CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model

Abstract

Background: CD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model.

Methods: The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments.

Results: We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells.

Conclusions: The study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).

Keywords: B-cell lymphoma; Bispecific antibody; CD47; Cancer immunotherapy; Dendritic cells; Macrophages; NK cells; Tumor microenvironment.

Conflict of interest statement

All authors are current or former employees of Light Chain Bioscience/Novimmune SA, excepted DC. GD is presently employee of Ichnos Sciences Biotherapeutics SA. All the authors declare that their employment does not alter their adherence to the policies of Cancer Immunology, Immunotherapy journal.

© 2022. The Author(s).

Figures

Fig. 1

NI-1701 modulates the tumor microenvironment by promoting accumulation of immune cells associated with anti-tumorigenic functions and triggers enhanced engulfment of tumor cells by TAMs and monocytes. Immunodeficient NOD SCID mice were implanted with Raji GFPhi lymphoma tumor cells and treatment was initiated when tumor volume reached 100 mm3. Tumors were excised 14 and 25 days after treatment initiation to evaluate impact of NI-1701 on the tumor microenvironment. a Design of the experiment. b Mean tumor volume at days 14 and 25 after treatment with human IgG1 isotype control Ab or NI-1701. Data are presented as mean ± SD, with n = 7–9 mice per group. c Analysis by flow cytometry of tumor-infiltrating total mouse leukocytes, myeloid cell subsets (TAMs, monocytes, G-MDSCs, dendritic cells) and NK cells. Gating strategy to identify subpopulations in the TME is displayed in Additional file 1: Fig. S1. Data are presented as mean ± SD, with n = 4–9 mice per group. d Analysis by flow cytometry of MHCII+ TAMs. e Representative flow cytometry plots of phagocytic TAMs or monocytes, determined as F4/80+GFP+ or Ly6C+GFP+ respectively, from Raji GFPhi tumors dissected 14 days after treatment initiation. The threshold of GFP positivity and GFP internalization in TAMs and monocytes were determined based on methods and analysis depicted in supplementary experimental procedures. f The percentage of phagocytosis of GFPhi Raji tumor cells by TAMs and monocytes, assessed at day 14 and day 25 post-treatment initiation, is shown for each individual mouse. Data are presented as mean ± SD, with n = 4–9 mice per group. Significance was determined by unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Targeted transcriptomic analysis of tumor-infiltrating mouse leukocytes reveals upregulation of proinflammatory cytokine and chemokine genes upon NI-1701 treatment. Mouse CD45+ tumor-infiltrating leukocytes were sorted from tumors 14 days after treatment initiation with NI-1701 or hIgG1 control and submitted to Nanostring transcriptomic analysis using the mouse Myeloid Innate Immunity panel (n = 4 mice per group) a Volcano plot of differentially expressed genes in tumor-infiltrating leukocytes between hIgG1 versus NI-1701. Vertical grey lines correspond to absolute log2 fold change > 0.76 (i.e., fold-change > 1.7) and horizontal dot line correspond to a p value of p = 0.05. Grey zone corresponds to statistically non-significant changes and red and blue dots to significantly upregulated and downregulated genes, respectively. b Heatmap depicting row z scores of normalized mRNA count of significantly up-regulated and down-regulated genes with fold change > 1.7

Fig. 3

Both macrophages and NK cells are important effector cells for NI-1701 antitumor efficacy in Raji lymphoma xenograft model. a Design of the macrophage depletion experiment. b Tumor volume follow-up of CB17-SCID mice carrying Raji tumors treated with i.p. injection of NI-1701 and i.v. injection of clodronate liposomes. n = 5 mice per group. c Design of the NK cell depletion experiment. d Tumor volume follow-up of NOD SCID mice carrying Raji tumors treated with i.v. injection of NI-1701 and i.p. injection of anti-Asialo GM1 antibody. n = 8 mice per group. For b and d mean ± SEM is shown. Significance was determined at endpoint by one-way ANOVA test. *p < 0.05, ***p < 0.001, ****p < 0.0001

Fig. 4

NI-1701 induces tumor cell phagocytosis by bone-marrow-derived dendritic cells and promotes cross-priming of CD8+ T cells. Bone marrow-derived mouse dendritic cells (CD11c+) from BALB/c mice were cocultured for 2 h 30 (a) or 24 h (b) with Raji GFPhi tumor cells (1:1 ratio) in the presence of hIgG1 or NI-1701. Representative flow cytometry plots for hIgG1 and NI-1701 treated groups are depicted, and the percentage of phagocytosis is indicated (left panel). Phagocytic events were confirmed by imaging flow cytometry acquisition in the CD11c+GFP+ gate with tumor cells in green fluorescence and CD11c+ dendritic cells in red fluorescence (middle panel). The mean percentage of phagocytosis at 2 h 30 ± SD is shown (right panel), n = 5 independent experiments. b Percentage of residual Raji GFPhi tumor cells after 24 h of coculture with CD11c+ DCs was determined by the analysis of remaining GFP+ tumor cells within the total live cell gate. n = 5 experiments. c Bone-marrow derived dendritic cells from BALB/c mice were cocultured overnight with hemagglutinin-expressing Raji tumor cells (Raji HA-GFP) in the presence of human IgG1 or NI-1701 (ratio 1:1). The next day, HA-specific CD8+ T cells from CL-4 transgenic mice (bearing HA-specific TCR on CD8+ T cells) labelled with CellTrace violet were added (ratio 1:5). Analysis of CD8+ T cell proliferation was performed 3 days later. Plots represent an illustration of proliferating CD8+ T cells (left panel). Mean ± SD was calculated from 4 independent experiments (right panel). Significance was determined by unpaired t test. *p < 0.05, ****p < 0.0001