Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint implicated in T cell exhaustion and a potential therapeutic target in glioblastoma (GBM). We conducted a multicenter, open-label, phase 1 study with sequential allocation to evaluate the safety and preliminary activity of the anti-LAG-3 antibody relatlimab, administered alone or with the anti-programmed cell death protein 1 (PD-1) antibody nivolumab, in patients with recurrent GBM. Forty-six patients were treated (23 per cohort). The primary endpoint of safety was met, with maximum tolerated doses of 800 mg relatlimab for monotherapy and 160 mg relatlimab/240 mg nivolumab for combination therapy. Treatment-related grade 3–4 adverse events occurred in 6 of 23 patients receiving combination therapy and were not observed with monotherapy. Neoadjuvant administration was associated with increased intratumoral CD8 + T cell infiltration for both monotherapy and combination therapy. Exploratory analyses suggested that tumors with elevated baseline interferon signaling and increased T cell clonality were enriched among patients with durable responses to combination therapy.
Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint implicated in T cell exhaustion and a potential therapeutic target in glioblastoma (GBM). We conducted a multicenter, open-label, phase 1 study with sequential allocation to evaluate the safety and preliminary activity of the anti-LAG-3 antibody relatlimab, administered alone or with the anti-programmed cell death protein 1 (PD-1) antibody nivolumab, in patients with recurrent GBM. Forty-six patients were treated (23 per cohort). The primary endpoint of safety was met, with maximum tolerated doses of 800 mg relatlimab for monotherapy and 160 mg relatlimab/240 mg nivolumab for combination therapy. Treatment-related grade 3–4 adverse events occurred in 6 of 23 patients receiving combination therapy and were not observed with monotherapy. Neoadjuvant administration was associated with increased intratumoral CD8 + T cell infiltration for both monotherapy and combination therapy. Exploratory analyses suggested that tumors with elevated baseline interferon signaling and increased T cell clonality were enriched among patients with durable responses to combination therapy. Twelve-month overall survival was 34.8% with relatlimab alone and 52.2% with combination therapy; however, this study was not designed to assess efficacy. These findings demonstrate an acceptable safety profile and provide preliminary immunologic and clinical signals supporting further evaluation of LAG-3 blockade in GBM. ClinicalTrials.gov identifier: NCT02658981 .
GBM is refractory to standard oncologic treatments with median survival of 14 months 1 despite maximal safe resection, aggressive radiotherapy and temozolomide chemotherapy. Immuno-oncologic therapies targeting immune checkpoint molecules PD-1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have not improved outcomes for GBM 2 , 3 , 4 , although this strategy has substantially improved survival in many other cancer types 5 , 6 , 7 . Barriers to effectiveness of these checkpoint inhibitor therapies may include multiple factors, including a tumor microenvironment (TME) defined by profound immune cell exhaustion and marked by the elevated expression of multiple other checkpoint molecules, such as LAG-3 (CD223) 8 , 9 . In the present study, we assessed the safety and immunologic impact of targeting the LAG-3 checkpoint with or without concurrent targeting of the PD-1 checkpoint.
In cancer, LAG-3 is thought to promote immunosuppression by decreasing the proliferation of CD4 + effector T cells, inhibiting CD8 + T cell antitumoral cytotoxic function and recruiting regulatory T (T reg ) cells 10 , 11 . LAG-3 upregulation in the TME is associated with poor treatment outcomes, and its upregulation after anti-PD-1 monoclonal antibody (mAb) therapy has implicated it as a mediator of acquired immune checkpoint inhibition resistance 12 . The recently reported RELATIVITY-047 trial in patients with metastatic melanoma evaluated combination immune checkpoint blockade and demonstrated greater than two-fold improvement in progression-free survival (PFS) with relatlimab plus nivolumab compared with nivolumab alone (10.12 months versus 4.63 months) 13 . Preclinical data in murine glioma models suggest that this combination may be effective in GBM as well 10 .
Here we present the results of an open-label, multicenter phase 1 trial testing anti-LAG-3 mAb (relatlimab, BMS-986016) alone or in combination with anti-PD-1 mAb (nivolumab, BMS-936558) in patients with first-time recurrent GBM ( NCT02658981 ). Our primary endpoint was safety to determine a maximum tolerated dose (MTD) for relatlimab given either as monotherapy or combined with nivolumab in patients with recurrent GBM. A window of opportunity arm where patients received either neoadjuvant relatlimab monotherapy or combination therapy with nivolumab prior to surgery was also included. Secondary objectives were site-determined 1-year PFS and overall survival (OS) rates as well as radiographic response per modified Response Assessment in Neuro-Oncology (mRANO) criteria. Exploratory objectives for this study involved cellular and molecular assessments of relatlimab alone and in combination with nivolumab on infiltrating immune cell populations in the context of survival outcomes.
A total of 46 patients were enrolled in the Adult Brain Tumor Consortium (ABTC) 1501 trial. All patients had histopathologic confirmation of GBM at diagnosis and had radiographically diagnosed recurrence after prior standard treatment with radiation therapy and temozolomide. Of those patients, 9 of 46 (19.6%) had undergone previous subtotal resection, and 37 of 46 (80.4%) had received gross total resection for their surgery at time of diagnosis. The first patient was enrolled on 9 September 2016, and the last patient was enrolled on 29 April 2020. The trial is completed. Clinical characteristics of all 46 patients are described in Table 1 .
Patients were sequentially allocated to either relatlimab monotherapy or anti-CD137 mAb therapy initially as part of the ABTC 1501 trial (Fig. 1 ). Once the MTD of relatlimab monotherapy was determined, combination therapy was started with eventually 23 patients receiving relatlimab monotherapy (17 adjuvant, six neoadjuvant) at starting dose 80 mg and subsequent dose escalation to 800 mg. Twenty-three patients were given combination relatlimab and nivolumab therapy (16 adjuvant, seven neoadjuvant), with relatlimab dosages escalating from 80 mg to 160 mg and nivolumab dosage remaining at 240 mg (Fig. 1 ). Neoadjuvant therapy was given within 10 ± 3 days prior to the date of surgery. All patients who received neoadjuvant therapy went on to also have adjuvant therapy with the same treatment schema as adjuvant-only patients. All patients were required to receive at least one dose of the intervention treatment to qualify for the study.
Schematic overview of the multicenter phase 1 trial design illustrating sequential allocation and dose escalation of relatlimab. Patients were enrolled across three study components: adjuvant monotherapy dose escalation, adjuvant combination therapy dose escalation and neoadjuvant therapy followed by adjuvant treatment. Neoadjuvant therapy consisted of a single dose administered prior to surgical resection.
Following National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0 guidelines, grade 1–4 adverse events observed with relatlimab monotherapy and with relatlimab/nivolumab combination therapy are summarized in Table 2 . All reported adverse events were deemed by site investigators to be possibly, probably or definitely attributable to the investigational agents. No patients treated with relatlimab monotherapy experienced a dose-limiting toxicity (DLT) (0/23, 0%) (Table 2 ), whereas 6 of 23 patients (26%) receiving combination therapy developed a DLT (Table 2 ). These events included worsened cerebral edema in 2 patients treated with 80 mg or 160 mg relatlimab, respectively, in combination with 240 mg nivolumab; grade 3 muscle weakness in 1 patient receiving 80 mg relatlimab plus 240 mg nivolumab; grade 3 hypertension in 1 patient receiving 160 mg relatlimab plus 240 mg nivolumab; grade 3 syncope in 1 patient receiving 160 mg relatlimab plus 240 mg nivolumab; and grade 3 thyroiditis in 1 patient receiving neoadjuvant 160 mg relatlimab plus 240 mg nivolumab (Table 2 ). Of note, both patients who experienced worsened cerebral edema were put on a short course of dexamethasone for symptomatic control.
Tumor response to treatment was evaluated under mRANO criteria 14 to account for pseudoprogression and delayed responses to immunotherapy by requiring confirmation of progression for a 6-month posttreatment evaluation window (Extended Data Fig. 1 ). Several patients who underwent relatlimab and nivolumab combination therapy demonstrated persistent or increasing contrast enhancement within the first 3 months of starting treatment, followed by near-total resolution of all enhancing disease that corresponded with prolonged OS. Although many patients had a significant period of disease control as assessed by imaging, upon central review no patients exhibited a confirmed partial response or complete response. Pseudoprogression, defined as initial progressive enhancement within the first 6 months of treatment followed by disease stability or regression on the confirmation scan, was noted in eight of 17 patients in the adjuvant relatlimab group, in nine of 16 patients in the adjuvant combination relatlimab and nivolumab group, in five of six patients in the neoadjuvant relatlimab group and in three of seven patients in the neoadjuvant combination relatlimab and nivolumab group per mRANO guidelines (Extended Data Fig. 1 ).
To correlate immunological changes in the TME with patients who had clinical response to LAG-3 blockade, we used several immune correlative studies, including immunofluorescence, gene expression profiling, T cell clonality assays and spatial proteomics through multiplexed ion beam imaging by time-of-flight (MIBI-TOF).
Neoadjuvant therapy involving relatlimab demonstrated increased infiltration of CD8 + T cells into the TME at the time of surgical resection (Fig. 2a,b ). When comparing neoadjuvant treatment samples with archival samples without immunotherapy treatment, exposure to relatlimab and nivolumab combination therapy in particular was associated with an increase in the number of infiltrating CD8 + T cells as well as upregulation of LAG-3 and PD-1 expression (Fig. 2b ).
a , Representative mIF images from a patient treated with neoadjuvant relatlimab/nivolumab, showing nuclear staining (DAPI) and expression of CD8, PD-1 and LAG-3. b , Comparative mIF images from paired tumor samples obtained before treatment and after neoadjuvant combination therapy. c – f , Correlation of intratumoral CD8 + T cell subsets (CD8 + ( c ), CD8 + PD-1 + ( d ), CD8 + LAG-3 + ( e ) and CD8 + PD-1 + LAG-3 + ( f )) expressing PD-1 and/or LAG-3 with OS; patients receiving adjuvant anti-LAG-3 and anti-PD-1 therapy who survived longer than the median are highlighted. g , h , Quantification of PD-1 and LAG-3 expression patterns on CD8 + T cells (CD8 + LAG-3 + ( g ) and CD8 + PD-1 + LAG-3 + ( h )) in patients before and after neoadjuvant (neoadj.) anti-PD-1 and anti-LAG-3 therapy. A, archival; P, protocol.
However, correlating the immunophenotype of the TME to clinical response demonstrates that infiltration of PD-1 + , LAG-3 + or PD-1 + LAG-3 + T cells in the resected tumor samples do not correlate with survival among patients receiving relatlimab monotherapy or combination relatlimab/nivolumab therapy (Fig. 2c–f ). Of note, although PD-1 and LAG-3 are typically co-expressed on T cells in the setting of exhaustion 15 , neoadjuvant therapy resulted in CD8 + T cell recruitment with both LAG-3 and PD-1 co-expression as well as some cells with only LAG-3 or PD-1 expression (Fig. 2g,h ).
To investigate baseline differential gene expression between long-term survivors (OS > 24 months) and short-term survivors (OS P 3a,b ). Gene set enrichment analysis (GSEA) of type I (IFNα) and type II (IFNγ) interferon signaling pathways demonstrated enrichment in responder patients, reaching significance with Wald test statistics ( P 3c ).
a , Heatmap showing normalized gene expression of genes with differential expression ( P 24 months) and short-term survivors (OS b , Volcano plot displaying log 2 (fold change) versus −log 10 ( P value) for baseline differential gene expression. Differential expression analysis was performed using NanoString nSolver Advanced Analysis. Statistical significance was assessed using two-sided t -tests, and P c , GSEA showing enrichment of type I (IFNα) and type II (IFNγ) interferon signaling pathways in long-term survivors at baseline. d , Heatmap showing normalized gene expression of genes with differential expression ( P e , Volcano plot displaying log 2 (fold change) versus −log 10 ( P value) for differential gene expression before and after neoadjuvant treatment. Statistical significance was assessed using two-sided t -tests, and P f , GSEA pathway analysis demonstrating enrichment of interferon signaling pathways after neoadjuvant treatment.
To further explore transcriptional changes induced by neoadjuvant treatment, we performed gene expression profiling on FFPE biopsy specimens obtained from three patients before and after neoadjuvant relatlimab and nivolumab combination therapy. This analysis identified 67 DEGs ( P 3d,e ). Multiple genes associated with interferon signaling and antigen presentation, including CXCL9, CCL5, GZMA, STAT1 and HLA family members, showed consistent trends toward upregulation in patients after treatment, with Wald test statistics showing significance ( P 3f ).
We analyzed T cell receptor (TCR) sequences from FFPE specimens of four long-term responders (patients surviving >24 months) and five non-responders (patients surviving P 2a ). This likely reflects a targeted immune response against specific tumor antigens. To assess T cell expansion in circulation after immunotherapy, we analyzed TCRβ sequences from peripheral circulating T cells in one responder patient and in one non-responder patient. Compared with the non-responder patient, the long-term survivor demonstrated expansion of intratumoral T cell clones (Extended Data Fig. 2b ). In the responder patient, peripheral T cell expansion was noted with as few as two immune checkpoint inhibitor infusions, indicating that these T cells had robust proliferative potential (Extended Data Fig. 2c ).
Given our findings of increased T cell clonality, we also found that there was upregulation of genes associated with antigen presentation on myeloid cells that was positively correlated with long-term survivors (Fig. 4a,b ). Myeloid cells capable of antigen presentation can directly activate T cells with clonal expansion 16 . We performed MIBI-TOF analysis on four patient samples and found that there was robust intratumoral infiltration of CD68 + macrophages throughout the entire tumor with concentrated infiltration of lymphoid cells, largely consisting of B cells, in perivascular spaces (Fig. 4c,d ). This observation is consistent with previous studies reporting increased B cell infiltration in the TME after immune checkpoint blockade, including PD-1-directed therapies 17 . Further phenotypic resolution of B cell subsets was limited by tissue availability and marker constraints. Highlighting the importance of the myeloid compartment for immunotherapy response, patients who received neoadjuvant relatlimab with or without nivolumab exposure, as well as those who went on to demonstrate long-term response, exhibited a substantial presence of myeloid cells, including CD68 + myeloid cells and activated microglia. Conversely, short-term survivors had a dearth of intratumoral microglia at the time of surgery while maintaining a population of immunosuppressive CD163 + myeloid cells (Fig. 4e ). When comparing this sample of patients to a larger cohort of 15 standard-of-care patients with GBM, long-term survivors and the neoadjuvant patient had a pattern of increased microglia, non-immunosuppressed myeloid and lymphoid cell populations when compared with all other patient populations (Fig. 4f ).