Anti-CD73 antibody activates human B cells, enhances humoral responses and induces redistribution of B cells in patients with cancer

Abstract

Background: CD73 is widely expressed on immune cells playing a critical role in immunomodulatory functions including cell adhesion and migration, as a costimulatory molecule for T cells and in production of adenosine. The function of CD73 expressed on B cells has not been fully characterized. Mupadolimab is an anti-human CD73 antibody that activates B cells. We evaluated the characteristics of this antibody and its effects on immune cells in vitro and in vivo.

Methods: Mupadolimab binding to CD73, inhibition of CD73 enzymatic activity, and effects on lymphocyte activation were evaluated in vitro by measuring changes in immunophenotype by flow cytometry. Cryogenic-transmission electron microscopy was used to determine epitope binding. Effects on human B cells in vivo were evaluated in immunodeficient NSG-SGM3 mice immunized with SARS-CoV-2 and influenza viral antigens. Safety and immune effects were evaluated in the completed dose escalation portion of a phase 1 trial conducted in patients with cancer.

Results: Mupadolimab binds to a unique epitope on CD73^POS B cells resulting in their activation and differentiation through B cell receptor signaling pathways. Mupadolimab induces expression of CD69, CD83, CD86 and MHC class II on B cells along with morphological transformation into plasmablasts and expression of CD27, CD38 and CD138. These effects are independent of adenosine. Mupadolimab binds to the N-terminal of CD73 in the closed position and competitively inhibits substrate binding. Mupadolimab enhanced antigen specific antibody response to SARS-CoV-2 spike protein and influenza hemagglutinin in humanized mouse models. Mupadolimab was evaluated as a monotherapy in a phase 1 trial (NCT03454451) in 34 patients with advanced cancer and demonstrated binding to CD73^POS circulating cells and transient reduction in the number of B cells, with return of CD73^NEG B cells with memory phenotype. No dose-limiting toxicities or changes in serum immunoglobulins were seen.

Conclusions: Mupadolimab activates B cells and stimulates the production of antigen specific antibodies. The effects in patients with cancer suggest that activated, CD69^POS B cells redistribute to lymphoid tissues. Minor tumor regression was observed in several patients. These results support further investigation of mupadolimab as an immunotherapy for cancer and its potential use as a vaccine adjuvant.

Trial registration number: NCT03454451.

Keywords: B-lymphocytes; immunomodulation; immunotherapy.

Figure 1

Mupadolimab blocks CD73 enzymatic activity and restores T cell proliferation and cytokine production in an AMP-mediated immunosuppressive environment. (A) CD73 catalytic activity was measured with MDA-MB-231 cells or MDA-MB-231 CD73 CRISPR cells in the presence of mupadolimab, MEDI9447, or 1 mM APCP by adding 250 mM AMP and measuring phosphate levels in the cell culture supernatant. (B) Human PBMCs were incubated with AMP in the presence of mupadolimab, MEDI9447, or isotype control antibody at the saturating concentration of 300 nM. After 4 hours of incubation, the AMP remaining in the supernatant was quantified using the CellTiterGlo assay as described in the methods. Percentage of inhibition was plotted. Data represent nine individual donors and error bars represent mean±SD ****p<0.0001 as determined by t-test. (C) T cell proliferation for 13 donors and (D) IFN-γ production for 6 donors treated with 500 nM mupadolimab or MEDI9447 or 890 nM isotype control. Data for (C) and (D) normalized across donors. Error bars represent mean±SD *p<0.05, ***p<0.001, ****p<0.0001 as determined by t-test. APCP, adenosine 5’-(α,β-methylene) diphosphate; PBMCs, peripheral blood mononuclear cells.

Figure 2

Cryo-TEM analysis of CD73 and mupadolimab complex. (A) Represented 2D classification results of CD73-mupadolimab 2:2 complex. (B) The interaction between mupadolimab CDR and CD73. The heavy chain and light chain of CDR are shown in is blue and pink, respectively. CD73 is colored in wheat. The calcium ions are shown as green sphere. (C) 3D models of CD73 and the Fab region of the antibody based on density map. The N- and C-terminal domain of the CD73 dimer were shown in gray and wheat, respectively. The mupadolimab Fab is shown in green. Residues involved in the catalytic site of CD73 are shown in red. Due to the high flexibility and low density of the half of the structure, the constructed model only contains one Fab molecule.

Figure 3

Mupadolimab directly activates B lymphocytes and induces maturation into antibody secreting plasmablasts in vitro. (A) Purified B cells from 3 to 5 healthy donors were incubated overnight with human IgG₁ isotype control or mupadolimab (10 μg/mL) or anti-IgM microbeads, a positive control for BCR stimulation. Expression of activation markers CD69, CD83, CD86, or MHC-II was measured on viable cells by flow cytometry. Error bars represent mean±SD *p<0.05, **p<0.01, and ****p<0.0001 as determined by t-test. (B) Time dependent increases in the expression of CD27, IgG, IgM, CD38, and CD138 on purified B cells cultured in the presence of mupadolimab or isotype control (1 µg/mL). Mean fluorescence intensity (MFI) was determined for each marker and was normalized to the untreated control for each donor. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by two-way ANOVA. (C) B cell activation is unique to mupadolimab as other anti-CD73 antibodies do not induce CD69 expression. Expression of CD69 (MFI) was measured by flow cytometry. (D) Representative images of purified B cells cultured with isotype control (left panel) or mupadolimab for 2 days. (E) Purified B cells were incubated overnight with 10 μg/mL human IgG₁ isotype control or mupadolimab or equimolar mupadolimab Fab. CD69 and CD73 were measured on B cells by flow cytometry. (F) Human PBMCs were incubated overnight with a fixed concentration of mupadolimab (10 μg/mL) along with ibrutinib or vehicle control over a range of concentrations. Expression of CD69 on B cells (CD19^POSCD3^NEG) was measured by flow cytometry. (G) Human PBMCs from healthy donors (n=2) were incubated for 15 min with 10 μg/mL human IgG₁ isotype control, mupadolimab, or anti-IgG+anti IgM, a positive control for BCR stimulation. Phospho-ERK was measured by flow cytometry on B cells, CD4+T cells, and CD8+T cells. (H) Human PBMCs were incubated overnight with 10 μg/mL mupadolimab with or without NECA over a range of concentrations. expression of CD69 on B cells (CD19^POSCD3^NEG) was measured by flow cytometry and MFI is reported. ANOVA, analysis of variance; BCR, B cell receptor; NECA, 5’-(N-Ethylcarbox-amido) adenosine.

Figure 4

Mupadolimab activates human B cells to secrete immunoglobulin and cytokines associated with B cell activation. (A) Human PBMCs were incubated for 6 days with 1 mg/mL human IgG₁ isotype control or mupadolimab. IgG₁ and IgM secreted into the culture supernatant was quantified by ELISA. IgG-k was not measured as addition of mupadolimab, an IgG1-k antibody, would have confounded the results. Data are represented as mean±SEM of 6 independent experiments, each in duplicate. Purified B cells were incubated for 5 days with 10 µg/mL human IgG₁ isotype control or mupadolimab or anti-IgM microbeads, a positive control for BCR stimulation. Concentration of CCL2 (B), CCL3 (C), CCl4 (D) and CCL22 (E) in supernatant were measured by ELISA. Two representative donors are reported and data are represented as mean±SD of duplicate measurements. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by two-tailed, unpaired t-test.

Figure 5

Mupadolimab enhances antigen-specific responses in preclinical animal model. NSG-SGM3 mice were immunized with an emulsion of full-length spike protein from SARS-CoV-2 plus Freund’s incomplete adjuvant. Mice were then randomized into two treatment groups: half the animals (n=4) were dosed daily (i.p.) with 10 mg/kg mupadolimab, and half the animals (n=4) were dosed daily with 10 mg/kg hIgG₁. Animals were cheek bled on days 1 (pre-treatment), 4, 8, 11, and 15 to assess anti-spike immunoglobulin (A), anti-nucleocapsid immunoglobulin (B) as negative control overtime by ELISA. (C) NSG-SGM3 mice were immunized with an emulsion of influenza H1N1 hemagglutinin plus Freund’s incomplete adjuvant. Anti-influenza antibody level was assessed overtime by MSD platform after treatment with mupadolimab, hIgG₁, or PBS. Error bars represent mean±SD. P values are shown whenever results achieved statistical significance. PBS, phosphate buffered saline.

Figure 6

B cell dynamics in patients with advanced cancer treated with mupadolimab. (A) Mupadolimab induces rapid change in peripheral blood B cells, returning to pretreatment baseline by day 21. Results are shown as fold change from baseline for patients receiving up to 12 mg/kg. Results between 0.5 hour and 21 days were statistically significant at 3 mg/kg (*p<0.05) and 12 mg/kg (**p<0.01). (B) An increased frequency of memory B cells (CD27^POS IgD^NEG) is observed at doses ≥3 mg/kg. Each symbol represents a treated patient, with lines connecting paired samples. (C) Representative examples of BCR repertoire diversification in 3 patients with cancer 21 days after mupadolimab treatment with 12 mg/kg (RCC patient) or 18 mg/kg (patients with colorectal cancer). The Y axis represents clones increased in frequency and the X axis indicates clones that decreased in frequency. Red dots indicate clones that were significantly expanded or exclusively observed following treatment with mupadolimab. BCR, B cell receptor; RCC, renal cell cancer.

Figure 7

Mupadolimab antitumor activity. The waterfall plot shows the best change in the sum of the longest dimensions (SLD) for each patient.