagen fibrils produced by the fibroblasts:

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(The image is a screenshot from Dr Turley’s talk at CICON18 last month).

It follows that a combination of a TGF beta inhibitor and a PD-(L)-1 inhibitor for the treatment of bladder and perhaps other cancers should be used in patients whose tumors show the immune excluded TME phenotype, and perhaps also show a fibroblast signature in that exclusion zone. Indeed, in a recent paper, gene expression profiling of melanoma patients was used to demonstrate that a CD8-related gene signature could predict response to immuno-therapy – but only if the TGF beta signature was low (link).

There are other immunotherapy resistance pathways – some we know and some are yet to be discovered. We should eventually be able in future to pair specific pathway targeting drugs to tumors whose profile includes that pathway’s signature – this has been done, retrospectively, with VEGF inhibitors and anti-PD-(L)-1 therapeutics. This will require a more comprehensive analysis of biopsy tissue beyond CD8+ T cell count and PD-1 or PD-L1 expression – perhaps immunohistochemistry and gene transcript profiling – but these are relatively simple technologies to develop, and adaptable for a hospital clinical lab settings. Not every next generation immuno-therapeutic will succeed as the clinical prosecution becomes more targeted, but some certainly will (we might remain hopeful about adenosine pathway inhibitors, STING agonists, and oncolytic virus therapeutics, to name a few examples).

Another driver of success will be cross-talk with other technologies within immuno-oncology – notably cell therapy (eg. CAR-T) and oncolytic virus technologies. We have already seen the successful adaptation of cytokines, 4-1BB signaling, OX40 signaling and other T cell stimulation pathways into CAR T cell designs, and the nascent use of PD-1 and TGF beta signaling domains in cell therapy strategies designed to thwart immuno-suppression (we should note here that CAR T cells, like tumor infiltrating T cells, will face  barriers to activity in different tumor indications). The example of local (and potentially safer) cytokine secretion by engineered CAR-T cells has helped drive the enormous interest in localized cytokine technologies. Most recently, the combination of CAR-T, oncolytic virus and immune checkpoint therapy has shown remarkable preclinical activity.

SITC 2018 – #SITC18 on Twitter – will feature sessions on  immunotherapy resistance and response, the tumor microenvironment, novel cytokines and other therapeutics, cell-based therapies, and lessons from immuno-oncology trials (often, what went wrong). We can expect lots of new information, much of it now focused on understanding how better to deploy the many next generation immuno-therapeutics that have been developed.

So, I would argue that “radical optimism” for next generation immunotherapy and immunotherapy combinations is warranted, despite a year or more of clinical setbacks. Much of the underlying science is sound and it is targeted clinical translation that is often lagging behind. Progress will have to come from sophisticated exploratory endpoint analysis (who responded, and why), sophisticated clinical trial inclusion criteria (who to enroll, and why) and eventually, personalized therapeutic application at the level of the indication and eventually the patient.

In the meantime, stay tuned.

The Tumor Microenvironment “Big Tent” series continues (part 4)

 

The Tumor Microenvironment (TME) series to date is assembled here http://www.sugarconebiotech.com/?s=big+tent containing parts 1-3

I’m happy to point you to the most recent content, posted on Slideshare: http://www.slideshare.net/PaulDRennert/im-vacs-2015-rennert-v2

In this deck I review the challenges of the TME particularly with reference to Pancreatic and Ovarian cancers. A few targets are shown below.

Feedback most welcome.

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“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)

Immune Checkpoint Conference Interview (PDF)

Hit the link to see the full text (PDF) of Paul Rennert’s interview by Fiona Mistri, representing ICI15. The Immune Checkpoint Conference is being held next month in Boston.

Paul Rennert SugarCone Biotech LLC

The Big Tent: Halozyme is Targeting the Tumor Microenvironment, part 3 of an occasional series.

Many drug development programs claim to be truly unique and novel. It’s a mixed message really – complete novelty implies (or ensures) a high level of risk. It’s a bit difficult to attract early investment to such programs and maintain investor interest going forward. When we work with companies raising money, or are raising money ourselves, we are constantly trying to minimize risks, plural, as risks represent diverse aspects of a program or company: technology risk, biology risk, clinical risk, commercial risk, to highlight just a few. Companies that can move novel programs forward while derisking them in multiple areas certainly warrant our attention – for the scientific thesis and the investment thesis. We recently wrote about Innate Pharma, a company with first-in-class programs targeting NK cell immune checkpoint pathways (link 1). This is a good example of a company that has shed biology and clinical risks as the partnership with Bristol-Myers Squibb (BMS) continues to grow. The entire second tier of antibody-drug conjugate linker/payload companies (Redwood, Igenica, Mersana, Catalent and many others) will remain technology risk-heavy until each individual company either secures partnerships that eventually move ADCs into the clinic, or get their themselves. We could go on and on.

A few weeks ago I asked for companies and programs targeting the tumor microenvironment. Among the responses I got these:

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william gerber HA

Halozyme (Nasdaq: HALO) has a lead program that is very novel and I think scientifically is very interesting and has understood biological risk. We’ll talk about other risk elements in a bit, but science first. PEGPH20 is a pegylated version of the company’s approved recombinant human hyaluronidase (rHuPH20; brand name Hylenex). Hylenex is licensed to several partners, and provides a steady income stream from royalties. Hyaluronidase catalyzes the random hydrolysis of 1,4-linkages between 2-acetamido-2-deoxy-b-D-glucose and D-glucose residues in hyaluronan (HA), a constituent of the ECM. Hyaluronidase increases tissue permeability and as used locally (sc) to improve drug distribution. In the systemic tumor setting we have the interesting hypothesis that some tumor types use HA to create a cell impermeable “wall” around tumor cells or the tumor mass. The best-characterized tumor in this sense is pancreatic cancer, which is encased in an ECM that resists penetration by therapeutics and cells.

HALO is running a Phase 1/2 clinical program in PEGPH20 in patients with previously untreated metastatic pancreatic cancer. A completed Phase 1 clinical trial assessed the safety and tolerability of PEGPH20 treatment in patients with solid tumor malignancies refractory to prior therapies. A Phase 2 trial, built off a Phase 1b run-in, is underway in metastatic pancreatic cancer. The cohorts are standard of care (gemcitabine) with PEGPH20 or with placebo. An on-target toxicity (muscle spasm/pain) was addressed in a trial in which patients were pre-dosed with dexamethasone. At ASCO 2013, HALO presented data from the Phase 1b clinical study of PEGPH20 in combination with gemcitabine for the treatment of patients (n=28, 24 evaluable) with previously untreated stage IV metastatic pancreatic ductal adenocarcinoma (link 2). Patients received doses of PEGPH20 (1.0, 1.6 and 3.0 µg/kg) twice weekly for four weeks, then weekly thereafter, in combination with gemcitabine, IV. The RECIST 1.1 ORR (overall response rate = complete response (CR) + partial response (PR)) was 42% percent at the two higher doses. Subsequent exploratory analyses suggested better progression free survival (PFS) and overall survival (OS) in patients with high levels of tumor HA compared to patients with low levels of tumor HA. This has led the company to embark on the development of a companion diagnostic to enable pre-selection of patients.

Other clinical studies include a Phase 2 multicenter, randomized clinical trial first-line therapy trial of PEGPH20 in patients with stage IV metastatic pancreatic cancer. Patients were randomized to gemcitabine plus nab-paclitaxel with or without PEGPH20. The primary endpoint is PFS. SWOG has sponsored a Phase 1b/2 randomized clinical trial of PEGPH20 in combination with modified FOLFIRINOX chemotherapy compared to mFOLFIRINOX treatment alone in patients with metastatic pancreatic adenocarcinoma. MSKCC is sponsoring a trial +/- cetuximab. A full trial list is shown here:

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In October (2014) the FDA granted Orphan Drug designation for PEGPH20 for the treatment of pancreatic cancer. OK, so what do we see here? The therapeutic hypothesis is compelling, that disassembling the tumor-shielding ECM will be helpful (see link 3). Would this work as monotherapy? Perhaps, but that is not being tested, since keeping standard of care (SOC) on-board is important for these patients. But if we consider the impact of a disrupted architecture, I think we would argue that monotherapy, or at least interesting combination therapies, could be considered. The mechanisms of action are complex and include physical disruption of the tumor microarchitecture, disruption of aberrant circulation and interstitial pressure in the tumor, disruption of zones of hypoxia, and other effects. Look at this figure from the preclinical study (pancreatic cancer, mouse model):

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Panel A shows the dosing regimen (with gemicitabine), B shows the impact on pressure within the tumor and C v D shows representative tumors from the control and treated animals. Another recent paper discusses the vascular effects in detail (link 4). With the obvious leakage and loss of tissue integrity it makes sense to argue for combination with chemotherapy or antibody therapy, as in the cetuximab combo trial show above, from the MSKCC. One might also postulate that the collapse in pressure and increased access to the interstitial space might allow better penetrance by lymphocytes, allowing consideration of immune checkpoint combinations. But lets look closer:

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I left the figure legend in place so I don’t have to repeat the details, which show a reduction in smooth muscle actin (A v B) and collagen (D v E). Basically this figure suggests that the structural elements of the tumor microenvironment have collapsed. Given the impact on ECM components, I would predict that  you would see adverse impact on myeloid cell populations, inducing the TAM and MDSC populations discussed earlier (another link). I’d have loved to see a panel with PEGPH20 alone as I’ll bet you would see some impact with the monotherapy.

So if we go back to our three-legged stool model…

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… now we are dead-on the microenvironment piece, and perhaps an obvious complement to the other 2 legs.

All well and good but the proof is in the clinic, and so is the risk. We talked about diverse risks earlier – here we have clinical risk (efficacy/toxicity) and commercial risk (is it good enough). HALO is presenting at the ASCO GI meeting with abstracts to come out from under embargo on January 12, 2015. The abstracts will include an update on the clinical trial NCT01453153, phase 1/2 +/- gemcitabine in metastatic pancreatic cancer. Presentation of median OS data is rumored (but n.b. I’ve not confirmed with the company). I’m excited by the prospects here, and hope we see some nice results…

… because the science makes sense.

stay tuned.

The Big Tent: Tumor Microenvironment Targets Heat Up – part 2 of an occasional series

I recently asked folks for their favorite hot targets in the tumor microenvironment space. Among a flurry of responses I got these two related answers:

mcbio CSF-1R

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These responses from @mcbio316 and @Festivus159 were very timely, given what happened 4 days later (and a big shout-out to mcbio, whose post had preceded this):

Bristol-Myers Squibb and Five Prime Therapeutics Announce Exclusive Clinical Collaboration to Evaluate the Combination of Investigational Immunotherapies Opdivo (nivolumab) and FPA008 in Six Tumor Types

Five Prime Therapeutics, Inc. November 24, 2014 8:59 AM GlobeNewswire

  • NEW YORK and SOUTH SAN FRANCISCO, Calif., Nov. 24, 2014 (GLOBE NEWSWIRE) – Bristol-Myers Squibb Company (BMY) and Five Prime Therapeutics, Inc. (FPRX) today announced that they have entered into an exclusive clinical collaboration agreement to evaluate the safety, tolerability and preliminary efficacy of combining Opdivo (nivolumab), Bristol-Myers Squibb’s investigational PD-1 (programmed death-1) immune checkpoint inhibitor, with FPA008, Five Prime’s monoclonal antibody that inhibits colony stimulating factor-1 receptor (CSF1R). The Phase 1a/1b study will evaluate the combination of Opdivo and FPA008 as a potential treatment option for patients with non-small cell lung cancer (NSCLC), melanoma, head and neck cancer, pancreatic cancer, colorectal cancer and malignant glioma. Bristol-Myers Squibb has proposed the name Opdivo, which, if approved by health authorities, will serve as the trademark for nivolumab.

So BMS will immediately move FPA008, but all measures an early stage and largely unproven therapeutic, into combination therapy trials with nivolumab for the treatment of solid tumors. Not to be outdone, Roche has already positioned it’s CSF1R targeted therapeutic, as noted by @jq1234t:

 JQ screen shot

There are a number of interesting questions to answer here: What does CSF1R do, why is it so interesting, how does it impact the tumor microenvironment, how are these trials being done and (a favorite of mine), who else has assets in development?

CSF1R is the receptor for macrophage-colony-stimulating factor (aka M-CSF or CSF-1). The receptor is a control node for macrophage differentiation. CSF1R also serves as a receptor for the monocyte survival factor IL-34. Although the ultimate outcome depends on many factors, signaling through CSF1R is necessary for myeloid lineage precursor cell differentiation into macrophages, and it is this feature that interests us in the tumor microenvironment setting. We cannot gloss over the fact that this is a pleiotropic and complex biological system but it is safe to say that by the time we are confronted by an immunosuppressed tumor (as in the case of combo therapy with anti-PD-1/PD-L1 therapeutics), our pathway focus is on tumor associated macrophages (TAM), their impact on the tumor microenvironment and their susceptibility to CSF1R-targeted therapy.

Roche poached this figure that I’m now borrowing (with fair reference to Chen and Mellman, 2013).

Roche version Cancer Immunity Cycle

In the original figure (see Immunity 39: http://dx.doi.org/10.1016/j.immuni.2013.07.012 – an open access article), Chen and Mellman placed the PD-1 pathway inhibitors with a variety of microenvironmental modulators (IDO1, Arginase, TGFb) that together prevent, in distinct ways, cancer cell death. The Roche version of the figure, reproduced above, has been modified to include CSF1R among other targets in the “killing cancer cells” category.

Broad strokes, what does this mean? TAM, the tumor associated macrophages mentioned above, are dependent on CSF1R signaling. TAM are myeloid lineage-derived cells that are co-opted by the resident tumor as part of it’s microenvironmental support system. TAM are potently angiogenic, remodel the stroma (extracellular matrix and related components) and are immunosuppressive. Among the plethora of critical factors produced by TAM we find the hypoxia response proteins and growth factors that drive angiogenesis, tissue remodeling and immunosuppression, i.e. HIF2a, MMP-9, EGF, VEGF and TGFbeta, cytokines that can maintain this response in a chronic state (IL-10, IL-4) and chemokines that attract myeloid cells and regulatory T cells (CCL22, CCXL8). The TAM population can be directly regulated by tumor cell secretion of CSF-1, thus the importance of the CSF1R target. Multiple labs have produced preclinical data showing that anti-CSF1R antibody therapy can rapidly and effectively deplete tumors of the TAM population, and that this depletion has an impact on tumor growth and survival.

Clinical development to date is scattered. The FPRX program began with a Phase 1 trial in healthy volunteers and rheumatoid arthritis patients (NCT01962337) reflecting the role of diverse macrophage populations in inflammation and autoimmunity. Indeed the FPRX website states “we are currently conducting nonclinical research in areas such as idiopathic pulmonary fibrosis, lupus nephritis and other inflammatory disorders to identify a second target indication by the end of 2014″ although this may be trumped by the BMS deal. That trial reported safety and pharmacodynamic endpoints at AACR earlier this year. FPA008 was well-tolerated at all dose levels tested and the drug impacted inflammatory macrophage numbers and, interestingly, bone turnover (this latter effect due to the control of osteoclast differentiation by CSF1R, an important feature in bone metastasis settings perhaps).

In contrast Roche has been testing it’s antibody in a rare disease (a form of giant cell tumor) that is caused by a  t(1;2) translocation resulting in fusion of COL6A3 and M-CSF genes encoding for CSF1. The tumor is characterized by CSF1R+ cells. Roche reported that RG7155 had the following activities (Reis et al. 2014. Cell 25: 846–859):

- Anti-CSF-1R antibody depletes tumor-associated macrophages in cancer patients

- CSF-1R inhibitor shows clinical activity in diffuse-type giant cell tumor patients

- CSF-1R signaling inhibition increases lymphocyte infiltration in cancer patients

That last highlight referring to an effect on immunosuppression and refers to a relative increase of CD8+ T cells versus CD4+ FoxP3+ T regulatory cells, thus feeding the enthusiasm for combination therapy with anti-PD-1/PD-L1 therapeutics. More data is available in their ASCO abstract (http://meetinglibrary.asco.org/content/131522-144).

Other clinical stage antibodies include IMC-CS4 from Eli Lilly, in Phase 1 for advanced solid tumors (NCT01346358), ARRY-282 from Array BioPharma and Celgene, which had completed a Phase 1 trial in advanced solid tumors (NCT01316822) before the program was terminated, AMG 820 from Amgen with a completed Phase 1 study in advanced malignancies (NCT01444404), and others. Preclinical programs are visible at many small companies, both private and public, and include small molecule inhibitors of the receptor, e.g the Ambit and Plexxikon programs.

While the enthusiasm seems warranted by the preclinical modeling data and the (to date) apparent tolerability of the antibody therapies, I did receive this one note of caution from @Boston_Biotech:

Bos Biotech screenshot

Nuances, indeed. It is important to consider a few possible issues. First, blockade of CSF1R in mice led to the pronounced and sustained upregulation of CSF-1, and drug doses had to be kept high in order to “drug-through” this level of ligand to block the receptor. Rebound activity at trough or upon drug cessation could be a big problem, as has been described for other systems, including CCL2 blockade in breast cancer models (leading to abundant metastasis). Sticking with breast cancer, it has been reported that blocking CSF1R leads to upregulation of GM-CSF signaling, changing the composition perhaps (but not the stability) of the tumor microenvironment. Finally, as always, we cannot yet see what efficacy drugs will have as monotherapies (its too early) while we race ahead to combo therapies. While its all hands on deck to get these assets into patients, they won’t all work and certainly can’t be sure they will do no harm. However, that said, I think targeting components of the tumor microenvironment, including TAM, is our next best step forward, and I certainly will enjoy watching the data unfold.

next time … what wraps pancreatic cancer up so tight that you can’t treat it until it explodes in a deadly metastasis fireball?

cool stuff.

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

Creating a New Therapeutic Focus

In 2012 we were engaged by a large local biotech company to evaluate a new therapeutic area. This effort was driven by the desire of the client to move aggressively into a new suite of diseases. We began by doing a deep dive into the client’s existing portfolio in order to identify assets already in development that could be directed to novel diseases. Concurrently we began a comprehensive review of preclinical and clinical stage assets available for partnering or in-licensing. Finally we engaged in pathological pathway analysis to identify novel targets for discovery programs. This effort, initiated and completed within two quarters, led to the eventual acquisition of a private company and its Phase 2 clinical stage assets, for nearly 100MM $USD. Based on our analyses the client also started several new discovery and preclinical development programs to complement the clinical stage acquisition.

Cautionary Tales from Human Microbiome Frontier

The concept of symbiotic microbiomes (yes, plural) influencing our health seems now, in hindsight, to be obvious, and the fact that the science has caught up to the folk medicine has all sorts of people buzzing. Some of the buzz is well informed (see below), some not, but all in all we are making progress understanding a few of the ways in which our vast mucosal environment interacts with the outside world. At the same time its fair to say that we know very little yet, and have a long way to go. Some recent findings drive this point home.

We can think of the frontier mentioned in the title in two ways. One, maybe obvious, is to think about the frontier of science, as this is where we find ourselves as the technology to do the some of this work was not widely available until recently (e.g. affordable deep sequencing). More subtly, we can think of the mucosal environments – oral, pulmonary, digestive, excretory, reproductive – as frontier environments where self interacts with non-self in an exploratory manner, that is, not confrontational a priori. There is a lot at stake: pathogen recognition and defense, nutrient uptake, metabolic regulation, waste disposal, on and on.

It makes sense that there are tightly controlled and very complex rules of engagement. The new findings I want to review touch on some of these rules and suggest layers of control and organization that we really don’t understand yet. Secondarily, we can study these systems with an eye on drug discovery.

Back to back papers in the December 16/26 double issue of Nature identify a critical pathway for the development of regulatory T cells (Tregs) in the gut. Data from the Ohno lab in Japan and the Rudensky lab in NYC paint broadly similar stories of the role of the specific commensal bacteria in fostering Tregs (see references 1 and 2, below). Both papers show that the fatty acid butyrate stimulates the development of Tregs. This in itself is not a new finding. Butyrate is a major energy source in mammalian metabolism and not surprisingly it’s production is driven by commensal bacteria, notably the abundant Clostridia class of bacteria (some species within Clostridia are pathogenic, but that’s a different story). Again, it’s not particularly surprising that one of the most abundant mammalian commensals gives off good vibes in the form of fatty acids that support a quiet immune system. The papers differ in some curious ways, in particular, the Ohno paper states that the induction of Tregs was limited to the gut, while the Rudensky papers highlight Treg production in the lymph nodes and spleen, but not the colon. Regardless, the reason these papers made it into Nature is that they identify the mechanism by which butyrate induces Treg differentiation, and this is by inhibiting a histone deacetylase (HDAC IIa) thereby allowing for the specific acetylation (and therefore activation) of DNA elements that support Treg differentiation, notably at the FoxP3 promoter and enhancer.

Cool.

But before we all run out and start swallowing a bunch of butyrate capsules and subject ourselves to butyrate enemas (yes, both are available), lets be clear about what these papers are saying and what they are not saying. First, we are dealing here with inbred mouse strains on carefully defined diets. Translation of the results to outbred humans on diverse diets is not so straightforward. That said, the results support eating a high fiber diet, which will yield plenty of butyrate and related fatty acids. Second, the papers agree on one thing very specifically, which is that the generation of Tregs in the gut is a local phenomena, specific to the colon (large intestine, south of the caecum). This makes sense of course, as that is where the Clostridia are cranking out the fatty acids. The application of these findings to colonic disease, notably Ulcerative Colitis, is worth exploring. But broadening the scope to include general health, well-being and immune serenity is not warranted – despite the pile on by the Supplements and Wellness Industries.

A very different story just came out in PNAS (reference 3), and this one concerns the response of different populations to a gut pathogen found in the gastric mucosa (lining of the stomach). The bacterium Helicobacter pylori is found in about half of the human population worldwide. H. pylori is a causative agent of gastric adenocarcinoma in a small percentage of the people who are infected, less than 1%, although hotspots are known. One such hotspot was studied by a team from Vanderbilt who found that the higher incidence of H. pylori induced precancerous inflammation correlated with the presence of a European strain of the bacterium infecting an Amerindian population in Columbia. In contrast, an African strain of H. pylori infecting the descendants of African slaves nearby did not cause inflammation and cancerous lesions. The investigators conclude that H. pylori is mainly pathogenic when it occurs in a population distinct from that with which it co-evolved. So, a fine line between commensal and pathogen is drawn.

Ok, one more.

The gut microbiome has been implicated in the development of Th17 effector T cells, at least in mice. This is interesting in light of where we started, with the generation of Treg cells, since in some ways Tregs and Th17s are the result of different developmental pathways that T cells take. Note that the first two studies reviewed were focused on extrathymic (in that case, colon-specific) Treg generation. Mice that are raised with no pathogens in their environment, including their food, which is irradiated, don’t develop very many Th17s as a percentage of the total T cell population. Since Th17 cells are associated with diseases (including rheumatoid arthritis (RA), psoriatic arthritis (PA), psoriasis, inflammatory bowel disease) it seems reasonable to ask whether a Th17 inducing microbiota is linked to any particular disease. Littman’s lab at the Rockefeller in NY has done exactly that (reference 4). Newly diagnosed RA patients were found to carry the intestinal bacterium Prevotella copri at much higher levels (75%) than PA patients (37%) or healthy control patients (21%). This association of a specific pathogen with an autoimmune/chronic inflammatory disease is very striking. When mice were infected with a rodent-compatible strain of P. copri they developed pronounced intestinal inflammation, but not arthritis. Still, the intestinal inflammation was associated with the induction of Th17 cells, and so the hypothesis that this may underlie more systemic inflammation (e.g. RA) is still reasonable.

There are some problems with the story. The clinical development of IL-17 targeting drugs has shown that these do very well in PA and psoriasis, perhaps in inflammatory bowel disease, but they have failed to show sufficient benefit so far in RA. So at the level of drug discovery the link of an intestinal pathogen to Th17 T cells producing IL-17 and then to the disease, RA, seems to falter.

Thinking more broadly, the application of microbiome studies to drug development is in its infancy, and I think there is some reason for optimism as these studies become more sophisticated. The H. pylori and P. copri studies mentioned make it clear that many factors influence the response of a given population or individual to their microbioma. One interesting approach, the use of fecal transplantation to treat severe diarrhea and also Crohn’s disease, has made it into early clinical trials. Isolation of the critical components that reset the immune system in the local (inflammatory bowels diseases) and systemic (RA and other non-gut inflammatory diseases) settings is going to take significant time and effort, so we’ll have to stay tuned.

References
1) Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells, Nature, http://www.nature.com/nature/journal/v504/n7480/full/nature12721.html
2) Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation, Nature, http://www.nature.com/nature/journal/v504/n7480/full/nature12726.html
3) Human and Helicobacter pylori coevolution shapes the risk of gastric disease, PNAShttp://www.pnas.org/content/early/2014/01/08/1318093111
4) Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis, elife, http://elife.elifesciences.org/content/2/e01202

Cells underlying fibrosis: emerging themes

by Paul D. Rennert, 28 December 2013
A few weeks ago a paper describing the origin of cells responsible for dermal wound healing showed up in Nature, with a beautiful cover picture showing distinct cell lineages in skin. I actually missed the paper when it came out, so thanks to my friend Katherine Turner (ex-GI, ex-Biogen, now at Scholar Rock) for pointing it out.
The abstract is here, the full text requires a subscription. The paper is from Fiona Watt’s group at the Center for Stem Cells and Regenerative Medicine, King’s College, London.
The paper convincingly shows that the dermal layer of the skin is comprised of two distinct fibroblast lineages. Some context: the dermis is the layer of the skin that lies below the epidermis and includes the upper dermis (the dermal papilla) and the hypodermis (lower dermis). The major cells types residing in the dermis are fibroblasts, which along with the extracellular matrix they produce, form the connective tissue. Other prominent cells include macrophages and adipocytes (fat cells). There are also resident dendritic cells, Mast cells, T cells and other immune system cell types. Other structures include the hair follicles, sweat glands, oil glands, blood vessel endothelium, lymphatic vessels, and nerve endings.
Ok, so why is this paper interesting?
What the authors demonstrate is that the upper and lower dermis originate from distinct lineages of fibroblasts. This is shown using lineage tracing techniques and cell transfer assays. The lineage tracing assays required the use of multiple promoter/expression transgenic mice, and is elegantly and robustly done. The data show that late in mouse embryonic development (day 16.5) the dermis becomes fate-restricted: the Lrig1+ fibroblasts become the cells of the upper dermis and Dlk1+ cells give rise to the lower dermis. Here I’ve just picked a few of the markers they used and have grossly simplified the developmental story because I’m more interested in what they did next.
Having established that there are 2 different fibroblast lineages in the mouse dermis, the investigators asked a pretty simple question: what happens when you wound the skin in the adult mouse? Cellular wound-healing models have been widely used as in vitro models of fibroblast migration, ECM production and differentiation into myofibroblasts (so called because they express smooth muscle actin, a marker of myocytes/smooth muscle cells). Wound healing more broadly is a process of local repair and remodeling. “Deranged” or unregulated wound-healing is one model of fibrosis.
Skin or dermal fibrosis is a highly active area of study. Important fibrotic diseases of the skin include systemic sclerosis (scleroderma), amyloidosis, nephrogenic systemic fibrosis, mixed connective tissue disease, wound-healing fibrosis and others. Scleroderma is modeled in mouse by creating chemical insults on the skin, for example with bleomycin. Such models mimic the effect of chronic wounding, thus the connection to wound healing assays. It has been further demonstrated that many of the processes and pathways involved in dermal wound-healing are dysregulated in skin fibrosis, for example ECM production, TGFbeta production, and CTGF activity among many others.
While there is a good deal of consensus as to the mediators of the fibrotic process, the source of the activated cells that drive fibrosis has been controversial. This is a critical question to address, as effective anti-fibrotic therapies should target the pathogenic cell types. Without getting into immune cells, resident macrophages and Mast cells and the like, we can now at least carefully address this question regarding the source of activated fibroblasts. One model proposes that migrating fibrocytes traffic into wounded/fibrotic sites from the circulation. Another model, widely held, proposes that pericytes, which are mesenchymal cells that support blood vessels, somehow migrate away from endothelium and become myofibroblasts, thus supporting wound healing (and by extension contributing to fibrotic diseases). Finally, it has been proposed that myofibroblasts are derived from local (resident) fibroblasts that migrate into the wound and differentiate.
The Watt’s paper shows clearly that the Dlk1+ fibroblasts of the lower dermis migrate into the wound and that a proportion of these cells express smooth muscle actin and are therefore myofibroblasts. These cells produce collagen, the classic ECM protein that characterizes fibrosis, in this case simply deployed to help close the wound. After the wound is closed the epidermal layer is reformed and only then does the upper dermis reform.
These findings allow for the formulation of specific questions regarding the lower dermal fibroblasts:
- what signals activate and then shut down the lower dermal fibroblast to myofibroblast differentiation process?
- are these signals dysregulated in fibrosis?
- do these same cells deploy in response to fibrotic injury (e.g. with bleomycin)?
- do these cells interact with inflammatory cells, thought to contribute to the pathogenic fibrotic response?
- can we identify actionable targets on this cell type to prevent or decrease the fibrotic response?
Importantly this paper encourages us to ask whether similar resident fibroblast or other mesenchymal cell populations contribute to fibrotic disease in other organs such as the kidney, liver, heart and perhaps even lung. If so, the validity of the circulating fibrocyte and differentiating pericyte models might be reevaluated. Notably, linage tracking was used in support of a pericyte model of myofibroblast differentiation in a similar dermal/muscle injury model as detailed in a paper published in 2012 in Nature Medicine (abstract here). I suspect that squaring these studies will take a careful review of the markers used to identify different cell populations and a detailed look at the injury protocols. I’ll update as more info becomes available.
This is a classic example of the beautiful work that comes from an active and competitive research area and we can and look forward to further studies that will illuminate the processes of wound healing and fibrosis. Such work will certainly support and advance successful fibrosis drug development.
you may leave a comment or contact me:
rennertp@sugarconebiotech.com
on Twitter @PDRennert
cheers-
Paul