Monthly Archives: April 2020

The next great drug hunt, part 2: WHAT RECENT PAPERS TELLS US ABOUT TGF-beta-MEDIATED IMMUNOSUPPRESSION (and what they don’t)

Back in 2017 I put together a presentation I informally called “The Big Stick Talk” for a series of local immuno-oncology and investor conferences, including at one of my favorite venues, the Jefferies IO conference hosted by Birin Amin which is always a great event. You can find the slides here (IO combos). The premise of the presentation was that resistance to immunotherapeutics (anti-PD-1, anti-PD-L1, anti-CTLA4) was driven by pathways that controlled complex biologies – TGF-β, beta-catenin and VEGF – and that other targets (eg. TIM-3, LAG-3, IDO, etc) were secondary features. It followed that drug development targeting the secondary phenomena were likely doomed to failure – and here we are now, 4 years later, with our focus back on those big biological pathways.

Today: the TGF-β story revisited.

As noted in the prior post (link), there are three distinct isoforms of TGF-β, and all three signal through the TGF receptor complex. All isoforms are expressed in an inactive form, bound as prodomains to the latency-associated peptide (LAP). As discussed last time, one way that active TGF-β isoforms 1 and 3 can be released or exposed to the receptor complex is via the action of specific alpha-v integrins that bind to an amino acid consensus sequence (RGD) on LAP and induce a conformational change when the integrin is activated.

The latent TGF-β complex (TGF-β/LAP) becomes more complicated with the addition of proteins that can covalently bind to the latent complex. This even larger latent complex (LLC) comes in a number of different forms depending on the identity of the covalently bound protein. Two proteins (LTBP1 and LTBP3) are used when the LLC is bound to the extracellular matrix (ECM): imagine a cell secreting and placing a protein complex, just so, onto ECM (collagen, fibrinogen, GAGs, elastin et al). Of course the ECM is also created by cells. Two different proteins, GARP (LRRC32) and LRRC33, hold the LLC on the surface of specific immune cells: regulatory T cells and differentiated myeloid cells.

That’s just by way of introducing this complex biology.

Of the three isoforms, isoform 2 is most often cited as the cause of the toxicity seen when pan-TGF-β inhibitors were used clinically, and drug development efforts avoid this isoform. The new paper from the team at Scholar Rock (link to paper) focuses on a specific isoform, TGF-β1, thereby avoiding β-2 altogether but also not targeting β-3. The rationale is that β-1 is the critical isoform in most cancers and is the specific cause of TGF-β-mediated immunosuppression in those cancers. In an analysis of mRNA expression data from the TCGA database, the authors found that TGF-β1 was the predominant isoform in most human cancers and further that β1 expression correlated with a gene signature of resistance to checkpoint inhibition (called IPRES) in 7 different cancer types for which immune checkpoint inhibitors have been approved.

The authors present an antibody, SRK-181, that binds to the TGF-β/LAP complex and prevents TGF-β from becoming activated. Remarkably, the antibody was screened so that it would block alpha-v integrin mediated activation of the latent complex in all 4 LLC contexts (ie. complexed with LTBP1, LTBP3, GARP or LLRC33). To be clear, the antibody only binds the complex, not to free TGF-β1. The binding potency is very good, in the low pM range. The activity of the antibody was assessed in cellular assays. The cell-based assay is very clever and deserves some explanation. Using a LN229 glioblastoma cell line that endogenously express LTBP1 and LTBP3 they transfected in TGF-β1 or TGF-β3-encoding plasmids. This allows these cells to now make LLCs that can be deposited on ECM. In order to also make LLCs that can stay on the cell surface they then co-transfected in either GARP or LLRC33 expression plasmids. Now the cells could be used to make all 4 of the LLCs.

Now, here it gets fun. As detailed in the last post, αvβ8 is an integrin that can regulate the release of TGF-β from the latent state. In the prior paper (found here), it was demonstrated that when “sprung” from a GARP-containing LLC, the TGF-β was not released from the cell surface but remained tethered to the complex in such a way as to be able to activate TGF-β-receptors on a second cell (remember, that was a two-cell assay). So, LN229 cells express the αvβ8 integrin and can activate latent TGF-β1 complexes. Therefore once the LLCs are expressed, either into the extracellular matrix or on the cell surface, they can be activated by endogenous αvβ8 integrins expressed in cis (ie. on the same cell).

To measure the activity of released TGF-β1 the transfected LN229 cells were co-cultured with a reporter cell that expresses luciferase then the TGF-β-receptor complex is activated. In this assay the antibody inhibited TGF-β1 release from all four LLCs and blocked activation of the reporter cells that express TGF-β-receptors. An interesting question to ask here is whether the TGF-β1 was actually released and was in solution or remained tethered, and whether this was different for each of the LLCs made. A simple bilayer culture system in which the reporter cells are physically separated from the LN229 cells would answer this question.

Ok but that’s enough on the assay – the biology is also very interesting. I want to focus just on the tumor microenvironment findings, although the in vitro assays and the in vivo tumor growth data are also of interest. The key finding is that the combination of an anti-PD-1 antibody and the anti-TGF-β/LAP complex antibody improved tumor control by a mechanism that includes an increase of the tumor by CD8-positive T cells. In contrast to the data reported by the Genentech group (https://www.nature.com/articles/nature25501) this was not obviously due to redistribution of the T cells from an “immune exclusion” zone, set up by cancer-associated fibroblasts and attendant collagen matrix. Rather the effect was associated with a decrease in myeloid lineage cells and an influx of T cells from the vasculature. So, not surprisingly, there may be two different TGF-β-dependent biologies at work in the two tumor models used (MBT-2 tumors in this study and EMT-6 tumors in the Genentech study). One reasonable explanation is the difference in TGF-β isoform expression between the two models. MBT-2 tumors only express isoform β-1 whereas EMT-6 tumor express both isoforms 1 and 3. Somewhat confusing though is the finding reported here that in their hands, the Scholar Rock group found a similar result (decrease of myeloid suppression and influx of T cell across the vasculature) using EMT-6 tumors as they did using MBT-2 tumors (shown in the supplemental data). In the Genentech paper an impact on a myeloid cell signature was not detected while fibroblast genes associated with matrix remodeling were significantly reduced. Importantly, in the Genentech study a pan-anti-TGF-β antibody was used, that would be able to block both isoforms. Whether there is differential contribution of isoforms 1 and 3 to the immunosuppressive biology seen in these syngeneic models is not known. The authors do note that the MBT-2 and EMT-6 tumors used expressed specific LLCs, with LRRC33 and LTBP1 being the most highly expressed. However, whether these different LLCs contain different TGF-β isoforms in various cells types within EMT-6 tumors (eg. fibroblasts, myeloid cells and endothelium) is not described. Since GARP does not appear to be present in these tumor types, a role for TGF-β-1 expressed by Tregs is ruled out. Clearly there is more to learn from these models and their translatability to the human tumor setting.

There is emerging clinical evidence of the relevance of the TGF-β pathway to cancer therapy. Preliminary clinical data from studies using M7824, an anti-PD-1-TGF-βR2-TRAP protein, reported responses across several indications including a complete response in cervical cancer and partial responses in pancreatic and anal cancers (link) An expansion cohort study of patients with advanced NSCLC (n = 80) treated with M7824 in the second-line setting showed an objective response rate of 86% in the subgroup with high PD-L1 tumor expression (link 2). In an expansion cohort of 30 patients with pretreated advanced biliary tract carcinomas, M7824 monotherapy demonstrated a 23.3% response rate, with some durable responses (link 3). This TRAP protein is stated to be selective for the blockade of TGF-β1 and TGF-β3 isoforms and of course also blocks the PD-1 pathway. In the cell therapy space, encoded inhibition of TGF-β (eg. by using a dominant-negative TGF-β-R on the CAR T cell) is one of a variety of methods being explored to increase the efficacy of CAR T cells in solid tumor therapy. Our publication (here) provides much more discussion regarding strategies for successful solid tumor cell therapy.

In summary the regulation of TGF-β activity remains a fascinating area for drug development. If we see benefits in line with those achieved in some cancers by combining anti-PD-1 therapy with anti-VEGF therapy, we will indeed have picked up another big stick, and learned how to use it.

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.