BACKGROUND:Chimeric antigen receptor (CAR) T-cell therapy is the standard of care for relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL). However, more than half of patients fail to achieve durable remission. Identifying predictive biomarkers within the CAR T-cell infusion product (IP) may guide strategies to improve clinical outcomes. PATIENTS AND METHODS:This single-center observational study, conducted at Lausanne University Hospital (CHUV), Switzerland, analyzed IPs from 13 patients with R/R DLBCL who underwent standard-of-care CAR T-cell therapy. A 39-marker mass cytometry panel was used to compare phenotypic and functional markers between long-term responders (R) and nonresponders (NR). Unsupervised and supervised analytic approaches were applied to IP data, and longitudinal peripheral blood samples were collected over 30 days after infusion to track CAR T-cell subpopulation dynamics. RESULTS:At a median follow-up of 13.5 months, median progression-free survival (PFS) was 13.3 months [95% confidence interval (CI) 9.7-24.3 months] in R (n = 8) versus 3.5 months (95% CI 0.5-5.4 months) in NR (n = 5) (hazard ratio 56.67, 95% CI 7.3-439.3, P = 0.0001). A CD3+CXCR3+CD7+ CAR T-cell subpopulation-found in both CD4+ and CD8+ compartments-was significantly enriched in R. These cells showed increased expression of perforin, granzyme B, and NKG2D (restricted to CD8+ cells). In contrast, NR had a higher frequency of CXCR3+CD7+LAG3+ CAR T-cells. Surface expression of CD3, CD7, CXCR3, and NKG2D were higher in R, whereas LAG3, Ki67, and CD71 were elevated in NR. A predictive cut-off ratio of CD3+CXCR3+CD7+LAG3+CAR+ T-cells <0.83 and CD3+CXCR3+CD7+NKG2D+CAR+ T-cells >1.034 yielded a predictive accuracy of 0.92. Serum CXCL9 and CXCL10 concentrations did not differ between groups. CONCLUSIONS:Enrichment of CD7+CXCR3+ CAR T-cells alongside elevated NKG2D expression in R, in contrast to higher LAG3 and CD71 in NR, emerged as potentially robust correlates of therapeutic outcome. Although derived from a small, hypothesis-generating cohort, these findings suggest that targeted analysis of IP composition may inform the development of biomarker-driven strategies to optimize CAR T-cell products and improve the likelihood of durable remission in R/R DLBCL.

The immune-related adverse events associated with chimeric antigen receptor (CAR)-T cell therapy result in substantial morbidity as well as considerable cost to the health-care system, and can limit the use of these treatments. Current therapeutic strategies to manage immune-related adverse events include interleukin-6 receptor (IL-6R) blockade and corticosteroids. However, because these interventions do not always address the side effects, nor prevent progression to higher grades of adverse events, new approaches are needed. A deeper understanding of the cell types involved, and their associated signalling pathways, cellular metabolism and differentiation states, should provide the basis for alternative strategies. To preserve treatment efficacy, cytokine-mediated toxicity needs to be uncoupled from CAR-T cell function, expansion, long-term persistence and memory formation. This may be achieved by targeting CAR or independent cytokine signalling axes transiently, and through novel T cell engineering strategies, such as low-affinity CAR-T cells, reversible on-off switches and versatile adaptor systems. We summarize the current management of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, and review T cell- and myeloid cell-intrinsic druggable targets and cellular engineering strategies to develop safer CAR-T cells.

Adoptive cell therapy (ACT) using ex vivo–expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell–intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8 + TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor–reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network–based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

BACKGROUND:Experience-Based Co-Design (EBCD) is a multi-stage participatory action research process which was developed originally to increase patient involvement in service improvement initiatives. This viewpoint article serves as a reflection on the researchers' experiences, focusing on the application and feasibility of participatory approaches, particularly co-design, in the specific context of early-phase clinical trials. METHODS:We reflect on the opportunities and challenges of applying EBCD in a new context of early-phase clinical trials in oncology where experimental treatments are increasingly perceived as a therapeutic option and, in certain instances, their efficacy may lead to accelerated approval facilitating a swifter integration into standard care. RESULTS:We propose that the opportunity of applying EBCD in such trials lies in improving the delivery of person-centered care, care coordination, and support during the transition from experimental to standard care. Three potential challenges when applying EBCD in early-phase clinical trials are discussed related to: the need for standardization in trial processes; planning EBCD in a context of high uncertainty; and vulnerability of patient populations. CONCLUSION:Integrating EBCD into early-phase oncology trials presents an opportunity to enhance person-centered care and can lead to simultaneous improvements in care processes and therapeutic development. PATIENT OR PUBLIC CONTRIBUTION:This article has been developed with the collaboration of a patient partner who serves on the advisory board of our ongoing EBCD study in early clinical trials.

Adoptive cell therapy (ACT) using in vitro expanded tumor-infiltrating lymphocytes (TILs) has inconsistent clinical responses. To better understand determinants of therapeutic success, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients using single-cell RNA and T cell receptor (TCR) sequencing. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, and these were more effectively mobilized upon in vitro expansion, yielding products enriched in tumor-specific CD8+ cells that preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes and with cell products mostly composed of blood-borne clonotypes that persisted in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost tumor-associated transcriptional signatures, including exhaustion, and responders exhibited an intermediate exhausted effector state after TIL engraftment in the tumor, suggesting functional reinvigoration. Our findings provide insight into the nature and dynamics of tumor-specific clonotypes associated with clinical response to TIL-ACT, with implications for treatment optimization.

Cellular therapy using genetically modified T lymphocytes expressing synthetic receptors, known as CAR (Chimeric Antigen Receptor), has revolutionized the treatment of certain hematologic malignancies. This success has led to exploring the same approach in the treatment of severe autoimmune diseases refractory to conventional therapies. Initial results in systemic lupus erythematosus have shown complete remissions that appear to persist over time. Consequently, there is a growing number of ongoing clinical trials. In this review, we discuss the rationale behind the use of CAR-T therapies, the targeted autoimmune diseases, and the associated risks.

Cellular therapy using genetically modified T lymphocytes expressing synthetic receptors, known as CAR (Chimeric Antigen Receptor), has revolutionized the treatment of certain hematologic malignancies. This success has led to exploring the same approach in the treatment of severe autoimmune diseases refractory to conventional therapies. Initial results in systemic lupus erythematosus have shown complete remissions that appear to persist over time. Consequently, there is a growing number of ongoing clinical trials. In this review, we discuss the rationale behind the use of CAR-T therapies, the targeted autoimmune diseases, and the associated risks.

Background. CAR T-cell therapy is the standard of care for R/R DLBCL, but more than half of patients fail to achieve long-term remission. Identification of cellular biomarkers in CAR T-cell infusion products (IPs) that predict complete remission beyond six months may guide the development of strategies to improve outcomes. Methods. IPs from 13 R/R DLBCL patients were analyzed using a 39-marker mass cytometry panel, comparing cell populations between long-term responders (R) and non-responders (NR). Both unsupervised and supervised analyses were performed. Longitudinal blood samples were analyzed for 30 days to track CAR T-cell subpopulation dynamics. Results. At a median follow-up of 13.5 months, median progression-free survival (PFS) was 13.3 months (95% CI: 9.7-24.3) in R (n=8) versus 3.5 months (95% CI: 0.5-5.4) in NR (n=5). The HR for PFS was 56.67 (95% CI: 7.3-439.3; P=0.0001). A subset of CD3+CXCR3+CD7+ CAR T-cells found in both CD4+ and CD8+ populations was significantly enriched in R and expressed higher levels of perforin, granzyme B, and NKG2D (restricted to CD8+). NR had more CXCR3+CD7+LAG3+ CAR T-cells. CD3, CD7, CXCR3, and NKG2D cell surface levels were higher in R, whereas LAG3, Ki67, and CD71 were elevated in NR. A predictive cut-off ratio of CD3+CXCR3+CD7+LAG3+CAR+ T-cells <0.83 and CD3+CXCR3+CD7+NKG2D+CAR+ T-cells >1.034 yielded a predictive accuracy of 0.92. Serum CXCL9 and CXCL10 levels were not different between groups. Conclusions. Increased frequency of CAR T-cells expressing CD7, CXCR3 and NKG2D in R versus LAG3 and CD71 in NR emerged as strong correlates of therapeutic outcome. ### Competing Interest Statement MO received honoraria and speaker fees from Moderna, Roche and BMS. C.A. holds patents and provisional patent applications in the field of engineered T cell therapies. C.A. receives licensing fees and royalties from Immatics (through previous institution Baylor College of Medicine), participated in advisory boards for Kite/ Gilead, Janssen and Celgene/ BMS, received sponsored travel from Gilead (through current institution Lausanne University Hospital (CHUV). G.C. has received honoraria from Bristol-Myers Squibb. CHUV has received honoraria for advisory services provided by G.C. to Iovance and EVIR. G.C. has received royalties from the University of Pennsylvania for CAR T cell therapy licensed to Novartis and Tmunity Therapeutics. G.C. has received royalties from the Ludwig Institute for Cancer Research, UNIL and CHUV for NeoTIL intellectual property previously licensed to Tigen Pharma. G.C. is inventor in technologies related to T cell expansion and engineering for T cell therapy. RS participated in advisory boards for Janssen, Celgene/ BMS, AbbVie, Takeda and Incyte. ### Funding Statement This study did not receive any dedicated funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Cantonal Ethics Committee (CER-VD) of the Vaud Canton (Switzerland) gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The datasets supporting the results of this study are not publicly available. Requests for access to the dataset will be granted upon reasonable request to the principal investigator. Study data will be managed, stored, shared, and archived according to CHUV standard operating procedures to ensure the continued quality, integrity, and utility of the data.

Adoptive cell therapy (ACT) using ex vivo expanded tumor-infiltrating T lymphocytes (TILs) can mediate responses in metastatic melanoma, but long-term efficacy remains limited to a fraction of patients. Here we interrogated tumor-microenvironment (TME) cellular states and interactions of longitudinal samples from 13 metastatic melanoma patients treated with TIL-ACT in our clinical study ([NCT03475134][1]). We performed single-cell RNA-seq and spatial proteomic analyses in pre- and post-ACT tumor tissues and showed that responders exhibited higher tumor cell-intrinsic immunogenicity. Also, endogenous CD8+ TILs and myeloid cells of responders were characterized by increased cytotoxicity, exhaustion and costimulation and type-I IFN signaling, respectively. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders have rich baseline intratumoral and stromal tumor-reactive T-cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study reveals CD8+ T-cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.One-Sentence Summary Response to adoptive TIL therapy in melanoma is determined by CD8+ TIL-myeloid cell networks### Competing Interest StatementSZ is currently an employee of F. Hoffmann-La Roche. RD has intermittent, project focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dhome (MSD), Bristol-Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre, Sun Pharma, Sanofi, Catalym, Second Genome, Regeneron, Alligator, T3 Pharma, MaxiVAX SA, Pfizer and touchIME outside the submitted work. OM has consulting/advisory roles for Bristol Myers Squibb, MSD, Roche, Novartis, Amgen, Pierre Fabre, Neracare, research grants from Bristol Myers Squibb, MSD, Amgen. PC, consultant advisor or paid speaker for Bristol Myers Squibb, MSD, Novartis, Pierre Fabre, Amgen, Nektar, has received research funding from Bristol Myers Squibb, Pierre Fabre. GC has received grants from Celgene, Boehringer-Ingelheim, Roche, Bristol Myers Squibb, Iovance Therapeutics and Kite Pharma. The institution GC is affiliated with has received fees for GC participation on advisory boards or for presentation at a company-sponsored symposium from Genentech, Roche, Bristol Myers Squibb, AstraZeneca, NextCure, Geneos Tx and Sanofi/Avensis. GC holds patents around antibodies and receives royalties from the University of Pennsylvania regarding technology licensed to Novartis. DDL, AH, and GC are inventors on patent applications filed by the Ludwig Institute for Cancer Research Ltd pertaining to the subject matter disclosed herein and such patent applications have been licensed to Tigen Pharma SA. All the other authors have no conflict of interest to declare. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03475134&atom=%2Fbiorxiv%2Fearly%2F2022%2F12%2F23%2F2022.12.23.519261.atom

Adoptive cell therapies (ACT) have demonstrated promise in the treatment of patients with cancer, leading to long-lasting responses and, in some cases, even cure. Technological advances have brought these individualized therapies closer to reality, establishing them as credible therapeutic option. However, to date, few efforts have been made to understand patients' experience during ACT trials. Patient-reported outcomes (PROs) and patient-reported outcome measures (PROMs), which are instruments used to report PROs, are increasingly being used in oncology to capture patients' perspective, provide real-world data on treatment safety, and support decision-making processes, such as health economic decisions. Due to the inherent complexity of ACT, the inclusion of PROMs in this field remains limited. In this commentary, we discuss the benefit of capturing PROs in ACT trials, the challenges of PROM administration and collection, and we propose simple and actionable recommendations to promote their adoption in ACT trials.

2533 Background: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has demonstrated a curative potential for patients with metastatic melanoma (MM). Nevertheless, activity remains unsatisfactory in many patients, requiring development of biomarkers that predict therapeutic efficacy. We report results of a single-center phase I study to assess feasibility, safety and efficacy of TIL-ACT in MM patients (NCT03475134). Methods: Patients with MM refractory to at least one prior line of therapy received TIL therapy with lymphodepleting chemotherapy before T-cell infusion, followed by high-dose interleukin-2. RDG- and FDG-PET imaging was performed before and after TIL infusion. Multispectral immuno-fluorescence (mIF) imaging and bulk-RNA sequencing (Seq) were performed on tumor samples pre-ACT and post-ACT (day+30 and upon progression). Single-cell RNA-Seq and TCR-Seq were performed on pre-ACT tumor and ACT product, as well as on tumor-reactive and neoantigen-specific TILs and on longitudinal blood samples. Results: As of 02/02/2021, thirteen patients (enrolled between March 2018 and December 2020) have successfully completed TIL-ACT therapy, with a median follow-up of 9.5 months (IQR 3.0 -24.6). Median age was 53 years (range 20-69) and all were previously treated with PD-1 based blockade. Median number of TILs infused was 55.0 x109 cells (range 12.8-84.7). The best overall response rate by RECIST 1.1 and disease control rate in evaluable patients was 41.7% (5/12) and 50% (6/12) respectively at 3 months. Two patients have an ongoing near-complete response at 3 years. Up to data cut-off, 10 patients have progressed by RECIST v1.1, with median PFS of 4.8 months (95% CI 1.5 - 9.6), while median OS is not reached. mIF revealed biomarkers of response, which may allow proper identification of patients in subsequent studies. In addition, deep sequencing of bulk and neoepitope-specific TIL clonotypes highlighted transcriptomic signatures revealing cell programs regulating in vitro expansion, in vivo blood persistence as well as tumor infiltration post-ACT. RGD-PET data will also be presented. Conclusions: We demonstrate reproducibility of TIL-ACT in our center, consistently with previous reports. Comprehensive translational studies reveal immune correlates of clinical responses that contribute to the understanding of mechanisms of TIL potency and will guide the development of next-generation cell products. Clinical trial information: NCT03475134.

Background Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) is a promising experimental immunotherapy that has shown high objective responses in patients with melanoma. Current protocols use a lymphodepletive chemotherapy before infusion of ex vivo expanded TILs, followed by high-dose interleukin-2 (IL-2). Treatment-related toxicities are mainly attributable to the chemotherapy regimen and to the high-dose IL-2 and are generally reversible. Neurological side effects have rarely been described. Nevertheless, due to improvements in cell production techniques and due to combinations with other immunomodulating molecules, side effects not previously described may be encountered. Case presentation We report the case of a 53-year-old heavily pretreated patient with melanoma who developed Guillain-Barré syndrome (GBS) 19 days after ACT using autologous TILs, given in the context of a phase I trial. He presented with dorsal back pain, unsteady gait and numbness in hands and feet. Lumbar puncture showed albuminocytological dissociation, and nerve conduction studies revealed prolonged distal motor latencies in median, ulnar, tibial and peroneal nerves, compatible with a GBS. The patient was treated with intravenous immunoglobulins and intensive neurological rehabilitation, with progressive and full recovery at 21 months post-TIL-ACT. Concomitant to the onset of GBS, a cytomegalovirus reactivation on immunosuppression was detected and considered as the most plausible cause of this neurological side effect. Conclusion We describe for the first time a case of GBS occurring shortly after TIL-ACT for melanoma, even though we could not identify with certainty the triggering agent. The report of such rare cases is of extreme importance to build on the knowledge of immune cellular therapies and their specific spectrum of toxicities.

Background aims Several studies report on Good Manufacturing Process (GMP)-compliant manufacturing protocols for the ex vivo expansion of tumor-infiltrating lymphocytes (TILs) for the treatment of patients with refractory melanoma and other solid malignancies. Further opportunities for improvements in terms of ergonomy and operating time have been identified. Methods To enable GMP-compliant TILs production for adoptive cell therapy needs, a simple automated and reproducible protocol for TILs manufacturing with the use of a closed system was developed and implemented at the authors' institution. Results This protocol enabled significant operating time reduction during TILs expansion while allowing the generation of high-quality TILs products. Conclusions A simplified and efficient method of TILs expansion will enable the broadening of individualized tumor therapy and will increase patients' access to state-of-the-art TILs adoptive cell therapy treatment.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) has been tested in advanced melanoma patients at various centers. We conducted a systematic review and meta-analysis to assess its efficacy on previously treated advanced metastatic cutaneous melanoma. The PubMed electronic database was searched from inception to 17 December 2018 to identify studies administering TIL-ACT and recombinant interleukin-2 (IL-2) following non-myeloablative chemotherapy in previously treated metastatic melanoma patients. Objective response rate (ORR) was the primary end point. Secondary end points were complete response rate (CRR), overall survival (OS), duration of response (DOR) and toxicity. Pooled estimates were derived from fixed or random effect models, depending on the amount of heterogeneity detected. Analysis was carried out separately for high dose (HD) and low dose (LD) IL-2. Sensitivity analyses were carried out. Among 1211 records screened, 13 studies (published 1988 - 2016) were eligible for meta-analysis. Among 410 heavily pretreated patients (some with brain metastasis), 332 received HD-IL-2 and 78 LD-IL-2. The pooled overall ORR estimate was 41% [95% confidence interval (CI) 35% to 48%], and the overall CRR was 12% (95% CI 7% to 16%). For the HD-IL-2 group, the ORR was 43% (95% CI 36% to 50%), while for the LD-IL-2 it was 35% (95% CI 25% to 45%). Corresponding pooled estimates for CRR were 14% (95% CI 7% to 20%) and 7% (95% CI 1% to 12%). The majority of HD-IL-2 complete responders (27/28) remained in remission during the extent of follow-up after CR (median 40 months). Sensitivity analyses yielded similar results. Higher number of infused cells was associated with a favorable response. The ORR for HD-IL-2 compared favorably with the nivolumab/ipilimumab combination following anti-PD-1 failure. TIL-ACT therapy, especially when combined with HD-IL-2, achieves durable clinical benefit and warrants further investigation. We discuss the current position of TIL-ACT in the therapy of advanced melanoma, particularly in the era of immune checkpoint blockade therapy, and review future opportunities for improvement of this approach.

Objective: Pain undertreatment, or oligoanalgesia, is frequent in the emergency department (ED), with major medical, ethical, and financial implications. Across different hospitals, healthcare providers have been reported to differ considerably in the ways in which they recognise and manage pain, with some prescribing analgesics far less frequently than others. However, factors that could explain this variability remain poorly understood. Here, we used neuroscience approaches for neural signal modelling to investigate whether individual decisions in the ED could be explained in terms of brain patterns related to empathy, risk-taking, and error monitoring. Methods: For 15 months, we monitored the pain management behaviour of 70 ED nurses at triage, and subsequently invited 33 to a neuroimaging study involving three well-established tasks probing relevant cognitive and affective dimensions. Univariate and multivariate regressions were used to predict pain management decisions from neural activity during these tasks. Results: We found that the brain signal recorded when empathising with others predicted the frequency with which nurses documented pain in their patients. In addition, neural activity sensitive to errors and negative outcomes predicted the frequency with which nurses denied analgesia by registering potential side-effects. Conclusions: These results highlight the multiple processes underlying pain management, and suggest that the neural representations of others' states and one's errors play a key role in individual treatment decisions. Neuroscience models of social cognition and decision-making are a powerful tool to explain clinical behaviour and might be used to guide future educational programs to improve pain management in ED.

New oncological approaches using immune checkpoint inhibitors aim to reinvigorate lymphocytes against the tumor. The prognosis of cancer has been significantly improved by these treatments, which nonetheless lead to a new spectrum of immune-related side effects. The most frequent are skin rash, colitis, thyroid dysfunction, hypophysitis, hepatitis and pneumonitis. Early detection of these toxicities is crucial to determine the etiology and to introduce a temporary immunosuppressive therapy, allowing resolution of toxicity in most of cases. A multidisciplinary team is essential for optimal management.