Prevention of Acute Graft-Versus-Host Disease in Human/Mouse Xenogeneic SCID Mouse Model by Humanized Anti-CD74 Monoclonal Antibody, Milatuzumab

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Prevention and treatment of GVHD remains a major challenge, because current T-cell depletion and mainstay immunosuppressive therapies compromise preexisting T-cell immunity, leading to severe infections and disease relapse. Thus, novel anti-GVHD agents that can spare protective T-cell memory are critically needed. Milatuzumab (hLL1) is a humanized anti-CD74 antagonist IgG1_κ monoclonal antibody (mAb) currently under clinical investigation as a therapeutic mAb for relapsed or refractory B-cell malignancies. Since CD74 is widely expressed in both hematopoietic and non-hematopoietic antigen-presenting cells (APCs), and because host APCs, especially non-hematopoietic APCs, play an important role in initiating GVHD, whereas donor APCs contribute and are required to maximize GVHD, we reasoned milatuzumab could affect recipient or donor APCs, thereby modulating GVHD. We report herein the in vitro effect of milatuzumab on the survival and function of human blood APCs and T cells, including CMV-specific T cells, and the in vivo therapeutic efficacy on preventing acute GVHD in a humanized SCID mouse model.

The effects of milatuzumab on APCs and T cells in human peripheral blood mononuclear cells (PBMCs) were assessed by multi-color flow cytometry. The effect of milatuzumab on the proliferation of T cells and T cell subsets in allogeneic mixed lymphocyte reactions (allo-MLRs) was measured by CFSE labeling of allogeneic PBMCs that were mixed to each other and cultured for 11 days before flow cytometric analysis of the proliferating cells that lost fluorescence. The impact of milatuzumab on preexisting anti-viral T-cell immunity was evaluated by intracellular staining of CD3⁺CD8⁺IFN-γ⁺T cells in allo-MLRs upon stimulation with HLA-A2-restricted cytomegalovirus (CMV) pp65 peptide (NLVPMVATV). The therapeutic effect of milatuzumab on acute GVHD was evaluated in a human PBMC-transplanted SCID mouse model, in which GVHD is developed and mediated by engrafted human T cells and DCs.

Milatuzumab moderately reduces the number of myeloid DC type 1 (mDC1), myeloid DC type 2 (mDC2), and B cells in PBMCs, but has little effect on plasmacytoid DC (pDC), monocytes, or T cells, which correlates with the level of CD74 expression on these cells. As a consequence, milatuzumab inhibits the proliferation of total T cells, CD4 and CD8 T cell subsets in allo-MLRs. In a human/mouse xenogeneic SCID mouse model, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses the serum levels of human interferon-g and IL-5, eliminates the infiltration of human lymphocytes in GVHD target organs (lung, liver and spleen), and significantly promotes the survival of animals (90% versus 20% for controls, P=0.0012). Furthermore, exposure to milatuzumab does not affect the number of CMV-specific, IFN-γ-producing, human CD8⁺ T cells in allo-MLRs.

CD74 is a potential target for antibody-mediated mitigation of GVHD, as demonstrated by the encouraging results obtained with milatuzumab. Further exploration of milatuzumab as a new therapeutic agent for GVHD is warranted.

Cardillo:Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.