Targeting T Cells for enhancing anti-tumor immunity in pediatric brain tumors.

Targeting T Cells for enhancing anti-tumor immunity in pediatric brain tumors. is sponsored by National Cancer Institute (NCI). This is an active research award (R37CA288506) from the National Cancer Institute focusing on targeting T cells to enhance anti-tumor immunity in pediatric brain tumors.

Central nervous system (CNS) tumors are the most common, and deadliest, cancers in children1.") Diffuse intrinsic pontine glioma (DIPG) is an aggressive tumor that arises in the brainstem and comprises 10–20% of pediatric brain tumors2.") with a poor prognosis and a median overall survival (OS) of 11 months3.")

,4.") Recurrent CNS cancers are likewise typically incurable with significant treatment-related morbidity5.") highlighting the need for new therapeutic approaches.

T cell therapies have shown potential against viral infections and hematological malignancies6.") , but are thus far less effective in solid tumors, although some encouraging results have been recently published7.") ,8.")

The polyclonal nature of multi-antigen-specific T cells9.") may help overcome some potential obstacles, such as intratumoral heterogeneity. Our group has previously shown that tumor-associated antigen T (TAA-T) cells can safely induce prolonged disease stabilization in nonCNS pediatric solid tumors10.")

, and peripherally infused chimeric antigen receptor (CAR)-T cells have been shown to cross the blood–brain barrier in high-grade glioma (HGG)11.") ,12.") Further, WT1, PRAME and survivin are widely expressed across pediatric brain tumors13.")

,14.") ,15.") ,16.")

,17.") We therefore evaluated, in a phase 1 dose-escalation trial (NCT03652545), the safety and feasibility of intravenous infusion of multi-antigen-specific T cells targeting these tumor-associated antigens (TAAs) in children with newly diagnosed DIPG or recurrent brain tumors. The study met its primary end points, and as secondary end points, clinical responses were observed, including a durable complete remission.

### Study design, end points and patient characteristics Pediatric and young adult patients with CNS malignancies were enrolled in a phase 1 adaptive dose-finding study entitled ‘Research on Multi-antigen T cell Infusion Against Neuro-oncologic Disease (ReMIND)’ from 18 September 2018 to 18 July 2024.

The primary end points of the ReMIND trial were the safety and feasibility of intravenously administered TAA‑T therapy and determination of the maximum tolerated dose (MTD).

Pre‑specified secondary end points included preliminary clinical efficacy, assessed by objective tumor response, progression-free survival (PFS) and OS, as well as in vivo persistence of infused TAA‑T cells and systemic immune activation (circulating cytokine and lymphocyte profiles); correlation of clinical outcomes with immune parameters; evaluation of droplet digital PCR for detection of peripheral markers of disease; and characterization of manufactured TAA‑T products (assessment of antigen specificity, in vitro activity and comparison with tumor antigen expression in patient samples).

Immune correlative analyses were intended to be exploratory. Some of these correlates, particularly lymphocyte phenotyping, PCR‑based peripheral disease monitoring, and comparative analyses of TAA‑T product specificity relative to tumor antigen expression, are not reported in this paper.

Key eligibility requirements included age 6 months to 80 years, newly diagnosed radiographic DIPG (arm A) or relapsed/refractory/recurrent nonbrainstem high-grade CNS malignancy (arms B/C). Patients were required to have completed standard upfront therapy, to have adequate functional status (performance score ≥60) and baseline organ function, and to have met protocol-defined washout periods with anticipated steroid use of <0.

4 mg kg−1 day−1 at the time of T cell infusion. In October 2022, a protocol amendment further required patients enrolled on arm A to receive their first TAA-T infusion within 5 months of initiating standard radiotherapy given the risk of early disease progression. Patients enrolled on arms B/C could not have evidence of uncal herniation or significant midline shift, nor a substantial brainstem disease component.

A total of 33 patients were infused intravenously with an autologous T cell product at three dose levels (DLs): 2 × 10 7 cells per m 2 per dose (DL1), 4 × 10 7 cells per m 2 per dose (DL2) and 8 × 10 7 cells per m 2 per dose (DL3) (Fig. 1a,b). Blood for TAA-T cell manufacture was collected via venipuncture.

Arms A and B were dose-finding and did not include pre-infusion lymphodepleting conditioning, whereas arm C was added as an expansion at the confirmed arm B MTD and included fludarabine/cyclophosphamide lymphodepletion before the first infusion.

TAA-T cells were manufactured by culturing antigen-presenting cells (APCs) with peptide libraries spanning the tumor-associated proteins WT1, PRAME and survivin; autologous T cells were then co-cultured with the peptide-loaded APCs, and the final TAA-T products were assessed for phenotype by flow cytometry and specificity by interferon (IFN)γ ELISpot. **Fig. 1: Clinical trial overview.

** **a**, Patients enrolled and treated on ReMIND as shown by a Consolidated Standards of Reporting Trials (CONSORT) diagram. ‘Procured’ refers to completion of blood collection for TAA-T product manufacturing. ‘TAA-T products generated’ refers to the number of patients for which a final TAA-T product meeting criteria for clinical administration was available.

‘Manufacturing failure’ is defined here as failure to manufacture sufficient TAA-T cells to at least meet DL1 or products with out-of-specification (OOS) release testing results. All 33 patients infused were evaluable for DLT analysis. **b**, DLs used for dose escalation, assigned as per mCRM, independently in arms A and B.

Not applicable to arm C, which only studied a single dose level determined by arm B’s MTD. Participants receiving subsequent infusion cycles were assigned to the same dose level, when feasible (no intra-patient dose escalation). F/U, follow-up; LD, lymphodepleting chemotherapy; LTFU, long-term follow-up through final study visit (day 365 after final infusion of TAA-T).

**c**, Each point represents the outcome of an individual manufacturing run. For some participants, more than one run was completed; therefore, although 48 patients underwent procurement, 58 manufacturing runs are represented. Violin plots depict distributions, with dashed horizontal lines indicating the medians and horizontal dotted lines indicating the upper and lower quartiles.

Statistical comparisons were performed using a two-sided Mann–Whitney _U_-test between the number (no.) of doses produced at DL1 before (pre) and after (post) clinical protocol and manufacturing modifications. The median number of doses produced at DL1 was 3. 8 (_n_ = 44 runs) pre-modifications versus 11.

0 (_n_ = 14 runs) post-modifications (_P_ = 0. 0099; Hodges–Lehmann estimate of median difference = 6. 2; _U_ = 168; 95.

21% CI of difference 1. 0–11. 2).

**_P_< 0. 01. Overall, 16 patients with DIPG were enrolled on arm A, and 11 of these patients were infused a median of 3.

5 months (range, 1. 1–4. 3) after radiotherapy and 5.

6 months (range, 3. 0–9. 3) from diagnosis (Fig.

1a and Table 1). Seven infused participants did not undergo biopsy, including one radiation-induced DIPG. Among the four participants who underwent biopsy (P11, P13, P27 and P38), molecular profiling indicated H3K27M mutations in H3F3A in all, TP53 mutation in three (P13, P27 and P38) and a single occurrence of PTPRZ1-MET fusion (P11).

Five patients were not infused due to insufficient TAA-T expansion for clinical use (_n_ = 1), out-of-specification release testing result (_n_ = 1), withdrawal of consent (_n_ = 1), ineligibility (_n_ = 1) and death (_n_ = 1). Of the patients enrolled on arm A who received TAA-T infusions, the median age was 5. 5 years (range, 2–14) with four male and seven female participants.

**Table 1 Summary of patients enrolled and infused by arm** A total of 28 patients with relapsed, refractory or recurrent high-risk nonbrainstem CNS malignancies were enrolled on arm B, and 18 of these patients were infused (Fig. 1a and Table 1). Two patients were not procured due to ineligibility, and eight procured patients were not infused due to insufficient TAA-T expansion (_n_ = 6) or ineligibility (_n_ = 2).

Of the arm B patients who received TAA-T infusions, the median age was 13 years (range, 3–31) with ten male and eight female patients. Diagnoses included HGG (_n_ = 7), medulloblastoma (MB; _n_ = 6), ependymoma (EPN; _n_ = 4) and pineoblastoma (PB; _n_ = 1). In this cohort, 72.

2% (_n_ = 13) had metastatic disease. Patients had a median of two previous relapses/recurrences (range 0–4), and a median of six previous therapy courses for their disease (range 1–17). Seven patients were enrolled on arm C, and four of these patients were infused (Fig.

1a and Table 1). Three patients were not infused due to insufficient TAA-T expansion for assigned DL (_n_ = 2) and death (_n_ = 1). Of the infused patients, the median age was 13.

5 years (range 8–17), with two male and two female patients. Diagnoses included HGG (_n_ = 2), MB (_n_ = 1) and astroblastoma (AB; _n_ = 1); 50% (_n_ = 2) had metastatic disease. Each patient’s specific diagnosis, molecular/disease characteristics (if known), age at diagnosis, age at enrollment, number/type of previous treatments, and number of previous relapses can be found in Table 2.

Due to the limited sample size in arm C precluding meaningful inter-arm comparisons, and as arms B and C included the same recurrent/relapsed nonbrainstem CNS tumor patient population, these two arms were combined for all pooled analyses. **Table 2 Characteristics of infused participants** ### TAA-T cell product characteristics and manufacturing feasibility Of 48 patients who were procured, 41 (85.

4%) had autologous TAA-T cell products manufactured at DL1 or higher for infusion (Fig. 1a). Nine (18.

8%) required dose reassignment to a lower dose level due to inadequate initial yield, prompting optimization of procurement and manufacturing methodologies (Methods, Manufacture of TAA-T products). As part of these changes, the median starting blood collection volume was 110 ml for the first 41. 7% of procured participants and 200 ml for the remaining patients.

After implementation of all procurement and manufacturing modifications, 100% (_n_ = 14) of subsequently procured patients met DL1 or above for at least one infusion, without repeat manufacturing attempts. To assess the feasibility of supporting multiple infusions, we then evaluated the number of doses available at DL1 per manufacturing run before (_n_ = 44) and after (_n_ = 14) these modifications.

The median number of available doses increased significantly after modifications from 3. 8 to 11. 0 (_P_< 0.

01; Fig. 1c). Of these latter 14 manufacturing runs, 71.

4% (_n_ = 10) resulted in at least one full dose available at DL3. TAA-T cell products were primarily comprised of CD3+ T cells with a mean of 92. 4% and median of 96.

9% (range, 31. 3–99. 6%) (Extended Data Fig.

1a–g). We observed heterogenous reactivity to TAA peptides across manufactured TAA-T cell infusion products (Extended Data Fig. 1h,i), and product specificity was determined based on IFNγ ELISpot as described in Methods (Supplementary Table 1).

### Treatment details, safety profile and toxicities The median number of infusions was two for both arm A (range, 1–5) and arm B (range, 1–4), with three arm B patients receiving four infusions. Arm C patients received a median of 2. 5 infusions (range, 1–6; Extended Data Tables 1 and 2).

In arm A, 54. 5% (_n_ = 6) of patients discontinued treatment due to clinical and/or radiographic progression, 9% (_n_ = 1) due to performance status beneath the inclusion criterion threshold, and 36. 4% (_n_ = 4) due to depletion of all TAA-T products.

In arms B and C, 50% (_n_ = 11) of patients first discontinued treatment due to depletion of TAA-T product, 45. 5% (_n_ = 10) due to clinical and/or radiographic progression and 4. 5% (_n_ = 1) due to withdrawal of consent (Supplementary Table 2).

Safety was assessed according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), v. 5. 0.

Treatment was generally well tolerated in all arms, with 88% (293 of 332) of all adverse events (AEs) being low grade. Arms A and B independently dose-escalated to the same modified continual reassessment (mCRM)-estimated MTD and recommended phase 2 dose (RP2D): DL3. The most common treatment-emergent AEs (TEAEs), irrespective of attribution, included fatigue, headache and vomiting, with some differences in frequency between arms.

In arm A, 72. 7% (8 of 11) of participants experienced headache (grade 1, _n_ = 7; grade 2, _n_ = 1) and 45. 5% (5 of 11) experienced nausea/vomiting or fatigue/lethargy (for both, grade 1, _n_ = 3; grade 2, _n_ = 1; grade 3, _n_ = 1).

In arms B and C, fatigue/lethargy (50. 0%, 11 of 22 participants; grade 1, _n_ = 10; grade 2, _n_ = 1) and headache (31. 8%, 7 of 22 participants; grade 1, _n_ = 6; grade 3, _n_ = 1) were most frequent (Fig.

2a,b). Arms B and C had similar AE profiles, except for transient, grade 3 cytopenias, which were seen in 100% (four of four) of patients in arm C. These were expected, owing to lymphodepleting conditioning and were thus excluded from combined safety analyses.

**Fig. 2: Adverse event profile of TAA-T infusions. ** **a**,**b**, TEAEs, defined as new or worsening events following TAA-T infusion, irrespective of investigator attribution to TAA-T therapy, that occurred in ≥10% of patients for a given stratum are shown for arm A (**a**) and arms B/C (**b**).

Each bar represents the number of patients who experienced the given AE shown as CTCAE preferred term; however, for nausea/vomiting, rhinorrhea/nasal congestion and fatigue/lethargy, terms were grouped on the _y_ axis for clarity. Bars are stacked by maximum CTCAE grade experienced per patient for the given term.

Percentages refer to the proportion of treated patients within each arm who experienced the respective AE and the _x_ axis is also scaled to the total number of patients treated in each group (_n_ = 11 for arm A; _n_ = 22 for arms B/C). Across the study, grade ≥3 TEAEs at least possibly related to TAA-T therapy occurred in 9% of patients (_n_ = 3).

In arm A, these included brainstem edema, hydrocephalus and respiratory failure (all associated with patient P27). In arms B and C, grade ≥3 ataxia, muscle weakness lower limb, memory impairment, headache and cerebral edema were observed (associated with patients P02 and P42) (Extended Data Table 3). Two of these events were classified as serious AEs (SAEs).

One SAE, a grade 5 event in arm A at DL2, was considered a dose-limiting toxicity (DLT). No additional DLTs occurred in any arm. The DLT was observed in a patient with DIPG (P27), who had a pre-infusion tumor area of 2,585 mm 2 and was infused 5.

7 months from diagnosis. This patient developed hydrocephalus, tumor edema and respiratory distress 10 days after TAA-T infusion and did not improve despite placement of a ventriculoperitoneal shunt and increased steroid dose (4 mg dexamethasone bolus on days 1 and 3 of admission, in addition to pre-existing maintenance dose of 1. 5 mg day−1).

Imaging was consistent with progressive disease (PD). Nonetheless, this patient’s death prompted a pause in accrual, and treatment eligibility was amended to require neurological stability for at least 2 weeks (previously 1 week) with a shorter timeline between radiation and first infusion to ensure TAA-T administration preceded the typical window for disease progression.

Following P27’s DLT, two additional patients were treated on arm A at DL2, and three additional patients at DL3, all without DLT. One patient with progressive thalamic diffuse midline glioma (DMG), who had a pre-infusion tumor area of 3,496 mm 2 was treated in arm B at DL1 and experienced a grade 3 SAE 16 days post-infusion (P42).

This SAE consisted of MRI-documented cerebral edema accompanied by grade 3 headache, grade 1 vomiting, new-onset photophobia and tremor. Subsequent clinical worsening prompted treatment with bevacizumab, upon which, these events returned to baseline. Retrospectively, all infused participants were evaluated for cytokine release syndrome (CRS) using the American Society for Transplantation and Cellular Therapy (ASTCT) consensus criteria.

One participant had a temperature of 38. 1 °C on day 6 post-infusion, with normal blood pressure and oxygen saturation, and no other AEs; this was coded as grade 1 CRS. Two other participants were febrile (≥38 °C), but were not classified as experiencing CRS, as their fevers had infectious or post-surgical etiologies.

In arm A (DIPG), median PFS from time of diagnosis was 10. 5 months (range, 6. 2–19.

5), and median OS from time of diagnosis was 13. 7 months (range, 6. 2–32.

0), with no significant differences between DLs (Fig. 3a–c). Among the ten patients evaluable for objective radiographic response, best response post-infusion was stable disease (SD) in 60% (_n_ = 6; two of whom had radiographic features consistent with pseudoprogression on the date of best response) and PD in 40% (_n_ = 4; Extended Data Table 1).

**Fig. 3: Clinical responses in patients with diffuse intrinsic pontine glioma. ** **a**, Swimmer plot for arm A (DIPG) showing clinical responses post diagnosis and TAA-T infusion.

**b**, Kaplan–Meier curve showing PFS for arm A. **c**, Kaplan–Meier curve showing OS for arm A. Black dashed line indicates median historical survival OS.

In arms B/C, given the heterogeneity within the relapsed–refractory patient population, time zero for survival calculations was defined from the first TAA-T infusion, rather than diagnosis. Given these differing time origins, OS estimates are not directly comparable with arm A. Arm B/C median PFS was 5.

0 months (range, 0. 5–51. 6), and median OS post-TAA-T infusion was 12.

7 months (range, 6. 3–61. 1) (Fig.

4a–c). Ten patients were evaluable for objective radiographic response, including patients with HGG (_n_ = 4; pediatric glioblastoma _n_ = 1, DMG _n_ = 3), EPN (_n_ = 4) and MB (_n_ = 2). Best response was SD in 50% (_n_ = 5; two of whom had radiographic features consistent with pseudoprogression) and PD in 50% (_n_ = 5).

The median percent change in tumor area from baseline to best response was 10% (range, –93% to +91%; Fig. 4d and Extended Data Table 2). Among those with a best response of SD, four (80%) had a PFS of 5 months or longer (EPN _n_ = 1, MB _n_ = 2, HGG _n_ = 1).

One participant categorized as SD (P37) achieved a >90% lesion reduction; however, clinical deterioration occurred before confirmatory imaging could be obtained. Among patients with measurable disease, the patient with the smallest baseline tumor area (P25; 100 mm 2), diagnosed with subgroup 4 MYC-amplified MB, had an OS of 3. 5 years post-infusion.

**Fig. 4: Clinical responses for patients with relapsed/refractory or otherwise high-risk, nonbrainstem CNS tumors without or with lymphodepletive conditioning. ** **a**, Swimmer plot for arms B/C showing previous therapies and clinical responses post-TAA-T infusion.

**b**, Kaplan–Meier curve showing PFS for arms B/C. **c**, Kaplan–Meier curve showing OS for arms B/C. **d**, Waterfall plot showing percent change in target lesion area from the most recent MRI pre-TAA-T infusion to the time of best radiographic response post-infusion, for all arm B (blue) and arm C (yellow) patients with measurable disease evaluable for objective response.

Dashed horizontal lines indicate the thresholds for PD (+25%) and partial response (PR; −50%). The asterisk indicates a patient with a best response of SD with pseudoprogression. The dagger indicates a patient with >50% reduction in target lesion area, who was categorized as SD rather than PR due to clinical deterioration before confirmatory MRI.

**e**, P41 (arm C) pre-infusion and year 1 post-final-infusion MRI images, shown on post-contrast T1-weighted sequences, demonstrating a CR. Orange open squares have been overlaid on these images to denote the neuroanatomical region in which the participant’s enhancing lesion was present pre-infusion and absent at year 1 post-therapy.

Five patients with evidence of nonmeasurable disease at baseline were evaluable for nontarget response, including patients with HGG (_n_ = 2; pediatric glioblastoma _n_ = 1, DMG _n_ = 1), MB (_n_ = 2) and AB (_n_ = 1), with best responses of complete response (CR; _n_ = 1), SD (_n_ = 3) and PD (_n_ = 1).

Among patients with nonmeasurable and evaluable disease, one (P31) remains alive more than 4 years post-infusion despite PD, and another (P41) remains alive more than 3 years post-infusion with continued radiographic CR (Fig. 4a,d,e, Extended Data Table 2 and Supplementary Fig. 1).

Similarly, three of seven patients who had nonmeasurable nonevaluable disease pre-infusion survived 5 years post-infusion (P09) or remain alive without evidence of disease recurrence at over 2 and 4 years post-infusion (P35 and P36, respectively).

Brief clinical vignettes are provided for P41 to contextualize their radiographic CR, as well as for P35 and P36 to contextualize their prolonged, continued disease stability post-TAA-T therapy. Participant P41 enrolled on study as an 8-year-old child with an EWSR1-BEND2 rearranged AB involving the third ventricle. Upfront therapy consisted of resection and focal radiation.

At recurrence, the patient completed re-irradiation approximately 7 months before the first TAA-T infusion, followed by several cycles of low-dose chemotherapy completed 2 months before infusion. On baseline pre-infusion MRI, third-ventricular disease was present (T1-weighted post-contrast and T2-FLAIR images; Fig. 4e and Supplementary Fig.

1a), although the proportion of viable tumor was difficult to definitively confirm (Supplementary Fig. 1b). Participant P35 enrolled on study as a 14 year-old child with multiply relapsed World Health Organization (WHO) grade IV MB with anaplastic features, diagnosed at age 7 years and having experienced three previous recurrences.

Before enrollment, the patient had received 17 distinct disease-directed therapies, including two courses of radiotherapy, three surgical resections, six chemotherapy regimens, multiple courses of bevacizumab and previous immunotherapy. Following completion of TAA-T therapy, the patient entered a prolonged period of radiographic stability while off all additional antitumor treatments.

Participant P36 enrolled on study as a 14 year-old child with recurrent pediatric glioblastoma (WHO grade IV; H3-wild-type, IDH-mutant, CDK4-amplified, low tumor mutational burden) that progressed following standard chemoradiation and investigational PARP inhibitor-based therapy. The patient experienced radiographic progression approximately 3 months before TAA-T infusion and underwent repeat surgical resection followed by temozolomide.

During active TAA-T therapy, the patient did not receive any disease-directed concomitant therapies but began a few months of CDK4/6 inhibitor therapy following the final TAA-T cell infusion.

### Exploratory immunobiological correlates We performed T cell receptor (TCR) profiling to assess TAA-T cell persistence by longitudinally tracking product-derived clones in 3 of 11 infused patients in arm A, 12 of 18 patients in arm B, and 3 of 4 patients in arm C.

Product-derived clonotypes were detectable post-infusion, and in several patients, dominant clones from the TAA-T cell infusion product persisted in blood up until the collection time point most proximate to confirmed disease progression.

Evidence of clonal dynamics was apparent in some patients; the most common pattern detected was clonal expansion in week 2 post-infusion, followed by contraction at week 4 post-infusion (Extended Data Fig. 2 and Supplementary Figs. 2–4).

Global TCR repertoire in peripheral blood was evaluated using Simpson clonality in 13 of 18 patients in arm B, and 4 of 4 patients in arm C. This metric also demonstrated transient clonal expansion in week 2 post-infusion (from baseline/pre-infusion for arm B and from week 1 post-infusion for arm C), followed by clonal contraction at week 4 post-infusion in a subset of patients (Extended Data Fig.

2g), though this metric is not specific to product-derived clonotypes. Together, these data may reflect dynamic biological activity of T cells post-infusion.

We next evaluated systemic immune activation by measuring IL-6, IL-8 (also known as CXCL8), CCL2 (also known as MCP1) and CXCL10 (also known as IP-10) plasma concentrations in blood samples collected at baseline (pre-infusion), weeks 1, 2 and 4 post-infusion and also from a sample proximate to confirmed disease progression (Extended Data Fig. 3a–h).

We first examined these cytokines/chemokines across all treated patients by arm, accounting for inter-participant variability by analyzing longitudinal concentrations as log 2-transformed fold change relative to each participant’s own pre-infusion baseline. Across patients, plasma IL-6 and IL-8 log 2 fold changes were significantly increased at weeks 1 through 4 post-infusion (_P_< 0. 05; Extended Data Fig.

4a–d). Although individual kinetics varied, in arm A, among patients demonstrating increases relative to baseline (_n_ = 8), peak IL-6 and IL-8 log 2 fold changes occurred at a median of 2 and 3 weeks post-infusion, respectively. Similarly, among arm B/C patients with such increases (_n_ = 21 for IL-6 and _n_ = 19 for IL-8), peak fold changes occurred at a median of 2 weeks post-infusion.

A transient, significant increase in CCL2 at week 2 was demonstrated in arms B/C (day 14, _P_< 0. 05; Extended Data Fig. 4e; though not arm A; Supplementary Fig.

5a), and there were no changes in CXCL10 in any arm (Supplementary Fig. 5b). Taken as a whole, these data indicate a systemic inflammatory response without evidence of IFN-associated chemokine induction.

No significant differences were observed between arm A and arms B/C when comparing IL-6, IL-8, CCL2 and CXCL10 log 2 FCs (from baseline; Supplementary Fig. 5c); therefore, all arms were pooled to investigate cytokines/chemokines in the setting of AEs. We first examined these markers in the context of the possibly related SAEs described above.

For both SAEs, absolute maximum IL-6 concentrations remained <30 pg ml−1 at time points sampled before and after hospitalization (P42, 28. 9 pg ml−1; P27, 17 pg ml−1; Extended Data Figs. 3a,c and 4f); these values are below thresholds associated with severe inflammatory toxicity as reported previously in literature18.")

,19.") Because associations between inflammatory toxicities and plasma concentration thresholds are less established for IL-8, CCL2 and CXCL10, we evaluated these levels relative to the entire cohort of participants sampled at the corresponding time points (_n_ = 33; Extended Data Fig. 4f).

For P42, maximal pre-SAE plasma IL-8, CCL2 and CXCL10 concentrations were in the third quartile, top quartile and second quartile, respectively, with post-admission CXCL10 increasing to the 75th percentile. In contrast, for P27, the IL-8, CCL2 and CXCL10 concentrations in the pre-SAE sample were in the first, second and second quartiles, respectively, with post-admission IL-8 increasing to the second quartile.

Together, these data suggest that neither participant’s SAE was consistent with an IL-6-related mechanism, and that peri-SAE IL-8, CCL2 and CXCL10 levels were heterogeneous, limiting our interpretation of these as predictive biomarkers. Continuing these analyses across all participants, maximum TEAE CTCAE grade was associated with differences in baseline-normalized maximum IL-6 and IL-8 concentrations post-infusion (_P_< 0.

05), with IL-6 levels significantly higher in participants experiencing grade 2 versus grade 1 TEAEs (4,279 versus 197 pg ml−1; adjusted rank-based _P_< 0. 05) (Extended Data Fig. 5a,b).

Sensitivity analyses assessing outlier exclusion demonstrated that IL-8 findings, but not IL-6 findings, retained statistical significance (Supplementary Fig. 6a,b). In both primary and sensitivity analyses, no differences in maximum IL-6 or IL-8 concentration were observed between grade ≥3 versus lower-grade AEs.

Beyond AE severity, higher cytokine concentrations were associated with a greater number of grade ≥2 TEAEs, whereby patients with maximum IL-6 or IL-8 concentrations ≥500 pg ml−1 experienced more TEAEs than those with <500 pg ml−1 elevations of these markers (_P_< 0. 05; Extended Data Fig. 5c,d; robust to outlier exclusion, Supplementary Fig.

6c,d). Collectively, these findings indicate that elevated IL-6 and IL-8 levels are associated with moderate, but not severe, life-threatening or fatal TEAE burden. Among all TEAEs, maculopapular (MP) rash was associated with these inflammatory markers.

Maximum post-infusion IL-6 and IL-8 concentrations in patients with MP rash (_n_ = 5) versus without (_n_ = 26) were a median of 589 pg ml−1 (versus 9. 6) and 8,255 pg ml−1 (versus 258), respectively (_P_< 0. 01; Extended Data Fig.

5e,f; robust to outlier exclusion, Supplementary Fig. 6e,f). The median onset of MP rash was 21 days (range, 11–28) post-infusion.

The ReMIND phase 1 trial reports results from a systemic multi-antigen T cell therapy for the treatment of pediatric brain tumors.

While this study evaluates TAA-T therapy in CNS tumors, this multi-antigen TAA-T platform has been previously studied by our group in pediatric and adult nonCNS malignancies, including phase 1 trials using the same WT1/PRAME/survivin-targeting product in high-risk solid and hematologic tumors (NCT02789228, NCT02203903 and NCT03843294) with favorable preliminary safety profiles10.") ,20.") ,21.")

The present study also increased to a higher dose level, 8. 0 × 10 7 cells per m 2, given anticipated differences in T cell trafficking in the CNS. In this trial, we demonstrated that an autologous multi-antigen TAA-T therapy can generally be manufactured using peripheral blood from pediatric patients with high-risk CNS malignancies.

We also showed that TAA-T cell infusions are well tolerated when administered intravenously with and without prescribed lymphodepletion. Additionally, we established a model-estimated MTD and RP2D of 8 × 10 7 cells per m 2 per dose for future studies. Of note, the empiric MTD was not reached, as no dose level reached the threshold of unacceptable toxicity.

The majority of patients experienced only mild to moderate AEs. Only two patients experienced potentially related SAEs; both had the largest tumor areas represented in arms A and B (nonresectable brainstem and thalamic DMG, respectively).

This trial was not powered to evaluate efficacy, and although TAA-T cell therapy was associated with durable disease stabilization in some patients who would otherwise be expected to experience rapid progression, conclusions about clinical efficacy must await a more definitive trial.

However, those patients who do continue to remain without progression also harbored minimal residual tumor at baseline, suggesting that tumor burden may influence response. Theoretically, a higher effector cell-to-tumor ratio could favor immune-mediated control, although this could also be due to other variables. Three patients showed objective responses.

Patient P41 (AB with an EWSR1-BEND2 rearrangement) achieved a CR 1 year after completion of three TAA-T cell infusions and remains disease-free 3 years later. While this is a rare tumor subtype with an incompletely defined natural history, and OS may be somewhat better than other recurrent HGGs22.") , durable CRs to available therapies remain uncommon.

The delayed timing of best response relative to the final infusion raises the possibility that intravenously administered TAA-T cells may have persisted and/or initiated supportive immune processes that evolved over time. Patients P35 and P36 (recurrent MB and HGG, respectively) remain disease-free more than 2. 6 and 4.

3 years after TAA-T cell infusion. Overall, across arms B and C, most participants were heavily pretreated before enrollment, and some pursued additional therapies after study treatment, limiting the extent to which survival can be solely attributed to TAA-T cell therapy23: results of the three-arm biomarker-driven randomized trial in the first 230 patients from Europe and Australia. Neuro.

Oncol. 21, vi183 (2019).") Interpretation of pooled outcomes of arms B and C is further limited by the use of lymphodepletion in the latter arm, and the associated clinical and biological heterogeneity that may ensue.

Although our study presents an interesting signal of potential response, the exploratory nature of efficacy-related observations in this phase 1 study must not be overlooked. Arm A of the ReMIND trial permits cautious contextualization relative to recent phase 1 CAR-T cell trials for DIPG7.") ,8.")

,24.") ,25.") ,26.")

In contrast to the wholly systemic administration of T cells in ReMIND, the B7-H3 CAR-T cell study exclusively utilized the intracerebroventricular (ICV) route, and the GD2 CAR-T cell study primarily employed the same (although each patient’s first infusion was intravenous). Reported median OSs were 19. 8 and 17.

6 months,