Long-term Remissions Following CD20-Directed Chimeric Antigen Receptor–Adoptive T-cell Therapy

CD20-targeted CAR T-cell therapy in two lymphoma patients resulted in over 7 year-long remissions, even though CAR T cell activity was insufficient to induce B-cell aplasia, and residual disease remained. Anti-tumor antibody and T-cell responses as well as TCR clonal dynamics support the possibility of CAR T cell-initiated endogenous anti-lymphoma immune response.

Abstract

Chimeric antigen receptor (CAR) T-cell therapy produces high response rates in refractory B-cell non–Hodgkin lymphoma, but long-term data are minimal to date. In this study, we present long-term follow-up of a pilot trial testing a CD20-targeting third-generation CAR in patients with relapsed B-cell lymphomas following cyclophosphamide-only lymphodepletion. Two of the three patients in the trial, with mantle cell lymphoma and follicular lymphoma, had remissions lasting more than 7 years, though they ultimately relapsed. The absence of B-cell aplasia in both patients suggested a lack of functional CAR T-cell persistence, leading to the hypothesis that endogenous immune responses were responsible for these long-term remissions. Correlative immunologic analyses supported this hypothesis, with evidence of new humoral and cellular antitumor immune responses proximal to clinical response time points. Collectively, our results suggest that CAR T-cell therapy may facilitate epitope spreading and endogenous immune response formation in lymphomas.

Significance: Two of three patients treated with CD20-targeted CAR T-cell therapy had long-term remissions, with evidence of endogenous antitumor immune response formation. Further investigation is warranted to develop conditions that promote epitope spreading in lymphomas.

Introduction

Chimeric antigen receptor (CAR) T-cell therapy, in which autologous T cells are genetically modified ex vivo to redirect T-cell specificity toward a tumor-associated target antigen, has changed the landscape of management of lymphoid malignancies. Although initially used only in heavily pretreated patients with few other options, CAR T-cell therapy is now being used in earlier lines of therapy (1).

In patients with large B-cell lymphomas treated with CD19-targeted CAR T-cell therapy, about 40% of patients achieve sustained remissions that may represent cures. However, for patients with follicular lymphoma (FL) and mantle cell lymphoma (MCL), the follow-up is shorter and the durability of responses less clear (2–5). Without a convincing plateau on the progression-free survival curves, it is still unclear whether CAR T-cell therapy represents a curative option for these diseases, which are considered incurable with standard therapies. The reasons for treatment failure are under intense investigation, and potential causes include loss of target antigen on the tumor cells, intrinsic T-cell dysfunction, lack of functional CAR T-cell persistence (due to exhaustion, anergy, or immune rejection of CAR T cells), and potentially inhibitory factors within the tumor microenvironment (6).

The expansion of infused CAR T cells triggers activation of myeloid cells, resulting in the secretion of proinflammatory cytokines and creating a systemic inflammatory response, as well as activation of non–CAR T cells in the tumor microenvironment (7). This raises the question of whether antigen-presenting cells are able to present tumor neoantigens released by dying tumor cells, leading to priming of naïve tumor antigen–specific T cells and formation of an endogenous antitumor immune response. Indeed, instances of such epitope spreading have been documented in the setting of CAR T-cell therapy for solid tumors in both preclinical (8) and clinical (9–11) settings. Although such epitope spreading has been described to occur in preclinical mouse models of lymphoma (12), it has not yet been documented to occur in patients with B-cell malignancies treated with CAR T cells. Such a broadening of the immune response may contribute to the durability of remission, even after CAR T-cell persistence has waned.

We previously reported the results of a pilot trial testing a third-generation CAR targeting the CD20 antigen in patients with relapsed B-cell lymphomas (13). Here, we report long-term follow-up of three patients enrolled in the trial, two of whom had remissions lasting more than 7 years, despite an apparent lack of robust direct CAR T-cell activity based on poor in vivo expansion and lack of B-cell aplasia in all patients and stable disease at 1 month in the patient with measurable disease, suggesting endogenous antitumor immune responses.

Results

Clinical Follow-up of Each Study Patient

UPN-03 was diagnosed with stage IVA MCL after presenting with enlarging lymph nodes in his right neck and right groin. He was treated with four cycles of cyclophosphamide–doxorubicin–vincristine–prednisone (CHOP) chemotherapy followed by autologous stem cell transplant and achieved a molecular complete remission. He remained in remission for more than 12 years but relapsed with an isolated left flank soft tissue mass, confirmed by biopsy to be CD20⁺ MCL. He was 62 years old at the time of study enrollment, and his pretreatment PET-CT scan showed low-level 2-[18F]fluoro-2-deoxy-D-glucose (FDG) uptake in the tonsils, a mildly hypermetabolic small cervical lymph node, and an enlarging 2.2-cm mass in the left flank. Approximately 8 months from the time of his relapse, he was treated with a course of three CD20 CAR T-cell infusions after lymphodepletion with cyclophosphamide 1 g/m², followed by 14 days of twice daily low-dose s.c. IL2 injections, as previously described (Fig. 1A; ref. 13). He had a normal lactate dehydrogenase (LDH) and Eastern Cooperative Oncology Group (ECOG) performance status of 0, and serum cytokine levels are shown in Supplementary Table S1. Following CAR T-cell infusion, he did not develop any cytokine release syndrome (CRS) or immune effector cell–associated neurotoxicity syndrome. He underwent a study-related excisional biopsy 48 hours after his third CAR T-cell infusion, and a restaging PET-CT scan at 3 months postinfusion showed a complete response. He remained in complete remission for 2 years but relapsed with a 4-cm subcutaneous left flank mass, confirmed by biopsy to be recurrent CD20⁺ MCL. He was treated with 40 Gy radiotherapy to the left flank, and a restaging CT scan showed a residual 3.2 cm thickening of the left latissimus dorsi muscle with a new adjacent 2.9-cm soft tissue focus (Fig. 1B). The patient requested infusion of his remaining cryopreserved CAR T cells, and the protocol was amended and approved by the FDA and the Institutional Review Board to allow a fourth infusion.

Figure 1.

Clinical response summary. A, Timeline of UPN-03 (MCL) and UPN-04 (FL) disease course relative to CAR-T infusions. UPN-03 received a fourth infusion 2+ years after his first series of infusions. B, Products of the largest diameters of the indicated tumor sites at various time points after the last CAR T-cell infusion. C, MRD testing was performed on peripheral blood samples at the indicated time points. Deep sequencing of the BCR locus (IGH and IGL for UPN-03 and IGH for UPN-04) was performed, and the frequency of the dominant clones present in the tumor biopsy at each time point is represented as a percentage of nucleated cells. A value of “0.0001” indicates an undetectable sample, as 0 cannot be plotted on a log scale. XRT, radiotherapy.

Approximately 29 months after his initial CAR T-cell infusions, he had a normal LDH and ECOG performance score of 0 and again underwent lymphodepletion with cyclophosphamide 1 g/m² followed 2 days later by a single infusion of 1.8 × 10⁹ CAR T cells/m² (total dose 3.79 × 10⁹ cells), followed by 14 days of twice daily low-dose s.c. IL2 injections. Six weeks postinfusion, he developed a suspected local recurrence based on the emergence of a palpable nodule at the margin of the radiation field in the left flank. The lesion was not biopsied, but there was an increase in one of the minimal residual disease (MRD) subclones at that time (Fig. 1C). The nodule subsequently resolved approximately 3 weeks later. He remained in clinical remission for approximately 8 years (Fig. 1A), albeit with detectable MRD by high-throughput next-generation sequencing (NGS) of the immunoglobulin heavy chain (IGH) and immunoglobulin κ loci (Fig. 1C), until he developed a palpable mass in his chest, with MRI and PET-CT showing an FDG-avid subcutaneous chest mass and axillary adenopathy. Surgical biopsy of an axillary node showed CD20⁺ MCL.

UPN-04 was diagnosed with grade 2 FL after presenting with left neck pain and constitutional symptoms. Staging revealed extensive lymphadenopathy including bulky abdominal and retroperitoneal adenopathy along with marrow and spleen involvement. He was treated with six cycles of R-CHOP and achieved a complete remission. This was followed by six doses of rituximab maintenance given every 3 months, but approximately 18 months after completion of R-CHOP, during maintenance therapy, he developed recurrent adenopathy that was confirmed by biopsy to be CD20⁺ FL.

After study enrollment and CAR T-cell manufacturing, he had a normal LDH and an ECOG performance score of 1, and baseline cytokine levels are shown in Supplementary Table S1. He received treatment with cyclophosphamide lymphodepletion, three infusions of CD20 CAR T cells, and low-dose s.c. IL2 injections with no development of CRS or immune effector cell–associated neurotoxicity syndrome (Fig. 1A). He had no response at 1 month after his last CAR T-cell infusion but achieved a partial metabolic response on PET-CT scan at 3 months after the infusion, with decreased metabolic activity in multiple left cervical, posterior auricular, right axillary, right epitrochlear, and right inguinal lymph nodes. About 2 weeks after the PET-CT scan, the patient noted a significant decrease in his cervical adenopathy, and physical examination confirmed a decrease in size of the most prominent cervical node from 2.5 cm at the time of the PET-CT to 1.5 cm a month later. However, by 12 months after treatment, there was evidence of progressive disease, with modest enlargement of cervical and inguinal lymph nodes (Fig. 1B). One year later, a scan demonstrated enlargement of chest and pelvic lymph nodes with resolution of the cervical lymph nodes. Approximately 2.5 years postinfusion, in the absence of any additional therapy after the CAR T-cell infusions and IL2 injections, he noted complete spontaneous resolution of all palpable lymph nodes, and a CT scan at 35 months posttreatment confirmed a near-complete remission, though he continued to have detectable MRD. NGS testing showed that although two of the top three subclones present in the lymph node biopsy became undetectable, one remained detectable (Fig. 1C). He remained in clinical remission until approximately 7 years postinfusion, when he was found to have a left supraclavicular lymph node on CT scan. After several months of observation, he developed rapidly progressive disease, with extensive diffuse adenopathy, splenomegaly, and leukemic involvement. He had a normal LDH and no areas of very high FDG uptake on PET scan that were suspicious for histologic transformation, and a peripheral blood flow cytometry was consistent with CD20⁺ FL.

UPN-02, the third patient treated in the trial, died of progressive MCL and myelodysplastic syndrome approximately 26 months post–CAR T-cell treatment.

CAR T-cell Persistence and B-cell Aplasia

To determine whether CAR T cells were responsible for the late clinical responses, we evaluated CAR T-cell persistence. We attempted to evaluate CAR T-cell persistence in UPN-03 after the fourth infusion using qPCR, but the results were not considered reliable given the nonspecific signal seen in the negative control sample, and lack of remaining material precluded additional testing. Patient UPN-04 had previously been shown to have low (<1%) levels of CAR T cells by qPCR after the infusions, which were detectable up to 9 months later, but became undetectable by 1 year after treatment (13). Both patients also had evidence of CAR T-cell infiltration in the tumor by qPCR, albeit at low levels (≤1%; ref. 13). By NGS testing of T-cell receptor β (TCRβ) sequences within the infused CAR T-cell product for UPN-04, most CAR T-cell clones were undetectable by 3 months postinfusion (Fig. 2A). Both patients had previously been shown not to have achieved B-cell aplasia following CAR T-cell therapy (13) and demonstrated ongoing recovery of the B-cell repertoire over time (Fig. 2B), indicating a lack of functional CAR T-cell persistence. We also evaluated overlap of infused TCR sequences between the infused CAR T-cell product and the posttreatment tumor biopsy in UPN-04, which showed that TCRβ sequences from the CAR-T product were present but comprised a tiny minority of all TCRβ sequences in the tumor (Fig. 2C), consistent with the previous qPCR data (13).

Figure 2.

Lack of CAR-T persistence with B-cell recovery over time and minimal CAR T-cell presence in tumor. A, The top 30 most abundant TCR sequences within the CAR T-cell infusion product for patient UPN-04 (representing 94.3% of all the TCR clonotypes within the CAR product), as determined by NGS TCRβ analysis. Clonotype frequency of these sequences over time is shown. The value of “0.00001” represents an undetectable sample. B, B-cell recovery over time after the most recent CAR T-cell infusion for UPN-03 and UPN-04. Patient PBMCs at the time points shown were analyzed for clonal IGH or IGK/IGL analysis using the immunoSEQ NGS assay (Adaptive Biotechnologies). The numbers of total productive sequences identified, representing the BCR repertoire, are shown. C, Overlapping TCRβ sequences between the CAR T-cell infusion product and post–CAR T-cell infusion lymph node biopsy in patient UPN-04 were determined by immunoSEQ. The 14 common sequences shown in the Venn diagram represent 53.8% of the productive TCRβ sequence reads in the CAR-T infusion product and 0.086% of productive TCRβ sequence reads in the tumor biopsy.

Endogenous Antitumor Immune Responses

In the absence of robust CAR T-cell expansion and persistence, we hypothesized that the late clinical responses were caused by endogenous antitumor T-cell responses, potentially triggered by the CAR T-cell infusions. To evaluate antitumor T-cell activity, we conducted cellular enzyme-linked immunospot (ELISpot) assays in which peripheral blood mononuclear cells (PBMC) collected from both UPN-03 and UPN-04 were coincubated with autologous tumor cells. Both patients exhibited spikes in IFNγ-secreting cells at time points proximal to their clinical responses (Fig. 3A), suggesting an increased frequency of tumor-reactive T cells.

Figure 3.

Development of antitumor immune responses. A, IFNγ ELISpot assays using patient PBMCs from serial time points, incubated with either autologous tumor cells or media, were performed to assess antitumor T-cell responses. Red arrows denote timing of clinical responses. Error bars represent the SEM of triplicate wells. Comparisons were made using an unpaired two-tailed t test. B, Immunoblotting analysis to detect humoral antitumor immune responses was performed using lysates from patient tumor cells, incubated with 1:1,000 diluted patient serum obtained at the indicated time points relative to the last infusion of CD20 CAR T cells, and probed with a secondary antihuman IgG antibody. C, NGS TCRβ analysis to identify differentially expressed TCR sequences compared with the apheresis sample (FDR <0.05) for patient UPN-04. Clonotype frequency of these sequences is represented as log₂FC from the apheresis sample. D, Multiparameter flow cytometry was performed on a single-cell suspension of a tumor biopsy obtained 24 hours after the final CAR T-cell infusion for UPN-04, with the identification of intratumoral immune cell subsets. A total of 499,885 viable singlet events were collected. The gating strategy is shown in Supplementary Fig. S3. E, mIHC of UPN-04 posttreatment tumor biopsy using a 6-plex panel containing the antibodies shown at right plus DAPI nuclear stain. Panel 1: ×5 magnification demonstrating FL with the pink/purple nodules surrounded by a mixed population of CD4⁺ and CD8⁺ T cells. Panel 2: ×20 magnification of the interfollicular region adjacent to a neoplastic follicle from the yellow box gate in the first panel, showing the complex interactions between T cells, monocytes, and DCs. Panel 3: ×40 magnification of the interfollicular region highlighted in the yellow box in the second panel but restricted to CD8 and CLEC9A markers, showing the interactions between CD8⁺ T cells and CLEC9A⁺ DCs. Panel 4: ×40 magnification of the region highlighted in the second panel, restricted to CD4 and CLEC9A markers. Note the relatively increased percentages of CD4⁺ cells and that both CD4⁺ and CD8⁺ T cells are seen in close contact with CLEC9A⁺ DCs. The figure has been enhanced (brightness/contrast) for easier viewing. log₂FC, log₂-transformed fold change; Treg, regulatory T cells. MDSC, myeloid-derived suppressor cells; mDC, myeloid dendritic cells; T_FH, T-follicular helper cells; T_FR, T-follicular regulatory cells.

Additionally, we hypothesized that the endogenous immune responses might include T cell–dependent antibody responses. We performed Western blot assays incubating postinfusion serum with autologous tumor cell lysates and found evidence of new bands at postclinical response time points in both patients, suggesting the formation of new antitumor antibodies (Fig. 3B).

The absence of persistent functional CAR T cells suggested that endogenous antitumor immune responses, perhaps as a result of epitope spreading after CAR-T infusions, were responsible for the prolonged clinical remissions. We hypothesized that the frequencies of endogenous T cells specific for tumor-associated antigens would be increased around the time of these late clinical remissions.

We tested this in UPN-04 by performing high-throughput sequencing of the rearranged TCRβ locus to characterize and quantify the T-cell repertoire in the peripheral blood at various time points, as well as in the tumor. We identified 16 clonal TCR sequences that were statistically significantly increased in abundance at 3 and/or 35 months (the nearest time points following his clinical remissions), with 9 of these sequences also being present in the tumor biopsy and 2 significantly enriched in the tumor (Fisher exact FDR < 0.05; Fig. 3C; Supplementary Table S2), suggesting tumor specificity. These sequences were unique and not present in the CAR T-cell infusion product. We searched published datasets of TCRβ complementarity-determining region 3 (CDR3) sequences of known specificity (14–16) and discovered that one was described as recognizing a Cytomegalovirus epitope, another was confirmed to be reactive against an unknown tumor-associated antigen in non–small cell lung cancer, and the remaining sequences did not match known TCRβ CDR3 sequences in these databases (Supplementary Table S2). Additionally, we performed studies of UPN-04’s immediate posttreatment tumor biopsy to determine whether there was a cellular infiltrate compatible with the formation of antitumor immune responses after the series of CAR T-cell infusions. Multiparameter flow cytometry demonstrated a large number of infiltrating immune cells, which were primarily CD4⁺ T cells, but with subsets of CD8⁺ T cells and a small number of antigen-presenting cells present as well (Fig. 3D). Based on our previously published qPCR data (13) showing <1% CAR T cells in the tumor and the new TCRβ data showing 14 of 18,267 productive TCRβ sequences (comprising a frequency of 0.086% of all sequence reads; Fig. 2C), the vast majority of the T cells in the tumor represent non–CAR T cells. To evaluate the spatial relationship of these immune cells within the tumor microenvironment, we performed multiplex IHC (mIHC) of this tumor biopsy, which revealed close proximity of CD4⁺ and CD8⁺ T cells to CLEC9A-expressing dendritic cells (DC) within interfollicular T-cell zones of the nodal tumor (Fig. 3E), marking a subset of CD141⁺ DCs that are proficient at antigen cross-presentation to CD8⁺ T cells, suggesting that the conditions to support T-cell priming were present (17).

Discussion

Long-term follow-up of patients with large B-cell lymphomas treated with CD19-targeted CAR T cells shows plateaus on progression-free survival curves that suggest a large subset of patients is cured with this treatment. However, the durability of complete remissions after CAR T-cell therapy for MCL and FL is less clear because these diseases, unlike large B-cell lymphoma, are not curable with standard therapies, often exhibit a pattern of delayed relapse, and have shorter follow-up data to date. Here, we presented long-term follow-up data of two patients with MCL and FL treated with CD20-targeted CAR T cells and demonstrated clinical remissions lasting more than 7 years.

In these patients, we observed suboptimal CAR T-cell expansion and persistence, lack of B-cell aplasia, and a delayed response time that further argues against a direct CAR T-cell effect. Our correlative studies suggest that humoral and cellular endogenous immune responses were responsible for the prolonged clinical remissions. These may have been triggered by initial suboptimal CAR T-cell responses, leading to epitope spreading. mIHC in a post-treatment tumor biopsy showed infiltration of CD4⁺ and CD8⁺ cells in close contact with CLEC9A-expressing DCs in interfollicular zones of the node, supporting the hypothesis that endogenous immune responses may contribute to the durability of CAR T-cell responses.

Both patients in our study were MRD⁺ during remissions, indicating tumor control rather than eradication. Indeed, both patients ultimately relapsed, suggesting that their diseases evolved to escape endogenous responses. It is possible that several years of MRD⁺ disease without clinical relapse reflects the presence of a quiescent lymphoma stem cell reservoir population that is inherently resistant to immunosurveillance (or residing in a protected niche) but with proliferative progeny cells that are susceptible to immune destruction and quickly eliminated (18, 19). Both patients relapsed with CD20⁺ disease, ruling out antigen loss as a cause of resistance and further arguing against these remissions being directly mediated by CAR T cells.

It is noteworthy that UPN-04 experienced initial relapse of his FL within 18 months of first-line R-CHOP therapy, during maintenance. Disease progression within 24 months of completing induction chemotherapy has been shown to be associated with poor outcomes in FL (20), and thus, his 7-year remission suggests the possibility that CAR T-cell therapy altered the trajectory of his disease. UPN-03 had a prolonged remission of his MCL after initial therapy and consolidative autologous stem cell transplant but relapsed less than 2 years after his first series of CAR T-cell infusions. The 8+ years of remission he experienced after his fourth infusion of CAR T cells also argues against an effect from chemotherapy only or indolent disease biology.

Our lymphodepletion regimen prior to CAR T-cell infusion did not incorporate fludarabine. The use of fludarabine, which is highly toxic to T cells, may reduce the likelihood of endogenous immune responses, and thus, future investigations to develop lymphodepletion regimens or adjuvant treatments that promote effective CAR T-cell therapy while permitting formation of endogenous immune responses may be warranted.

Although the collective data are highly suggestive of an endogenous antitumor immune response triggered by the CAR T-cell infusions, they do not definitively prove this, and we cannot exclude the possibility that other factors, such as the IL2 injections in combination with cyclophosphamide, may have elicited immune responses against the tumor cells independent of the CAR T cells. We noted that patient UPN-03 had radiotherapy prior to the CAR T-cell infusion, so it is possible that the antitumor immunity arose from an abscopal effect, though given the 5-month interval between the radiation and postinfusion tumor response, with intervening cyclophosphamide, the possibility that the immune response formed after CAR T-cell infusion seems more plausible. We also cannot formally exclude the possibility that the TCR sequences we identified that increased at the time of clinical responses represented low-frequency CAR T-cell clonotypes that were not sampled within the infusion product, though the disappearance by 3 months of the vast majority of other CAR T-cell clones makes this unlikely. We also could not determine the antigen specificity of most of these TCRs, and although they were present in the tumor, it remains a possibility that they could be responding to nontumor antigens. Indeed, a database search revealed that one of the sequences seems to represent a Cytomegalovirus-specific TCR, though the others were not known to be associated with virus-specific TCRs, and one was associated with a tumor-reactive TCR in the context of lung cancer. Finally, we cannot exclude that other immune cells such as NK cells could account for the IFNγ spikes. These caveats notwithstanding, our data, together with previous demonstrations of epitope spreading in CAR T-cell and TCR T-cell trials for solid tumors and preclinical mouse models of lymphomas, suggest that the emergence of endogenous antitumor responses following CAR T-cell therapy may be more common than previously appreciated (9–12, 21, 22). The long-term remissions observed in this study suggest a benefit of developing strategies that enhance the formation of endogenous immune responses following CAR T-cell therapy, which may improve response rates and prevent relapse.

Methods

Patients and Treatment

The trial design, eligibility criteria, and treatment plan were previously reported (13). The study (NCT00621452) was approved by the Fred Hutchinson Cancer Center Institutional Review Board, and all patients provided written informed consent in accordance with the Declaration of Helsinki. The protocol was modified to allow an additional infusion of cryopreserved CAR T cells at the request of patient UPN-03.

ELISpot Assay to Detect Cellular Antitumor Responses

To detect tumor-specific T cells, cellular ELISpot assays were performed according to the manufacturer’s recommendations (eBioscience). Anti-IFNγ antibody–coated plates were washed and blocked with RPMI1640 with 10% FBS for 1 hour at room temperature. PBMCs from each time point to be tested were suspended in RPMI1640 and plated in 96-well plates at 0.4 × 10⁶ cells/well. Autologous tumor cells (cryopreserved as unsorted single-cell suspensions from an excisional biopsy obtained 24–48 hours after the third CAR T-cell infusion) were resuspended in RPMI1640 and added at 0.1 × 10⁶ cells/well (UPN-04) or 0.2 × 10⁶ cells/well (UPN-03). PBMCs were stimulated with phytohemagglutinin (10 µL/mL, 0.05 × 10⁶ cells/well) as a positive control or RPMI1640 (0.1 × 10⁶ cells/well) as a negative control. After 22 to 24 hours of incubation at 37°C, ELISpot assays were developed according to the manufacturer’s recommendations. Cellular composition of the PBMCs (where available) is listed in Supplementary Table S3. Spots were visualized using aminoethyl carbazole substrate solution, and reactions were stopped using multiple water rinses. ELISpot plates were allowed to dry, stored protected from light, counted using a Bioreader 3000 PRO (BIOSYS, Miami, FL), and analyzed using EazyReader software. Data are presented as spot-forming units per 10⁶ cells. Cellular composition of the tumor biopsy for patient UPN-03 is shown in Supplementary Figs. S1 and S2 and for UPN-04 in Fig. 3D and Supplementary Fig. S3.

Immunoblot Assay to Detect Humoral Antitumor Responses

An assay to detect tumor-reactive antibodies was derived from a previously published protocol (9). Autologous patient-derived tumor cells were thawed and washed, and then viable cells were separated by Ficoll-Paque. Viable patient tumor cells were lysed using an NP40 lysis buffer (Boston BioProducts, Ashland, MA) at 4°C. Extracts were collected and purified by centrifugation at 15,000 × g for 10 minutes. Cell lysates from cells were prepared for the immunoblot assay, and lysates from equal numbers of cells were separated by 4% to 12% Tris-glycine SDS-PAGE and transferred to polyvinylidene difluoride transfer membrane. The membranes were blocked with 5% nonfat milk prepared with tris-buffered saline and Tween 20 (TBST) (20 mmol/L Tris, 137 mmol/L NaCl, and 0.1% Tween 20). The membranes were incubated with patient serum (1:1,000 dilution) for 24 hours at 4°C. After overnight incubation, the membranes were washed and incubated with horseradish peroxidase–conjugated anti–human IgG (H + L; Jackson ImmunoResearch Laboratory) for 2 hours at room temperature. Bands were visualized using an enhanced chemiluminescence plus Western blotting system (Pierce).

MRD Testing

MRD testing along with B-cell receptor (BCR) locus sequencing was performed using the clonoSEQ (for UPN-03) or immunoSEQ (for UPN-04) assay (Adaptive Biotechnologies, Seattle, WA; refs. 23, 24). IGH and immunoglobulin light chain (IGL) loci were sequenced for UPN-03, and the IGH locus was sequenced for UPN-04.

Multiparameter Flow Cytometry

Cryopreserved single-cell suspensions from an excisional malignant lymph node biopsy obtained 24 (from patient UPN-04) or 48 (from patient UPN-03) hours after the third CAR T-cell infusion were thawed and washed. The cells were blocked with anti-FcγR (Human TruStain FcX; BioLegend) prior to surface staining. After 10 minutes of incubation at 4°C, the cells were washed and stained with antibodies against the following antigens (clone name in parentheses) for 1 hour at 4°C: CD3 (UCHT1), CD4 (SK3), CD5 (UCHT2), CD8 (RPA-T8), CD10 (HI10a), CD14 (M5E2), CD19 (SJ25C1), CD20 (2H7), CD24 (ML5), CD25 (2A3), CD33 (WM53; all from BioLegend), CD38 (HIT2), CD56 (NCAM16.2), CD127 (HIL-7R-M21), HLA-DR (G46-6), IgG κ (G20-193), and IgG λ (TB28-2; all from eBioscience), CXCR3 (1C6/CXCR3), CXCR5 (RF8B2), CCR6 (11A9), and PD1 (EH12.1), as well as a live/dead stain. All antibodies were obtained from BD Biosciences unless otherwise specified. Cells were analyzed on a FACSymphony analyzer (BD Biosciences), and all data were analyzed using FlowJo software (BD Biosciences).

Analysis of the TCR Repertoire

NGS of the TCRβ locus CDR3 region was performed on genomic DNA harvested from PBMCs in serial postinfusion blood samples, the CAR-T product, and the posttreatment tumor biopsy obtained for patient UPN-04 using the immunoSEQ assay (Adaptive Biotechnologies). Data were analyzed using the R package LymphoSeq (https://bioconductor.org/packages/LymphoSeq; ref. 25). After identifying and quantifying the productive sequences (sequences in frame and without an early stop codon), we ran a differential abundance analysis performing a Fisher exact test to calculate differential abundance of each sequence between two time points and report the log₂-transformed fold change, P value, and adjusted P value, in which the adjustment for multiple comparisons was conducted using the Benjamini and Hochberg method to control the FDR. We also queried several published datasets (14–16) to determine whether any of our identified TCRβ CDR3 sequences of interest matched TCRs of known specificity.

mIHC

Formalin-fixed, paraffin-embedded tissues from the excisional lymph node biopsy 24 hours after the last infusion for patient UPN-04 were used to make 4-µm sections and were stained for CD8, CD11b, CD19, CD4, CLEC9A, and HLA-DR.

The formalin-fixed, paraffin-embedded tissues were sectioned at 4 microns onto positively charged slides and baked for 1 hour at 60°C. The slides were then dewaxed and stained on a Leica BOND-RX autostainer (Leica, Buffalo Grove, IL) using Leica BOND reagents for dewaxing (Dewax Solution), antigen retrieval and antibody stripping (Epitope Retrieval Solution 2), and rinsing after each step (BOND Wash Solution). See Supplementary Table S4 for the staining panel used. A high-stringency wash was performed after the secondary and tertiary applications using high-salt TBST solution (0.05 mol/L Tris, 0.3 mol/L NaCl, and 0.1% Tween-20, pH 7.2–7.6).

Antigen retrieval and antibody stripping steps were performed at 100°C, with all other steps at ambient temperature. Endogenous peroxidase was blocked with 3% H₂O₂ for 8 minutes, followed by protein blocking with tris-buffered saline with Tween 20 (TCT) buffer (0.05 mol/L Tris, 0.15 mol/L NaCl, 0.25% casein, 0.1% Tween 20, pH 7.6 ± 0.1) for 30 minutes. The first primary antibody (position 1) was applied for 60 minutes, followed by the secondary antibody application for 10 minutes and the application of the tertiary tyramide signal amplification reagent (Opal fluor, Akoya Biosciences, Menlo Park, CA) for 10 minutes. The primary and secondary antibodies were stripped with retrieval solution for 20 minutes before repeating the process with the second primary antibody (position 2) starting with a new application of 3% H₂O₂. The process was repeated until all positions were completed; however, there was no stripping step after the last position. Slides were removed from the autostainer and stained with Spectral DAPI (Akoya Biosciences) for 5 minutes, rinsed for 5 minutes, and coverslipped with ProLong Gold antifade reagent (Invitrogen/Life Technologies, Grand Island, NY).

Slides were cured for 24 hours at room temperature in the dark, and representative images from each slide were acquired on the Akoya Vectra 3.0 automated imaging system. Images were spectrally unmixed using Akoya inForm software and exported as multi-image TIFFs for use in the HALO Link image management system (Indica Labs, Corrales, NM).

Cellular analysis of the images was then performed using HALO image analysis software. After the cells were visualized based on nuclear and cytoplasmic stains, the software measured the mean pixel fluorescence intensity in the applicable compartments of each cell (e.g., CD8 in the cytoplasmic compartment). A mean intensity threshold above background was used to determine positivity for each fluorochrome, thereby defining cells as either positive or negative for each marker. The positive cell data were then used to define colocalized populations and to perform spatial analysis.

Data Availability

The data generated in this study are available upon request from the corresponding author. The TCR and BCR sequencing data are available through the Adaptive Biotechnologies immuneACCESS database at https://clients.adaptivebiotech.com/pub/mo-2024-1-bcd; https://clients.adaptivebiotech.com/pub/mo-2024-2-bcd; https://clients.adaptivebiotech.com/pub/mo-2024-3-bcd. The TCR sequence data from reference #14 are available in the R package LymphoSeqDB (https://bioconductor.org/packages/release/data/annotation/html/LymphoSeqDB.html). The TCR sequence data from reference #15 can be found at https://digitalcommons.providence.org/sitc2018/2/.

Supplementary Material

Table S1

Table S1. Baseline cytokine levels (prior to lymphodepletion) for UPN-03 and UPN-04.

Table S2

Table S2. Differentially expressed TCR-β CDR3 sequences increased at 3 and/or 35 month timepoints in patient UPN-04

Table S3

Table S3. Cellular composition of post-infusion timepoints.

Table S4

Table S4. Staining panel used for multiplex immunohistochemistry

Supplementary Figures

Figure S1. Cellular composition of the post-treatment tumor biopsy of patient UPN-03. Figure S2. Gating strategy for multiparameter flow cytometry for UPN-03. Figure S3. Gating strategy for multiparameter flow cytometry for UPN-04.

Authors’ Disclosures

I.R. Kirsch reports personal fees and other support from Adaptive Biotechnologies during the conduct of the study, as well as personal fees and other support from Adaptive Biotechnologies outside the submitted work. R. Gottardo reports other support from Ozette Technologies, Takeda, Sanofi, and Arcellx and grants from 10X Genomics and Owkin outside the submitted work, as well as a patent for markers, methods and systems for identifying cell populations, and diagnosing, monitoring, predicting, and treating conditions issued. K.S. Smythe reports other support from Exicure and X4 Pharmaceuticals and personal fees and other support from Sensei Bio outside the submitted work. A. Greenbaum reports grants from ASH during the conduct of the study. D.J. Green reports grants and other support from Juno Therapeutics, personal fees from GlaxoSmithKline and Ensoma Therapeutics, grants and personal fees from Janssen Biotech, and grants from SpringWorks Therapeutics, Sanofi, Seattle Genetics, Cellectar Biosciences, and Celgene outside the submitted work, as well as a patent for 62/582,270 issued to the Fred Hutchinson Cancer Center and a patent for 62/582,308 issued to Juno Therapeutics. D.G. Maloney reports personal fees from Genentech during the conduct of the study; grants and personal fees from Bristol Myers Squibb, Celgene, Juno Therapeutics, and Kite Pharma, grants from Legend Biotech, personal fees from Caribou Biosciences, Janssen, Chimeric Therapeutics, Bristol Myers Squibb, Genentech, Gilead, and Novartis, and personal fees and other support from A2 Biotherapeutics and NAVAN Technologies outside the submitted work; and a patent for Juno Therapeutics and Bristol Myers Squibb licensed and with royalties paid. B.G. Till reports grants from the Damon Runyon Cancer Research Foundation, Fred Hutchinson Cancer Center, David and Patricia Giuliani Family Foundation, and NIH during the conduct of the study; grants and personal fees from Mustang Bio, grants from Bristol Myers Squibb and Juno, and personal fees from Proteios Technology outside the submitted work; and a patent for Mustang Bio issued, licensed, and with royalties paid. No disclosures were reported by the other authors.

Authors’ Contributions

G. Mo: Conceptualization, data curation, formal analysis, supervision, funding acquisition, investigation, visualization, methodology, writing–original draft, writing–review and editing. S.Y. Lee: Data curation, formal analysis, investigation, visualization, writing–original draft, writing–review and editing. D.G. Coffey: Formal analysis, investigation, methodology, writing–original draft, writing–review and editing. V. Voillet: Formal analysis, investigation, methodology, writing–review and editing. I.R. Kirsch: Formal analysis, investigation, writing–review and editing. R. Gottardo: Formal analysis, investigation, writing–review and editing. K.S. Smythe: Investigation, visualization, writing–review and editing. C.C.S. Yeung: Formal analysis, investigation, visualization, writing–review and editing. A. Greenbaum: Investigation, visualization, writing–review and editing. D.J. Green: Formal analysis, investigation, visualization, writing–review and editing. D.G. Maloney: Resources, investigation, writing–review and editing. B.G. Till: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, investigation, visualization, methodology, writing–original draft, writing–review and editing.