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)
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:
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:
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.
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:
Part 2 – The Border Wars.
One of the fascinating aspects of the toxicity of immune checkpoint therapeutics is that it is a lot of is triggered at the border between self and non-self, where non-self is everything that the immune system must encounter and sort through continuously. The sorting serves to identify pathogens and ignore non-pathogens among the myriad components of the microfauna and flora that inhabit these borders. The “sampling” of these ecosystems is continuous and highly reactive – one glass of unpurified water taken on foreign soil will teach you this lesson pretty quickly. When the immune system is unrestrained by blockade of CTLA4 and/or PD-1 it is not surprising that we see the breakdown of immune tolerance in these border zones.
There are three major surfaces where toxicity has been an issue: the skin, the gut mucosa, and the airspaces of the lung. Ipilimumab treatment can cause pretty intense inflammation of the skin, generally dismissed in the clinical trial literature as “rash”. In a pooled analysis of nearly 1500 patients enrolled in various ipilimumab clinical trials, 45% developed dermatological AEs considered drug related, and 2.6% (so 39 people) developed severe symptoms rating a grade 3-4 (where grade 5 is lethal) (see Tarhani, A. Scientifica 2013, Article ID 857519). A fair amount of the milder skin AEs can be ascribed to an anti-melan-A response, as this antigen is abundant in melanoma, the setting for the clinical development trials. In the Phase 3 registrational trials dermatologic AEs were reported in more than 40% of patients in the ipilimumab arms, and there were very severe AEs that cannot be ascribed to an anti-melan-A (i.e melanocyte) immune response. This is from Tarhani’s review of patients in the ipilimumab + gp100 (vaccine) and ipilimumab monotherapy arms having dermatological irAEs,
“of these, 2.1% and 1.5%, respectively, were grade 3 or higher … Severe, life threatening, or fatal immune-mediated dermatitis (Stevens- Johnson syndrome, toxic epidermal necrolysis, … full thickness dermal ulceration, or necrotic, bullous, or hemorrhagic manifestations; grade 3–5) occurred in 13 of 511 (2.5%) patients treated with ipilimumab. One patient (0.2%) died as a result of toxic epidermal necrolysis, and one additional patient required hospitalization for severe dermatitis… .”
That’s some rash. We note in passing that dermatologic AEs were see in a phase 2 trial of ipilimumab plus chemotherapy in non-small cell lung cancer (NSCLC) and so this is certainly not limited to the melanoma setting. PD-1 pathway antagonists also cause skin inflammation in both the melanoma and other settings, similarly suggesting that what we are seeing here are immune responses to antigenic stimulation that is normally immunologically inert. Nivolumab-induced dermatologic toxicity can be severe, but is less common than seen with ipilimumab therapy.
The issue of skin toxicity is well known clinically, and there are established treatment protocols requiring cessation of therapy and treatment with anti-inflammatories, usually steroids (i.e the REMS protocols). The gastrointestinal (GI, “gut”) AEs are also common, can arise suddenly, be resistant to therapy (corticosteroids, and rarely, anti-TNF antibody), and are of significant concern. Returning to the pooled analysis of ~1500 ipilimumab patients we see roughly half of the patients developing GI symptoms (this includes diarrhea). If we focus on grade 3/4 SAEs we have 10-12% of patients with GI disorders that include colitis, enterocolitis, intestinal perforations etc that can proceed to lethal septic complications. Of note, inflammatory infiltrates in the intestines include abundant T cells and neutrophils, showing that acute ongoing inflammation is occurring. GI toxicity is less common and less severe in nivolumab-treated patients, and this is true also of Merck’s anti-PD-1 antibody pembrolizumab and the anti-PD-L1 antibody MPDL3280A from Roche. Colitis is generally not a big issue, for example, GI SAEs are seen in less than 1% of nivolumab-treated patients. We might conclude here that other pathways are maintaining tolerance in the gut mucosa when the PD-1 pathway is blocked.
A different picture emerges when we consider AEs in the lung. Pulmonary toxicity is rare in the context of ipilimumab monotherapy, with only scattered case reports in the literature (see Voskens et al for a review of rare ipilimumab-induced AEs: link). Anti-PD-1 pathway therapeutics, particularly nivolumab, are associated with pneumonitis, which is inflammation of the lung tissues. In the monotherapy setting, both nivolumab and pembrolizumab causes pneumonitis in 3-4% of patients – the condition is generally mild and treatable. Of note this AE rate is consistent across indications (e.g. melanoma, renal cell). The anti-PD-L1 antibodies (Roche’s MPDL3280A and Astra Zeneca’s MEDI4736) have not been associated with pneumonitis to date, perhaps reflecting a unique profile. The recent data from the anti-PD-L1 antibody MEDI4736 trial in NSCLC presented a tolerable profile. While response rate was low, significant numbers of patients remained on therapy with stable disease (ASCO 2014, Abstract #3002).
More worrisome is the pneumonitis rate and severity in combination therapy particularly in the NSCLC setting where diminished lung function is already a concern (smokers with lung cancer can’t breathe). When nivolumab was combined with platinum-based chemotherapy in NSCLC the SAE rate jumped to 45%, with notable findings of grade 3/4 pneumonitis (7%) and acute renal failure (5%) (ASCO 2014, Abstract #8113). Nivolumab plus erlotinib was not associated with pneumonitis (ASCO 2014, Abstract #8022) but response rates were low as well suggesting that these therapies were not particularly additive. The combination of nivolumab with ipilimumab was most worrisome, with grade 3/4 pneumonitis (6%) now seen along with grade 3/4 SAEs of skin (4%), GI (16%) and others (16%) (ASCO 2014, Abstract #8023). Most problematic is that 35% of patients discontinued, and between 3 to 5 patients died due to drug related SAEs including respiratory failure (caused by severe colitis), epidermal necrolysis (in a patient with multiple SAEs) and pulmonary hemorrhage (pneumonitis). As indicated above, the anti-PD-L1 antibody MEDI4736 may better suited for combination therapy. A combo trial in NSCLS with anti-CTLA4 mAb tremelimumab is enrolling, so we’ll wait and see.
It’s fair at this point to take a step back and say “so what?” These are close to terminal patients with deadly cancers usually highly refractory to treatment, and we cannot expect a free ride. The unmet need is acute and urgent, and these therapeutics offer potential cures and increase in life expectancy – as shown very clearly in last weeks early termination of the Phase 3 trial of nivolumab versus dacarbazine due to the obvious overall survival advantage offered by nivolumab (see John Carroll’s story in Fierce Biotech here: link)
The problem is that the response rates we are seeing are generally low, the discontinuation rates high, and for anti-CTLA4 and anti-PD-1 therapeutics there is no clear consensus regarding the use of biomarkers to select patients most likely to respond. Therefore the actual percent penetrance of therapy in the patient cohorts becomes quite low. For those relatively few patients who respond well the outcomes can be sustained and robust. It is critical however to get these response rates up. The blockbuster combination of nivolumab plus ipilimumab in metastatic melanoma gives us a sense of what is possible, if the drugs are tolerable. It is also critical to understand how and why immune therapy can make subsequent therapy intolerable, as we’ve seen in case reports, or conversely, how and why prior therapies can cause such problems for patients going onto an immune therapeutic (see that Voskens review mentioned above). We’ve seen some the issues that can bedevil combinations in metastatic melanoma (with vemurafenib) and in renal cell carcinoma clinical trials (pazopanib) When we look at all of the combination clinical trials underway with these agents we have to wonder what surprises lay in store.
Part 3 – The Fifth Column.
The fifth column refers to enemies lurking within the boundaries of the state, in this case the human body. These are a mixed collection of AEs that can be difficult to understand. While we are used to see liver and kidney inflammation in the setting of cancer therapy, it remains a bit mysterious that immune checkpoint therapy can cause severe inflammatory responses in these organs, the most notable is probably the induction of hepatitis in patients treated with ipilimumab. Even weirder (for me anyway) are the endocrinopathies, headlined by pituitary inflammation, seen with both CTLA4 and PD-1 directed immunotherapies. Primary thyroid inflammation is also seen although less frequently. These are of course autoimmune targets in this setting, but the triggers are obscure, as is also almost always true in autoimmune disease. Somewhat remarkable is the emergence of a sometimes fatal but normally very rare condition known as autoimmune hypophysitis or lymphocytic hypophysitis, which is inflammation of the pituitary gland. Hypophysitis is a unique toxicity of immune checkpoint inhibitors, and has been been seen in patients treated with ipilimumab, tremelimumab, and nivolumab. Because the pituitary sits in the middle of the limbic hypothalamic-pituitary-adrenal axis effects on the thymus and adrenal gland are also noted, with adrenal insufficiency being a severe and life-threatening complication. It must be stressed that the frequency of this AE is stunningly high, reaching 17% in some trials, as the disease has been described only very rarely, with a good deal less than 1000 cases ever known prior to the introduction of immune checkpoint therapeutics.
So we won’t dwell on this, as clinicians now know what to watch for, and treatment paradigms have been developed. As mentioned earlier, treatment generally involves initiation of steroids to control to autoimmune response, and cessation of immune checkpoint therapy.
Let’s return to the consideration of combination therapy, which I think we all agree is essential if we are really to expand use of immune therapeutics in the treatment of these difficult cancers. Great hope has been placed in the combination of CTLA4 and PD-1 targeting agents with “safe” immune checkpoint modulators, notably the IDO-inhibitor from Incyte. We have very little information to date, but it is notable that the dose limiting toxicity in the first combination trial of ipilimumab and INCB024360 from Incyte (INCY) was liver damage as measured by ATL elevation. It may be that merely piling on ways of disrupting Treg activity will not help with the toxicity profile; in fact, one might make the prediction that this approach will make things worse in some settings.
We’ve remarked in passing on the apparently mild safety profile of the anti-PD-L1 inhibitors compared to the PD-1 inhibitors. This makes some sense, as the ligands are expressed by the target tumor cells, and this may be the main sink for the injected antibody, i.e. antibody may not be evenly bio-distributed but rather predominantly localized to the tumors. The concordance of anti-PD-L1 antibody activity with tumor PD-L1 expression is consistent with a direct and localized effect. The fact that there is less consistent concordance of anti-PD-1 antibody activity with PD-1 expression by tumor-infiltrating T cells suggests less specificity in the induced immune response, and this may be why we see autoimmune toxicity in the nivolumab setting. As CTLA-4 is exclusively T cell expressed, the same seems to hold true for anti-CTLA4 antibody therapy. So combining these may not be the most ideal way forward.
We will discuss alternative approaches next time, but first there is some new data on novel immune checkpoint therapies to consider. These are the TNF receptor superfamily proteins that we discussed last month (link): 4-1BB, CD27, OX40 and GITR. There is admittedly very little data to date. Pfizer’s (PFE) anti-4-1BB antibody PF-05082566 reached a safe dose in Phase 1 without undue toxicity signals (ASCO 2014, Abstract #3007). Pfizer disclosed combination trials with rituximab in Non-Hodgkin Lymphoma (NHL) and pembrolizumab (anti-PD-1). The BMY antibody urelumab was tolerated through its’ dose escalation cohorts, and ex vivo analysis showed activation of CD8+ T cells and NK cells (ASCO 2014, Abstract #3017). The Celldex anti-CD27 mAb also has demonstrated safe dose escalation, although to date without signs of clinical activity (ASCO 2014, Abstracts #3024 and #3027). Celldex (CLDX) claims planned studies in combination with nivolumab, ipilimumab, and the targeted therapeutics darafenib and trametinib.
As we discussed in an earlier post, 4-1BB, CD27, OX40 and GITR are evolutionarily closely related receptors. Biomarker studies such as the one performed in the urelumab trial will be essential in understanding how these immune stimulatory pathways will differentiate clinically and which will be safe in combination settings. We’ve reviewed the biology of this superfamily recently (see these posts) so won’t do so again until we get some more clinical data.
Next we will introduce some novel targets in the TNF receptor superfamily, revisit some apoptotic pathway “influencers”, and will swing back around to PD-1 and PD-L1 in some other solid tumor settings (not necessarily in that order).
stay tuned.
One of the puzzles in thinking about the available costimulatory receptors on T cells (and NK cells) is the unsettling number of them. Sticking just to the TNF receptor superfamily (TNFRSF) we have OX40 (discussed earlier), 4-1BB, GITR, CD27, and also the TNF receptors themselves (1 and 2), the lymphotoxin beta-receptor, HVEM, and TNFRSF25. There may be some I’ve forgotten. As noted in part 1 OX40, GITR, 4-1BB and CD27 are evolutionary cousins, as are their cognate ligands. Why did the immune system evolve such a complexity of T cell costimulators?
The answer is not entirely clear although the expression patterns and kinetics of expression suggest some rationale for understanding the number of different receptors. Also, as it’s understood that all the TNF receptors signal via NF-kB, Jun and p38, we might see these receptors either compete (for signaling proteins) or cooperate. All of the available genetic and pharmacologic data suggest they cooperate or even synergize, thereby powering the T cell response when needed. Since T cell responses (and immune responses generally) are so dangerous when dysregulated, the multiplicity of on and off switches presumably allows for redundancy of control.
As we said previously, OX40 comes on slow and easy, starting about 12 hours after TCR stimulation, and riding along for up to 96 hours. This is in vitro, cell culture data … so lets recognize that in vivo, in response to the chaotic presentation of antigen, the population of T cells is likely to be turning over, proliferating … so it’s unlikely we will see a finely tuned kinetic response in the real world, as regards the population of responding cells. Nonetheless we can focus on a single T cell, just the one. And we’ve guessed it will be expressing OX40 say from day 2 to day 5 after activation. Lets ignore the fact that activated effector T cells are likely dividing more rapidly than every 5 days, and just ask the simple question – of the other TNFSF receptors, what else is expressed and when, and on what T cell and other cell types?
Cue 4-1BB.
4-1BB is also expressed following T cell activation, and is expressed on other cell types also. 4-1BB expression come up much more quickly after T cell activation, within a few hours, and wanes after several days. This receptor is critical to supporting activated T cell proliferation, differentiation and survival. Expression on T cells may be coincident with OX40, but the consequences of engaging the receptor with an agonist antibody are different. 4-1BB preferentially supports the proliferation and survival of CD8+ T cells, although at least in some settings the activity of CD4+ T cells is also stimulated through 4-1BB. Much of the anti-tumor activity of 4-1BB agonist antibodies in preclinical studies can be traced to stimulation of NK cells although this depends of the tumor type and model used. Less well understood is the role on 4-1BB on other cells types. This receptor is also found on DCs, macrophages, granulocytes, and Tregs. Expression has also been described on vascular endothelium and on some tumor cells. The role of 4-1BB is confusing, with various studies showing expansion of the Treg subset and others suggesting that 4-1BB dampens Treg responses, perhaps via direct effects on DCs. The 4-1BB gene-knockout mouse shows aspects of autoimmune disorders (at least in the mouse strains tested), suggesting a role for 4-1BB in maintaining immune homeostasis following activation. 4-1BB knockout mice have trouble handling tumor challenge, and at least some spontaneously develop B cell lymphomas as they age. It is all a bit complicated.
Regardless, there are two antibodies that can provide some early clinical data. Bristol-Myers Squibb (BMY) started development of the BMS-663513 antibody quite early, before the immunotherapy wave had really gotten started. BMS-663513 is a specific anti-4-1BB agonist antibody, isotype IgG4. The antibody ran into toxicity issues in a phase 2 trial of metastatic melanoma in 2011, leading to a halt of three trials with that antibody. As the toxicity was correlated with dose, BMY has restarted the clinical campaign with BMS-663513, now called urelumab to establish a safe and efficacious dose. A monotherapy trial is being run in patients with advanced/metastatic solid tumors or with relapsed/refractory Non-Hodgkin Lymphoma (NHL). A second trial in NHL is being run in combination with rituximab treatment. A third trial in advanced/metastatic colorectal and head and neck cancers is being run in combination with cetuximab (anti-EGFR). Pfizer’s (PFE) PF-05082566 is an agonist anti-4-1BB antibody (IgG2 isotype). One trial is listed at clinicaltrials.gov, a 3×3 dose escalation phase 1 trial run as monotherapy in patients with advanced cancers, and as combination therapy with rituximab in NHL.
At the June ASCO meeting there will be updates on both the BMY and PFE programs. BMY has a presentation focused on mechanism of action and biomarker analyses. Abstract #3017 outlines the goal of monitoring the immune status of 4 patients prior to and during the phase 1 study of urelumab (BMS-663513: clinical trial NCT01471210). The antibody was given every 3 weeks and the analysis presents results through 3 cycles. PBMCs were isolated from whole blood, and stimulated for 4 hours with PMA/ionomycin to activate lymphocytes. There was an increase in CD8 T cells up to 41% and NK cells up to 62%. CD4 T cells decreased by as much as 23% and regulatory CD4 T cells decreased by as much as 18% comparing the 3rd cycle to baseline. The results are consistent with a preferential impact on CD8+ T cells and NK cells. The level of the cytokines GM-CSF and IFNgamma were increased.
The PFE study (abstract #3007) describes very early data from clinical trial NCT01307267. Patients received PF-05082566 IV every 4 weeks (one cycle) with an 8 week period for assessment of dose-limiting toxicity (DLT) and radiographic analysis of tumor burden (RECIST 1.1). 27 patients were up to 0.3 mg/kg, the highest dose reported in the abstract. The majority of patients had either colorectal cancer (n=11) or Merkel cell carcinoma (n=6), the rest were a collection of solid tumor patients and 2 patients with B cell lymphoma. 25/27 patients completed the DLT assessment period (first cycle). No DLT was established but only 7 patients remain on therapy. All discontinuations from treatment were due to disease progression. A best overall response of stable disease was observed in 6 patients. No duration data is supplied.
These two early trials suggest that safe dose levels can be achieved, that a mechanism of action can be confirmed (urelumab: expansion of CD8+ T cells and NK cells), and that some clinical response can be observed (PF-05082566: stable disease). That’s a pretty good picture coming out of Phase 1. As preclinical data suggest that 4-1BB is most effective in various combination formats, it will be interesting to monitor advances in the rituximab and cetuximab co-therapy arms of these trials. Several potent combinations arising in the preclinical literature include 4-1BB with immune checkpoint inhibition (CTLA4 or the PD-1 pathway) and in combination with agonist OX40 antibody therapy.
Lets get back to the Treg cells, whose function is to suppress immune responses, primarily those of CD4+ T cells. These express 4-1BB constitutively, although it’s not clear how or if they are responding to treatment with agonist anti-4-1BB antibodies. Let’s turn to a different pathway known to have a profound effect on Tregs, the Glucocorticoid-Induced TNFR Related gene (GITR). GITR was first identified as a regulatory T-cell marker and was shown to play a critical role in breaking T cell tolerance by direct suppression of Treg activity. The preclinical evaluation of GITR produced some very striking data, including in combination settings in which anti-GITR antibodies essentially synergized with other immune checkpoint therapeutics to eliminate established tumors. Such combinations have included PD-1 blockade and CTLA4 blockade. GITR agonism is also synergistic with chemotherapy in preclinical models. Clinical development of GITR antibodies has been slow. A program initiated at TolerX and reborn at GITR, Inc., is recruiting for a phase 1 trial in advanced melanoma and other solid tumors. The antibody is TRX518 (NCT01239134). Merck is advancing a phase 1 study with the anti-GITR antibody MK-4166, although this trial (NCT02132754) is not yet recruiting patients. Review articles have mentioned an ongoing clinical program at MSKCC – this is one of the three sites enrolling patients in the TRX518 trial.
To the extent that the driving mechanism of action (MOA) of GITR stimulation is shown to be downregulation of Treg activity, this pathway should be a good candidate for combination therapy with 4-1BB, OX40 or CD27 agonists (see below) as well as with the CTLA4 and PD-1 pathway antagonists. If the MOA in human cancer patients is different or more complex than proposed, different combinations may be more or less attractive.
One last receptor – CD27.
The costimulatory molecule CD27 is constitutively expressed on most effector T cells, memory B cells, and an NK cell subset. So its expression may also overlap with those of the other receptors. CD27 appears to be important for sustained T cell effector function and also the development of T cell memory. CD27 is a marker of memory T cells, conversely, it is low or absent on Tregs. More broadly, CD27 supports germinal center formation that drives B cell maturation and the differentiation of plasma cells that produce high affinity antibodies, and is also important in driving the cytolytic activity of some NK cells.
Although there is a large preclinical literature on CD27 and its ligand CD70, there are few antibodies in clinical development. There are several historical explanations for this I think. CD70 is expressed at high levels on certain tumor types, particularly renal cell carcinoma (RCC). Much effort has gone into the development of cell-depleting antibodies targeting CD70. This expression pattern also called into question the relevance of CD27 in controlling tumor growth, as the ligand would be expected to stimulate immune responses. We now know that RCC and other solid tumors expresses high levels of PD-L1, and likely disables immune responses via this pathway. Not surprisingly then, one of the ongoing clinical efforts is a combination trial of nivolumab, the anti-PD-1 antibody from BMY with CDX-1127, an anti-CD27 antibody from Celldex (CLDX) in a collaboration announced by the 2 companies last week. In the meantime we have 2 abstracts from CLDX at ASCO in June to look forward to. In a 3×3 phase 1 dose escalation study of B cell lymphoma patients the drug was well tolerated with weekly IV dosing, and there were signs of clinical response, including a durable complete response in one patient with advanced refractory disease (abstract #3024; clinical trial NCT01460134). In the same trial, solid tumor patients were treated in a dose escalation phase and then an expansion phase (RCC and melanoma). The drug was well tolerated, there were preliminary signs of clinical response, and measureable activation of the immune system (Abstract #3027). With the potential to support memory T cell differentiation, CD27 may provide an important additional signal to drive long term tumor control. We’ll have to wait and see.
So, we have 4 receptors with overlapping activities and we have multiple antibodies in various stages of development. There will be plenty to learn about these targets and their roles in the future of combination immunotherapy. One of the most promising paths forward is the analysis of immune checkpoint and costimulatory proteins on tumor infiltrating lymphocytes (TILs). It seems very likely that the makeup of the tumor cell defense against the immune system will diverge between tumor types, and perhaps between patients with the same tumor types, or even with the same patient tumor at different times, or in different metastatic locations. Profiling TILs, and perhaps sentinel lymph nodes, for the expression patterns of lymphocytes and antigen presenting cells is likely to help guide combination therapy.
We’ll come back to that. And we’ve not forgotten those NK cells either.
stay tuned.