Blockade of the CD47/SIRPα checkpoint axis potentiates the macrophage-mediated antitumor efficacy of tafasitamab

Abstract

Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47, on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamab- mediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy control lymph nodes. CRISPR-mediated CD47 overexpression affected tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. A combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro-generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor-targeting immunotherapies such as tafasitamab and suggest that there is value in exploring the combination in the clinic.

Figure 1.

Increased expression of CD47 and SIRPα in diffuse large B-cell lymphoma. (A) Expression of CD47 on B cells (CD19⁺) was quantified using confocal microscopy on tonsils as benign controls (white circles) (N=7) or diffuse large B-cell lymphoma (DLBCL) specimens (red circles) (N=9). (B) SIRPα on macrophages (CD68⁺) was quantified using confocal microscopy on tonsils as benign controls (white circles) (N=7) or DLBCL specimens (red circles) (N=9). Regions of interest matching B cells or macrophages were segmented using Zeiss-software (ZEN 2.6) or the open-source software QuPath (https://qupath.github.io), and the mean fluorescence intensity of CD47 or SIRPα was assessed for each region of interest. Data for 25 cells from each donor were plotted for CD47 or SIRPα. Lines show the mean value. (C, D) Bone marrow biopsies from healthy controls (N=8, white circles) or DLBCL patients with tumor infiltration (N=9, red circles) were analyzed for (C) the expression of CD47 on B cells/lymphoma cells (CD19⁺/ CD20⁺) or (D) SIRPα on macrophages/lymphoma-associated macrophages (CD163⁺/CD15^–). The graphs (C, D) show the result of five independent experiments. Each dot represents a tested donor. A nonparametric Mann-Whitney U test was performed. Lines show the mean value. MFI: mean fluorescence intensity; BM: bone marrow; LAM: lymphoma-associated macrophages.

Figure 2.

The CD47-SIRPα axis impairs tafasitamab-mediated phagocytosis. (A) In vitro-differentiated macrophages were analyzed for SIRPα expression by flow cytometry before they were used for assays of antibody-dependent cellular phagocytosis (ADCP) (N=13). Tafasitamab-induced phagocytosis of HT, Toledo and U2946 cell lines was correlated with SIRPα expression on macrophages. A simple linear model was fitted to the data and the R value was calculated. (B) The CRISPR/Cas9 system was used to alter gene expression in Toledo cells. CD47 expression on CD47 knock out (CD47^KO, light gray), CD47 overexpressing (CD47^high, red) and wild-type Toledo (control, dark gray) cells, determined by flow cytometry (N=5). The dotted line represents the isotype control. MFI is defined as median fluorescence intensity. The bar chart shows average MFI values of three independent measurements. Error bars show the standard error of the mean. A two-tailed paired Student t test was performed. (C) Tafasitamab-mediated phagocytosis of control, CD47^KO or CD47^high cells measured by flow cytometry. Lines show the mean value. The graph shows the result of five independent experiments. A two-tailed paired Student t test was performed. (D) Immunofluorescence of in vitro-differentiated macrophages co-cultured with different cell lines (control, CD47^KO, CD47^high) and tafasitamab (1 µg/mL) for 3 h. Lymphoma cells lines were stained with Cytolight Rapid Green Dye (green), macrophages were stained with CD11b-APC (red). The microscopy images show representative sections of five experiments. (E) Quantification of phagocytosis of Toledo cells (control, dark gray) and the genetically modified cells (CD47^KO: light gray; CD47^high: red) by confocal microscopy. One hundred macrophages per condition were counted and the percentage with ingested lymphoma cells was calculated. The graph shows the result of five independent experiments. A two-tailed paired Student t test was performed. Lines show the mean value. Scale bar: 20 µm.

Figure 3.

Blocking CD47 increases tafasitamab-mediated phagocytosis. (A) For quantification of phagocytosis of different diffuse large B-cell lymphoma (DLBCL) cell lines (Toledo, U2946, HT) by confocal microscopy, 100 macrophages per condition were counted and the percentage with ingested lymphoma cells was calculated. Lines show the mean value. The graphs show the results of five independent experiments. A two-tailed paired Student t test was performed. (B) Percentage phagocytosis of lymphoma cell lines using in vitro-differentiated macrophages as effector cells, in the presence or absence of tafasitamab and anti-CD47 monoclonal antibody. Phagocytosis was measured with flow cytometry. The diagrams show the results of several independent experiments (Toledo: N=11; U2946: N=23; HT: N=12). A two-tailed paired Student t test was performed. Lines show the mean value. (C) Phagocytosis of lymphoma cell lines in the presence or absence of tafasitamab and/or anti-CD47 monoclonal antibody for 6 h. Incubation and measurements were performed in the absence (white circles) or presence of tafasitamab (dark gray circles) or anti-CD47 (light gray circles) or a combination of both (red circles). Measurements and analysis were conducted using an Incucyte^® Live Cell Imaging system microscopy and Incucyte^® 2022A software. Total red signal per image was evaluated for all conditions and timepoints. The course of total red signal is shown in the plots for three cell lines. The graph shows a representative result of three independent experiments. Ab: antibody; mAb: monoclonal antibody; RCU: red mean intensity object average.

Figure 4.

Blockade of CD47 enhances tafasitamab-mediated phagocytosis of lymphoma-associated macrophages. (A) Schematic illustration of flow cytometric isolation of lymphoma-associated macrophages (LAM) and primary lymphoma cells from bone marrow (BM) (N=11) and lymph nodes (LN) (N=6) of patients with diffuse large B-cell lymphoma (DLBCL). (B) Representative gating strategy and fluorescence activated cell sorting plots for determination of phagocytosis. Isolated lymphoma cells were stained with CPD and co-cultured with isolated LAM (gate R1). Macrophage effector cells were counterstained with an anti-CD11b antibody and absolute numbers of remaining CD11b^–/CPD⁺ lymphoma cells were determined using counting beads (gate R2). (C) Phagocytosis of primary lymphoma cells by LAM isolated from DLBCL patients’ BM (left graph) or LN (right graph), in the presence or absence of tafasitamab and anti-CD47 monoclonal antibody. Phagocytosis was measured by flow cytometry. The graphs show the results of several independent experiments (LAM BM: N=11; LAM LN: N=6). A two-tailed paired Student t test was performed. Lines show the mean value. SSC: side scatter; FSC: forward scatter; Ab: antibody; mAb: monoclonal antibody.

Figure 5.

A combination of tafasitamab and anti-CD47 increases the elimination of CD19^low-expressing lymphoma cells. (A) CD19^low (blue histogram) or CD19^high (red histogram) Toledo cells were isolated by flow activated cytometric cell sorting (FACS). (B, C) Sorted cell populations were immediately incubated with in vitro-differentiated macrophages and phagocytosis in the presence or absence of tafasitamab and/or anti-CD47 was determined after 3 h by flow cytometry (B) or confocal microscopy (C). The graphs in (B) summarize the results of eight macrophage donors tested by FACS in eight independent experiments. A two-tailed paired Student t test was performed. Lines show the mean value. (C) CD19^low and CD19^high Toledo cells were stained with Cytolight Rapid Green Dye (green), macrophages were stained with CD11b-APC (red) and phagocytosis was analyzed by confocal microscopy. Images were taken after washing steps, removing non-adherent and/or non-phagocytosed cells. The microscopy images show representative sections of five experiments performed. Scale bar: 20 µm. (D) For quantification of phagocytosis of sorted CD19^low (upper panel) or CD19^high (lower panel) Toledo cells by confocal microscopy, 100 macrophages per condition were counted and the percentage with ingested lymphoma cells was calculated. The graphs show the result of five independent experiments. A two-tailed paired Student t test was performed. Lines show the mean value. MFI: mean fluorescence intensity; Ab: antibody; mAb: monoclonal antibody;

Figure 6.

Combination treatment with tafasitamab and anti-CD47 antibody prolongs survival and decelerates tumor growth in mice. (A) Schematic illustration of the disseminated xenograft model. Ramos cells were injected intravenously on day 0. Treatment with tafasitamab, anti-CD47 (clone B6H12) and the combination commenced on day 5 and was continued until day 21, followed by continuous monitoring of animals for signs of morbidity until day 99. (B) Kaplan–Meier plot of the disseminated survival model following treatment with tafasitamab, CD47 monoclonal antibody or the combination. Tafasitamab vs. control: P<0.0001, anti-CD47 vs. control: P<0.0001, tafasitamab vs. tafasitamab + anti-CD47: P<0.0001, anti-CD47 vs. tafasitamab + anti-CD47: P=0.035. (C) Schematic illustration of the flank xenograft model. Ramos cells were injected subcutaneously into the right flank of each mouse on day 0. Treatment with tafasitamab, anti-CD47 (clone B6H12) monoclonal antibody was started and continued for up to 4 weeks; the study terminated on day 55. (D) Spider plots showing tumor growth curves for individual animals (N=15) for each treatment group. (E) Kaplan-Meier plot showing animal survival until a final tumor volume of 1,500 mm was reached. Tafasitamab vs. control: 0.0095, anti-CD47 vs. control: P<0.0001, tafasitamab vs. tafasitamab + anti-CD47 monoclonal antibody: P<0.0001; anti-CD47 vs. tafasitamab + anti-CD47: P=0.0017.