Orca-T Promotes T-Cell Activation in Hematologic Malignancies

Higher expression of the activation markers FOXP3 and Helios in CD4-positive T-conventional cells (Tcons) at 3 weeks after receiving Orca-T or unmanipulated peripheral blood stem cell (PBSC) grafts may be a sign of early T-cell activation risk in patients with hematologic malignancies, and additional research could help correlate these markers with risk of graft-vs-host disease (GVHD) and other clinical outcomes for this patient population, according to Cameron S. Bader, PhD.

Notably, findings from the study presented at the 2025 Transplantation and Cellular Therapy Meetings demonstrated that a higher rate of patients who received Orca-T had CD4-positive, FOXP3- and Helios-expressing cells at 3 weeks compared with those who received unmanipulated PBSC grafts. The study data also showed that activated CD4-, FOXP3-, and Helios-positive Tcons at 3 weeks were likely to remain activated at 3 months after treatment.

“There’s room for a lot of improvement [in the transplant field], and Orca-T closes the gap a bit more than some other [treatments] I’ve seen,” Bader, a postdoctoral scholar at Stanford Medicine in California, said in an interview with OncLive®.

In the interview, Bader discussed the development and potential clinical benefits of Orca-T for patients with hematologic malignancies; findings from a study evaluating the prevalence of FOXP3 and Helios expression in Orca-T and unmanipulated PBSC graft recipients; and the next steps for validating these findings in more patients and exploring their applicability across different treatment strategies.

OncLive: How does Orca-T differ from traditional allogeneic stem cells?

Bader: The history of Orca-T goes back to at least a Nature Medicine paper by Robert Negrin, MD, of Stanford Medicine, who was one of my co-mentors, showing that transplanting T-regulatory cells [Tregs] along with Tcons can protect against GVHD without abrogating graft-vs-leukemia or graft-vs-tumor effects. Clinical translation takes a long time. It took until approximately the mid-2010s for this [research] to be translated into humans and developed into what is Orca-T today.

Orca-T is considered a precision immunotherapy in which patients receive a selected CD34-positive graft on day 0, which is the day of the transplant. They also receive Tregs that have been purified on that same day, and those cells are given approximately 2 days to engraft before the Tcons are transplanted. The other key difference between an unmanipulated PBSC graft and Orca-T is that an unmanipulated graft will typically be normalized to whatever number of stem cells the patient is set to receive. However many T cells come along with that graft [will be] how many they receive. In an Orca-T immunotherapy graft, patients instead receive a target of 3 million Tregs and 3 million Tcons. [Data from] phase 1b and phase 2 trials have shown that single-agent GVHD prophylaxis results in reduced rates of acute and chronic GVHD compared with unmanipulated PBSC grafts.

What was the rationale for conducting research evaluating the correlation between FOXP3/Helios expression in CD4-positive T cells and patient outcomes with Orca-T?

Going back to 2003, it’s been known that Tregs can suppress immune reactions, but the exact methodology of how they do that is both multifactorial and a bit unknown. In this study, we wanted to figure out this mechanism in Orca-T immunotherapy. What is happening to the T cells that potentially prevents GVHD but allows for graft-vs-tumor effects to occur?

We started with a discovery experiment. We took a subset of patients and captured their T cells for single-cell RNAseq, which is a strategy to look at all the genes that are expressed in these immune cells to identify differences in gene expression between patients who received Orca-T vs those who received unmanipulated grafts. We found that the T cells are not that different between the 2 groups, but there were a few things that stood out as being different. Since there were only a few things that were different, we thought [these differences] were important.

One of those populations of cells was CD4-positive Tcons, which expressed high levels of FOXP3 and Helios. This is a population that would probably otherwise be missed if you were looking by traditional flow cytometry because these are 2 transcription factors that are generally associated with Tregs, which, by definition, are CD25 positive. The population we found these in was CD25 negative; therefore, by definition, [these cells did not meet] not the classical definition of a Treg. This was interesting to us. We recapitulated this in another set of 8 patients using a [flow cytometry]. We showed that this [finding] was technologically agnostic; it wasn’t just an artifact of what we were doing [with single-cell RNAseq].

Then, we looked in another number of patients at the protein level, because sometimes the RNA at the transcriptional level doesn’t always get all the way through to the protein level. We unfortunately can’t do single-cell RNAseq in all our patients due to the high cost, but flow cytometry is a little more doable with large numbers of patients. We looked at an additional 20 patients, and we found that [our previous finding] was true. In the CD4 Tcons that were CD25 negative, patients receiving Orca-T had higher frequencies of cells expressing FOXP3 and Helios compared with those receiving unmanipulated grafts 3 weeks after the transplant.

What are the potential implications of these findings, and what could they mean for patients’ long-term outcomes when their cells express these markers?

At the moment, there’s no predictive biomarker for patients who are going to exhibit either acute or chronic GVHD. There’s been a lot of work trying to find innate biomarkers. This [research] is still in the early stages. You can’t call [FOXP3/Helios] a predictive biomarker at this point, but we are interested. We found that 3 weeks after transplant, [patients who received Orca-T] had higher frequencies of [FOXP3- and Helios-expressing] cells, and otherwise there’s not a huge number of differences in terms of the phenotype of these T cells.

Unfortunately, this study was conducted alongside a trial, and that trial ended, so we ran out of patients [in whom] we could examine [the expressions of FOXP3 and Helios], so we didn’t have the power to compare [rates of] GVHD with our metric of interest: FOXP3- and Helios-expressing cells. However, we were interested because patients who had higher expression of [FOXP3- and Helios-expressing] cells 3 weeks after transplant had higher activation markers on their T cells when we looked 3 months after their transplant. This was interesting because in general, patients who received Orca-T had higher levels of these activation markers to begin with. That is a treatment-related effect.

However, this was also true in the unmanipulated graft cohort. When we pooled the patients together, it didn’t matter which treatment they received. This correlation was true [in both populations]. This is interesting because FOXP3 and Helios are known to be relatively immunosuppressive or immunomodulatory. It was surprising that we saw that [high expression of these markers] could be correlated with higher activation of T cells later.

When we looked at the 3-month measurements, we had a greater number of patients in whom we had measured this, so we were able to then look at patients who developed acute GVHD based upon whether they had high or low frequencies of these activated T cells. In these preliminary data, patients who had lower activation of their T cells 3 months after transplant had greater incidences of grade 2 to 4 acute GVHD—what we would call moderate-to-severe acute GVHD—[as well as] relapse; [overall, we looked for] negative outcomes within the first 100 days. Using the transitive property, there’s the proposition that the higher the frequency of FOXP3- and Helios-expressing Tcons a patient has 3 weeks after transplant, the lower the risk of relapse and acute GVHD later.

This leads to the next steps [of research]. First, we have to make sure this is true in more patients receiving Orca-T and [and patients receiving unmanipulated PBSC grafts] to see if this is broadly applicable. In the bone marrow transplant field, patients receive numerous different conditioning regimens, graft types, etc. The more patients we can look at who receive different types of conditioning and transplantation strategies, the more confident we can be that this is a real effect.

If these findings regarding FOXP3 and Helios expression are applicable across the transplant field, how might this research help improve outcomes for patients who have a high incidence of non-relapse mortality, GVHD, and other transplant-associated complications?

If [FOXP3 and Helios] end up being decent biomarkers for patients who will have negative outcomes in the first 100 days, that would be incredible. There’s a big belief in the field that there is a standard therapy that every patient gets, no matter who they are. If there was any inkling for the patients who may need a bit more immunosuppression because they’re about to get acute GVHD, or for those who may not at be risk of acute GVHD and they’re receiving immunosuppression for no reason [and experiencing] negative adverse effects, having a biomarker [to tell us] that, whether specific to this condition or not, would be valuable for treating physicians to tailor personalized medicine that is optimal for each patient.

One of the huge benefits of this [research] is that we are looking 3 weeks after the transplant. This is an early [time point] right after patients have been engrafted. This is one of the more difficult things to do. One of the reasons why [researchers often] don’t look at that time point is because patients’ T cells are rare [at that moment]. It’s an important time point, though, because typically it precedes acute GVHD.

Most people [consider ‘early’] to be the first 100 days. However, at that point, oftentimes it’s too late. The patient has already gotten acute GVHD, and then you’re looking at predictive values for chronic GVHD or relapse, [which are] longer-term outcomes.

We also hope to look at patients receiving post-transplant cyclophosphamide. With the Orca-T graft, we have additional trials that are ongoing involving memory cell selection. Additionally, we have another trial that’s [evaluating a] reduced-intensity conditioning regimen for older patients. Looking in those trials for this subset of [FOXP3- and Helios-expressing] cells would be valuable to see if [our findings are also] applicable in those settings.

Maybe there will be a day in the future where we [decide on a] graft strategy, then look at [the patient’s] FOXP3- and Helios-expressing Tcons 3 weeks after the transplant, and then we’ll tailor immunosuppression based upon [those findings]. That would be an ideal world. At the Transplantation & Cellular Therapy Meetings, Sergio A. Giralt, MD, [of Memorial Sloan Kettering Cancer Center], said: ‘We’re all going toward what we can call the perfect transplant.’ The perfect transplant is going to be different for everybody. However, the hope is that we’ll get a bit closer to the perfect transplant.

If data with Orca-T continue to be positive, what could the introduction of this immunotherapy into clinical practice mean for this patient population?

Results from the phase 3 [Precision-T] trial [NCT05316701] will be presented at the 2025 EBMT Annual Meeting, so that will be something to look forward to. Additionally, the allogeneic stem cell transplantation field is relatively young in terms of disease studies. The 1940s is when this all began. At the same time, the drugs that are typically used [in this field today], such as calcineurin inhibitors and methotrexate, are drugs from the 1980s and the 1960s, respectively. We have newer generations of these drugs, but they’re still old drugs, and the improvement in patient outcomes has been slow and stepwise.

There has been improvement. You can still look at when [in history a] patient was transplanted, and outcomes have slowly improved over time. However, Orca-T has the potential to be a bit bigger of a step forward in improvements in patient outcomes.

We have found that rates of relapse in general, at least in previous trials, are not all that different [between Orca-T and other agents], but [we have seen] remarkably low rates of acute and chronic GVHD [with Orca-T]. Patients [who receive Orca-T] also have low rates of non-relapse mortality. When you can go to a patient and say, ‘this is going to be a safe transplant,’ [that’s important]. These outcomes oftentimes lead to mortality, but [they] also have significant morbidity, which is oftentimes overlooked.

I’ve also looked at patients’ peripheral blood 3 weeks after transplant, and these [findings] aren’t necessarily correlative with clinical outcomes. However, Orca-T is called a precision graft, and I see that when I look at the patients’ blood and the phenotype of their cells. It’s predictable and consistent from patient to patient. We’ve compared [Orca-T] with other methods of GVHD prophylaxis, whether it be standard tacrolimus and methotrexate or post-transplant cyclophosphamide. Patients are so highly varied that [their outcomes are] not predictable. Having a bit more predictability [in a field] that’s unpredictable will be helpful for the treating physicians and for peace of mind for the patients.

References

Bader CS, Killian S, Le CT, et al. FOXP3 and Helios expressing CD4+ T conventional cells correlate with T cell activation after Orca-T allogeneic T cell immunotherapy. Presented at: 2025 Transplantation and Cellular Therapy Meetings; February 12-15, 2025; Honolulu, HI. Abstract 87.