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Andrea Miyahira: Hello, everyone. I'm Andrea Miyahira and I'm the Senior Director of Global Research and Scientific Communications at PCF. Today I'm joined by Dr. John Lee, an Assistant Professor at the University of Washington and Fred Hutch Cancer Center. Dr. Lee and colleagues recently published the paper, Targeting Advanced Prostate Cancer with STEAP1 Chimeric Antigen Receptor T Cell and Tumor-Localized IL-12 Immunotherapy in Nature Communications. John, thanks for joining us to discuss your study today.

John Lee: Thanks for having me. So I'm going to go over some of the key points of this work and I first wanted to make a note that this work was funded by Prostate Cancer Foundation led by Vipul Bhatia, who is a Prostate Cancer Foundation Young Investigator. And a lot of this work is due to a Prostate Cancer Foundation Challenge Award that was awarded to Dr. Nelson and I in 2019.

So the focus of our work is a protein called six transmembrane epithelial antigen of the prostate 1, or STEAP1. And STEAP1 is a protein that's been known to be enriched in prostate cancers. It was first reported about 20 years ago and it's really the focus of a significant therapeutic development in prostate cancer. STEAP family members are integral member proteins with enzymatic metalloreductase activity. They form homo- or hetero-trimeric structures with other family members. And they demonstrate limited expression except in the prostate gland. Furthermore, STEAP1 has been implicated in cancer cell proliferation, invasion and EMT. And so this is really why many companies have been focused on developing therapies against this target.

Specifically, Genentech had developed an ADC or antibody-drug conjugate called Vandortuzumab vedotin. This was unfortunately discontinued in 2017 after a phase one clinical trial. But this hasn't stopped further development. Amgen, for instance, it developed a bispecific T cell-redirecting antibody called the AMG 509. It's currently under investigation in a phase one clinical trial.

And so we had identified STEAP1 previously in a paper published in 2018 where we characterized the landscape of cell surface proteins expressed in prostate cancer and found that STEAP1 was highly enriched in AR-positive prostate cancers. And so the first step that we took was to really examine STEAP1 expression relative to the well known target in prostate cancer, prostate specific membrane antigen, in a cohort of lethal metastatic castrate-resistant prostate cancer patients. And so we leveraged a tissue microarray consisting of 44 patients who had multiple metastases collected at the time of rapid autopsy. And so these included multiple metastatic sites, including the bone, lymph node and visceral organs. And we conducted immuno chemical studies of PSMA as well as STEAP1. And what we saw was that there were some pores or tissues that did not express PSMA, but expressed STEAP1 and in other tissues we saw that there was heterogeneity of PSMA expression where some regions expressed PSMA and others did not, whether it was more homogenous expression of STEAP1.

So when we tabulated the degree of expression, we found that 87% of the tissues expressed STEAP1, where only about 60.5% of the tissues expressed PSMA. And when we look at it from the patient level, what these charts show are individual metastatic sites for a patient and how much STEAP1 or below how much PSMA is expressed. And so what you can see is that there is more homogenous expression of STEAP1 across metastatic sites within patients relative to PSMA. And you can see that this is tabulated on the right where high STEAP1 represents homogenous expression and now it's found in about 70% of the patients. Whereas heterogeneous STEAP1 expression was only found in about 30%. And when we do the same analysis with PSMA, we see that high PSMA or homogenous PSMA expression is only seen in about 45%. Heterogeneous expression in 32% and there were about 23% of cases where ether was no PSMA seen at all in the tissue.

And so based on this broad expression we decided to pursue the development of a STEAP1 CAR T cell therapy. And so we want to do this because CAR T cell therapy is a living drug. And it can be engineered for potency as well as specificity and memory. And so this is the design of our second generation STEAP1 CAR T cell construct. And we initially engineered and optimized this in cell line models and ultimately identified a candidate that we took forward into mouse studies. And this is one of these mouse studies where we've taken the C4-2B line injected into the tail vein of mice to establish metastatic tumors and then treated with untransduced T cells or STEAP1 CAR T cells. And you can see from the bioluminescence imaging that in the context of STEAP1 CAR T cell therapy that many of these tumor lesions actually disappeared by about four weeks and this is quantified dint his chart here.

We also took a more aggressive prostate cancer cell line called 22Rv1 and performed a similar experiment. In this context, the mice treated with untransduced T cells also succumbed to the disease within about four to five weeks. Whereas ones that were treated with STEAP1 CAR T cells, while these mice were not cured, we were actually able to delay the progression of the disease quite significantly, such that they were surviving for about 90 to 100 days. And so in our studies we were able to identify that these were very potent, but in studies I'm not showing you here, we also identified that these are very reactive and sensitive to most STEAP1 antigen density conditions.

And so one of the primary concerns for us was to really establish the pre-clinical safety of this treatment. And because our STEAP1 CAR only reacts against the human STEAP1 and not the mouse STEAP1, we went ahead and actually engineered a humanized STEAP1 mouse where we actually knocked in this human STEAP1 gene into the mouse STEAP1 mucous. And then once these mice were generated, we formed a tissue survey to evaluate where the human STEAP1 was expressed. And reassuringly we found that the human STEAP1 was expressed most highly in the prostate, which is the same pattern that we see in humans. And so we were able to then take these mice and established this syngenetic mouse prostate cancer tumor model and then perform studies to evaluate not only the efficacy in terms of treating these tumors, but also to look at safety. And so in this context we were able to see that in the treatment with these mouse STEAP1 CAR T cells causes a reduction, albeit, temporarily in tumor burden and does lead to an extension of survival. But most importantly, we saw no gross toxicities or any evidence of on-target tumor toxicities in organs that we defined here in the prostate or in the adrenal glands.

So we wanted to understand what might be the mechanisms of resistance to STEAP1 CAR T cell therapy. So we went back to these tissues from these human and mouse as well as mouse and mouse tumor models, and what we saw as a recurrent mechanism of resistance was actually loss of STEAP1 expression. And that's shown here in these immunochemical studies.

And we then wanted to figure out how STEAP1 antigen loss may actually impact the anti-tumor activity and the change in the tumor micro environment. And so we first used some cell lines in which we had engineered deletion of STEAP1 or rescued STEAP1 expression and performed RNA sequencing analysis to examine gene expression. And when we did this one of the pathways that emerged was antigen processing and presentation. Here's a heat map showing a variety of genes associated with antigen presentation processing that are modulated by deletion or add back of STEAP1.

And so we went back to our tumors and asked is there an impact on these tumors related to antigen processing presentation when there's antigen loss in the context of STEAP1 CAR T cell therapy? And indeed when we looked our 22Rv1 tumors, we found that after treatment with STEAP1 CAR T cells and STEAP1 antigen loss, there was a loss of HLA ADC expression. And then similarly in our mouse, RM9 prostate cancer tumors, we saw a loss of BETA-2-microglobulin expression indicating that there was a deficit in antigen presentation occurring in these STEAP1 antigen loss tumors.

So this really suggested that STEAP1 antigen loss can actually enhance tumor immuno invasion through down regulation of this process thereby leading to less visibility of these tumors to the host immune system. So we wanted to figure out whether there might be a strategy to overcome this and we collaborated with a colleague, Jun Ishihara at Imperial College London, who developed this collagen-binding domain IL-12 cytokine. So IL-12 is inflammatory cytokine that's been shown to have pretty significant FI tumor effects. The problem with this is that when it's been taken into clinical trials in the past, systemic delivery of IL-12, it's actually been quite toxic. And so the use of a collagen-inviting domain allows for localization of IL-12 to the tumor by binding to exposed tumor vasculature.

So we applied to this RM9 prostate cancer model and this prostate cancer model is resistant to immune checkpoint therapy or anti PD1 therapy based on this graph. But when we applied CBD-IL-12 you could see pretty significant anti-tumor effects. And so we analyzed the tumor microenvironment of these tumors and could see that there is a significant remodeling of the tumor immune microenvironment with an increase of CDA-positive T cells, increase of monocyte and macrophages as well as some reduction in population of neutrophils. When we zoned in on the tumor cells themselves, we saw that CBD-IL-12 could actually induce the expression of BETA2 microglobulin and other MHC class one molecules indicating perhaps this could rescue the effect seen with STEAP1 antigen loss.

And so we conducted experiments where combined STEAP1 CART T therapy with CBD-IL-12 therapy and in that context we actually saw an extension of survival, certainly not a cure. But most importantly we saw in immunochemical studies that when we introduced CBD-IL-12 that indeed we could rescue the expression of BETA-2-microglobulin in these tumors as well as increasing the influx of T cells within these tumors. And then lastly when we performed TCR sequencing to look at clonality of the TCRs present within the tumors, we actually saw that there was an expansion of the TCR repertoire indicating that CBD-IL-12 is not only bringing these T Cells into [inaudible 00:11:02] but probably broadening the immune response as well.

And so the basic conclusions of the study were that STEAP1 is broadly expressed in lethal metastatic castrate-resistant prostate cancer with less heterogeneous expression relative to PSMA. We've developed a second generation STEAP1 CAR T cell therapy with potent antigen specific anti-tumor activity. We've provided evidence preliminarily of preclinical safety of this therapy in a humanized STEAP1 mouse model. And then we focus on STEAP1 antigen loss as a current mechanism of resistance. And this is associated with downregulation of the antigen prostate presentation. And we offer CBD-IL-12 combined with STEAP1 CAR T cell therapy as a means to broaden the anti-tumor immune response to help combat the sequela of STEAP1 antigen loss.

Andrea Miyahira: Wonderful. Thank you for sharing that. Just some questions for discussion. What regulates expression of STEAP1 in prostate cancer and is there a way to increase or maintain its expression?

John Lee: Yeah. That's a great question. So there have been a couple of studies performed in the last decade or so. So in 2014, Jason Lewis in the Journal of Nuclear Medicine performed a series of studies looking at whether modulation of AR expression as well as castration or treatment with Enzalutamide could have an effect on STEAP1 expression. It had been suggested from prior studies that STEAP1 is androgen regulated. And so indeed many of those studies performed by Dr. Lewis and his group confirmed that indeed that it is androgen regulated. There are some additional hints, so for instance, STEAP1 is highly expressed in Ewing's sarcoma. And Ewing's sarcoma is driven by translocation in the EWS-FLI-1 gene. And FLI-1 is actually a transcription factor. And so the interesting parallel with prostate cancer is that in prostate cancer we also see very common translocations in transcription factors like HER, ETV1 and ETV4. So there is a possibility that these transcription factors in prostate cancer can also play a role in regulation.

Adam Sharp, in 2019 published a paper in The Journal Clinical Investigation indicating that actually RV7 also seems to be a regulator of STEAP1 correction. And then there's also been some recent work indicating that DNA methylation may associate with STEAP1 expression. And so to answer the second part of the question. It's possible that antigen inhibitors could relieve some of that methylation-induced inhibition of STEAP1 expression. But that has yet to be shown in experiment.

Andrea Miyahira: Okay. Awesome. So when you start thinking about translating this to patients, what toxicities might you expect with STEAP1 CAR T cells?

John Lee: Yeah. So based on the expression profiles that we've seen, the primary place that STEAP1 appears to be expressed is in the normal prostate, specifically in the normal prostate luminal epithelial cells. And so certainly in a large number of men who have metastatic prostate cancer, they may have previously had their prostates removed. In others, they may have been diagnosed de novo metastatic disease. But certainly, one place that we'd be concerned about potential adverse effects would be the prostate, whether an infusion of CAR T cells could induce some sort of inflammatory prostatitis.

Andrea Miyahira: All right. And what are your plans to translate these CAR T cells to patients?

John Lee: So we've been fortunate to be working with the NCI Experimental Therapeutic Program for about a year and a half. And so they've actually been working on developing processes for manufacturing. As well as also conducting IND-enabling toxicity studies on our behalf with a plan to actually initiate a phase one clinical trial here at Fred Hutch and we're anticipating an IND submission actually at the end of this year with the trial actually opening to accrual in early 2024.

Andrea Miyahira: Okay. So very exciting and this is such great work. Thanks for coming on to share today.

John Lee: Thank you.