Weekly Digests
‹ Back to July

Enhancing ACT with o9R

July 27, 2022

Adoptive T cell therapy has come a long way in recent years, but it typically requires lymphodepleting pre-treatment, which can limit patient eligibility, and its efficacy is still limited in many patients. In recent efforts to improve ACT, particularly for solid tumors, Kalbasi, Siurala, and Su et al. engineered T cells with orthogonal cytokine receptors – mutated versions of native cytokine receptors that selectively bind mutated versions of their corresponding cytokines. In this case, the engineered receptors were chimeric, containing an extracellular domain of orthogonal IL- 2R (oIL-2R) (which binds only to an orthogonal version of IL-2), and various intracellular domains from receptors for IL-2, IL-4, IL-7, IL-9, or IL-21. This allowed the researchers to use previously developed oIL-2R/oIL-2 cytokine pairs to induce and study the effects of different γc cytokine receptors in adoptively transferred engineered T cells, without inducing systemic cytokine signaling. Their findings were recently published in Nature.

Stimulation of chimeric orthogonal receptors with oIL-2 fused to mouse serum albumin (MSA-IL-2) induced patterns of phospho-STAT signaling that were consistent with known signaling for each γc cytokine receptor, without interfering with wild-type IL-2-induced STAT signaling. Cells expressing the receptor with the intracellular domain for IL-9R (o9R) showed increased STAT1, STAT3, and STAT5 phosphorylation, consistent with natural IL-9R signaling, which has only a limited role in T cell biology. These cells also showed increased expression of CD62L, Fas, and Sca-1, consistent with a stem cell memory (Tscm) phenotype, and reduced proliferation compared to control o2R T cells (containing the IL-2R intracellular domain).

Looking more closely at o9R signaling, the researchers incorporated the o9R receptors into pmel (gp100-specific) T cells and adoptively transferred them into B16 melanoma-bearing mice without lymphodepleting pre-treatment. In this setting, both o9R and o2R cells expanded and showed proliferation and STAT signaling profiles that were consistent with in vitro observations. Compared to o2R cells, o9R cells treated with MSA-oIL-2 induced more consistent antitumor effects and prolonged survival comparable to the effects of pmel T cells with lymphodepletion. o9R cells treated with MSA-oIL-2 were also superior within the context of lymphodepletion.

Given that oIL-2 is highly orthogonal and does not interact with endogenous cells, the researchers hypothesized that dosing with oIL-2 would be more tolerable than with IL-2, which becomes toxic after about 5 days. Indeed, in a prolonged dosing regimen of 25 days, no clinical toxicity was observed, and o9R T cells without lymphodepletion induced better antitumor results than pmel ACT with lymphodepletion and 5 days of MSA-IL-2.

Investigating the underlying mechanisms behind the superior antitumor efficacy of o9R pmel T cells, despite their weaker capacity for proliferation, the researchers found that at five days after transfer, o9R cells showed greater tumor infiltration and made up a higher percentage of CD45+ TILs compared to o2R cells. They were also enriched for clusters associated with T cell activation, including a cluster that expressed CD39, PD-1, and TBET. In vitro, o9R showed a higher cytotoxic capacity and increased IFNγ production compared to o2R cells.

RNAseq analysis showed that compared to o2R pmel T cells, o9R cells were enriched for genes associated with a Tscm phenotype, as well as for genes associated with activation and effector functions, which are typically excluded from the Tscm phenotype. A transcriptomic analysis of transcription factor pathway enrichment showed significant enrichment for genes associated with STAT1 and STAT3, as expected, as well as genes associated with JUN. This was accompanied by an increase in the ratio of Jun to Fos expression, which indicates tumor-specific T cells that are resistant to tumor-mediated exhaustion. In line with this, genes associated with T cell dysfunction were downregulated. Whether these features represented a distinct T cell phenotype or heterogeneity was unclear.

Looking specifically at CD62L expression, the researcher saw that while expression levels were higher on o9R cells in draining lymph nodes and spleens, they were comparable on o9R and o2R cells in the tumor, suggesting that antigen-specific T cell activation may dominate in the TME.

Kalbasi, Siurala, and Su et al. next tested the effects of o9R in CAR T cells and found that CAR-o9R T cells resembled o9R pmel T cells, showing characteristic STAT phosphorylation, reduced proliferation, and increased stemness, activation, effector functions, and antitumor activity in vitro. Using lymphodepleted mice bearing immunotherapy-resistant pancreatic tumors expressing mesothelin, the researchers tested adoptive transfer of mesothelin-specific CAR T cells engineered with o9R. In contrast to previous experiments, in which MSA-oIL-2 was administered systemically, the researchers switched to adenovirus-vectored intratumoral delivery of oIL-2 (Ad-oIL-2) to ensure high concentrations of oIL-2 in the tumor. In this setting, CAR-o9R T cells plus Ad-oIL-2 induced early toxicity, with 5 of 12 mice dying by day 10. However, 6 of 12 mice achieved complete regressions, which appeared to be more durable than the complete regressions (8 out of 12 mice) induced by CAR-o2R cells plus Ad-oIL-2.

Evaluating the high toxicity, which was consistent with immune effector cell-associated neurotoxicity (ICANs), the researchers found evidence of on-target, off-tumor activity against mesothelin-expressing meningeal cells, as well as increased mesothelin expression, which can occur in the context of inflammation. Evidence of cytokine release syndrome and tumor lysis syndrome were not observed, suggesting that the toxicity was mostly related to the specificity of the CAR.

Interestingly, when the researchers tested the same treatment without lymphodepletion, the toxicity of CAR-o9R was reduced. In this setting, the CAR-o9R cells outperformed the CAR-o2R cells, inducing more complete regressions and prolonging survival. Compared to the pmel T cell experiments, tumor infiltration of transferred cells was less pronounced, possibly as a result of intratumoral, rather than systemic delivery of oIL-2. Still though, the frequency of intratumoral CAR-o9R cells expressing IFNγ in the tumor was increased, suggesting that enhanced potency, rather than enhanced infiltration, may play a more important role in the improved antitumor efficacy.

Finally, to evaluate the translational potential of this approach, the researchers engineered human o9R and o2R T cells specific for NY-ESO-1. When treated with human oIL-2, the human o9R and o2R cells demonstrated characteristics that were consistent with what was seen in mouse cells. Differences between the two cell types persisted even after four challenges with a NY-ESO-1+ cell line, with the ho9R cells retaining a greater frequency of CD45RA+CD27+ and Tscm cells, and expressing higher levels of CD62L and CXCR3. Upon stimulation, ho9R cells were more cytotoxic and polyfunctional. Similar results were also observed in mesothelin-specific CAR-ho9R and CAR-ho2R cells.

Overall, these results show the potential to enhance adoptive T cell therapies using orthogonal cytokine and cytokine receptors pairs, and particularly, the o9R receptor paired with oIL-2, which enhanced antitumor immunity in a variety of models. However, given the enhanced potency and the potential for on-target, off-tumor toxicity, it will be important to incorporate additional safety features into future systems.

Write-up and image by Lauren Hitchings


Kalbasi A, Siurala M, Su LL, Tariveranmoshabad M, Picton LK, Ravikumar P, Li P, Lin JX, Escuin-Ordinas H, Da T, Kremer SV, Sun AL, Castelli S, Agarwal S, Scholler J, Song D, Rommel PC, Radaelli E, Young RM, Leonard WJ, Ribas A, June CH, Garcia KC. Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature. 2022 Jul.

In the Spotlight...

Aging-associated and CD4 T-cell–dependent ectopic CXCL13 activation predisposes to anti–PD-1 therapy-induced adverse events

Tsukamoto et al. studied the effects of aging on immune response mechanisms associated with immune-related adverse events (irAEs) following ICB therapy. In an irAE mouse tumor model, anti-PD-1 therapy in aged, but not young mice resulted in irAE-like IgG-mediated multiorgan toxicity due to ectopic buildup of tertiary lymphoid-like structures containing CD4+ T cells and B cells. Mechanistically, CD4+ T cells stimulated IL-21 production in irAE organs, resulting in CXCL13 activation. Toxicity was reduced by B cell depletion, or by blockade of IL-21 or CXCL13. Systemic increases in CXCL13 and T cell IL-21 correlated with irAE incidence in ICB-treated patients.

Contributed by Katherine Turner

Local delivery of low-dose anti-CTLA-4 to the melanoma lymphatic basin leads to systemic T(reg) reduction and effector T cell activation

Based on a phase 1 dose escalation study, Pul and Notohardjo et al. reported that a single intradermal injection of tremelimumab (anti-CTLA-4) at the primary tumor excision site of patients with clinical stage I/II melanoma was safe and well tolerated. Intradermal anti-CTLA-4 systemically decreased activated Treg frequencies, increased conventional T cell activation and central memory conversion, and induced migratory dendritic cell subset activation in the sentinel LN. Systemic melanoma-specific T cell responses against NY-ESO-1 and MART-1 detected in responders were associated with increased T cell activation and memory T cell differentiation.

Contributed by Shishir Pant

Dendritic cell-mediated cross presentation of tumor-derived peptides is biased against plasma membrane proteins

Fessenden, Stopfer, and Chatterjee et al. showed that MHC-I-associated peptides cross-presented by BM-DCs co-cultured with irradiated murine tumor cells were preferentially derived from cytoplasmic and not plasma membrane proteins. When antigen was limiting, intracellular processing and ex vivo T cell-specific recognition of antigen cross-presented by cDC2/moDC, but not cDC1, were biased toward cytoplasmic proteins. Analyses of two cohorts of patients with melanoma showed that reduced survival and poor response to ICB therapy correlated with the proportion of membrane-derived neoantigens, particularly when tumors had low mutational burden.

Contributed by Paula Hochman

mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells

Lipo-based nanoparticles (LNPs) containing mRNA efficiently elicit immune responses, possess adjuvant activity, and induce in situ antigen expression. Bevers et al. optimized the molar ratio and composition of the 4 LNP components (ionizable lipid, phospholipid, cholesterol, and PEGylated lipid) for elicitation of CD8+ T cell responses following i.v. administration of an mRNA vaccine encoding the E7 oncoprotein. The optimized mRNA-LNP increased transfection and activation of splenic immune cells (in mice and non-human primates), and elicited strong antitumor responses (in a TC-1 mouse model). CD8+ T cell responses depended on IFNAR signaling, phagocytes, and B cells.

Contributed by Margot O’Toole

Pre-encoded responsiveness to type I interferon in the peripheral immune system defines outcome of PD1 blockade therapy

Boukhaled et al. used mass cytometry and multi-omics analysis to establish physiological cell states of response to IFN-I that can predict outcomes of anti-PD-1 therapy. Patients with pre-therapy hyporesponsiveness to IFN-Is among their peripheral blood CD4+ and CD8+ effector T cells show improved responses to anti-PD-1 therapy and longer OS, whereas hyperresponsiveness to IFN-I was associated with treatment failure. IFN-I responsiveness was not associated with differences in tumor inflammation or mediated by IFN-I-stimulated genes (ISGs), but was associated with distinct gene expression and pre-existing chromatin accessibility for transcription factors at the basal state.

Contributed by Shishir Pant

Spatial delivery of immune cues to lymph nodes to define therapeutic outcomes in cancer vaccination

Through intra-lymph node (LN) injection, Andorko et al. localized OVA antigen and/or poly(I:C) adjuvant loaded in PLGA microparticles to distinct LNs, where they were taken up by DCs and macrophages. At an equivalent vaccine dose, systemic OVA-specific CD8+ T cell responses were strongest when a half-dose was delivered each to two LNs (“two”), compared to a full dose to one LN (“one”) or split antigen and adjuvant to separate LNs (“split”). OVA alone induced local LN OVA-specific T cells, but these did not circulate. Both one- and two-LN delivery protected from OVA+ tumor challenge; split only worked when adjuvant was delivered to the tumor-draining LN.

Contributed by Alex Najibi

Everything New this Week In...

Close Modal

Small change for you. Big change for us!

This Thanksgiving season, show your support for cancer research by donating your change.

In less than a minute, link your credit card with our partner RoundUp App.

Every purchase you make with that card will be rounded up and the change will be donated to ACIR.

All transactions are securely made through Stripe.