Monthly Archives: April 2015

Celgene and friends…

The ever-nimble company Celgene (NASDAQ: CELG) was back in the news last week, signing a sweetheart deal with AstraZeneca (NYSE: AZN) that brings access to a Phase III immune checkpoint therapeutic.

The deal between Celgene and Astra Zeneca is remarkable for balancing the relative strengths and weaknesses of each company. For AZN the deal enhances the competitive reach of the anti-PD-L1 mAb MEDI4736, now backed by a rich war chest and the potential for combination therapy with Celgene’s myeloma and hematologic malignancy portfolio. Notably, these diseases have remained relatively indifferent to monotherapy with immune checkpoint therapeutics, with a few exceptions. Refractory multiple myeloma, an indication that Celgene dominates, is particularly resistant to monotherapy with immune checkpoint therapeutics and the bet is that efficacy will be seen when MEDI4736 is paired with Celgene’s approved drugs lenalidomide and pomalidomide, among others. The deal may also ultimately bring access to first line solid tumor patients in the form of a MEDI4736 combination with Celgene’s Abraxane, a synergy that has been overlooked I think.

For Celgene this is an overdue move into the immunotherapy space and reflects their willingness to spend their way into contention and expand market dominance from multiple myeloma into other hematological malignancies, a counter of sorts to Abbvie’s buyout of Pharmacyclics and it’s B cell cancer blockbuster Imbruvica. Celgene has already made forays into the immuno-oncology space with it’s in-licensing of Inhibrx’ anti-CD47 antibody and the deal with VentiRx and Array Biopharma to develop VTX-2337, a TLR8 agonist but these are much earlier stage assets. It is reasonable to predict that Celgene will also move quickly to acquire additional assets in the immune checkpoint space.

I’d expect to see both AZN and Celgene aggressively pursue additional deals. AZN did exactly that with Juno Therapeutics (combining CAR T and anti-PD-L1 therapies) and Innate Pharma in separate deals two weeks ago. It is interesting to speculate that there is additional synergy between the Innate Pharma programs and the Celgene programs that could be explored later on.

More importantly I think, and looking ahead, both AZN and Celgene are building multiplatform immuno-oncology companies, with cellular therapeutics (CAR-T and TCR), antibody therapies, targeted therapeutics, and immune checkpoint therapeutics – a broad reach across the evolving oncology clinical landscape, similar to the position Novartis has built for itself. Both AZN and Celgene floated that their deal could grow into a larger collaboration, if so, this means that similar deals in this space will have to become bigger and broader just to keep up. Merck and Pfizer’s recently announced collaboration is a good example of a deal not quite juicy enough to be transformative in the way the AZN/Celgene hookup may be for both of these companies. Novartis recently stated that they are looking for acquisitions in the 2-5 billion dollar range – this gives us a sense of scale required. We are entering a phase of “go big or go home” and the winners will dominate oncology clinical care.

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:


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.

RNAi Screening and Immunotherapy Target Discovery – Truly Translational Research Is Hard

We are constantly told that target ‘X’ is a novel checkpoint inhibitor, and then target ‘Y’, and don’t forget ‘Z’! We hold such claims to a high standard of validation, but press releases often outrun the science. So let’s look at some recent papers in this light.

RNAi screening has been around for several decades and yet like its’ distant cousin, transcript profiling (Txp), it is a technology that only rarely produces actionable, translational, data. Large scale screening of transcription whether using positive (Txp) or negative (RNAi) readouts can, in the wrong hands, produce a lot of false hits. Two recent papers approach this problem carefully. These are both RNAi based screens that use constrained pools and built in redundancy to limit false positives.

Last year an elegantly crafted paper appeared in Nature. The paper was a local tour-de-force with contributions from Dana Farber, the Koch and Whitehead Institutes at MIT, the Broad Institute (MIT and Harvard), and the Novartis Inc Biomedical Research and Genomics Institutes.

Here is the reference:

In vivo Discovery of Immunotherapy Targets in the Tumor Microenvironment. 2014. Nature 506: 52–57. doi:10.1038/nature12988.

Why highlight this paper, nearly a year later? We’ll get to that in a bit. First let’s take a look at the data. The paper poses a provocative question: can novel regulatory switches controlling T cell function in immunosuppressive tumors be identified in vivo?

The background to the work is laid out in the introduction, summarized here:

1) Cytotoxic T cells play a central role in immune-mediated control of cancer because they specifically detect and eliminate cancer cells following TCR-mediated recognition of tumor-derived peptides bound to MHC.

2) Infiltration of both the tumor center and the invasive tumor margin by CD8+ cytotoxic T cells correlates with a favorable prognosis regardless of the extent of tumor invasion and local lymph node involvement.

3) In the majority of patient tumors this anti-tumor immune defense is blocked by immunosuppressive cell populations recruited to the tumor microenvironment, including regulatory T cells, immature myeloid cell populations and tumor-associated macrophages.

4) Highly complex interactions among a variety of different cell types – e.g. tumor cells, immune cells and stromal cells – in the tumor microenvironment contribute to clinical outcome.

5) Such complex cellular interactions are best modeled in vivo.

To tackle this question the authors devised a screen using in vivo pooled short-hairpin RNA (shRNA), shRNAs are artificial RNA molecules containing a tight hairpin turn that silences target gene expression via RNA interference (RNAi). A lentiviral vector is used to target the construct to genomic DNA so that it is integrated.

Two screens were devised. One focused on genes over-expressed in anergic or exhausted T cells. 255 genes were represented by an average of 5 independent shRNAs each. This screen was further split into 2 different pools. The second screen comprised shRNAs targeting kinases and phosphatases, again with an average of 5 shRNAs per target. Thus these pools were both constrained in size and armed with built-in redundancy.

The technique takes advantage of the extensive proliferative capacity of T cells following TCR triggering of the TCR by a tumor-associated antigen. shRNAs capable of restoring CD8 T cell function by targeting negative regulators may be uncovered because they will be over-represented in the T cell compartment upon expansion. The shRNAs within a pool can be quantified by deep sequencing of the shRNA cassette from tumors and secondary lymphoid organs as compared to control tissues.

The relatively simple experimental design was to collect naive T cells from the mice that matched the mice used for the melanoma modeling. The T cells were pretreated for two days with IL-7 and IL-15 prior to lentiviral vector-mediated infection with shRNA pools. The transduced T cells were injected into B6 mice bearing day 14 B16-Ova tumors. After 7 days, T cells were purified from tumors and secondary lymphoid organs (spleen, tumor-draining and irrelevant lymph nodes) for isolation of genomic DNA, followed by PCR amplification of the shRNA cassette. The representation of shRNAs was then quantified in different tissues by Illumina sequencing.

The analysis focused on genes whose shRNAs were over-represented in tumor samples but not spleen, a secondary lymphoid organ, or other organs. Substantial T cell accumulation in tumors was observed for a number of shRNAs, despite the immunosuppressive environment. These shRNAs represent putative novel immune modulatory pathways active in tumor setting.

In this paper the authors highlight two results with the highest degree of specific enrichment in T cell populations isolated from tumors: Cblb (an E3 ubiquitin ligase that induces T cell receptor internalization) and Ppp2r2d (a phosphatase, not previously studied in T cells). The most enriched shRNAs from this study are shown here (Figure 2 in the paper):

 screen 1

 Note that the statistical significance shown (*: P<0.05; **: P<0.01) is in comparison to control LacZ, and the first 7-10 hits are probably statistically indistinguishable although I didn’t check. Regardless the phosphatase Ppp2r2d sits to the far left on the graph, with a whopping 16x increase in T cells isolated from tumors versus those isolated from spleen.

The authors proceed to confirm the Ppp2r2d hit and explore its’ cellular mechanism of action. This phosphatase pathway controls T cell proliferation and cytokine expression, and its’ blockade by shRNA leads to the accumulation of tumor responsive IFNg-secreting CD8+ T cells. PP2A proteins are a family of phosphatase complexes with catalytc, scaffolding and regulatory subunits. Ppp2r2d is a regulatory subunit that functions by controlling cellular localization and substrate. Specifically, Ppp2r2d directs PP2A to Cdk1 substrates during cell division to inhibit mitotic entry and induce mitotic exit. This is therefore a mechanism underlying the control of T cell proliferation. PP2A also regulates cell apoptosis via BAD-induced cell death and PP2A phosphatases have been shown to interact with the cytoplasmic domains of CD28, CTLA-4 and the NF-κB regulator Carma1. It is not known which regulatory subunits are required for these activities although one might hypothesize a role for Ppp2r2d.

Other interesting hits include Arhgap5, a RHO GTPase previously shown to be important in T cell responses after TCR engagement; SMAD2, a signaling molecule downstream of the highly immunosuppressive TGF-b receptor, and Cbl-b, an E3 ligase that controls TCR responsiveness. Cbl-b is the subject of a recent review (Front Oncol. 2015, 11:58. doi: 10.3389/fonc.2015.00058). In T cells, Cbl-b negatively regulates activation signals through TCR or pattern recognition receptors, and its’ activity is regulated in response to TGF-b signaling (we might refer back to SMAD2 here). cblb-gene-deficient mice spontaneously develop autoimmunity and are highly susceptible to experimental autoimmunity and gene association studies have linked Cbl-b with several human autoimmune diseases. On the other hand cblb knockout CD8(+) T cells are hyper-responsive to TCR and CD28 stimulation and are in part protected against immunosuppression induced by TGF-β, at least in vitro. In vivo, cblb-gene deficiency contributes to tumor rejection due to highly active CD8 T cell and NK cell activity. The role of cbl-b in NK cell anti-tumor responses has received considerable attention. In a paper by Penninger and colleagues it was shown that the innate immune TAM tyrosine kinase receptors (Tyro3, Axl and Mer) are ubiquitylation substrates for Cbl-b. Targeting cblb allowed increased NK cell activation mediated by these receptors, and has led to increased appreciation of the TAMs as potential targets in immunotherapy. Finally, and this is a consistent theme in the immune checkpoint space, Cbl-b activity has also been associated with anti-viral immunity and T cell exhaustion due to chronic activation.

While there is also interest in targeting Cbl-b as a strategy to enhance anti-cancer immunity, we note that Cbl-b is expressed in all leukocyte subsets and regulates multiple signaling pathways in T cells, NK cells, B cells, myeloid cells and dendritic cells. Thus targeting this widely expressed protein may require specific targeting techniques. Apeiron targets Cbl-b in PBMCs using siRNA electroporation – essentially boosting the immune competence of these cells ex vivo, and then putting them back in the patient. There are several POC Cbl-b inhibitor compounds in academic labs whose use thus far appears limited to rodent studies.

Lots of pathways present themselves as interesting drug development targets. A classic example of such a pathway would include a cell surface signaling receptor, the receptor tyrosine kinase, and the downstream intracellular tyrosine kinases. We have lots of examples: VEGFR, EGFR and the like. Prosecution of pathogenic pathways keeps expanding however, to include ever more diverse targets. This brings us to the protein phosphatases, and the reason we’ve returned to the nearly year-old Nature paper highlighted in Part 1.

I’ve highlighted the kinases as an exemplary drug-targeting pathway in part because their mechanism of action is the exact opposite of the phosphatases. Kinases add phosphate groups to specific segments of proteins that are intracellular; phosphatases strip these same phosphate groups away. Since phosphorylation is a critical component of cell signaling cascades leading to gene transcription, regulation of the process is critical to maintain cellular homeostasis and also allow rapid cellular responses. Thus in T cells phosphorylation of ZAP70 immediately upon TCR engagement is a canonical first step in T cell activation. Dephosphorylation of ZAP70 is accomplished by the low molecular weight phosphatase among many other mechanisms. Of note downstream mediators of TCR signaling are independently regulated. As just one example, CTLA4 utilizes PP2A and the SHP phosphatases to inhibit AKT phosphorylation while PD-1 acts via Src homology-2 (SH2) domain-containing phosphatases to block PI3-K activation, among other targets. Targeting phosphatases specifically has been very difficult – these proteins perform diverse functions and are downstream of many different signals.

A recent paper demonstrates the feasibility of targeting specific PP2A regulatory subunits, and this is why we have returned to the shRNA screening paper a year later.

Here is the reference:

Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit. 2015. 348: 239-242. doi: 10.1126/science.aaa4484.

In this paper the authors tackle a different PP2A pathway, and describe a compound identified as Sephin1 (selective inhibitor of a holophosphatase), a small molecule inhibitor of Ppp1r15a. This is a simple molecule indeed:


 While likely lacking in traditional drug-like development properties, the compound was orally available and concentrated specifically in the CNS while disappearing rapidly from plasma circulation – in this case a beneficial profile, as the targeted pathology models result from derangement of the mis-folded protein response in the CNS, a cellular process regulated by PP2A activities. Specifically, Ppp1r15b acts to prolong a beneficial adaptive phospho-signaling pathway that protects cells from otherwise lethal protein misfolding stress. This cellular biology is counter-regulated by the stress-induced regulatory subunit Ppp1r15a. The trick then was to inhibit Ppp1r15a specifically, without inhibiting the related protein Ppp1r15b. Serphin1 safely prevented CNS motor, morphological, and molecular defects of two distinct protein-misfolding diseases in mice, Charcot-Marie-Tooth 1B, and amyotrophic lateral sclerosis. Thus this paper demonstrated that 2 closely related regulatory subunits of phosphatases could be the target of successful proof-of-concept drug discovery.

The second RNAi screening paper leads us to a more tractable target. In this paper, the screening is done in vitro, using a tumor cell line. Such experiments are notorious for producing results that cannot be reproduced or confirmed, so the authors step carefully, building in layers of controls, as we’ll see.

Here is the reference:

A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes. 2015. EMBO Mol Med 7: 450–463. doi: 10.15252/emmm.201404414).

The paper is from the Beckhove lab in Heidelberg – they’ve done a lot of this type of work. The screen utilizes MCF7 breast cancer cell lysis as the read-out. Cell lysis was triggered by either survivin-specific activated T cells or T cell engaging bi-specific antibodies that cross-linked CD3 on activated T cells to EpCAM on tumor cells (CD3 x EpCAM bispecific). MCF7 (luciferase+) cells were transfected with siRNAs then cocultured with CD8+ T cells. The luciferase serves as a marker of tumor cell death. The screen was focused on a library of 520 genes coding for transmembrane and cell surface proteins, with the goal of identifying antibody targets. Controls included scrambled RNAi constructs (negative controls) and PD-L1, Ceacam-6 and Galectin-3 (positive controls). Furthermore, the screen was run 3 times, independently.

Here is a snapshot of the reported results.

 screen 2

 The hits uncovered using this approach included CCR9, a chemokine receptor widely reported to be important in tumor cell resistance and metastasis. The authors trace the activity in the assay from the tumor-specific knockdown of CCR9 to increased activation of STAT signaling in T cells, by an unknown mechanism. This resulted in changes in gene expression in those T cells that correlated with T cell effector functions including increased production of IFN-g, IL-2, and TNF). The authors report they have generated experimental validation for GLIPR1L1 (poorly known) and GHSR (a hypermethylated gene in some cancers) in addition to CCR9. On the flip side, hits such as Frizzled D3 (FZD3) may or may not have translational relevance, as biology relevant to the experimental system is not readily deconstructed. This lab has done similar work in pancreatic cancer and will present results next week on a gene/protein that is unknown to me (TONI1, AACR abstract #245).

These papers present interesting and careful approaches to the use of RNA-targeting screens, a field long bedeviled by false promise. Such techniques, and the application of other gene-targeting methods, may yet uncover novel immune modulation targets. As always it is critical to view such papers and results with care and maintain a healthy degree of skepticism. That said, both of these papers have presented reasonable results suitable for further hypothesis testing.

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: