First-in-human phase 1 study of budigalimab, an anti-PD-1 inhibitor, in patients with non-small cell lung cancer and head and neck squamous cell carcinoma

Publisher Copyright: © 2021, The Author(s).Background: Budigalimab is a humanized, recombinant immunoglobulin G1 monoclonal antibody targeting programmed cell death protein 1 (PD-1). We present the safety, efficacy, pharmacokinetic (PK), and pharmacodynamic data from patients enrolled in the head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) expansion cohorts of the phase 1 first-in-human study of budigalimab monotherapy (NCT03000257; registered 15 December 2016). Patients and methods: Patients with recurrent/metastatic HNSCC or locally advanced/metastatic NSCLC naive to PD-1/PD-1-ligand inhibitors were enrolled; patients were not selected on the basis of oncogene driver mutations or PD-L1 status. Budigalimab was administered at 250 mg intravenously Q2W or 500 mg intravenously Q4W until disease progression/unacceptable toxicity. The primary endpoints were safety and PK; the secondary endpoint was efficacy. Exploratory endpoints included biomarker assessments. Results: In total, 81 patients were enrolled (HNSCC: N = 41 [PD-L1 positive: n = 19]; NSCLC: N = 40 [PD-L1 positive: n = 16]); median treatment duration was 72 days (range, 1–617) and 71 days (range, 1–490) for the HNSCC and NSCLC cohorts, respectively. The most frequent grade ≥ 3 treatment-emergent adverse event was anemia (HNSCC: n = 9, 22%; NSCLC: n = 5, 13%). Both dosing regimens had comparable drug exposure and increased interferon gamma-induced chemokines, monokine induced by gamma interferon, and interferon-gamma-inducible protein 10. Objective response rates were 13% (90% CI, 5.1–24.5) in the HNSCC cohort and 19% (90% CI, 9.2–32.6) in the NSCLC cohort. Median progression-free survival was 3.6 months (95% CI, 1.7–4.7) and 1.9 months (95% CI, 1.7–3.7) in the HNSCC and NSCLC cohorts. Conclusions: The safety, efficacy and biomarker profiles of budigalimab are similar to other PD-1 inhibitors. Development of budigalimab in combination with novel anticancer agents is ongoing.Peer reviewe

Src Kinase and Syk Activation Initiate PI3K Signaling by a Chimeric Latent Membrane Protein 1 in Epstein-Barr Virus (EBV)+ B Cell Lymphomas

<div><p>The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.</p> </div

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Additional file 3. Percentage of RSV specific CD3+CD4+ and CD3+CD8+ responses

NGFR-LMP1 Activates Src Family Kinases.

<p><i>(A)</i> Cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with indicated antibodies. <i>(B)</i> NGFR-LMP1 signaling or BCR signaling (IgM) was triggered for the indicated amount of time. Lysates were separated on SDS-PAGE gels, transferred to nitrocellulose, and probed for the indicated proteins by western blotting. <i>(C)</i> Cells were pretreated for 1 hour with DMSO control (−) or the Src inhibitor PP2 (+) before triggering NGFR-LMP1 signaling or BCR signaling for 5 minutes in BL41.NGFR-LMP1 cells as described. Lysates were separated on SDS-PAGE gels, transferred to nitrocellulose, and probed for the indicated proteins by western blotting. For all western blots, the closest molecular weight marker (e.g. 64 kDa) to the protein of interest is indicated to the right of each blot. <i>(D)</i> Cells were pretreated for 1 hour with DMSO control (−) or the Src inhibitor PP2 (+) before induction of NGFR-LMP1 signaling. Cells were then plated at 0.5×10⁶/ml in the presence of inhibitor. At 24 hours, supernatants were harvested and assayed for IL-10 by ELISA. IL-10 production was normalized to the number of viable cells at the time of supernatant harvest. Data is expressed as the mean percent of IL-10 produced for each group (compared to control) with standard deviations and is representative of 3–4 replicate experiments.</p

NGFR-LMP1 Activates Syk Upstream of PI3K and IL-10.

<p><i>(A)</i> Lysates from R406-treated cells (as indicated) were immunoprecipitated with anti-Syk antibodies. Washed immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and blotted for pSyk and Syk. JC62 is shown here as a representative cell line. <i>(B)</i> NGFR-LMP1 crosslinking was triggered for 5 minutes as indicated, in the presence of DMSO control (−) or the Syk inhibitor R406 (+). Lysates were separated on SDS-PAGE gels, transferred to nitrocellulose, and probed for the indicated proteins by western blotting. The closest molecular weight marker (e.g. 64 kDa) to the protein of interest is indicated to the right of each blot. <i>(C)</i> Cells were pretreated for 1 hour with DMSO control (−) or the Syk inhibitor R406 (+) before induction of NGFR-LMP1 signaling. Cells were then plated at 0.5×10⁶/ml in the presence of inhibitor. At 24 hours, supernatants were harvested and assayed for IL-10 by ELISA. IL-10 production was normalized to the number of viable cells at the time of supernatant harvest. Data is expressed as the mean percent of IL-10 produced for each group (compared to control) with standard deviations and is representative of 3–4 replicate experiments.</p

NGFR-LMP1 Activates, but Does Not Physically Interact with, Syk.

<p><i>(A)</i> NGFR-LMP1 crosslinking or BCR crosslinking was triggered as described for 5 minutes. Lysates were separated on SDS-PAGE gels, transferred to nitrocellulose, and probed for the indicated phospho-proteins by western blotting. Blots were then stripped and reprobed for total protein levels. <i>(B)</i> Schematic and amino acid sequence of the C-terminal signaling tail of LMP1. TRAF binding motifs (PXQXT; black diamonds) and TRADD-interacting tyrosines (Y385, Y385; black circles) are noted in bold. C-terminal activating regions (CTAR), CTAR1 (E194 to G232) and CTAR2 (S361 to D386), are noted in underlined sections and in light grey shading. Potential JAK3 interaction sites, Box1 (P275 to P283, P302 to L316) and Box2 (P331 to K341), are noted in underlined sections and in dark grey shading. <i>(C)</i> Cells were treated with anti-NGFR and goat anti-mouse Ig to induce NGFR-LMP1 signaling for 5 minutes as indicated. Lysates from 10×10⁶ cells were immunoprecipitated with anti-LMP1. Washed immunoprecipitates and flow through were separated by SDS-PAGE, transferred to nitrocellulose, and blotted for Syk, TRAF3, LMP1, and actin as a loading control (41 = BL41, B95 = BL41.B95, N-L = BL41.NGFR-LMP1). <i>(D)</i> Immunoprecipitation with either mIgG isotype control or anti-LMP1 Abs and western blotting of lysates was performed as described in <i>(C)</i>. For all western blots, the closest molecular weight marker (e.g. 64 kDa) to the protein of interest is indicated to the right of each blot.</p

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Additional file 4. Workflow to perform viSNE analysis on antigen-specific cells

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Additional file 1. Mock subtracted IFNÎł responses at Day 1, at Day 8 undepleted, CD4 depleted and CD8 depleted

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Additional file 2. CyTOF workflow