These could be leveraged potentially in different ways, they may achieve priming, which is the generation of new immune recognition of tumor neoantigens, or they may help to simply promote existing immune response against cancers, or may help just to overcome sites of immune resistance. An agent that we've worked extensively on in preclinical and early-phase clinical studies now, NM600 is developed by my colleagues, Reinier Hernandez and Jamey Weichert, selectively delivers radiation to tumor cells using differences in metabolism in lipids, and particularly in the composition of lipid membranes and tumors. And in preclinical studies, Ravi Patel, who's now at the University of Pittsburgh, was in the lab as a postdoc at the time, showed that a relatively low dose of NM600 delivering an absorbed dose of two and a half gray in the tumor microenvironment, when combined with immune checkpoint blockade, led to a synergistic effect at both local tumor site and in the secondary rejection of a re-engrafted tumor cell promoting improved survival. And interestingly, dose escalation, while still below toxic levels, was potentially counterproductive. This was our first insight into the possibility that optimal dosing in combination with radioligand therapies and immunotherapy may not be the same or optimal approach to optimizing monotherapy dosing.
When we did deep sequencing of the TCR repertoire, Ravi observed no evidence of immune priming with this beta emitter Y90, but rather just propagation of existing TCRs that were present prior to treatment. So, we did not see priming with this combination. We've now launched a phase-one study that's opened a dose-escalation study, because of the risks for both potentially advantageous effects, but also potentially detrimental effects, this study will be done in patients who have developed immune-unconfirmed progression of disease. And so, these patients may not have much to lose. Patients who have begun to show progression on checkpoint blockade only have about a 5% chance that they will actually go on to respond to that agent, almost independent of whatever type of cancer they may have. And because this agent is taken up across tumor types, it's open to patients with any type of cancer on checkpoint blockade with immune-unconfirmed progressive disease. In the lab, we've taken that initial data and then begun to look at what the different effects of different isotopes are, this is challenging because as you all know, when you change an isotope, you change many parameters, not a single effect dose rate or the half-life, not an effect on range alone, and not an effect on the type of radiation, but all of these simultaneously.
In an initial look at this, Caroline Kerr, a very talented graduate student, now a resident in radiation oncology at Johns Hopkins, looked at one of the key effects that has been shown to deliver positive effects of radiation in the tumor microenvironment, that's the activation of a type-one interferon response downstream of the cGAS-STING pathway. And she observed that regardless of the isotope, we could see this pathway activated by radiopharmaceuticals as we had seen previously with external beam radiation. However, the time course of that, as you might imagine, was sensitive to the dose rate, or the half-life of the agent, and you could phenocopy that by basically giving radiation from an external beam source with approximately the same dose rate over time, but what you could not do was also phenocopy the BED effects of the alpha emitter. And so, the alpha emitter always had a higher effect even when we phenocopied the dose rate, which was not surprising. Basically, BED still mattered in this immunogenic effect.
In a subsequent publication, Caroline looked at what isotope might be better in combination with checkpoint blockade in different models. And this is one of those studies for a graduate student that was initially a big pain because it showed data in different models that was opposite, and Caroline did a lot of work to try to understand why that might be. And in relatively immunogenic MC38 model, the combination of a beta emitter was more effective with checkpoint blockade, in a relatively poorly immunogenic model, B78 melanoma, which is not responsive or has de novo resistance to checkpoint blockade, the alpha emitter was more effective. And this was puzzling. After a number of studies, what we came to observe was that the beta emitters were more effective in promoting activation of existing T cells. So, we took the T cells that were present and co-cultured those with tumor cells that had been treated with a beta emitter, we saw greater production of interferon gamma by those T cells, consistent with greater activation of T cells that were already present. And so, it was effective in a model that was immunogenic like MC38.
Conversely, when we looked in a poorly immunogenic model, only the alpha emitter, when we did single-cell sequencing of tumor-infiltrating CD45-positive immune cells, and we looked at CD8 T cells, effector T cells, as well as conserved TCR sequences that were not only shown on clonally expanded T cells, but also on effector memory cells, as a point of evidence to suggest these were the immune cells that had been clonally expanded, activated, and developed memory. We found conserved sequences in eight different TCR clonotypes only after the actinium treatment in combination with checkpoint blockade. In all other treatment groups, we saw only seven clonotypes. So, this was evidence of immune priming following an alpha emitter in combination with checkpoint blockade, and it suggested a difference in the outcome of the immune modulation with the alpha emitter compared to the betas in this model. It also suggested the potential of why that might be more effective in a poorly immunogenic model because it may overcome the mechanism of resistance, which is lack of immune recognition, as opposed to acquired phenotypic exhaustion of T cells, which is what we overcame with the beta emitter. That was a nice story that came together in a publication, however the picture I think is maybe more complicated than that, and I'm not sure that I can answer your question yet today, but I hope that we'll get there.
One of the things that we vary when we change an isotope, as I mentioned, is the range. And when you change the range, you change the dose heterogeneity within the tumor microenvironment. And separate from the studies I showed you, we recently published with brachytherapy that dose heterogeneity has a big effect on whether we achieve immune priming with external beam radiation or with brachytherapy. So, another talented graduate student in the lab, in unpublished studies, has begun to look at the effects of dose heterogeneity from radiopharmaceuticals. And to do this, we use FAP-targeted therapy, but we've used it just as a model where we exogenously express FAP on tumor cells. So, this is not how FAP would normally be targeted. And we created either tumors that were purely FAP-positive or those that were mixed, as a way to control dose heterogeneity. And what we observed was that in a FAP tumor model, we got much more heterogeneity of dose on a dose-volume histogram when we had a mixed model as you would expect. Because the different effects of radiation are dose-dependent, if you have a more homogeneous model in that model at different mean tumor doses, you could activate one mechanism at a time.
You could upregulate MHC at high doses, you could upregulate interferon response at a moderate dose. However, with a heterogeneous dose where you have peaks and troughs that are spread out in one tumor microenvironment, in that model, you could activate all of these mechanisms simultaneously. And we've previously shown this with other approaches, like brachytherapy. What we observed that was very interesting, which was counter to what I would have predicted from a radiobiological standpoint, was that a more heterogeneous model responded better to radiopharmaceuticals than a more homogeneous model, and that that effect went away if you depleted CD8 T cells. And so, dose heterogeneity was important and actually promoted response even in the absence of checkpoint blockade, but it was immune dependent. Mechanisms that we had studied previously were observed here as well. We saw these initially with brachytherapy and confirmed them here.
Tumor draining lymph nodes, if you gave a higher mean dose, if you did not preserve low dose regions because of a homogeneous distribution, you had a detrimental effect on the migration of dendritic cells to tumor-draining lymph nodes. So, the value of dose heterogeneity was potentially preserving cold spots to preserve functional immunity. And if you did that, that heterogeneous distribution allowed you to then see more infiltration later on by CD8 T cells. And here with a beta emitter, when you artificially promoted dose heterogeneity, you could see evidence of immune priming. So, this suggested possibly the result that we got with the alpha emitter, may not be specific to the alpha particle itself, but to the short range of the alpha particle, the ability to preserve cold dose regions. This brought together some seemingly disparate findings for us over the past few years. In different models, we've seen different isotopes have different optimal doses, and the same isotope sometimes has different optimal doses across models. And we believe that this reflects a unifying hypothesis, which is that the optimal dose to deliver radiation when combined with checkpoint blockade may be that which does not induce lymphopenia, and which preserves some low dose regions that stay below two gray, while still giving high dose in some portions.
And for different isotopes and different models, that's going to occur at a different mean tumor dose, but biologically that leads to a consistent effect across all of the different diseases we've seen. So, to summarize, radiopharmaceuticals may in preclinical studies affect the immunogenicity in ways that could promote immune response, there's different mechanisms that contribute to this. Dosimetry is critical, I believe, to the understanding and the optimizing of these effects. And as the previous speaker alluded to, I think some of the biggest opportunities may be that we induce radiation specific immune responses that could present new targets for immune combination. Thank you.