TGFβ is known to play a role in immunosuppression and resistance to immune checkpoint blockade, however, efforts to block TGFβ to enhance immunotherapy have been limited by cardiotoxicity. TGFβ exists in three isoforms – TGFβ1, TGFβ2, and TGFβ3 – and while TGFβ2 and TGFβ3 have been linked to cardiac function, TGFβ1 has not. Based on this evidence, Martin and Datta et al. investigated the role of TGFβ1 in tumors, in line with the possibility of selectively blocking TGFβ1 activation to enhance immunotherapy while circumventing toxicity. Their results were recently published in Science Translational Medicine.
First investigating the role of TGFβ1 in tumors, Martin and Datta et al. analyzed data from The Cancer Genome Atlas and found that TGFB1 was the most prevalent TGFβ isoform expressed in many types of human cancers, including seven of the cancer types for which PD-1 or PD-L1 checkpoint blockades are approved (with the exception of invasive breast carcinoma, which also expressed high levels of TGFβ3). Of the three TGFβ isoforms, TGFβ1 most consistently and most significantly correlated with TGFβ pathway activation in tumors and with a signature of innate anti-PD-1 resistance, suggesting that selective inhibition of TGFβ1 might be sufficient to overcome TGFβ pathway-driven resistance to checkpoint blockade in multiple cancer types.
In order to selectively block TGFβ1, Martin and Datta et al. focused on latent TGFβ complexes, which consist of the mature growth factor still associated with its prodomain and kept in an inactive state by one of several different binding proteins. When activated by integrins, the prodomain is cleaved off and the latent complexes release the active growth factor. Since the prodomain shows larger sequence dissimilarity than the active growth factor, targeting the latent complex offers a better opportunity to selectively target one isoform. The researchers screened a naive human antibody library to identify an antibody capable of binding latent TGFβ1 complexes and inhibiting the integrin-triggered release of active growth factors. Following affinity maturation and antibody engineering, the researchers identified SRK-181, a fully human IgG4/kappa antibody that binds the latent TGFβ1 complex with high affinity. SRK-181 showed little to no binding to TGFβ2 or TGFβ3, demonstrating its selectivity for TGFβ1. Looking more closely at the binding site of SRK-181 on latent TGFβ1 complexes, Martin and Datta et al. found that SRK-181 does not interact with the actual growth factor, but instead binds to a region on the latent complex that varies between TGFβ isoforms, and that this binding allosterically prevents integrin-dependent TGFβ1 activation.
To assess the biological effects of selectively blocking the activation of latent TGFβ1, the researchers examined the effects of SRK-181 on Tregs, which typically express high levels of TGFβ1 upon TCR stimulation and harness TGFβ signaling to suppress the proliferation of effector CD4+ T cells. As expected, Tregs showed reduced suppressive activity in the presence of SRK-181.
Next, the researchers aimed to determine whether selective inhibition of latent TGFβ1 using SRK-181 could overcome TGFβ pathway-driven resistance to checkpoint blockade. Martin and Datta et al. identified three mouse tumor models – MBT-2 bladder cancer, S91 melanoma, and EMT-6 breast carcinoma – that were resistant to typical doses of anti-PD-1 and showed evidence of immune exclusion, active TGFβ signaling, and TGFβ1 isoform expression.
In the MBT-2 model, anti-PD-1 alone had no effect, but combination with a chimeric version of SRK-181 (SRK-181-mIgG1) induced responses in 4 of 14 mice at a low dose (3 complete responses) and 8 of 14 mice at a high dose (5 complete responses). Of the 12 animals that responded to anti-PD-1 plus SRK-181-mIgG1, 10 remained tumor-free for 7 weeks after treatment was discontinued and were protected from rechallenge. In the S91 melanoma model, 75% of mice responded to combination treatment, a significant survival advantage was observed at all dose levels, and mice with no measurable tumors at the time of treatment discontinuation remained tumor-free. Mice that responded, but still had measurable tumor masses at the time of treatment discontinuation, had mixed long-term responses, with some tumors clearing, some remaining small but palpable, and others, mostly from the low-dose treatment group, growing. In the EMT-6 breast carcinoma model, in which TGFβ1 and TGFβ3 mRNA were expressed at similar levels, a high portion of durable responses and a survival benefit were observed, suggesting that expression of other TGFβ isoforms does not limit the efficacy of selective TGFβ1 targeting.
To better understand the mechanism by which SRK-181 sensitizes tumors to PD-1 blockade, Martin and Datta et al. harvested MBT-2 tumors ten days after treatment initiation to analyze the immune contexture. They found that the overall percentage of CD45+ cells was unchanged, however, the portion of CD8+ T cells increased ten-fold and evidence of CD8+ T cells clustering around blood vessels suggested that treatment may have lowered a TGFβ pathway-mediated vascular barrier to entry. Cytotoxic T cell activation markers perforin and granzyme B were also increased in the tumor, though the size of the IFNγ+CD8+ T cell population stayed consistent. CD11b+ cell representation, in particular among immunosuppressive M2-like macrophages and MDSCs, was reduced within the immune contexture. Interestingly, a significant increase in Tregs was also observed.
To determine whether SRK-181 circumvents the dose-limiting cardiotoxicity that has been observed in non-selective TGFβ-targeting strategies, Martin and Datta et al. performed a rat toxicology study comparing SRK-181 against a pan-TGFβ anti-growth factor antibody and an ALK5/TGFβRI kinase inhibitor. Mice receiving SRK-181 showed no signs of cardiac valvulopathy, cardiovascular lesions, or evidence of immune activation in tissue beds at any dose, while the other two treatment groups had heart valvulopathies characterized by heart valve thickening. This suggests that selective targeting of TGFβ1 may be safer than targeting all three TGFβ isoforms, and may be safe enough to administer at doses that could effectively enhance checkpoint blockade.
Overall, Martin and Datta et al. show that the TGFβ1 isoform plays a prominent role in the tumor microenvironment and in resistance to PD-1/PD-L1 checkpoint blockade across many human cancers. Using the SRK-181 antibody to selectively inhibit the activation of TGFβ1 is sufficient to alter the tumor immune contexture and overcome resistance to PD-1 checkpoint blockade in several mouse tumor models. Importantly, selective targeting of TGFβ1 circumvents cardiotoxicities that have been observed in treatment strategies that non-selectively target the TGFβ pathway, suggesting that this more focused strategy could one day be safely implemented to enhance responses to immune checkpoint blockade in patients.
by Lauren Hitchings
This week, lead author Thomas Schurpf answered our questions.
What prompted you to tackle this research question?
My motivation to target TGFβ1 in solid tumors has been two-fold. First, cancer patients who do not benefit from a durable response to checkpoint blockade therapy (CBT) often have only very limited treatment options left. When recent retrospective analyses of clinical tumor samples identified TGFβ signaling as a key pathway associated with primary CBT resistance, I wanted to test whether selective inhibition of TGFβ1, the most prevalent TGFβ isoform in solid tumors, may be sufficient to overcome resistance to CBT and enable more patients to benefit from it. The second motivation was the safety aspect. TGFβ has long been a challenging drug target due to dose-limiting toxicities observed with drugs that broadly target TGFβ signaling. We hypothesized that selectively targeting only one of the three TGFβ isoforms would have an improved safety profile, and we previously saw evidence to support that. To further confirm these hypotheses, we generated SRK-181, a highly potent, fully human monoclonal antibody that only binds and inhibits the latent TGFβ1 isoform. As a sneak peek for those who have not yet read the publication, we were able to demonstrate across multiple mouse tumor models that emulate primary CBT resistance that combination treatment with a highly selective inhibitor of TGFβ1 and CBT leads to tumor regression and survival benefit while avoiding the cardiotoxicity that has challenged traditional approaches.
What was the most surprising finding of this study for you?
While TGFβ1 is the most prevalent TGFβ isoform in the majority of solid human tumors, there are a few prominent exceptions. One of them is breast cancer, with a large percentage of tumors expressing TGFB3 mRNA in addition to TGFB1 mRNA, according to our TCGA database analysis. We identified EMT-6, a syngeneic mouse breast cancer model, which is resistant to therapeutic doses of anti-PD-1, has an immune-excluded phenotype, and expresses both TGFβ1 and TGFβ3 growth factors. You may expect that both TGFβ isoforms may be involved in driving TGFβ signaling and mediating immune exclusion in this breast model. Unexpectedly, we found that selective blockade of the TGFβ1 isoform with SRK-181-mIgG1 was sufficient to elicit a profound antitumor response when combined with anti-PD-1, despite the presence of TGFβ3. The observed antitumor combination effect was similar to what we and others have found with combinations of anti-PD-1 and broad TGFβ inhibition in the same tumor model, suggesting that selective targeting of the TGFβ1 isoform may overcome a key mechanism of primary resistance to CBT in a large number of solid tumor types.
What was the coolest thing you’ve learned (about) recently outside of work?
I recently learned about Jim Lahey’s no-knead bread baking approach and started baking my own bread. The method is really cool and takes very little effort, just a little bit of planning due to the slow rise periods. The results have been great, and very much to the delight of my family we now have easy access to fresh, flavorful bread with nice texture and crust! Looking at the baking aisles in the stores these days, it looks as if a lot of people have joined this bake-at-home trend.