Category Archives: integrins

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

biologics, immunotherapy, integrins, monoclonal antibodies, oncology, TGF beta, tumor microenvironment

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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 STX-100 was withdrawn from clinical development due to toxicity – more on this another time.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.

Cool Science: Pathways Driving Fibrosis and Related Drug Development

autotaxin, fibrosis, integrins, IPF, LPA and LPA receptors, TGFbeta

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December 10, 2013, by P.D. Rennert
Some recent interesting papers reveal new mechanisms for the regulation of fibrotic pathways, and suggest drug discovery opportunities.
Fibrosis is a huge medical problem and encompasses many different diseases across most organs and tissues: lung, kidney, liver, gut, vasculature, skin, ocular, cardiac, etc. Lets just assume upfront that we recognize that TGFbeta (TGFb) signaling is a critical pathologic pathway in the induction and persistence of the fibrotic state. I want to take that as a given. TGFb is also showing up more and more often in the context of tumor genetics, as befits a critical growth factor. From the drug development perspective, TGFb antagonism not yet been successful due to the toxicities encountered: TGFb is very pleiotropic and controls many normal functions as well. As a result several alternative means to target the TGFb pathway are under development for the treatment of fibrosis.
A very clever strategy for targeting TGFb was developed by researchers at Biogen, along with a top notch group of outside collaborators (see for example Horan et al. 2008). It was determined that activation of TGFb at sites of inflammation and injury, which are sites for the initiation or promotion of fibrosis, depended on an integrin, namely alphavbeta6 (avb6). Biogen developed a monoclonal antibody (mAb) to avb6 and subsequently outlicensed this mAb to Stromedix. I helped bring that mAb back into Biogen 5 years later (early 2012). By then the mAb, now called STX-100, was Phase 2 ready, a testament to the strength of the Stromedix team. Stromedix was acquired for 75MM upfront, plus milestones: a good deal for both companies. 
On the science side, how avb6 activated TGFb was not well understood, but this was thought to involve a conformational change in the beta6 strand of the integrin.
So lets jump ahead, or sideways anyway.
A parallel basic research and drug discovery effort had uncovered an interesting role for the bioactive lipid lysophosphatidic acid (LPA), and for the LPA receptors, in fibrosis. LPA receptor gene-deficient mice (LPAR knock-out or KO) were resistant to the development of fibrosis in a variety of models, and across diseases in different organ systems. This was interesting since it suggested that somehow LPA and the LPARs were regulating the TGFb pathway (see here, and here). How this occurred was not known, and the available data were correlative: when LPARs were blocked, disease was lessened and there was less TGFb, but was this specific to the LPA pathway or was it due to reduced disease activity? It turned out that the LPA/LPAR pathway interacted with integrin biology, in a couple of interesting ways. The first concerned signaling through one of the LPARs, LPAR2. LPARs are G-protein-coupled receptors, that is, they signal using G proteins. We won’t go into these, but its enough to say that signaling from LPAR2 through a specific G-protein induced a conformational change in the beta chain of avb6, and that this conformational change was associated with the ability of avb6 to activate TGFb. The second way has to do with the manner in which LPA is produced by the enzyme autotaxin, and we’ll come back to that later. This is not the only manner in which LPA impacts fibrosis, as was demonstrated by the Amira drugs that are selective antagonists for LPAR1 and perhaps also LPAR3 (see for example, here). This drug also blocked the development of fibrosis in multiple models, and showed activity in early clinical development for idiopathic pulmonary fibrosis (IPF). The data were suggestive enough that Bristol-Myers Squibb bought the company for $325MM upfront in 2011.
Now lets jump ahead.
In 2011 Tim Springer’s lab published a remarkable paper (Shi et al). What this paper explored, using structural biochemical and biophysical techniques, was how TGFb was kept in an inactive state by its own protein structure (called the “straightjacket”). Breaking this inactive state required distinct and critical interactions, one in which TGFb was embedded into the cell membrane in complex with latent TGFb binding proteins (LTBP), which in turn bound to the extracellular matrix, and the other by which TGFb interacted with the avb6 integrin. Productive binding of the integrin required interaction with cell’s the actin filaments, the protein fibers that hold the cell together and also rearrange themselves in order to allow the cell to move. We should note here that integrin:actin interaction is most often mediated by talins, the proteins that sort of cross link the actin filaments (think of rungs on a ladder as a simple picture). This complex interaction of TGFb, avb6, the cell membrane and the actin fibers imposed enough force on TGFb to break its lock, and free it for activation and release from the cell surface. Its as if you had to rearrange the walls of your house in order to open a window. This is a highly regulated system. So, how did avb6 accomplish this trick? Spinger’s lab didn’t give an answer – they were intently focused on the TGFb structure. But a recent paper on a different integrin provides a clue, and maybe brings us back to the LPAR pathway.
A few weeks ago Shen et al (in Nature) elucidated differential signaling by the integrin alphaIIb-beta3 (aIIb-b3), focusing on the interaction of the b3 subunit with talin and with a G-protein, Galpha13 (Ga13). They found a specific amino acid motif – EXE – mediated binding to the G-protein. This motif is found in many beta subunits, including b6. The protein domain in which this motif is found is within a region known to mediate binding to talin, and Shen et al. go on to show that the two binding phenomena are mutually exclusive. Importantly, interaction with the G-protein required binding to fibrinogen, a ligand for the aIIb-b3 integrin. Integrins transmit signals bidirectionally. Inside-out signalling, the process by which an integrin undergoes conformational change in response to ligand binding, is a talin-dependent mechanism. aIIb-b3 integrin ligation mediates platelet aggregation and triggers outside-in signalling, which requires Ga13 and greatly expands thrombus formation. Switching back to interaction with the G-protein corresponds to clot retraction. So this model proposes waves of signaling controlled in part by which component of the cellular machinery – talin or G-protein – is engaged.
It has been demonstrated in lung and kidney fibrosis models that avb6-mediated activation of TGFb can be triggered by the LPAR2 signaling through Galphaq (Gaq) to the Rho/Rac kinase pathway (see here, and here). The connecting dots from Rho/Rac kinase to avb6 activation are not yet described, by may work by regulating the interaction of avb6 with a G-protein (eg. Ga13) v talin or actin. In KO mouse experiments, Ga12/13 were shown to be required for basal TGFb activation in embryonic fibroblasts (MEFs), but not in LPAR2 transfected MEFs. The relevance of this data to the fibrotic condition however is unclear, and I think it remains worth exploring this link further.
There is an independent link between the LPA/LPAR2/avb6/TGFb pathway and integrin biology. The production of LPA is controlled by the enzyme autotaxin. In settings of tissue injury and inflammation, autotaxin and the LPARs are upregulated by various cells. In this way the LPA production machinery (autotaxin) and the responding receptors are operating in tandem. More remarkably, autotaxin is carried into the inflamed or injured tissue by activated cells – platelets, T cells, and likely other cells types. The manner in which autotaxin is transported is by binding to integrins with b1 or b3 subunits, carried on the cell surface and maneuvered into place by the coordinated action of the activated cell (see a review here). This has likely evolved to maximize the production of LPA at sites where it is required – the half life of LPA in circulation is very short, minutes only. Indeed the half-life of TGFb is even shorter, less than one minute.

So, this is a highly coordinated and highly regulated system, as might be expected when dealing with a potent growth factor like TGFb. The implications of the model for drug development are several. First it seems clear that LPA receptor antagonists that can target LPAR2 would be therapeutic and differentiated from the Amira assets now owned by BMS. The benefit of targeting avb6 was discussed earlier in the context of the Stromedix/Biogen deal. Another target that appears here is autotaxin, and several companies are pursuing autotaxin antagonists – I developed a beautiful compound series at X-Rx Discovery, as one example. Finally, there is the very exciting prospect of using the new structural information about TGFb and related proteins (its a big family) to design drugs that selectively block TGFb activation in the context of an activated cell response. This may be one of the things that Scholar Rock Therapeutics, a venture-company founded by Tim Springer and colleagues, is up to now. We’ll have to wait and see.
Bottom line: we are learning a lot about fibrosis pathophysiology, only a little of which is touched on here. With that knowledge will come a new wave of drug development, and ultimately we hope to provide relief for patients suffering from these chronic and very serious diseases.
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