Monthly Archives: December 2014

The Reading List – Day 1

Last week I posted a holiday reading list (link). I’ll be posting brief summaries of some of the more interesting papers over the next week or two.

Topic: Tumor Mutational Landscape

The Papers

Age related variants of variants occurred in the genes DNMT3A, TET2, and ASXL1 are associated with hematological malignancy risk - NEJM-1 and NEJM-2
Nature Genetics on the NEJM papers: Commentary

The Highlights

- Interesting papers on the power of whole exome and targeted gene sequencing – and a large number of patients followed for a long time – to uncover putative initiating cancer mutations. These can be termed initiating in the sense that they predate the occurrence of malignancy.

- Both studies identified somatic mutations in peripheral blood mononuclear cells that occurred more frequently in patients who developed hematological malignancies than those who did not.

- Here is a peak at some of the data. This first figure is from the paper out of Benjamin Ebert’s lab at Brigham & Women’s Hospital, Harvard Med School, Boston.

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On the left we see the 10 most commonly detected mutated genes, on the right the distribution of mutated genes per patient – overwhelmingly just one (Fig.2 from Jaiswal et al. 2014).

The second paper is from the McCarroll lab at the Broad Institute, MIT and Harvard, Cambridge, MA, across the river from Boston. Their list is a little different, as seen in this figure:

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Notably, however, the top three genes are the same (Fig. 2 from Genovese et al. 2014).

Many of the other mutations are in genes well studies in the context of hematological malignancies (JAK2, IDH2, MYD88) and other tumors (TP53, STAT3). The differences are mainly in the rarer mutations and reflect statistical noise, the difference in patient populations studied, or both.

- The top 3 somatically mutated genes are various types of DNA transcriptional regulators. This suggests that the drivers for these malignancies are actually the targets of the activity of DNMTA3 (a DNA methyltransferase), TET2 (a methylcytosine dioxygenase) and ASXL1 (an epigenetic regulator). All three had been previously identified as mutated genes in patients with various lymphoid and myeloid malignancies, however these new studies show the mutations to be present and stable years before cancer develops.

- These somatic mutations are one-hit wonders, that is, they are clonal, and very likely causative. The McCarroll group’s paper has a nice figure illustrating this principal:

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Thus clonal hematopoiesis evolves from one of the initiating mutations.

- A few other very interesting messages in these papers:

1) the number of mutations increases with age
2) mutations predict malignancy, but at a pretty low rate
3) mutations are associated with survival

The association with survival is not only due to the link with cancer: mutations in these transcriptional regulators appear cause other physiologies as well. The Ebert paper finds an association between age-related somatic mutations and cardiovascular disease for example. I’d caution here that this paper studies patients already predisposed to develop cardiovascular disease, and so this association might wash out in general population studies. Regardless, the relative risk of carrying one of these somatic mutations is not large, so it is not as if we should all run out and get these genes analyzed.

- Finally, and this has been known for a while, mutations in these transcriptional regulators are really problematic once cancer develops, and that is shown by the impact on overall survival post-diagnosis. This last point is very intriguing as it suggests that somatic mutations in transcription regulators do two distinct things, probably via action on distinct targets – one, predispose to hematologic oncogenesis and two, allow mutations to accumulate once oncogenesis has taken place.

Given that the relative risk of carrying any of these somatic mutations remains very low, what are we to do with the data? Genovese et al. anticipate this question, and offer the following (quoting here):

“Several important research directions could bring DNA sequencing for clonal hematopoiesis closer to clinical usefulness. First, some somatic mutations are likely to be associated with a particularly high risk of subsequent cancer; larger studies could identify such mutations. Second, single-cell analyses might identify high-risk combinations of mutations occurring in the same cells. Third, the sequencing of specific cell types might identify mutation–cell-type combinations with increased predictive value. Fourth, initial detection of clonal hematopoiesis might justify periodic screening for the presence of cooperating mutations at low allele frequencies that could presage cancer”.

I would add that a more detailed understanding of how somatic mutations in the DNMT3A, TET2, and ASXL1 genes trigger and support oncogenesis may yield downstream targets suitable for therapeutic intervention.

next topic: New Immunotherapy Papers

stay tuned.


Holiday Reading

some of the stuff we’re reviewing over the holiday break. N.b. paywalls ahead!  And at the very end, some current non-science favorites.

Tumor Mutational Landscape

Age related variants of variants occurred in three genes (DNMT3A, TET2, and ASXL1) are associated with hematological malignancy risk and

News and Views on the NEJM papers

using siRNA to identify driver genes in breast cancer


a primer on the role of PD-1 pathway inhibitors in Hodgkin’s Lymphoma, from Nat Rev Clin Oncol

the role of TILs and TIL-associated TNF in the survival of CRC patients

nivolumab in metastatic RCC, published data

resistance to T cells in melanoma (hint: they lose MHC expression)

interesting look at PD-L1 expression of the response of RCC to targeted therapies

it’s hard to control ipilimumab-induced tox 2353.abstract

IO combination review

tumor/microenvironment cross-talk mediated by microRNAs

functional blockade of miR-23a releases TILs in an ex vivo NSCLC assay

neutrophils, T cells and lung cancer

Given the new immunotherapy data in bladder cancer, a review of the molecular drivers of this tumor type is most welcome

MDSC requirements for survival

Gene Therapy and CAR T

Novel gene therapy methods puts a safety brake on a retrovirus-based vector

a new review of the CRISPR, Talen, and ZFN technologies for gene editing

NY-ESO-1 CAR T P1 results in solid tumors: long term follow-up and correlates of response

Targeted Therapies

A very timely primer of the role of different PI3K isoforms in diverse cancers

a Notch in the cancer treatment belt? Nope, a bit of a toxic mess made with anti-DLL4 antibody Demcizumab from OncoMed

IL-17 and colon cancer?

Hematological Malignancies

von Adrian and Sharpe tease apart Follicular Lymphoma

the role of one of gp130 in multiple myeloma

Fibrosis, Inflammation, Metabolism, MS

a brand new fibrosis review

the TRPV4 pathway, TGFbeta and IPF

The role of novel branched fatty acid esters of hydroxy fatty acids in Type 2 diabetes

will STING finally yield a useful target in lupus?

an animal model of JCV infection and PML

Investment and Deals

Pharma funding to pull programs out of the academic space

some color from NRDD on the Genentech + NewLink IDO-1 inhibitor deal

Also notable

300,000,000. A violent graphic lurid hypnotic novel of the dissolution of consciousness and the consequence of multiple realities converging within our unprepared empty minds and upon our decadent culture. Horrific and wonderful, but not for the squeamish.

Thug Kitchen – eat like you give a #$%@^. Fun, but you get the idea.

Death & Co: Modern Classic Cocktails. Drink like an adult.

The widget TIGIT

Genentech continues to work on TIGIT, so what the heck is this target? Lets have a look, but first, some context.

T cell constraint is a fundamental attribute of tumor-induced immunosuppression. CTLA4 and PD-1 are central regulators of this process, and antibody blockade of these pathways can restore anti-tumor responses. The state of T cell constraint (non-responsiveness) has been termed anergy in reference to CD4+ T cells and exhaustion in reference to CD8+ T cells. Exhausted CD8+ T cells have a recognizable T cell phenotype characterized by the expression of diverse inhibitory pathways and proteins, including PD-1, TIM-3, LAG-3 and TIGIT. Whether such a phenotype is absolutely selective for exhausted CD8s is a matter of debate, but is a good starting point for a discussion of the need for so many regulatory pathways.

Dual gene-deficient (knock-out) mice and the administration of blocking antibody combinations have shown that the inhibitory receptors can function synergistically to reject tumors in mouse models. The hypothesis that individual co-inhibitory receptors contribute distinct functions to collectively limit T cell responses has recently been tested in human cancer clinical trials, yielding the impressive result that co-blockade of CTLA4 and PD-1 has synergistic and beneficial anti-tumor activity. Such benefit comes with a toxicity cost, as pathological autoimmunity is revealed when the “brakes” come off the immune system.

Why does the T cell arm of immune system require so many different control pathways? This is a reasonable question, which can be answered somewhat glibly with the observation that uncontrolled immunity leads to autoimmune disease and/or chronic inflammation. Still, though, why are multiple breaks required? The working hypothesis is that one pathway (CTLA4) regulates T cell activation by CD28 that normally occurs in the spleen, lymph nodes, Peyer’s patches and other “secondary lymphoid organs” (the thymus, bone marrow and fetal liver are the major primary lymphoid organs). A second pathway (PD-1) is generally thought to regulate “peripheral” T cell activation at the sites of pathogen encounter – in this sense “peripheral” means outside of the lymphoid organs themselves, that is, in the tissues and circulation, or, in the case of cancer immunology, within the tumor. So, simplistically, there is one control pathway (CTLA4) in the house and another (PD-1) in the yard. The recent paper (link 1) describing the release of T cell recognition of tumor antigens upon CTLA4 blockade in melanoma suggests either cross-talk between the compartment (i.e. tumor beds have lymphatic or circulatory drainage to secondary lymphoid organs) or that the role of CTLA4 is more complex than we think.

What about the other control pathways? LAG-3 is a competitive regulator of CD4/MHCII antigen recognition activity and was shown to confer Treg function when transfected into naive CD4+ T cells. The expression of LAG-3 on CD8+ T cells (which are critical for anti-tumor activity) suggests a role in the interaction of CD4+ and CD8+ T cells. LAG-3 is also expressed on tumor cells and may mask tumors from immune recognition. LAG3/PD-1 doubly gene-deficient mice can reject poorly immunogenic tumors that wild-type mice cannot reject. However, the doubly deficient knockout mice also develop pathological and aggressive autoimmunity. These results show that these proteins have distinct roles in regulating immune responses.

TIM-3 has several immune regulatory activities, one of which is to suppress T cell recognition of phosphatidylserine, a molecule expressed on dead and dying cells but also on tumor cells. As with LAG-3 the combination of anti-PD-1 and anti-TIM-3 antibodies had enhanced anti-tumor efficacy in mouse tumor models when compared to either antibody alone.

And now we have TIGIT, an Ig superfamily protein and a member of the PVR/nectin family that includes CD226 (DNAM-1), CD96, CD112 (PVRL2), and CD155 (PVR), among others. The biology of this family of proteins is complex and a little intimidating. Genentech has been prosecuting this pathway for several years, and their new paper (link 2) has perhaps shed additional light on the biology and utility of this target.

One mechanism by which TIGIT modulated immune responses is via the interaction of TIGIT on T cells with CD155 expressed on immature or resting dendritic cells, which blocks maturation signals normally delivered by CD226, that is, TIGIT is a competitive inhibitor of the interaction of CD226 with CD155. The authors note that this system resembles the co-stimulatory/co-inhibitory receptor pair of CD28 and CTLA-4, where CTLA4 is a competitive inhibitor of the interaction of CD28 with B7-1/CD80 and B7-2/CD86. The expression pattern of the receptors is also similar: both TIGIT and CTLA-4 are induced upon cell activation, while the expression of CD226 and CD28 is constitutive.

As alluded to above, and noted explicitly by the Genentech team, the molecular and functional relationships between TIGIT and it’s various ligands/co-receptors are poorly characterized. Furthermore, TIGIT’s role in regulating CD8+ T cell responses and the mechanisms underlying such regulation are not known. Of note, antibodies to TIGIT or PD-L1 alone enhanced CD8+ T cell effector function in tumor-draining lymph nodes, but blockade of both receptors was required to allow activation of CD8+ T cells within the tumor microenvironment, as measured by IFNy production. The authors conclude that TIGIT is a critical and regulator of CD8+ T cell anti-tumor activity. The mechanism of action evoked to explain the role of TIGIT in the tumor setting was addressed using FRET and other analyses. The authors show that TIGIT interacts directly with CD226 to prevent homodimerization, a component of the interaction of CD226 with CD155.

There are a few things to consider here. The animal models were run with very high amounts of anti-TIGIT and anti-PD-L1 antibodies on board (10 mg/kg anti-PD-L1 and 25 mg/kg anti-TIGIT) given 3 times a week. That’s nearly a gram of antibody approximately every 2.5 days. While the anti-PD-L1 antibody used has a mutated Fc domain that cannot mediate direct cell killing by ADCC, the anti-TIGIT antibody used is a wild-type IgG2a isotype antibody and almost certainly mediates direct killing of TIGIT+ cells. While the in vitro FRET assays are suggestive of the proposed mechanism of action, what is actually occurring in vivo is less clear. TIGIT expression on NK cells is also worthy of further exploration.

So I have a doubt. Not that the pathway is important, but that we really have a good sense of how it functions, nor how antagonism of the pathway in patients will impact anti-tumor activity and baseline immune responses. Locally, Drs Vijay Kuchroo and Ana Anderson have done wonderful work on TIGIT biology, and no doubt one or more of the Cambridge immunotherapy companies is working on this target and exploring it’s utility in the tumor setting. Given the expression pattern of TIGIT in tumors – i.e. on PD-1+/TIM3+ “exhausted” T cells – it is certainly worth the effort to find out.

How to select patients who should respond to anti-TIGIT co-therapy (or anti-TIM-3 or anti-LAG-3) is a critical question, best left for another day.

stay tuned


Enumeral Biomedical lands a deal

Enumeral Biomedical Holdings, Inc (ENUM) announced today a collaboration with immunotherapy powerhouse Merck (MRK) to interrogate the tumor microenvironment in colorectal cancer tissues obtained directly from patients. The goal is to identify functional cellular responses to immuno-oncology therapies being developed by Merck.

The press release is here:

Tumor Neo-Epitopes

I’m asked a lot about the onco-vaccine field, and if immune checkpoint inhibitors will be the key to unlocking the potential of this long-suffering therapeutic class. The answer is never simple, since we are often looking at thin patient data that can contain compelling hints of efficacy – those immunized late-stage patients who not only regressed but stay in remission, month after month and year after year. The problem for companies and investors is that such observational data can be very misleading, and the vaccine candidates most often go on to fail in later and larger clinical trials, sometimes spectacularly. These big failures burden the field with a high evidentiary bar.

Data have emerged that suggest several issues with most vaccines, and these issues are both distinct and related.

At the end of November Nature published two interesting papers that asked a very simple question: what immunogenic antigens are present in common mouse tumor models. Yadav et al from Genentech and Immatics Biotechnologies (link 1) used a genome-wide exome and transcriptome sequence analyses, mass spectrometry and structural modeling to identify immunogenic neo-antigens in the widely used MC-38 and TRAMP-C1 mouse syngeneic tumor models. These models are considered poorly immunogenic in wild-type syngeneic (C57Bl6) mice. The sequencing analysis was used to identify mutated proteins that were present at >20% allelic frequency. From the MC-38 model, 1290 expressed mutations were identified of which 170 were considered to be neo-epitopes, that is, modeling suggested they would be expressed by MHCI and sufficient residues would be solvent exposed to allow immunogenicity. Only 67 expressed mutations were found in the TRAMP-C1 model, and of these 6 were considered to be potential neo-epitopes. Of this total of 170 (MC-38) and 6 (TRAMP-C1) only 6 bound MHC1 by Mass Spec, with a predicted IC50 for MHCI < 500nM. Of these, 3 were actually immunogenic in vivo (using C57Bl/6 mice) and could protect wild-type mice from tumor challenge. The neo-epitopes were found in the proteins Dpagt1, Reps1 and Adpgk. Here is a schematic of the filtering scheme:

 Screen Shot 2014-12-15 at 6.37.49 PM

Working the other way, the authors confirmed the immunogenicity of neo-epitope peptides by analyzing tumor-infiltrating lymphocytes (TIL) and staining with peptide–MHCI dextramers to identify bound T cells. CD8+ T cells specific for Reps1, Adpgk and Dpagt1 were enriched in the tumor. Using the Adpgk neo-epitope, the TIL were further investigated and found to express PD-1 and TIM-3, inhibitory receptors associated with anergic or “exhausted” CD8+ T cells, showing that the murine immune system had indeed recognized and responded to the neo-epitopes, a response that was then actively immunosuppressed in the tumor microenvironment. Importantly, none of the identified neo-epitopes would qualify as tumor-antigens, that is, they are not specifically overexpressed at a sufficient level to qualify. The neo-antigen-specific TIL are also pretty rare, suggesting that use of tumor lysate as an immunogen to elicit an anti-tumor response may “miss” by failing to present enough of the right antigen to the immune system.

We noted at the top that this paper came out of labs at Genentech and Immatics. An agreement between Roche/Genentech and Immatics will focus on the use of this technology. The two companies will develop new tumor-associated peptides (the neo-epitopes as cancer vaccine candidates, initially targeting gastric, prostate and non-small cell lung cancer. The most advanced candidate is IMA942, a peptide vaccine for the treatment of gastric cancer, in late preclinical development. Immatics CEO Paul Higham has publicly stated that a Phase I study of IMA942 with Roche’s PD-L1 inhibitor MPDL3280A in gastric cancer will be initiated soon. Immatics will also conduct research to identify neo-antigens for the additional indications. For those keeping score, Immatics received $17 million upfront, committed research funding, and potential milestones

The second work, led by Schreiber’s group at Wash U, used mouse models to ask a different but related question: what tumor antigens are recognized after immune checkpoint blockade with anti-PD-1 or anti-CTLA4 antibodies (link 2). The sarcoma lines d42m1-T3 and F244 were rejected in wild-type mice treated with either anti-PD-1 or anti-CTLA4 antibodies, in a CD4/CD8/IFNy/DC-dependent manner. As in the first paper, a filtering system built from diverse technologies was used to identify potential neo-epitopes. Mutations were identified by cDNA sequencing, translated to corresponding protein sequences, then tested against MHCI binding algorithms. Neo-epitopes were ranked by predicted median binding affinities and likelihood of productive immunoproteasome processing and antigen display. Using these methods, two MHCI restricted neo-epitopes were identified in Alg8 and Lama4.

As in the first paper, the authors then turned the system around, asking what neo-epitopes could be identified through analysis of TIL. Alg8 and Lama4 were found in tumor TIL and their frequency was increased by treatment with anti-PD-1 or anti-CTLA4. The neo-epitopes could successfully be used to induce anti-tumor immune responses. As in the first paper, these are not neo-epitopes that would qualify as tumor-antigens using the traditional criteria of selective and high expression.

So these are our two distinct but related issues with the current tumor vaccine landscape: those that have selected antigens have likely selected the wrong ones, while those that use lysates are likely too dilute.

Importantly, we can now compare these model systems to actual data from human patients treated with anti-CTLA4 antibody, as published recently in NEJM (link 3). The clinical group from Memorial Sloan Kettering Cancer Center  obtained tumor tissue from melanoma patients treated with anti-CTLA4 antibodies (ipilimumab or tremelimumab). As in the mouse study, whole-exome sequencing was performed, somatic mutations identified and potential neo-antigens were characterized. Here is their schematic:

Screen Shot 2014-12-16 at 9.42.56 AM

Baseline analyses showed that there was a significant difference in mutational load between patients with long-term clinical benefit and those with a minimal or no benefit, with higher mutational load associated with response. However the relationship was correlative, since some tumors with a high mutational burden were not responsive to anti-CLTA4 therapy. Using peptides predicted to bind to MHCI with a binding affinity ≤ 500 nM, the authors focused on mutated peptide sequences shared by multiple tumors and shared by patients having long-term clinical benefit. From this analysis a neo-epitope “signature” was derived, consisting of a distinct pattern of mutated peptide sequences. One of the peptide signatures identified matched an amino acid sequence in MART1, a known melanoma antigen. However the bulk of predicted neoantigens were tetrapeptide sequences shared across antigenic peptides, that is, they were encoded by diverse genes (you have to go into the huge supplemental data file to find this list, suffice to say it is very long). To make sense of this curious result the authors note that the some of the predicted sequences have high homology to viral and bacterial antigens, citing a CMV antigenic sequence as an example. They speculate, and here we quote from the paper: ” These data suggest that the neoepitopes in patients with strong clinical benefit from CTLA-4 blockade may resemble epitopes from pathogens that T cells are likely to recognize.”

The unstated null hypothesis is that there is no relationship between the shared tetrapeptide sequences and clinical response and that the association between the two phenomena is due to a productive immune system response to anti-CTLA4 antibody therapy which has released the anti-tumor response as well as many otherwise quiescent immune responses, such as those to pathogenic viruses and bacteria (and to self antigens, as shown by the autoimmune toxicity associated with anti-CTLA4 antibody treatment). The in vitro response assay data sheds no real light on this, since these assays cannot distinguish anti-tumor responses from other immune responses. So we are left with an intriguing correlation, and a nagging sense that only a very few of the vast number of predicted neo-epitopes will actually trigger bona-fide anti-tumor T cells responses. Indeed the weakness of the paper is the reliance on predictive rather than experimental identification of productive peptide/MHC interaction. As we say in the mouse studies the majority of predicted interactions are not confirmed experimentally.

Regardless, the paper is a remarkable and important step forward, and shows us (as do the mouse studies) the level of investigation required to identify neo-antigens that might expand be used to expand patient TIL populations, as we have discussed in other posts. Returning to onco-vaccines, these three papers together show us that neo-antigen anonymity, rarity and variability from patient to patient are critical issues that will need to be addressed if we are to efficiently develop this therapeutic class.

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:

Screen Shot 2014-12-05 at 7.04.52 AM

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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.

Last Week’s Immune Checkpoint Papers In Nature Are Complicated!

Last week we were treated to a barrage of good news regarding PD-1/PD-L1 therapeutics and the ability to select responders. The centerpiece was a trio of papers in Nature.

Powles et al. presented data on the use of MPDL3280A, an anti-PD-L1 IgG1 antibody that has been engineered to lack all ADCC function (link 1). The antibody blocks the interaction of PD-L1 with PD-1 and with CD80, two receptors found primarily on lymphocytes. The paper focused on the application of ’3280′ therapy in chemotherapy-resistant metastatic urothelial bladder cancer (UBC). Nearly all patients (93%) had failed platinum-based chemotherapy; 72% had failed 2 or more lines of prior therapy. 75% had visceral metastases, most had poor renal function and the majority (59%) had a performance score of 1 (very poor). In a word, these patients were incurable. Preliminary Phase 1 data demonstrating efficacy in UBC was presented at ASCO and led to breakthrough designation for ’3280 for the treatment of UBC in June 2014.

The original Phase 1 trial had enrolled UBC patients whose resection or biopsy tissue demonstrated the presence of tumor-infiltrating lymphocytes (TIL) with dark staining (score 2 or 3) for PD-L1. The expansion cohort allowed for the enrollment of patients whose tissue specimens contained TIL which were PD-L1 dim (score = 1) or negative. 205 patient tissues were analyzed (see table 1 in the paper). 67 patients were enrolled and evaluable with PD-L1 staining results as follows:

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A total of 17 patients responded and 16/17 responses were ongoing (i.e. durable) at the time of data cutoff. The longest duration of response was a remarkable 30 weeks in the cohort with the brightest PD-L1 TIL staining, although the range was broad (from 1 week to 30). Median duration of treatment was 9 weeks, so this is really an early snapshot. Regardless, the ability to invoke an anti-tumor response in a cohort of patients that are this ill, and deemed incurable, is remarkable.

With reference to the staining pattern of PD-L1 and the relevance of PD-L1 expression to successful response, the authors came to the following conclusions:

1) therapy triggered expansion of the circulation CD8+ T cell population, and transient elevation of IL-18 and IFNgamma was observed; these systemic changes reflect the proposed mechanism of action of ’3280 but did not correlate with response.

2) expression of PD-L1 on TIL, but not on tumor cells, was predictive of response to therapy. On note, this was true whether the available tissue sample was new acquired or archival (up to 10 years old). This suggests that there is an ongoing and futile immune response in these PD-L1+/TIL+ tumors. The lack of association with tumor PD-L1+ status is discussed more extensively in the companion paper (see below).

3) the efficacy of PD-L1-directed therapy in UBC and also NSCLC and melanoma, all tumors with very high mutational burdens, suggests that antigen diversity or antigen “burden” may be important for successful induction of an anti-tumor immune response in ’3280-treated patients.

The UBC cohort was part of a much larger clinical trial that included diverse solid tumors. A companion paper by Herbst et al. investigates the utility of PD-L1 TIL expression in other cohorts (link 2). The focus of this work is on the biomarker application, particularly with respect to PD-L1+ TIL staining, as defined in the prior paper. Patients (n=277) with advanced incurable cancers were enrolled in a ’3280 dose ranging study, given drug iv every 3 weeks. Across tumor types high PD-L1 expression on TIL, but not tumor cells, was associated with response and increased PFS. Note here that the PFS gain, while encouraging, does not suggest that we will see a high percentage of truly durable (“long tail”) responses in this particular patient population, even in those patients with PD-L1 bright (score of 3) staining:

Screen Shot 2014-12-04 at 9.27.50 AM

There were some interesting additional analyses. In NSCLC patients who had been smokers, 43% responded to therapy, while only 10% of non-smokers responded. Such data have been reported before, and are often taken to mean that the higher mutational burden seen in smokers with NSCLC biases their tumor toward immune recognition (this echoes the mutational diversity/mutational burden argument made in the Powles UBC paper). Sticking with NSCLC, 83% of patients with a PDL1+ TIL staining score of 3 (lots of cells and therefore dense/dark staining) responded versus 38% of patients with a PDL1+ TIL staining score of 2 (diffuse staining, fewer cells). Response was positively correlated with CTLA4+ staining on TIL, and negatively correlated with fractalkine expression. In melanoma (but not NSCLC or RCC) response was associated with elevated IFNgamma and IDO1 and CXCL9 that are induced by IFN gamma. Strikingly, positive anti-tumor responses were not associated with a measureable change in FoxP3 expression, suggesting the T regulatory T cells were not playing a role in the setting of ’3280 therapy.

What about the non-responders, as these make up the majority of the patients across indications? Progressing tumors were characterized into three classes:

1) few or no TIL present – “immune ignorance”

2) TIL present but little or no PD-L1 expression – “non-functional immune response”

3) TIL present and PD-L1+ but located on the edge of the tumor – “excluded infiltrate”

Missing here I think is an analysis of tumors with PD-L1+ TIL with high staining scores (2 or 3) that progressed, i.e. did not respond to therapy. It seem to me unlikely that these all fell into category “3″ above, so this analysis may be coming in a follow-up paper.

The authors make a very interesting point about this data, which is that they seem to refute the consensus model of “immune resistance” in which it is postulated that CD8+ T cells infiltrating tumors secrete IFNgamma and other cytokines that induce PD-L1 expression on the tumor cells themselves, and these tumor cells in turn produce factors that create an immunosuppressive environment that includes potently immunosuppressive, PD-L1 bright T regulatory cells. The “immune resistance” model further postulates that the expression of PD-L1 on tumor cells and T regulatory cells is responsible for shutting down CD8+ T cells by binding to PD-1.

There are several key messages in this paper – first, responses in these incurable patients are measureable and remarkable, if they respond (most do not). Second, CD8+/PD-L1+ TIL are highlighted as a potential prognostic indicator of the potential for response the ’3280 therapy. Finally, it is clear that other signals will have to be disabled or enhanced in order to induce a productive and durable immune response in more patients and/or move PD-1/PD-L1-directed therapies to front line.

Now, the final paper in this triad turns things upside down. Tumeh et al. analyzed tumor tissue samples from 46 metastatic melanoma patients treated with pembrolizumab, an anti-PD-1 antibody  (link 3). The analytic methods used are elegant and overlap but also extend the analyses used in the prior 2 papers: quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and TCR deep sequencing (NGS).

This paper is strictly about melanoma. The ORR in this small study was 48% (22/46). The authors focused on expression of PD-L1 on tumor cells and of PD-1 on CD8+ T cells. Doing so they come to strikingly different conclusions than the papers discussed above. Responders in this study had PD-1+ CD8 T cells massed on the tumor margin, adjacent to PD-1+ tumor cells. Response was associated with infiltration of the tumor by those CD8+ T cells, which also increased in density (proliferated). Therefore the paper specifically supports the “immune resistance” model in which tumor-expressed PD-L1 suppresses PD-1+ CD8 T cells. CD8 T cell proliferation was associated with expression of granzyme B within the tumor and phosphorylated STAT1 at the tumor margin where CD8+ T cells were infiltrating (phospho-STAT1 in induced by IFNgamma receptor signaling). Finally, response was associated with T cell (TCR) clonality, i.e. the fewer tumor antigens, and thus the lower the antigen burden that is invoking a response, the better. This is a different take than we got from the prior papers.

So, perhaps melanoma is distinctly different.

Aside from that, these papers provide critical take-home messages and perhaps even more critical questions to be addressed:

1) CD8 T cells are good. That’s pretty clear, whatever they are expressing. We can argue more about their geography, but if they are not present, you will not respond.

2) IFNgamma is good. We see this especially in the melanoma setting as detailed in two of the papers.

Neither of these conclusions is novel nor surprising.

3) Biomarker development beyond CD8+ T cell staining remains complex.

4) Regardless of their biomarker status most patients still do not respond and we do not know why. As we consider combination therapy, will other markers be used to further sort patients into rational combination buckets, or will this simply too complex to be useful?

5) Finally, what about those T regulatory cells we’ve been obsessed with for the last decade? These are hardly mentioned in the context of PD-1/PD-L1 therapeutics in the three studies.

next time:

>>> back to those tumor antigens? New papers, preclinical and clinical, shed some light… and

>>> those T regulatory cells may be important in some settings, but were betting on the tumor microenvironment to yield interesting new targets for therapy

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

 Screen Shot 2014-11-23 at 6.36.37 PM

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: – 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 (

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.