Targeting T cells with tetravalent bispecific antibodies for the treatment of graft versus host disease

• CD3 x CD3 tetravalent antibodies cure GVHD in xenogeneic mouse models and warrant further investigation in more clinically relevant models.

Allogeneic hematopoietic stem cell transplantation is an established treatment for hematological malignancies and some genetic diseases. Acute graft versus host disease (GVHD) is the most common and debilitating side effect with poor survival rates of 5-30% for severe cases. In this manuscript, we describe a tetravalent T cell-engaging bispecific antibody (BsAb) based on the IgG-[L]-scFv platform, with all four binding domains specific for CD3. In vitro, picomolar concentrations of the CD3×CD3 BsAb induced potent lysis of activated CD4 and CD8 T cells. In immunodeficient mice, where human T cells induced xenogeneic GVHD, administration of 0.1 µg BsAb per dose depleted the majority of T cells from the peripheral blood, and 10 µg per dose completely reversed established GVHD and achieved a 100% survival rate. In mice bearing NALM6-luc xenografts, treatment with CD3×CD19 BsAb and activated human T cells induced complete remission of the leukemia and all treated mice developed GVHD by 50 days post-treatment. CD3×CD3 BsAb (3 to 30 µg doses) reversed clinical signs of GVHD, allowing long term followup beyond 250 days. T cells were undetectable by PCR in 4/5 mice in the 30 µg CD3×CD3 BsAb group 180 days after leukemia injection, and complete necropsies on day 259 revealed no evidence of human T cells or leukemia cells. Curing GVHD allows for long-term follow up of tumor response heretofore impossible in humanized mouse models. Further studies are warranted to determine if the CD3×CD3 BsAb has potential for treating clinical GVHD and other autoimmune diseases in humans.