The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression

Abstract

Introduction: Targeting CD74 as the invariant chain of major histocompatibility complexes (MHC) became possible by the availability of a specific humanized monoclonal antibody, milatuzumab, which is under investigation in patients with hematological neoplasms. CD74 has been reported to regulate chemo-attractant migration of macrophages and dendritic cells, while the role of CD74 on peripheral naïve and memory B cells also expressing CD74 remains unknown. Therefore, the current study addressed the influence of milatuzumab on B-cell proliferation, chemo-attractant migration, and adhesion molecule expression.

Methods: Surface expression of CD74 on CD27- naïve and CD27+ memory B cells as well as other peripheral blood mononuclear cells (PBMCs) obtained from normals, including the co-expression of CD44, CXCR4, and the adhesion molecules CD62L, β7-integrin, β1-integrin and CD9 were studied after binding of milatuzumab using multicolor flow cytometry. The influence of the antibody on B-cell proliferation and migration was analyzed in vitro in detail.

Results: In addition to monocytes, milatuzumab also specifically bound to human peripheral B cells, with a higher intensity on CD27+ memory versus CD27- naïve B cells. The antibody reduced B-cell proliferation significantly but moderately, induced enhanced spontaneous and CXCL12-dependent migration together with changes in the expression of adhesion molecules, CD44, β7-integrin and CD62L, mainly of CD27- naïve B cells. This was independent of macrophage migration-inhibitory factor as a ligand of CD74/CD44 complexes.

Conclusions: Milatuzumab leads to modestly reduced proliferation, alterations in migration, and adhesion molecule expression preferentially of CD27- naïve B cells. It thus may be a candidate antibody for the autoimmune disease therapy by modifying B cell functions.

Figure 1

Surface expression of CD74, CD44, and CXCR4 on T cells, monocytes, and B cells. (A) Detection of CD74 with a commercially available FITC-labeled anti-CD74 antibody (n = 8) and PE-labeled milatuzumab (n = 9) on T cells, monocytes, and B cells. For each staining, representative histograms, including an isotype control or blocking experiment, are shown. Competitive blocking experiments were performed by using unlabeled milatuzumab (20-fold concentration). Significant differences were observed between the CD74 expression levels of T cells, monocytes, and B cells (Wilcoxon test), and specificity of the staining was confirmed. (B) Detection of CD74 (n = 10), CD44 (n = 8), and CXCR4 (n = 12) on CD27^- naïve and CD27⁺ memory B cells. These surface molecules showed a distinct expression profile between these B-cell subpopulations (Wilcoxon test). **P ≤ 0.01; ***P ≤ 0.001. BD, BD Biosciences; FITC, fluorescein isothiocyanate; MFI, (geometric) mean fluorescence intensity; PE, phycoerythrin.

Figure 2

Effects on proliferation of CD19⁺ B cells and macrophage migration inhibitory factor (MIF) concentration in vitro by milatuzumab. (A) Frequency of proliferated CD19⁺/CD3 ^-/CD14^- B cells according to their carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity. CFSE-labeled peripheral blood mononuclear cells were cultured for 7 days with or without milatuzumab or intravenous immunoglobulin (IVIG) at 37°C in 5% CO₂ and simultaneously stimulated with IL-2, IL-10, F(ab)₂, and CpG (n = 6). Addition of milatuzumab as well as IVIG resulted in a modest, but significant, inhibition of the proliferation (Wilcoxon test). For each condition, a representative histogram is shown. (B) The concentration of the chemokine MIF as a potential ligand of CD74 was tested in cell culture supernatants (n = 7), as described above, and showed no significant differences between the conditions (Wilcoxon test). (C) Proportion of dead CD19⁺ B cells, identified as high positive staining with DAPI (n = 3). There was no substantial influence observed by either IVIG or milatuzumab (Wilcoxon test). *P ≤ 0.05. CpG, cytosine-phosphatidyl-guanosine; DAPI, 4,6 diamidino-2-phenylindole; F(ab)₂, protein of two antigen-binding fragments; IL, interleukin; ns, not significant.

Figure 3

Influence of milatuzumab on the migration characteristics of CD19⁺ B cells and subsets in vitro. (A) Spontaneous baseline migration of B cells in a transwell migration assay. Peripheral blood mononuclear cells were incubated for 90 minutes at 37°C in 5% CO₂ with or without milatuzumab (n = 8) or IgG1/IVIG (n = 6) and allowed to migrate without any chemokine added. Percentages of migrated cells are shown. Migration was significantly enhanced (Wilcoxon test) by milatuzumab in CD19⁺ B cells, especially in CD27^- naïve B cells. (B) Effective CXCL12-dependent net migration determined for each individual by subtraction of the baseline migration - see (A) - from the total migration toward CXCL12 (not shown). A significant enhancement of the migration of CD19⁺ B cells, especially of CD27^- B cells by milatuzumab, was identified (Wilcoxon test). In control experiments, IgG1/IVIG had no influence on migration. *P ≤ 0.05; **P ≤ 0.01. CXCL, CXC motif ligand; IVIG, intravenous immunoglobulin; ns, not significant.

Figure 4

Surface expression of CD44, β7-integrin, and CD62L on CD19⁺ B cells was preferentially reduced by milatuzumab on CD27^- naïve B cells. (A) Surface expression of CD44 after incubation of peripheral blood mononuclear cells for 90 minutes at 37°C in 5% CO₂ with or without milatuzumab (n = 7) or IgG1/IVIG (n = 7). Milatuzumab led to a significant reduction of the expression of CD44 on CD19⁺ B cells, especially on CD27^- naïve B cells (Wilcoxon test). (B) Expression of β7-integrin was significantly reduced (Wilcoxon test) on CD19⁺ B cells, mainly confined to CD27^- naïve B cells after milatuzumab incubation (n = 8), but not influenced by IgG1/IVIG (n = 7). (C) Milatuzumab (n = 9) significantly reduced the expression of CD62L (Wilcoxon test) on CD19⁺ B cells, especially on CD27^- naïve B cells, whereas IgG1/IVIG (n = 8) did not modify this expression. The expression of the surface molecules CD44, β7-integrin, and CD62L on CD27⁺ memory B cells was not influenced by milatuzumab. *P ≤ 0.05; **P ≤ 0.01. IVIG, intravenous immunoglobulin; ns, not significant.