Category Archives: PD-L1

Immuno-oncology (IO) combination therapy- why the angst?

Thoughts triggered by discussions over the last month or two, perceived sentiment on social media, reaction to clinical updates, and pre-AACR butterflies.

In 2015 Gordon Freeman of the Dana Farber Cancer Institute, one of the discoverers of the PD-1/PD-L1 axis, rang me up and asked if I would help write a review with he and Kathleen Mahoney, an oncologist doing a research rotation in his lab. We ambitiously laid out the argument that PD-1/PD-L1 directed therapeutics would be the backbone of important combination therapies and reviewed the classes of potential combinatorial checkpoints ( We covered new immune checkpoint pathways within the Ig superfamily, T cell stimulatory receptors in the TNF receptor superfamily, stimulatory and inhibitory receptors on NK cells and macrophages, targets in the tumor microenvironment (TME), and so on. Importantly we also stopped to consider combinations with “traditional” cancer treatments, e.g. chemotherapy and radiation therapy, and also with “molecular” therapeutics, those directed to critical proteins that make cells cancerous. Regardless, it’s fair to say that we believed that pairing an anti-PD-1 mAb or an anti-PD-L1 mAb with another immuno-modulatory therapeutic would quickly yield impressive clinical results. A massive segment of the IO ecosystem (investors, oncologists, biopharma) shared this belief, and largely still does. Those stakeholders are betting clinical and R&D resources plus huge amounts of money on the promise of IO combinations. After all, the first IO combination of anti-CTLA4 mAb ipilimumab and anti-PD-1 mAb nivolumab has dramatically improved clinical response in advanced melanoma patients and to a lesser extent in advanced lung cancer patients. The downside is additive toxicity, and so the palpable feeling has been that new IO combinations would give a similar efficacy bump, perhaps even with less toxicity.

It’s now about two and a half years since we began drafting that paper and the inevitable letdown has set in. What happened? Let’s cover a few issues:

- Several marque IO combinations have been disappointing so far. Last year we saw unimpressive results from urelumab (anti-4-1BB) in combination with nivolumab (anti-PD-1) and of epacadostat (an IDO inhibitor) paired with pembrolizumab (anti-PD-1).

- Monotherapy trials of therapeutics directed to hot new targets (OX40, CSF1R, A2AR etc.) did not produce any dramatic results, forcing a reevaluation of the potential for truly transformative clinical synergy in the IO combination setting.

- These first two points also reminded the field of how limited preclinical mouse modeling can be.

- Combinations of standard of care with anti-CTLA4 mAb ipilimumab and with PD-1 pathway inhibitors have begun to show promising results, raising the efficacy bar in a variety of indications. There have been several startling examples: the combination of pembrolizumab plus chemotherapy in first line lung cancer, which doubled response rates over pembrolizumab alone; the combination of cobimetinib (a MEK inhibitor) with atezolizumab (anti-PD-L1 mAb) in colorectal cancer (MSS-type) which produced clinical responses in patient population generally non-responsive to anti-PD-1 pathway inhibition; the combination of atezolizumab plus bevacizumab (anti-VEGF) in renal cell carcinoma, showing promising early results; and so on.

- We can add the realization that relapses are a growing issue in the field, with approximately 30% of anti-CTLA4 or anti-PD-1 pathway treated patients eventually losing the anti-tumor response.

Note here that all of this is happening in a rapidly evolving landscape and is subject to snap-judgment reevaluation as clinical data continue to come in. For example, rumors that IDO inhibition is working well have been spreading in advance of the upcoming AACR conference. Indeed the clinical work on all of the immuno-modulatory pathways and IO combinations has increased, and the race to improve care in diverse indications continues. There will be additional success stories.

Why the perception of angst then? The sentiment has been summed up as “everything will work a little, so what do we research/fund/advance? How do we choose? How will we differentiate”? Such sentiment puts intense pressure on discovery, preclinical and early clinical programs to show robust benefit or, and perhaps this is easier, benefit in particular indications or clinical settings. I started thinking about this recently when a friend of mine walked me through a very pretty early stage program targeting a novel pathway. It was really quite impressive but it was also apparent that the hurdles the program would have to clear were considerable. Indeed it seemed likely that validation of the therapeutic hypothesis (that this particular inhibitor would be useful in IO) would not come from preclinical data in mice (no matter how pretty), nor from a Phase 1 dose escalation safety study, nor from a Phase 1 expansion cohort, but would require Phase 2 data from a combination study with an anti-PD-1 pathway therapeutic. That is, 5+ years from now, assuming all went smoothly. To advance such a therapeutic will take intense focus in order to build a fundable narrative, and will require stringent stage-gates along the way. Even then it will be very hard to pull it off. If this reminds you of the “valley of death” we used to talk about in the biotech realm, well, it should.

What should we look for to shake up this landscape? As mentioned, this is a rapidly evolving space. We have already seen a shift in language (“step on the gas” vs. “make a cold tumor hot” is one good example), but let’s list a few:

- “Cold tumors” have no immune response to stimulate. Making them “hot” is a hot field that includes oncolytic virus therapeutics, vaccines, “danger signals” (TLRs, STING, etc), and, to loop back around, chemotherapy and radiation therapy.

- Relapsed patients – as noted above we are seeing ~30% relapse rate in immunotherapy treated patients. Understanding the basis for relapse is a promising field and one that an emerging therapeutic could (and very likely will) productively target.

- Targeting the TME in cold tumors and in unresponsive tumors (the difference is the unresponsive tumors look like they should respond, in that they contain T cells). This is a vast field that covers tumor cell and stromal cell targets, secreted factors, tumor and T cell metabolism and on and on. One can imagine a setting in which a particular TME is characterized (by IHC, Txp or other means) and the appropriate immuno-modulatory therapeutics are applied. We see this paradigm emerging in some indications already. This would certainly be useful as a personalized medicine approach and could be an excellent way to position an emerging therapeutic.

We could go further to talk about the neoantigen composition of particular tumor types, the role of the underlying mutanome, the plasticity of the TME (it’s a chameleon), metabolic checkpoints, and other, potentially novel, targets.

All of this is under intense and active investigation and important data will emerge in time. Until then, nascent immunotherapy programs need to tell a clear and compelling story in order to attract the interest of investors, biopharma and ultimately, oncology clinical trialists. Those that fail to develop a compelling narrative are likely to struggle.

I’ll just end on a few narratives I really like for IO combinations going forward:

- the role of innate immunity in activating immune responses and expanding existing responses (e.g. immune primers like STING agonists and NK cell activators like lirilumab)

- the role of adenosine in maintaining an immunosuppressed (ie. non-responsive) TME (thus inhibitors of A2AR, CD39, CD73)

- the role of beta-catenin signaling in non-responsive tumors (while carefully selecting the mode of inhibition)

- the role of TGF-beta activity in resistance to PD-1 pathway therapeutics (again, with care in selecting the mode of inhibition)

of course at Aleta we’ve charted a different course, ever mindful of the need to focus where we see clear yet tractable unmet need. so we’ll see, starting with AACR in early April, kicking off an active medical conference season.

stay tuned.

Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival

A Few Day 1 Highlights

Ton Schumacher (Netherlands Cancer Institute), abstract IA04 ,has discovered a novel regulator of PDL1 expression called PD-L1M1. PD-L1M1 associates with PD-L1 and modulates the T cell inhibitory function of PD-L1. The protein is expressed ubiquitously, so unclear if this finding has therapeutic implication.

Michael Peled and Adam Mor (NYU School of Medicine), abstract A059, had a poster on molecules that interact with the cytoplasmic tail of PD-1 using high resolution Mass Spec. Two proteins were highlighted on their poster: EFHD2 and SH2D1A. EFHD2 co-localized with PD-1 and was essential for clustering and signal transduction (thus, ablation of EFHD2 blocks PD-1 mediated inhibitory activity). SH2D1A had the opposite function as evidenced by increased PD-1 inhibitory signaling when SH2D1A was knocked down and reduced PD-1 inhibitory signaling when overexpressed. SH2D1A physically competed with SHP2 for access to the PD-1 cytoplasmic tail.

Dario Vignali (U. Pitt School of Medicine), abstract IA05, focused on several emerging immune checkpoints. The first, IL-35, was investigated using anti-IL-35 antibody in various tumor models, with very nice results (similar to anti-PD-1). I liked the neuropilin story – this is a Sema4a binding protein and was offered up as a central control node for Treg activity. NRP1 controls Treg T cell expression of IFNgamma, acting in cis and in trans (so self-regulation and neighborhood regulation). Of interest he identified subsets of melanoma and H&N cancer patients having high levels of NRP1 in the TME, so this is perhaps an actionable finding.

Susan Kaech (Yale Univ Med School), abstract 1A07, presented data showing that the PEPCK overexpression ups the anti-tumor activity of T cells in the TME, thus showing that T cells – if given the tools – can co-opt the same metabolic pathways (lactate, fatty acids) used by tumor cells in the tumor microenvironment (TME). A consequence of this metabolic checkpoint is the upregulation of PD-1 via fatty acid signaling through the PPARs, delta I think. Of interest is that the metabolic switch is supported by gross upregulation of CD36, a fatty acid active transporter, on T cells in the TME.

Greg Delgoffe (U Pitt Cancer Inst), abstract IA08, picked up this general theme, demonstrating that T cells dividing in the TME rapidly lose mitochondrial (MT) mass, and therefore their ability to metabolize glucose ( a T cells preferred energy source). This is a failure of MT biogenesis, due to the downregulation of PGC1alpha, which is required for the process. In the TME, T cell PGC1alpha expression is regulated by AKT – robust AKT signaling leads to PGC1alpha downregulation. If note, PGC1alpha transgenic T cells retain high proliferative activity, do not lose MT, and are highly active Teffector cells.

“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)

The twisted tale of neoantigens and anti-tumor immune responses

Two papers out this week add to a pile of data addressing the role of neoantigens in tumor therapy. While these papers address tumor neoantigen “load” in the context of immune checkpoint therapy the results have implications for TIL therapeutics, TCR therapeutics and onco-vaccine development.

A really dramatic paper from diverse groups at the University of Pennsylvania and their collaborators, just published in Nature (link-1), explores the complex interplay of radiation therapy and anti-CTLA4 antibody therapy (ipilimumab, from BMS) in patients with stage IV metastatic melanoma (relapsed or previously untreated). In this Phase 1/2 clinical trial (NCT01497808) patients with multiple melanoma metastases received various doses of radiation therapy delivered to a single metastasis, termed the “index lesion”. They then received 4 doses of ipilimumab (3 mg/kg, i.v., once every 3 weeks) and non-irradiated lesions were evaluated within 2 months of the last dose.

Although the sample size reported is small (n=22) some interesting lessons emerged from the study. The response rate was low, and the progression free survival (PFS: 3.8 months) and overall survival (OS: 10.7 months) data bear this out. It appears that just shy of 40% of patients were still alive at ~30 months (see Figure 1c in the paper). It is too early to tell if there will be a “long-tail” effect going forward. In the original ipilimumab study a very small percentage of patients lived for a very long time, “pulling” the PFS and OS curves to the right. Regardless, most patients in this study did not respond and the questions posed in this paper are directed to the mechanisms of resistance.

The mouse B16-F10 melanoma model was used to model resistance. Mice with tumors were locally irradiated then treated with an anti-mouse-CTLA4 antibody, to mimic the clinical trial. Only 17% of the treated mice responded. Two predictors of response/non-response were elucidated: 1) the ratio of effector T cells (Teff) to regulatory T cells (Treg) and 2) a gene signature in the tumor cells that is dominated by the expression of PD-L1 and IFNgamma regulated genes. In short, if the melanoma cells are expressing PD-L1 and the tumor infiltrating lymphocyte (TIL) population is dominated by Tregs (which are PD-1+), then the radiation + anti-CTLA4 therapy failed.

To further subset TIL into active Teff versus non-responsive “exhausted” Teff, the authors used an expression profile of PD-1+/Eomes+ to identify exhausted Teff and PD-1+/Eomes+/Ki67+/GzmB+ for active Teff. Importantly, exhausted Teff could be reanimated upon treatment with PD-1 pathway antagonists: anti-PD-1 antibody or anti-PD-L1 antibody. This reanimation led to an improved CD8+ Teff/Treg ratio and led to tumor control in the majority of the mice (up to 80%) when the treatment consisted of irradiation plus anti-CTLA4 plus anti-PD-L1. Of note, radiation plus anti-PD-L1 did not achieve this effect; the triple therapy was required (see Figure 2d).

The striking conclusion is that upregulation of PD-L1 on tumor cells can subvert the effect of anti-CTLA4 antibody therapy, and this therefore qualifies as a mechanism of resistance.

What about the role of irradiation? In both the patients and the mouse model irradiation was local, not systemic. Further, this local irradiation was required to achieve complete responses in the mouse model. What is going on here? Irradiation was linked to a modest increase in TIL infiltration of melanoma tumors in the mouse model, but sequencing of the T cell receptors (TCR) revealed that there was an increase in the diversity of TCRs, meaning that more antigens were being recognized and responded to by TIL after irradiation. In this context then, anti-CTLA4 reduced the Treg population, anti-PD-L1 allowed CD8+ TIL expansion, and irradiation set the antigenic landscape for response.

Returning to the patients armed with this information from the mouse study, the authors find that low PD-L1 expression on the melanoma cells correlates with productive response to irradiation plus ipilimumab therapy, while PD-L1 high expressing tumors were associated with persistent T cell exhaustion. In addition, monitoring the state of the CD8+ T cell population (PD-1+/Eomes+ versus PD-1+/Eomes+/Ki67+/GzmB+) suggested that these phenotypes might be useful as peripheral blood biomarkers. The patient numbers are very small for this analysis however, which awaits further validation.

The conclusion: irradiation combined with ipilimumab plus anti-PD-L1 antibody therapy should be a productive therapeutic combination in PD-L1+ stage IV melanoma. Similar strategies may be beneficial in other solid tumor types. This is interesting news for companies developing anti-PD-L1 antibodies, including BMS-936559 (also from BMS), MPDL3280A (Roche/Genentech), MEDI4736 (AZN) and MSB0010718C (Merck Serono).

A second paper (link) bring our focus back to PD-1, in the context of non-small cell lung cancer (NSCLC). Using the anti-PD-1 antibody pembrolizumab (from Merck) a group from the Memorial Sloan-Kettering Cancer Center sought to determine correlates of response of NSCLC patients to anti-PD-1 therapy. Their findings again hone in on neoantigen load, as the best predictors of response were the non-synonymous mutational burden of tumors, including neoantigen burden and mutations in DNA repair pathways. What all this means is that mutations that change the amino acid sequence (thus, are non-synonymous) can produce neoantigens that can be recognized by CD8+ T cells; mutations in the DNA repair pathways increase the rate that such mutations go uncorrected by a cell.

The authors sequenced the exomes (expressed exons – these encode proteins) from tumors versus normal tissue, as a measure of non-synonymous mutational burden that could produce neoantigens. Patients were subsetted based on response: those with durable clinical benefit (DCB) and those with no durable benefit (NDB). High mutational burden was correlated with clinical efficacy: DCB patients averaged 302 such mutations, while NDB patients averaged 148; ORR, PFS and OS also tracked with mutational burden. In a validation cohorts the number of non-synonymous mutations was 244 (DCB) versus 125 (NDB).

Examination of the pattern of exome mutations across both cohorts was studied in an attempt to discern a pattern of response to pembrolizumab treatment. The mutational landscape was first refined using an algorithm that predicts neoepitopes that can be expressed in the context of each patients specific class I HLA repertoire – these are the molecules that bring antigens to cell surfaces and present them to T cells for recognition (I’m simplifying this process but that is the gist of it). The algorithm identified more potential neoepitopes in the DCB patient tumors than in the NDB cohort, more impressively, a dominant T cell epitope was identified in an individual patient using a high-throughput HLA multimer screen. At the start of therapy this T cell clone represented 0.005% of peripheral blood T cells, after therapy the population had risen 8-fold, to 0.04% of peripheral blood T cells. Note that most of this clone of T cell would be found in the tumor, not in circulation, so that 8-fold increase is impressive. The T cells were defined as activated CD8+ Teff cells by expression markers: CD45RA-/CCR7-/LAG3-. As in the first paper we discussed, it is useful that these markers of systemic response to immunotherapy treatment are being developed.

There is an interesting biology at work here. It is often noted that high mutational burden is associated with better outcome, for example to chemotherapy in ovarian cancer, and irrespective of therapy across different tumor types (link-2). This suggests that tumor neoepitopes are stimulating an ongoing immune response that is stifled by active immunosuppression, yet is still beneficial. Once unleashed by immune checkpoint blockade, the immune system can rapidly expand it’s efforts.

We recently reviewed the importance of neoantigens in anti-tumor therapy (link-3) although the focus then was on cellular therapeutics rather than on immune checkpoint modifiers such as anti-CTLA4 and anti-PD-1 or PD-L1 antibodies. We can mow add that our ability to track neoantigens and the immune response to neoantigens is opening new avenues for investigating immuno-oncology therapeutics and their efficacy.

Reading List – day 2, #JPM15 edition – The Power of Immunotherapy

BMY today announced that an open-label, randomized Phase 3 study (CheckMate-017; NCT0164200) evaluating Opdivo vs docetaxel in previously treated patients with advanced, squamous NSCLC was stopped early because an assessment concluded that the study met its endpoint, demonstrating superior overall survival in patients receiving Opdivo.

CheckMate-017 is a Phase 3, open-label, randomized study. Patients who had failed prior platinum doublet-based chemotherapy received either nivolumab 3 mg/kg intravenously every two weeks or docetaxel 75 mg/m2 intravenously every three weeks (N = 272, randomized).

The primary endpoint is overall survival. Secondary endpoints include objective response rate and progression free survival. The initial time frame was 38 months from enrollment. The trial opened in 2012 and was scheduled for primary outcome measurement in January 2016, so this halt is a year early. An association between PD-L1 expression and efficacy measures (ORR, OS PFS) will be explored post hoc.

Arms Assigned Interventions
Experimental: Arm A: Nivolumab

Nivolumab 3 mg/kg solution intravenously every 2 weeks until documented disease progression, discontinuation due to toxicity, withdrawal of consent or the study ends

Biological: Nivolumab

Other Name: BMS-936558

Experimental: Arm B: Docetaxel

Docetaxel 75 mg/m2 solution intravenously every 3 weeks until documented disease progression, discontinuation due to toxicity, withdrawal of consent or the study ends

Drug: Docetaxel

Other Name: Taxotere®

Key Inclusion Criteria:

  • Adult subjects with Stage IIIB/IV disease or with recurrent or progressive squamous cell NSCLC who present with disease following multimodal therapy (radiation therapy, surgical resection or definitive chemoradiation therapy for locally advanced disease)
  • Disease recurrence or progression during/after one prior platinum doublet-based chemotherapy regimen for advanced or metastatic disease
  • Evaluable by imaging (CT/MRI) per RECIST 1.1 criteria
  • ECOG performance status ≤1
  • Formalin fixed, paraffin-embedded tumor tissue block or unstained slides of tumor sample (archival or recent) available for biomarker evaluation. Biopsy is excisional, incisional or core needle.

Key Exclusion Criteria:

  • Subjects with untreated central nervous system (CNS) metastases are excluded. Subjects are eligible if CNS metastases are treated and subjects are neurologically returned to baseline for at least 2 weeks prior to enrollment. In addition, subjects must be either off corticosteroids, or on a stable or decreasing dose of ≤10 mg daily prednisone (or equivalent)
  • Subjects with active, known or suspected autoimmune disease (except for type I diabetes mellitus, hypothyroidism only requiring hormone replacement, vitiligo, psoriasis, or alopecia not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger).
  • Subjects with a condition requiring systemic treatment with either corticosteroids or other immunosuppressive medications within 14 days of randomization
  • Prior therapy with anti- PD-1, anti-PD-L1, anti- PD-L2, anti-CD137, or anti-CTLA-4 antibody (including ipilimumab or any other antibody or drug specifically targeting T-cell co-stimulation or checkpoint pathways)
  • Prior treatment with Docetaxel
  • Subjects with interstitial lung disease that is symptomatic or may interfere with the detection or management of suspected drug-related pulmonary toxicity
  • Treatment with any investigational agent within 14 days of first administration of study treatment

So stopping this trial early is great news. What can we anticipate in addition to the report of ORR, OS, PFS etc that we will likely get at ASCO? The answer lies in the details regarding the Checkmate-017 trial.

A few pointers:

1) there is no inclusion biomarker, ie., there is no specified use of PD-1 staining of biopsy tissue that puts patients into the trial. This is in line with the confusion surrounding use of PD-1 as a biomaker.

2) there is a requirement that pretreatment biopsy specimens be available, as these will be used retrospectively to associate response with expression of biomarkers, including PD-L1 (the PD-1 ligand). No doubt many other biomarkers will be explored.

3) if you have autoimmune disease or interstitial lung disease (a broad term) you are out of luck. So patients with RA, MS, IBD, lupus, and a whole host of other autoimmune diseases need not apply. If you have Type 1 Diabetes though, your good to go (which among other things reminds us just how damn puzzling T1D autoimmunity is).

4) you also cannot be immunosuppressed (corticosteroids) or have had prior treatment with with anti- PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, anti-CTLA-4 antibody (including ipilimumab), or docetaxel. This last one excludes patients who may have gotten docetaxel as second line therapy, which is a setting in which it is commonly used. This tells us that the risk of toxicity in patients is deemed too high.

5) the study halt, being based on efficacy, does not mention toxicity, so we’ll have to wait and see.

Now back to the reading list. In the context of biomarker investigation this story has some resonance:

Day 2 – Immunotherapy: back to those biomarkers of response

Genetic Evolution of T-cell Resistance in the Course of Melanoma Progression

Sucker et al 2014. Clin Cancer Res; 20(24); 6593–604

This interesting paper outlines a technique for tracking the evolution of immune resistance, an essential part of the so-called immune editing process, using in vitro analysis of patient-derived (PDX) samples.

Three consecutive melanoma lesions obtained within one year of developing stage IV disease were analyzed for their recognition by autologous T cells.

One skin and two lymph node metastases were initially analyzed for T-cell infiltration. Then, melanoma cell lines established from the respective lesions. T-cell–stimulatory capacity, expression of cell surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor–T-cell interactions were identified.

Sampled skin lesions were infiltrated by T cells. The T cell infiltrate was diminished in the lymph node metastatic samples which were found to be HLA class I–negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. This is an impressive response to avoid immune detection.

The study reveals a progressive loss in melanoma immunogenicity during metastasis. Screening tumors for this and other genetic alterations  that cause acquired immune resistance will be clinically relevant in terms of predicting patient responses and designing combinatorial approaches to immunotherapy.

Day 2 – Immunotherapy: back to those tox issues: it’s hard to control ipilimumab-induced tox

In the trial above we noted two things: no current corticosteroid use and no prior ipilimumab. Turns out these don’t play well together either

Min et el. 2014. Systemic high dose corticosteroid treatment does not improve the outcome of ipilimumab-related hypophysitis: a retrospective cohort study

Purpose To examine the onset and outcome of ipilimumab-related hypophysitis and the response to treatment with systemic high dose corticosteroids. Patient and Methods Twenty-five patients who developed ipilimumab-related hypophysitis were analyzed for the incidence, time to onset, time to resolution, frequency of resolution, and the effect of systemic high-dose corticosteroids on clinical outcome. To calculate the incidence, the total number (187) of patients with metastatic melanoma treated with ipilimumab at Dana-Farber Cancer Institute (DFCI) was retrieved from the DFCI oncology database. Comparisons between corticosteroid treatment groups were performed using Fisher’s exact test. The distributions of overall survival were based on the method of Kaplan-Meier. Results The overall incidence of ipilimumab-related hypophysitis was 13%, with a higher rate in males (16.1%) than females (8.7%). The median time to onset of hypophysitis after initiation of ipilimumab treatment was 9 weeks (range: 5-36 weeks). Resolution of pituitary enlargement, secondary adrenal insufficiency, secondary hypothyroidism, male secondary hypogonadism, and hyponatremia occurred in 73%, 0%, 64%, 45%, and 92% of patients, respectively. Systemic high dose corticosteroid treatment did not improve the outcome of hypophysitis as measured by resolution frequency and time to resolution. One-year overall survival in the cohort of patients was 83%, and while slightly higher in patients who did not receive high dose corticosteroids, there was no statistically significant difference between treatment arms. Conclusion Systemic high dose corticosteroid therapy in patients with ipilimumab-related hypophysitis may not be indicated. Instead, supportive treatment of hypophysitis-related hormone deficiencies with the corresponding hormone replacement should be given.

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:

Screen Shot 2014-12-04 at 11.14.57 AM

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

Side Effect Profiles of Immune Checkpoint Therapeutics – Parts 2 and 3

Part 2 – The Border Wars.

One of the fascinating aspects of the toxicity of immune checkpoint therapeutics is that it is a lot of is triggered at the border between self and non-self, where non-self is everything that the immune system must encounter and sort through continuously. The sorting serves to identify pathogens and ignore non-pathogens among the myriad components of the microfauna and flora that inhabit these borders. The “sampling” of these ecosystems is continuous and highly reactive – one glass of unpurified water taken on foreign soil will teach you this lesson pretty quickly. When the immune system is unrestrained by blockade of CTLA4 and/or PD-1 it is not surprising that we see the breakdown of immune tolerance in these border zones.

There are three major surfaces where toxicity has been an issue: the skin, the gut mucosa, and the airspaces of the lung. Ipilimumab treatment can cause pretty intense inflammation of the skin, generally dismissed in the clinical trial literature as “rash”. In a pooled analysis of nearly 1500 patients enrolled in various ipilimumab clinical trials, 45% developed dermatological AEs considered drug related, and 2.6% (so 39 people) developed severe symptoms rating a grade 3-4 (where grade 5 is lethal) (see Tarhani, A. Scientifica 2013, Article ID 857519). A fair amount of the milder skin AEs can be ascribed to an anti-melan-A response, as this antigen is abundant in melanoma, the setting for the clinical development trials. In the Phase 3 registrational trials dermatologic AEs were reported in more than 40% of patients in the ipilimumab arms, and there were very severe AEs that cannot be ascribed to an anti-melan-A (i.e melanocyte) immune response. This is from Tarhani’s review of patients in the ipilimumab + gp100 (vaccine) and ipilimumab monotherapy arms having dermatological irAEs,

“of these, 2.1% and 1.5%, respectively, were grade 3 or higher … Severe, life threatening, or fatal immune-mediated dermatitis (Stevens- Johnson syndrome, toxic epidermal necrolysis, … full thickness dermal ulceration, or necrotic, bullous, or hemorrhagic manifestations; grade 3–5) occurred in 13 of 511 (2.5%) patients treated with ipilimumab. One patient (0.2%) died as a result of toxic epidermal necrolysis, and one additional patient required hospitalization for severe dermatitis… .”

That’s some rash. We note in passing that dermatologic AEs were see in a phase 2 trial of ipilimumab plus chemotherapy in non-small cell lung cancer (NSCLC) and so this is certainly not limited to the melanoma setting. PD-1 pathway antagonists also cause skin inflammation in both the melanoma and other settings, similarly suggesting that what we are seeing here are immune responses to antigenic stimulation that is normally immunologically inert. Nivolumab-induced dermatologic toxicity can be severe, but is less common than seen with ipilimumab therapy.

The issue of skin toxicity is well known clinically, and there are established treatment protocols requiring cessation of therapy and treatment with anti-inflammatories, usually steroids (i.e the REMS protocols). The gastrointestinal (GI, “gut”) AEs are also common, can arise suddenly, be resistant to therapy (corticosteroids, and rarely, anti-TNF antibody), and are of significant concern. Returning to the pooled analysis of ~1500 ipilimumab patients we see roughly half of the patients developing GI symptoms (this includes diarrhea). If we focus on grade 3/4 SAEs we have 10-12% of patients with GI disorders that include colitis, enterocolitis, intestinal perforations etc that can proceed to lethal septic complications. Of note, inflammatory infiltrates in the intestines include abundant T cells and neutrophils, showing that acute ongoing inflammation is occurring. GI toxicity is less common and less severe in nivolumab-treated patients, and this is true also of Merck’s anti-PD-1 antibody pembrolizumab and the anti-PD-L1 antibody MPDL3280A from Roche. Colitis is generally not a big issue, for example, GI SAEs are seen in less than 1% of nivolumab-treated patients. We might conclude here that other pathways are maintaining tolerance in the gut mucosa when the PD-1 pathway is blocked.

A different picture emerges when we consider AEs in the lung. Pulmonary toxicity is rare in the context of ipilimumab monotherapy, with only scattered case reports in the literature (see Voskens et al for a review of rare ipilimumab-induced AEs: link). Anti-PD-1 pathway therapeutics, particularly nivolumab, are associated with pneumonitis, which is inflammation of the lung tissues. In the monotherapy setting, both nivolumab and pembrolizumab causes pneumonitis in 3-4% of patients – the condition is generally mild and treatable. Of note this AE rate is consistent across indications (e.g. melanoma, renal cell). The anti-PD-L1 antibodies (Roche’s MPDL3280A and Astra Zeneca’s MEDI4736) have not been associated with pneumonitis to date, perhaps reflecting a unique profile. The recent data from the anti-PD-L1 antibody MEDI4736 trial in NSCLC presented a tolerable profile. While response rate was low, significant numbers of patients remained on therapy with stable disease (ASCO 2014, Abstract #3002).

More worrisome is the pneumonitis rate and severity in combination therapy particularly in the NSCLC setting where diminished lung function is already a concern (smokers with lung cancer can’t breathe). When nivolumab was combined with platinum-based chemotherapy in NSCLC the SAE rate jumped to 45%, with notable findings of grade 3/4 pneumonitis (7%) and acute renal failure (5%) (ASCO 2014, Abstract #8113). Nivolumab plus erlotinib was not associated with pneumonitis (ASCO 2014, Abstract #8022) but response rates were low as well suggesting that these therapies were not particularly additive. The combination of nivolumab with ipilimumab was most worrisome, with grade 3/4 pneumonitis (6%) now seen along with grade 3/4 SAEs of skin (4%), GI (16%) and others (16%) (ASCO 2014, Abstract #8023). Most problematic is that 35% of patients discontinued, and between 3 to 5 patients died due to drug related SAEs including respiratory failure (caused by severe colitis), epidermal necrolysis (in a patient with multiple SAEs) and pulmonary hemorrhage (pneumonitis). As indicated above, the anti-PD-L1 antibody MEDI4736 may better suited for combination therapy. A combo trial in NSCLS with anti-CTLA4 mAb tremelimumab is enrolling, so we’ll wait and see.

It’s fair at this point to take a step back and say “so what?” These are close to terminal patients with deadly cancers usually highly refractory to treatment, and we cannot expect a free ride. The unmet need is acute and urgent, and these therapeutics offer potential cures and increase in life expectancy – as shown very clearly in last weeks early termination of the Phase 3 trial of nivolumab versus dacarbazine due to the obvious overall survival advantage offered by nivolumab (see John Carroll’s story in Fierce Biotech here: link)

The problem is that the response rates we are seeing are generally low, the discontinuation rates high, and for anti-CTLA4 and anti-PD-1 therapeutics there is no clear consensus regarding the use of biomarkers to select patients most likely to respond. Therefore the actual percent penetrance of therapy in the patient cohorts becomes quite low. For those relatively few patients who respond well the outcomes can be sustained and robust. It is critical however to get these response rates up. The blockbuster combination of nivolumab plus ipilimumab in metastatic melanoma gives us a sense of what is possible, if the drugs are tolerable. It is also critical to understand how and why immune therapy can make subsequent therapy intolerable, as we’ve seen in case reports, or conversely, how and why prior therapies can cause such problems for patients going onto an immune therapeutic (see that Voskens review mentioned above). We’ve seen some the issues that can bedevil combinations in metastatic melanoma (with vemurafenib) and in renal cell carcinoma clinical trials (pazopanib) When we look at all of the combination clinical trials underway with these agents we have to wonder what surprises lay in store.

Part 3 – The Fifth Column.

The fifth column refers to enemies lurking within the boundaries of the state, in this case the human body. These are a mixed collection of AEs that can be difficult to understand. While we are used to see liver and kidney inflammation in the setting of cancer therapy, it remains a bit mysterious that immune checkpoint therapy can cause severe inflammatory responses in these organs, the most notable is probably the induction of hepatitis in patients treated with ipilimumab. Even weirder (for me anyway) are the endocrinopathies, headlined by pituitary inflammation, seen with both CTLA4 and PD-1 directed immunotherapies. Primary thyroid inflammation is also seen although less frequently. These are of course autoimmune targets in this setting, but the triggers are obscure, as is also almost always true in autoimmune disease. Somewhat remarkable is the emergence of a sometimes fatal but normally very rare condition known as autoimmune hypophysitis or lymphocytic hypophysitis, which is inflammation of the pituitary gland. Hypophysitis is a unique toxicity of immune checkpoint inhibitors, and has been been seen in patients treated with ipilimumab, tremelimumab, and nivolumab. Because the pituitary sits in the middle of the limbic hypothalamic-pituitary-adrenal axis effects on the thymus and adrenal gland are also noted, with adrenal insufficiency being a severe and life-threatening complication. It must be stressed that the frequency of this AE is stunningly high, reaching 17% in some trials, as the disease has been described only very rarely, with a good deal less than 1000 cases ever known prior to the introduction of immune checkpoint therapeutics.

So we won’t dwell on this, as clinicians now know what to watch for, and treatment paradigms have been developed. As mentioned earlier, treatment generally involves initiation of steroids to control to autoimmune response, and cessation of immune checkpoint therapy.

Let’s return to the consideration of combination therapy, which I think we all agree is essential if we are really to expand use of immune therapeutics in the treatment of these difficult cancers. Great hope has been placed in the combination of CTLA4 and PD-1 targeting agents with “safe” immune checkpoint modulators, notably the IDO-inhibitor from Incyte. We have very little information to date, but it is notable that the dose limiting toxicity in the first combination trial of ipilimumab and INCB024360 from Incyte (INCY) was liver damage as measured by ATL elevation. It may be that merely piling on ways of disrupting Treg activity will not help with the toxicity profile; in fact, one might make the prediction that this approach will make things worse in some settings.

We’ve remarked in passing on the apparently mild safety profile of the anti-PD-L1 inhibitors compared to the PD-1 inhibitors. This makes some sense, as the ligands are expressed by the target tumor cells, and this may be the main sink for the injected antibody, i.e. antibody may not be evenly bio-distributed but rather predominantly localized to the tumors. The concordance of anti-PD-L1 antibody activity with tumor PD-L1 expression is consistent with a direct and localized effect. The fact that there is less consistent concordance of anti-PD-1 antibody activity with PD-1 expression by tumor-infiltrating T cells suggests less specificity in the induced immune response, and this may be why we see autoimmune toxicity in the nivolumab setting. As CTLA-4 is exclusively T cell expressed, the same seems to hold true for anti-CTLA4 antibody therapy. So combining these may not be the most ideal way forward.

We will discuss alternative approaches next time, but first there is some new data on novel immune checkpoint therapies to consider. These are the TNF receptor superfamily proteins that we discussed last month (link): 4-1BB, CD27, OX40 and GITR. There is admittedly very little data to date. Pfizer’s (PFE) anti-4-1BB antibody PF-05082566 reached a safe dose in Phase 1 without undue toxicity signals (ASCO 2014, Abstract #3007). Pfizer disclosed combination trials with rituximab in Non-Hodgkin Lymphoma (NHL) and pembrolizumab (anti-PD-1). The BMY antibody urelumab was tolerated through its’ dose escalation cohorts, and ex vivo analysis showed activation of CD8+ T cells and NK cells (ASCO 2014, Abstract #3017). The Celldex anti-CD27 mAb also has demonstrated safe dose escalation, although to date without signs of clinical activity (ASCO 2014, Abstracts #3024 and #3027). Celldex (CLDX) claims planned studies in combination with nivolumab, ipilimumab, and the targeted therapeutics darafenib and trametinib.

As we discussed in an earlier post, 4-1BB, CD27, OX40 and GITR are evolutionarily closely related receptors. Biomarker studies such as the one performed in the urelumab trial will be essential in understanding how these immune stimulatory pathways will differentiate clinically and which will be safe in combination settings. We’ve reviewed the biology of this superfamily recently (see these posts) so won’t do so again until we get some more clinical data.

Next we will introduce some novel targets in the TNF receptor superfamily, revisit some apoptotic pathway “influencers”, and will swing back around to PD-1 and PD-L1 in some other solid tumor settings (not necessarily in that order).

stay tuned.