Pharmacodynamics and molecular correlates of response to glofitamab in relapsed/refractory non-Hodgkin lymphoma

Abstract

Glofitamab, a novel CD20xCD3, T-cell-engaging bispecific antibody, exhibited single-agent activity in Study NP30179, a first-in-human, phase 1 trial in relapsed/refractory B-cell non-Hodgkin lymphoma. Preclinical studies showed that glofitamab leads to T-cell activation, proliferation, and tumor cell killing upon binding to CD20 on malignant cells. Here, we provide evidence of glofitamab's clinical activity, including pharmacodynamic profile, mode of action, and factors associated with clinical response, by evaluating biomarkers in patient samples from the dose-escalation part of this trial. Patients enrolled in Study NP30179 received single-dose obinutuzumab pretreatment (1000 mg) 7 days before IV glofitamab (5 µg-25 mg). Glofitamab treatment lasted ≤12 cycles once every 2 or 3 weeks. Blood samples were collected at predefined time points per the clinical protocol; T-cell populations were evaluated centrally by flow cytometry, and cytokine profiles were analyzed. Immunohistochemical and genomic biomarker analyses were performed on tumor biopsy samples. Pharmacodynamic modulation was observed with glofitamab treatment, including dose-dependent induction of cytokines, and T-cell margination, proliferation, and activation in peripheral blood. Gene expression analysis of pretreatment tumor biopsy samples indicated that tumor cell intrinsic factors such as TP53 signaling are associated with resistance to glofitamab, but they may also be interlinked with a diminished effector T-cell profile in resistant tumors and thus represent a poor prognostic factor per se. This integrative biomarker data analysis provides clinical evidence regarding glofitamab's mode of action, supports optimal biological dose selection, and will further guide clinical development. This trial was registered at www.clinicaltrials.gov as #NCT03075696.

© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

Figures

Graphical abstract

Figure 1.

T-cell margination after first glofitamab infusion is dose and response dependent. (A) Flow cytometric analysis of peripheral CD19+ B cells and CD3+ T cells before obinutuzumab pretreatment (C1D-7, predose) and before the first glofitamab infusion (C1D1, predose; n = 110 pairs). Dotted line indicates 5 cells/µL. (B) Graphs represent log2 fold change (Log2FC) from baseline (C1D1 predose) of peripheral CD8+ T-cell subsets at indicated time points during C1, as measured by flow cytometry. Error bars indicate confidence intervals, dotted lines indicate baseline levels, and dashed lines indicate twofold change from baseline. (C) Box plots (left) represent Log2FC from baseline (C1D1 predose) of peripheral CD3+ T cells at 6 hours’ post–end of infusion (6 H EOI; top) and end of C1 (bottom) time points, as measured by flow cytometry, in relation to the best overall response (BOR). Scatter plots (right) indicate the correlation between Log2FC from baseline (C1D1 predose) of peripheral CD3+ T cells and the administered glofitamab dose (milligrams) at 6 H EOI (top) and end of C1 (bottom) time points. Data in panels B and C are from n = 119 patients with evaluable flow cytometry data. Colors indicate BOR categories. P value represents CR vs PR/stable disease (SD)/progressive disease (PD) and was not adjusted for log(glofitamab dose) and IPI category. C, cycle; D, day; ND, not disclosed; RMSE, root mean square error.

Figure 2.

Induction of T-cell memory subsets and inflammatory cytokines are associated with the pharmacodynamic profile of glofitamab. (A) Graphs represent Log2 fold change (Log2FC) from baseline (C1D1 predose) of peripheral CD8+ T-cell subsets measured by flow cytometry on the first day (D1 predose) of the first 5 cycles. Error bars indicate confidence intervals. Data generated from n = 119 patients with evaluable flow cytometry data. (B) Plots show Log2FC from baseline (C1D1 predose) of CD8+ T-cell effector memory subsets measured by flow cytometry on the first day of cycle 3 (C3D1, predose) for the 4 to 25 mg dose cohort. The x-axes indicate the best overall response (BOR). Means of each response category are shown, and error bars indicate confidence intervals. P values >.05 for CR vs PR/stable disease (SD)/progressive disease (PD) and were not adjusted for log(glofitamab dose) and IPI category. (C) Plasma cytokine concentrations (pg/mL) of IFN-γ, IL-6, and IL-2 are shown at indicated time points, including before obinutuzumab pretreatment and during the first cycle before infusion, mid-infusion (MI), and end of infusion (EOI). Data generated from n = 119 patients with evaluable cytokine data. The y-axes are in logarithmic scales. Error bars indicate standard error of the mean. In panels A and B, dotted lines indicate baseline levels, and dashed lines indicate twofold change from baseline. 6 H EOI, 6 hours post-end of infusion; C, cycle; D, day; Gz, obinutuzumab; CD45RA−CD197−, Tem; CD45RA+CD197−, Temra.

Figure 3.

Glofitamab treatment induces spatial reorganization of CD8+ T cells in tumors. Images represent CD8+ (green), Ki67+ (pink), and 4′,6-diamidino-2-phenylindole (blue) immunofluorescence analysis of 3 DLBCL core biopsy specimens at baseline (BSL; before obinutuzumab pretreatment) and during treatment (OT). OT biopsy samples were taken on the first day of the second cycle (predose) (A and C) and on the eighth day of the third cycle (B). The insets in panel A indicate proliferative tumor cores (i), CD8+ T-cell area surrounding the core (ii), and area of necrosis (iii). The inset in panel B shows tumor cells (pink) interspersed and in close contact with CD8+ T cells (green). The OT biopsy in panel C is completely necrotic.

Figure 4.

Association of baseline blood biomarkers with response to glofitamab. (A) Box plots show the baseline (pre-obinutuzumab pretreatment [Gpt]) peripheral concentrations of CD3+, CD4+, and CD8+ T cells in relation to the best overall response (BOR) categories, as measured by flow cytometry (n = 75). P values >.05. (B) Box plots represent the baseline (pre-Gpt) plasma concentrations of CRP, IL-6, and IL-8 in relation to the BOR categories (n = 72). CRP (P = .004), IL-6 (P = .07), and IL-8 (P = .06) levels were lower at baseline in patients who achieved a CR compared with other response categories (PR, stable disease [SD], progressive disease [PD]).

Figure 5.

Association of baseline tissue biomarkers with response to glofitamab. Box plots demonstrate CD20 H score (immunohistochemistry; n = 59) (A) and percentage of total CD8+ T cells (out of total cells in tumor area; immunofluorescence; n = 51) (B) in baseline tumor biopsy specimens in relation to the best overall response (BOR) categories. P values >.05. In panel A, images represent H scores of 300 (i), 175 (ii), and 59 (iii). Statistical analyses were performed for CR vs PR/stable disease (SD)/progressive disease (PD) and adjusted for log(glofitamab dose) and IPI category.

Figure 6.

Association of gene expression signatures and mutational status of baseline tumors with response to glofitamab. (A) Bar plots show the distribution of COO classes in baseline biopsy samples (analyzed by RNA-sequencing) according to the best overall response (BOR) category (P = .9). (B) Bar plots represent the distribution of the BOR categories in tumor mutational burden (TMB) low subsets (<15 mutations/Mb) and high subsets (≥15 mutations/Mb), measured by targeted-sequencing in baseline biopsy samples (P = .95). Box plots show signature scores (RNA-sequencing) of baseline biopsy samples for CD8+ effector T cells (C), PD1 high (D), MYC (E), and TP53 (F) target genes in different BOR categories. Values above 0 indicate signature enrichments in each biopsy. (G) Bar plots represent distribution of the BOR categories in TP53 wild-type (WT) and mutant (mut) subsets measured by targeted-sequencing in baseline biopsy samples (P = .09). Statistical analyses in panels B to F were performed for CR vs PR/stable disease (SD)/progressive disease (PD) and adjusted for log(glofitamab dose) and IPI category. Data in panel A and panels C to F are generated from n = 35 patients with RNA-sequencing data, and in panels B and G from n = 33 patients with targeted sequencing data. ABC, activated B-cell; GCB, germinal center B-cell.