Category Archives: Pathways for Drug Development

T cell fitness and genetic engineering

This is a subject we have been thinking about in great detail and this publication in Cell was a trigger for me to start organizing those thoughts. Here is the full reference to the paper discussed: In press, Roth et al., Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies, Cell (2020).

My thanks to Mark Paris from Daiichi Sankyo for his tip to read this paper.

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This publication (https://doi.org/10.1016/j.cell.2020.03.039) is by Theodore Roth and colleagues from Alexander Marson’s lab at UCSF.  They present a nice technological advance, the development of a process by which a pool of genes are knocked into a locus, allowing one to examine the consequence of altering the responsiveness of a cell, in this case, a T cell. This type of work springs from a long lineage of genetic manipulation strategies, from random mutagenesis, to random then targeted gene knockouts (in cells and animals) and gene knockins (what we once called transgenics) and elegant gene-editing technologies (gene therapy, CRISPR/Cas-9, cell therapy, gene-delivery) and so on.

The focus in this paper is on optimizing T cell activity in the setting of solid tumors, something we think about every waking hour at Aleta Biotherapeutics (www.aletabio.com). So, let’s see what we’ve got here.

The pooled knockin strategy relies on two key elements – DNA barcoding, a well-developed technology that has its roots in high throughput library screening technologies, and locus targeting via HDR, which can be achieved using CRISPR/Cas9 and guide templates. Put these two things together and you now have the ability to mix and match genes of interest (following these via their specific barcodes) and place then into the desired locus – here that locus is the TRAC (the TCR locus). They also knocked in a defined TCR (for NY-ESO-1). So, this is a nice system with a known TCR and various immune modifications. There are some limitations. Only 2000-3000 base pairs will fit into the targeting vector (here using a non-viral method). It appears that only a fraction of the targeted T cells are functionally transfected (around 15% per Figure C and note that not every knocked-in cell has both the TCR and the extra gene). The expression level in primary human T cells is high, but I’m guessing expression is of limited duration (although at least 10 days, Figure S5). This is used here as a screening tool, where the goal is to identify critical pathways that reduce or enhance T cell activities (proliferation, effector function, release from immunosuppression).

The authors used a pooling approach to introduce one or two coding sequences from a short list of proteins implicated in T cell biology. Some sequences were modified to be dominant-negative or to be “switch receptors”, where the extracellular domain of the receptor is coupled to a T cell-relevant signaling component (eg. FAS-CD28, TGFβRII-4-1BB). Here are the components they used for their library:

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As we can see from the list there are interesting immune checkpoints, death receptors, cytokine receptors and signaling components that can be mixed and matched. The pool is made and transfected into primary T cells that are then put under selective pressure. The T cells that are enriched under that selective pressure are then analyzed by barcode sequencing to see who the “winners” are, as shown in this schematic from Figure 1A:

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The first screen was simple TCR stimulation (anti-CD3/anti-CD28) which rather robustly showed that a FAS truncation allowed for better cell proliferation (Figure 3B in the paper). This is an expected result – activated T cells undergo FAS-mediated cell death (activation-induced cell death, AICD) that is triggered by FAS-ligand expression, ie. activated T cells kill each other using this pathway. Since there are only T cells in this TCR stimulation culture a lot of other pathways are rendered irrelevant and therefore don’t appear (PD-1 for example):

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The key data are on the far right, showing a 2-4 fold increase in T cell number relative to input. The knockins in light blue showed a statistically meaningful increase vs. input number, across 4 different donor T cells (each circle is a different donor).

The second selective pressure was to stimulate the T cells in the presence of soluble TGFβ (see Figure 3D). As one might guess, the TGFβRII dominant-negative (dn) and switch receptors now come into play: TGFβRII-MyD88, TGFβRII-4-1BB, TGFβRII-dn. The FAS-dn and switch receptors are also represented as are two T cell proliferative components: the IL2RA and TCF-7 (aka TCF-1). These latter hits suggest that amping up T cell proliferation can allow the pool to outrun TGFβ-mediated immunosuppression, at least in vitro. Again, refer to Figure 3D in the paper for the results.

Several other selective pressures were applied in vitro, including tumor cytotoxicity using the NY-ESO-expressing melanoma cell line A375. Of more interest, the A375 cell line was used to establish a xenograft tumor in immunodeficient NSG mice, and the knockin pools of transfected T cells were injected into the mice after the tumor had established. A technical note here – 10 million T cells were injected, of which approximately 1 million were transfected – and 5 days later the tumors were removed and the TIL (tumor-infiltrating lymphocytes) were isolated by screening for the TCR. Bar-code analysis of the TCR-positive TIL allowed the team to identify which transfected T cells got in and expanded. This is tricky, because you’ve allowed time for extensive proliferation (so T cells that are dividing quickly will dominate) and you don’t know what you lost when the T cell pool encountered NY-ESO-positive tumor cells (did some die or did some traffic out of the tumor?). We should expect these data to be noisy and they are, but clear “winners” emerge, namely the TCF-7 transfectants, the TGFβRII-dn, and TGFβRII switch receptors with 4-1BB and also with the TLR signaling component MyD88. Since A375 melanoma cells do not make TGFβ (as far as I know) we have to assume that the T cells themselves are making this, and this is the TGFβ that is triggering these potent (NF-κB triggering) signaling components.

The TGFβRII-dn and switch receptors supported increased IL-2 and IFNγ production – note that IFNγ should have induced PD-L1 on the melanoma cells, but none of the PD-1 based cassettes had any notable effect (from Figure 6B):

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As with the PD-1 pathway, neither the FAS switch receptors nor the FAS-dn construct seemed to play a role in this setting. It’s not clear if FAS-L was upregulated in the tumor model, so that might explain the result.

There was a stark difference in T cell phenotype induced by TCF-7 versus the TGFβRII synthetic constructs. They are in fact polar opposites in some ways (CCR7 expression, Granzyme B expression, IFNγ expression – see Figure 6 E in the paper). Finally, the authors made a bona fide, polycistronic, TCR construct expressing the TGFβRII-4-1BB cassette or the TCF-7 sequence, used this to transduce donor T cells and then tested these for anti-tumor efficacy in vivo (Figure 7). T cells expressing the NY-ESO TCR and the TGFβRII-4-1BB cassette were able to clear the tumor completely. So that’s a very nice result.

Let’s put this into broader context. The table below is a small representation of the literature on genes associated with T cell anti-tumor responses, presented in no particular order. In the left column is the technology used to do the work, then the target, the result, the DOI if you want to read more and then some notes where applicable. I left off a lot of papers, my apologies to those labs.

Technology Target Result notes Reference
dominant-negative transgene FAS increased T cell persistence  and anti-tumor activity 10.1172/JCI121491
transgene overexpression c-Jun reversed tonic-signal induced exhaustion in T cells AP-1 driven 10.1038/s41586-019-1805-z
knockout  Reginase-1 increased T cell persistence, fitness, and anti-tumor activity > Batf and < PTPN2, SOCS1 10.1038/s41586-019-1821-z
knockout PTPN2 increased Lck, STAT5 signaling, and anti-tumor responses multiple papers 10.15252/embj.2019103637
disruption by random integration TET-2 improved CAR-CD19 clinical outcome   10.1172/JCI130144
CRISPR screen (CD8) Dhx37 increased tumor infiltration and effector function multiple papers 10.1016/j.cell.2019.07.044
dominant-negative transgene TGFβRII increased T cell proliferation, effector function, persistence, and anti-tumor activity multiple papers 10.1016/j.ymthe.2018.05.003
integration site association TGFβRII associated with positive clinical outcomes many other sites also identified 10.1172/JCI130144
pooled shRNA screen PP2r2d increased TCR activation, cytokine secretion, T cell trafficking into tumor   10.1038/nature12988
knockout NR4a complex increased CD8 effector T cell function and solid tumor control linked to Nf-kB, AP-1 activity, multiple papers 10.1038/s41586-019-0985-x
T cell profiling Tcf1/TCF-7 increased T cell stemness and anti-tumor activity (with anti-PD-1) multiple papers 10.1016/j.immuni.2018.11.014

I won’t go through all these but there are a few things to note here. One is the appearance of the three pathways we just discussed in the context of the pooled KI paper: FAS, TGFβRII and TCF-7. As mentioned earlier the FAS/FAS-L connection to AICD has been known for a long time, and that information has already been exploited in the context of CAR T cell engineering. Elaboration of the roles of TGFβ in mediating tumor resistance to immune therapy is a more recent advance, but now well established. As noted above I think one interesting question raised by this paper is the source of the TGFβ in the in vitro and in vivo tumor models. I’ve assumed this is T cell derived and understanding the trigger for TGFβ activation in these settings would be very interesting. The role of Tcf1 (aka TCF-7) in anti-tumor immunity has recently been explored in detail in the context of T cell “stemness” leading to the hypothesis that anti-PD-(L)-1 therapeutics work by releasing these T cell with stem-like properties, and allowing their maturity into effector T cell populations (see 10.1016/j.immuni.2018.12.021 and 10.1016/j.immuni.2018.11.014 for examples). It seems that in this knockin, enforcing TCF-7 (Tcf1) expression locked the T cells into a sort of limbo, proliferating, homing into the tumor, but failing to mature into effector cells with anti-tumor functions. A very interesting result. Development of a model in which canonical PD-1/PD-L1 immunosuppressive biology could be examined in order to probe for synergies would be a welcome next step.

Finally, word or two about some of the other targets. As shown in the paper, and as recently shown in the clinical setting (10.1126/science.aba7365), knockins are, at this time, an imperfect tool. Some of the targets listed in the table are associated with autoimmunity (eg. PTPN2) or T cell leukemia (eg. c-Jun, NR4a) and so care is needed when exploiting these targets. Safely engineering specific targets for improved cellular therapeutics will be an important advance on the road to durable and curative solid tumor therapy.

Stay tuned.

THE NEXT GREAT DRUG HUNT: Integrins, TGF-beta and Drug Development in Oncology and Fibrosis

PART 1: Integrin αvβ8

Advances in our understanding of the regulation and function of TGF-β is driving novel drug development for the treatment of diverse diseases. This is a field I’ve followed for a long time and of course in the development of cell therapeutics we (www.aletabio.com) always have an eye on immunosuppressive pathways – indeed, the immunotherapy and cell therapy fields cross-fertilize often and productively (see http://www.sugarconebiotech.com/?m=202002).

Several new papers in this space have caught my eye and I’m keen to share some key findings. This will be a multi-part post and today I want to talk about an integrin.

Long time readers will appreciate the importance of alpha v-integrin-mediated regulation of TGF-β release from the latent complex (http://www.sugarconebiotech.com/?p=1073). The model that first emerged around 2010 was elegant: various signaling pathways triggered GPCRs that could activate an integrin beta strand (paired with an alpha v integrin) and coordinate the release of TGF-β from the cell surface. Soluble TGF-β, free from restraint, could diffuse across nearby cells and trigger TGF-β-receptor activation. Three integrins have been linked to the regulation of TGF-β release: αvβ6, αvβ8 and αvβ3. The mechanism for releasing TGF-β from the latent complex on the cell surface requires a conformation change in the integrin structure. From this insight emerged diverse drug development efforts targeting specific integrins, targeting the ligands for specific GPCRs and so on. Notable examples include the anti-αvβ6 antibody STX-100 (Biogen), the autotaxin inhibitor GLPG1690 (Galapagos), small molecule inhibitors designed to block integrin conformational change, and isoform-specific anti-TGF-β biologics, among many others. The mechanism of action of these drugs includes reduction of free, active TGF-β and therefore reduced TGF-β-receptor signaling. STX-100 was withdrawn from clinical development due to toxicity – more on this another time. GLPG1690 is now in a Phase III trial (in IPF) having shown anti-fibrotic activity in earlier clinical trials – this drug has had an interesting life, originally partnered by Galapagos with Johnson & Johnson, later returned, and now part of the mega-partnership with Gilead. I’ve previously discussed these and many other drugs in development in the context of fibrosis pathogenesis (http://www.sugarconebiotech.com/?p=1073). We’ll look at novel TGF-β-directed antagonists and their role in immune-oncology in part 2, as part of a long-running thread (http://www.sugarconebiotech.com/?m=201811).

So back to integrins. The dogma that emerged based on work from disparate labs was that an activated integrin was required to release TGF-β from the latent (inactive) complex on cell surfaces, allowing for precise regulation of TGF-β activity. More specifically, this model refers to the release of two of the three isoforms of TGF-β – isoforms 1 and 3. Isoform 2 regulation is different and relies on physical force acting directly on cells to trigger release. Of note, isoform 2 antagonism contributes to the toxicity associated with pan-TGF-β blockade but does not appear to contribute significantly to disease pathology either in fibrosis or in oncology. Therefore, specifically antagonizing TGF-β-1/3 without antagonizing TGF-β-2 is ideal – and the model we’ve just outlined allows for this specificity by targeting specific integrins.

The model that alpha v integrins mediated release of free, active TGF-β has held firm for nearly a decade. Now however there is a fascinating update to this model that involves the αvβ8 integrin. Work from the labs of Yifan Cheng and Steve Nishimura at UCSF has revealed a novel mechanism of TGF-β regulation that has interesting implications for drug development (https://doi.org/10.1016/j.cell.2019.12.030). Uniquely, integrin αvβ8 lacks critical intracellular binding domains that allow an integrin to anchor to actin fibers within the cell. As a result, binding to αvβ8 does not cause the release of TGF-β from the latent complex on the cell surface but rather presents an active form of TGF-β on that cell surface, without release from the latent complex. Importantly the complex formed between αvβ8 and TGF-β is conformationally stable and relies (in their experimental system) on trans-interaction between one cell expressing αvβ8 and a second cell expressing TGF-β as displayed on a latent protein complex (here, containing the GARP protein), and expressing the TGF-β receptors. In this system TGF-β remains anchored to the GARP-complex, but the conformational rotation caused by αvβ8 binding allows anchored TGF-βto interact with TGF-β-RII, thereby recruiting TGF-β-RI and inducing signaling.

The focus on GARP (aka LRRC32) relates to this groups long-standing interest with T-regulatory cells, which uniquely express GARP. Biotech investors will recall the Abbvie/Argenx deal on this target, which is in clinical development (https://clinicaltrials.gov/ct2/show/NCT03821935). A related protein called LRRC33 has been discovered on myeloid lineage cells.

More important, in my view, is that αvβ8 is expressed widely on tumor cells and has been variably reported to correlate with metastases (depending on the indication). This suggests that one means that tumor cells have of inducing TGF-β activation on interacting cells (eg. lymphocytes, myeloid cells and perhaps stromal cells) is via αvβ8 activity. The dependent hypothesis would be that such activation is immunosuppressive for those tumor-interacting cells. This is consistent with the known effects of TGF-β on immune cells in particular, but also stromal cells like fibroblasts. As an aside I like this model as one way of accounting for the appearance of T-regulatory cells and myeloid lineage suppressor cells in the tumor microenvironment as result of, rather than the cause of, immunosuppression, that is, these cells may be epi-phenomena of broad TGF-β-mediated immunosuppression. This may in turn explain why targeting such cells as T-regs and MDSCs has been largely unsuccessful to date as a therapeutic strategy for cancer.

There are some other implications. As the authors point put, the integrin/TGF-β complex is stable, and the binding domain that mediates the interaction is buried with the protein complex. It is unclear whether anti-TGF-β antagonists that target the canonical integrin binding cleft would be able to access this site within the complex. It’s possible that some of these drugs (whether antibodies or small molecules) can’t work in this setting. On the other hand, antibodies to αvβ8 clearly prevent the complex from forming and should block TGF-β-mediated immunosuppressive signaling in settings where αvβ8 expression is dominant. An anti-αvβ8 antibody strategy is being pursued by Venn Therapeutics (disclosure: I sit on Venn’s SAB). Further, the structural features identified in the paper include well-defined pockets that might be suitable for small molecule drugs. Indeed, one of the structural features in the b8 protein, consisting of hydrophobic residues, appears to account for the differential binding of various integrins (β6, β1, β2, β4, β7) to TGF-β, a remarkable finding. Analyses of the differences between the structure of β8 and other β integrins has been extensive across laboratories (see https://www.nature.com/articles/s41467-019-13248-5 for another important paper). Small molecule drug discovery is well underway in this field (see for example Pliant Therapeutics and Morphic Therapeutics) and one might imagine that these novel results found an interested audience in many bio-pharma labs.

Next: what has Scholar Rock been up to, and what can we learn from their work?

Stay tuned.

Radical optimism: considering the future of immunotherapy

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

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

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

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

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

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

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

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

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

So why should we be optimistic?

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

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

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

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

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

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

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

In the meantime, stay tuned.

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

Enumeral update – guest post by Cokey Nguyen, VP, R&D

Paul’s introduction:  Enumeral has been sending ’round some interesting updates to several of their programs and I asked for some more detail. Below is a quick primer sent along by Cokey Nguyen. More detail is available in Enumeral’s recent 8K filings, including one that dropped this morning. Also the company will present this and other work at the AACR Tumor Microenvironment Meeting in January (http://www.aacr.org/Meetings/Pages/MeetingDetail.aspx?EventItemID=73#.VlyGS7_QO2k - see below).

New data from Enumeral, by Cokey Nguyen

PD-1 biology in human lung cancer is an active area of research, as these cancers have shown PD-1 blockade responsiveness in clinical trials.  Enumeral has a drug discovery effort aimed at generating novel anti-PD-1 antibodies to develop into potential therapeutic candidates.  Using a proprietary antibody discovery platform, two classes of PD-1 antagonist antibodies were discovered:  the canonical anti-PD-1 antibody which blocks PD-L1/PD-1 interactions and a second class of antibody which is non-competitive with PD-L1 binding to PD-1.  These antibodies were validated first in a pre-clinical model of NSCLC using NSG mice with a humanized immune system and a patient derived NSCLC xenograft (huNSG/PDX) (Figure 1).  Here either class of antibody demonstrated activity on par with pembrolizumab, confirming that PD-1 blockade can slow tumor growth.

Figure 1

Figure 1

In order to confirm these pre-clinical findings, Enumeral began proof of concept studies with NSCLC samples.  The first question was if resident TILs, as found in tumors, could be reinvigorated (Paukken and Wherry, 2015) or if PD-1 blockade is mainly a phenomenon that affects lymph node-specific T cells that have yet to traffic to the tumor.  In these studies, Enumeral found PD-1 blockade can, in fact, increase effector T cell function, as readout by IFNg, IL-12, TNFa and IL-6.  In addition, in a NSCLC sample that showed PD-1hi/TIM-3lo expression, PD-1 blockade strongly upregulated TIM-3 expression (~5% to ~30%, see Figure 2).

Figure 2

Screen Shot 2015-12-01 at 6.07.24 AM

In these NSCLC-based studies, it was also found that an anti-PD-1 antibody (C8) which does not bind to PD-1 in the same manner as nivolumab or pembrolizumab (PD-L1 binding site) displays differentiated biology:  increased IFNg production and significantly higher levels of IL-12 in these bulk (dissociated) tumor cultures (Figure 3).  As IL-12 is thought to be a myeloid derived cytokine, this mechanism of action is not yet well understood, but has been now observed in multiple NSCLC samples as well as in MLR assays.

Figure 3

Screen Shot 2015-12-01 at 6.08.35 AM

In these NSCLC studies, while a subset of patient samples demonstrates PD-1 blockade responsiveness, the co-expression of TIM-3 on NSCLC TILs suggests this is a validated path forward to increase the response rate in lung cancer.  As with the PD-1 program, armed with a substantial portfolio of diverse anti-TIM-3 binders, Enumeral is actively testing single and dual checkpoint blockade on primary human lung cancer samples.

Look for the companies 2 posters at AACR/TME in January

Screen Shot 2015-12-01 at 6.11.08 AM

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

 

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

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

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

Feedback most welcome.

Screen Shot 2015-08-27 at 7.20.21 AM

 

“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)

Brodalumab for Psoriasis – what a mess

Let’s agree that the headline “Suicide Stunner” – penned by John Carroll for FierceBiotech – can never auger anything but very bad news, and never more so then when it is used to describe clinical trial results. Released on the Friday before the long US holiday weekend, bookended to the announcement of positive news on it’s PSCK9 program, Amgen stated that it was walking away from an expensive co-development program with AstraZeneca, basically washing it’s hands of the anti-IL-17 receptor (IL-17R) antibody brodalumab because of suicidal tendencies and actual suicides that occurred in the Phase 3 psoriasis trials. Brodalumab is under development for the treatment of plaque psoriasis, psoriatic arthritis and axial spondyloarthritis. Amgen stated that they believed that the approval label for brodalumab would contain warning language regarding suicide risk, and this would limit the success of the drug. By using such language while pulling the plug Amgen has essentially put AstraZeneca in the position of having to prove to the FDA that there is no suicide risk.

Holy crap.

Note here that we are not talking about a psychiatric drug, where the risk of suicide might be the consequence of trying to re-align an aberrant central nervous system. Instead we are talking about a drug that targets autoimmune disorders by blocking the action of T cells. This is not a biology linked to psychiatric health, at least not as we understand it today (more on this later).

Backing up: in April 2012, AstraZeneca and Amgen announced a collaboration to jointly develop and commercialize five clinical-stage monoclonal antibodies from Amgen’s inflammation portfolio: AMG 139, AMG 157, AMG 181, AMG 557 and brodalumab (aka AMG 827). The drivers for the collaboration were Amgen’s biologics expertise, the strong respiratory, inflammation and asthma development expertise of MedImmune (AstraZeneca’s biologics division), AstraZeneca’s global commercial reach in respiratory and gastrointestinal diseases, and the shared resources of two experienced R&D organizations

Under the terms of the agreement, AstraZeneca paid Amgen a $50MM upfront payment and the companies shared development costs. The breakout was as follows: AstraZeneca was responsible for approximately 65 percent of costs for the 2012-2014 period, and the companies now split costs equally. Amgen was to book sales globally and retain a low single-digit royalty for brodalumab. Amgen retained a mid single-digit royalty for the rest of the portfolio with remaining profits to be shared equally between the partners.

It gets even more complicated. Amgen was to lead the development and commercialization of brodalumab (and AMG 557, see below). Amgen was to assume promotion responsibility for brodalumab in dermatology indications in North America, and in rheumatology in North America and Europe. AstraZeneca was to assume promotion responsibility in respiratory and dermatology indications ex-North America. AstraZeneca remains responsible for leading the development and commercialization of AMG 139, AMG 157 and AMG 181. We’ll touch on these other antibodies at the very end.

Back to brodalumab. On balance, Amgen was on the hook for the development and commercialization costs, direct, indirect and ongoing, for dermatology indications in the US and also rheumatology, which in this case refers to psoriatic arthritis and axial spondyloarthritis. On the other hand, AstraZeneca was on the hook for commercialization in respiratory indications worldwide, and dermatology ex-US. This is interesting because brodalumab failed in its’ respiratory indication, moderate to severe asthma, and failed late, in a Phase 2b patient subset trial. So, on balance, much of the overall development cost seems to have shifted back onto Amgen over time (this is not to say that the companies would not have changed terms mid-term, they may have).

Two weeks ago I chaired a session on “Biologics for Autoimmune Disease” at the PEGS conference on Boston. In my opening remarks I used psoriasis as an example of an indication in which we were making clear and important progress, including with IL-17-directed therapeutics. Indeed, psoriasis is now a “crowded” indication commercially, with antibodies and receptor fusion proteins targeting the TNFs, IL-6, IL-12, IL-17, and IL-23 pathways all showing at least some activity. Notably, IL-17 and IL-23 targeting drugs appear to offer the greatest benefit in clearing psoriatic plaques. These pathways intersect in myriad ways, not all of which are well understood. This cartoon shows the effector cytokines and the receptors are expressed by diverse cell types, including dendritic cells, macrophages, T cells, and keratinocytes in the dermis.

IL-17 and friends

In simplistic terms, IL-6 triggers IL-12 and IL-23, and IL-23 triggers IL-17. As mentioned, the IL-17 and IL-23 targeting agents have great efficacy in psoriasis. Amgen and AstraZeneca were preparing an NDA (new drug application) for FDA submission based on results from three large Phase 3 studies. Here are the listed Phase 3 programs for brodalumab:

broda 1

I suppose those Phase 3 studies in psoriatic arthritis will now be tabled or transferred to AstraZeneca. For the sake of completeness here are the earlier studies:

broda 2

Certainly the clinical program was a robust one. So, what went wrong? Amgen R&D head Sean Harper summed up Amgen’s thinking about the suicide issue in the press release: “During our preparation process for regulatory submissions, we came to believe that labeling requirements likely would limit the appropriate patient population for brodalumab.”

The news aggregator and commentary website UpdatesPlus had this to add, questioning whether this result was “bad luck, bad target or victim of brodalumab’s efficacy: Despite high efficacy in Phase 3 studies, whispers of suicidality associated with brodalumab started to emerge at AAD.  At the time Amgen suggested this was related to disease however the company refused to comment on total rates and whether events were seen across arms … The question is whether Amgen is being hyper-cautious or whether the risk of suicidality is especially concerning.  Questions also emerge around the cause of risk – is this a spurious cluster of events unrelated to brodalumab; is suicidality perhaps related to relapse from the excellent efficacy associated with brodalumab after withdrawal (remember most patients exhibited at least PASI 90 on treatment but durability was very poor upon withdrawal); or perhaps suicidality is related to blocking the IL-17RA (note that suicidality has not to our knowledge been reported for the IL-17A ligand mAb Cosentyx) … One final point is whether regulators will now reevaluate suicide risk of IL-17 related molecules as a class – much greater clarity of brodalumab data is required to make a judgement.” That’s quite a nice summary from UpdatesPlus.

FierceBiotech’s report added “AstraZeneca would face some stiff competition if it decides to move forward solo on the drug. Novartis is already well in front with its IL-17 program for secukinumab, approved in January as Cosentyx. Eli Lilly has also been racking up positive late-stage studies for its IL-17-blocking ixekizumab, trailed by Merck’s MK-3222 and Johnson & Johnson’s IL-23 inhibitor guselkumab.”

Still, brodalumab demonstrated remarkable efficacy in psoriasis – Amgen and AstraZeneca went so for as to include a PASI100 score in one of their trials, meaning 100% clearance of psoriatic plaques, and the drug would have shown well against the best of breed, which today is likely Novartis’ anti-IL-17 antibody secukinumab. It is crowded space however, with antagonists targeting multiple nodes in the IL-17/IL-23 axis, alongside the biologics mentioned earlier.

Here is the current landscape from CiteLine (including brodalumab):

CiteLine

All in all, a tough crowd, and one that Amgen likely felt it could not face with a compromised label.

Let’s go back to the question posed above: bad luck, bad target or victim of superior efficacy? “Bad luck” suggests a statistical fluke in the data, potentially caused by the generally higher rates of suicidal tendencies observed in the moderate to severe psoriasis patient population. “Victim of superior efficacy” is in a sense a related issue, since the suggestion is that the loss of responsiveness to the drug, or a relapse, triggers a suicidal response as plaques return. Neither of these statements is really formulated as a hypothesis, and it doesn’t matter, as we don’t have the actual trial data yet with which to perform hypothesis testing.

“Bad target” is the most worrisome suggestion, and this can be formulated as a hypothesis, formally, the null hypothesis is that targeting the IL-17 receptor does not cause suicidal tendencies. Unfortunately, we still can’t test the hypothesis, and it seems likely that having the actual data won’t really help, that is, the study is probably not powered to reject that particular null hypothesis. So, what do we know? A few things, as it turns out.

First is that a link between the immune system and the nervous system is well established, although much of the focus has been on the role of neuronal enervation on immune responses. But clinically at least, the picture is muddier than that. High dose IL-2 can cause neurotoxicity, even hallucinations, according to Dr. Kathleen Mahoney, an oncologist at Beth Israel Deaconess and the Dana Farber. But what is really interesting is what else happens: “Some IL-2 treated patients can have odd dreams, really crazy dreams, and they last for weeks after treatment, long past the time when IL-2 would still be present in the body”, Dr. Mahoney said. Interferon alpha therapy is associated with pathological (severe) fatigue and also depressive symptoms that develop after 4–8 weeks of treatment. Of note, preventive treatment with anti-depressants, in particular serotonin reuptake inhibition attenuates IFN-alpha-associated symptoms of depression, anxiety, and neurotoxicity. Some researchers have suggested (controversially) that anti-TNF antibodies can control depression. Such anecdotal clinical observations suggest that we really do not yet understand the immune system connection to CNS activity.

On the other hand, antagonism of cytokine activity, and particularly of the cytokines IL-6, IL-17 and IL-23, has not been associated with neurological symptoms. For example the anti-IL-6 receptor antibody tocilizumab has shown a positive impact in rheumatoid arthritis patients quality of life scoring, which includes fatigue, anxiety, depression and a number of other factors. More to the point, the anti-IL-17 antibody secukinumab, that targets the IL-17 ligand (rather than the receptor), has not shown a link to suicide.

Clearly more data are needed, and it would not be surprising if the FDA began a drug class review if the data in the brodalumab trials warrant. They could cast quite a wide net given the complexity of this pathway, which overlaps with IL-6, IL-12 and IL-23. This casts a pall over the dermatology and particularly the rheumatology landscape, which is really waiting for novel therapeutics to move them successfully into new and important indications such as lupus and Type-1 Diabetes. The IL-17/IL-23 axis was to be that next great hope, and with luck we will still see these drugs moving out of their core indications of psoriasis and inflammatory bowel disease into new indications.

One last thing.

Those other antibodies – where are they now? A quick scorecard:

snapshot

It is readily seen that none of these are beyond early Phase 2, so it’s fair to say that the rest of the Amgen/AstraZeneca partnership has a long way to go. I, for one, wish the ongoing collaboration the very best of luck, particularly in the lupus indications, where we can really use some good news.

stay tuned.

Some Adjacencies in Immuno-oncology

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

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

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

 immune viral

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

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

We’ll not review those subjects again today.

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

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

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

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

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

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

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

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

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

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

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

 graphical abstract

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

Ok, moving on.

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

Here is Bellicum’s cartoon:

 cidecar

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

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

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

ICI15 presentation is now available

Over 100 slides on immune checkpoint combination therapy, novel targets and drug development in immuno-oncology, created for a 3 hour workshop at ICI15 (link).

As always we work from indications to discovery and back again, keeping one eye on the rapid evolution of clinical practice in oncology and the other on novel targets and therapeutics.

on SlideShare now: