In laboratories, bispecific antibodies have been widely used for the retargeting of immune effector cells to tumor cells. In this case, one binding site is directed against a tumor-associated antigen (TAA) and the second antigen against a trigger molecule on the effector cells, such as for example CD3 on T cells (Kontermann, MABS 2012 (4) 182-197; Chames and Baty, MABS 2009 (1) 539-547; Moore et al. Blood 2011 (117) 4542-4551). The first bispecific antibodies targeting CD3 and a tumor cell associated antigen were of rodent nature and were produced using hybrid hybridomas (Liu et al. 1985 PNAS 82: 8648, Staerz et al. 1986 PNAS 83:1453, Lanzavecchia et al. 1987, Eur. J. Imm. 17:105). In these hybrid hybridomas the reassortment of Ig heavy and light chains resulted in the production of bispecific functional antibody molecules within a much larger pool of monospecific and non-functional bispecific antibodies resulting from heavy and light chain mispairing. Because of their double specificity, these functional bispecific antibodies were able to bridge murine and human cytotoxic T lymphocytes (CTL) to target cells and trigger cytotoxic function resulting in the lysis of tumor cells displaying the relevant antigen. However, the CD3×TAA bispecific IgG mediated induction of tumor cell lysis by polyclonal resting human T cells could not be achieved unless co-stimulation was provided by added exogenous IL-2 or anti-CD28 mAb. This is exemplified by the hybrid rat IgG2b/mouse IgG1 CD3×CD19 bispecific molecule that was able to induce lysis of the CD19 positive REH B-ALL tumor cell line by resting human T lymphocytes only upon co-administration of IL-2 (Haagen et al. 1995 Blood 85:3208). Zeidler et al. demonstrated using a similar rat IgG2b/mouse IgG2a CD3×Epcam molecule that bispecific IgG-induced lysis of Epcam-positive tumor cells could be achieved in mixed cell cultures comprising both peripheral blood mononuclear cells (PBMC) and tumor cells without addition of exogenous IL2 (Zeidler et al. 1999 J. Immunol. 163:1246). The authors claimed that the ‘third’ arm of the antibody, the Fc region, is causing this effect through interaction with Fcγ receptor-positive accessory cells present within the PBMC fraction. In particular, the strong activation potential was correlated to the hybrid subclass combination mouse IgG2a/rat IgG2b that, in contrast to other reported combinations (e.g., mouse IgG2a/mouse IgG1 or rat IgG2b/mouse IgG1), not only binds but also activates Fcγ receptor-positive accessory cells. This so-called triomAb CD3×Epcam bispecific antibody, also known as catumaxomab, has been developed clinically and has been registered in Europe for palliative treatment of abdominal tumors of epithelial origin. While this bispecific antibody has clearly demonstrated clinical efficacy, its rodent nature induces anti-product immune responses upon repetitive dosing and therefore prevents a widespread application of this format.
Alternative CD3×TAA formats have been explored to solve both the manufacturing issues and the immunogenicity problems associated with the hybrid rodent triomAb format. Such formats are often immunoglobulin-like molecules that deviate from full length human IgG molecules, and include molecules such as Dual-Affinity Re-Targeting (DART™) molecules that are developed by Macrogenics worldwide web at macrogenics.com/Platforms-DART.html, Bispecific T cell Engager (BiTE®) molecules that were developed by Micromet, now Amgen (Sheridan C, Nat Biotechnol. 2012 (30):300-1), Dual Variable Domain—immunoglobulin (DVD-Ig™) molecules that are developed by Abbott, and TandAb® RECRUIT molecules that are developed by Affirmed world wide web at affimed.com/tandab-recruit. It was demonstrated for one of these formats that successful retargeting of peripheral blood lymphocytes to lyse CD19-positive tumor cells using a CD3×CD19 diabody required pre-activation the of the peripheral blood T lymphocytes, now using anti-CD3 antibody plus human IL-2 (Kipriyanov et al. 1998 Int. J. Can. 77:763). Other formats, such as the bivalent single chain Fv CD3×TAA BiTE® format (Loffler et al. 2000 Blood 95:2098) do not require pre-activation of resting T cells and is able to induce antigen positive tumor cell lysis in vitro in an extremely efficient manner (Dreier et al. 2002 Int. J. Canc. 100:690). Additional studies using BiTE®s targeting different TAAs revealed that the potent efficacy of the BiTE® format was correlated to the antigen size and particularly to the distance of the epitope on the TAA to the tumor cell membrane (Bluemel et al. 2010 Cancer Immunol. Immunother. 59:1197). The effective formation of cytolytic T cell synapses was demonstrated for BiTE® molecules which is explained to form the structural basis for their potency (Offner et al. Molecular Immunology 2006 (43) 763-771) which is also believed to be linked to the small size of the BiTE® format. If size matters, this would suggest that larger molecules such as intact IgG would be too large to form effective cytolytic synapses. The CD3×CD19 BiTE®, blinatumomab, has demonstrated remarkable clinical efficacy in refractory non-Hodgkin lymphoma and acute lymphatic leukemia patients (Bargou et al. 2008 Science 321:974). Although the CD3×CD19 BiTE® displays very efficient tumor cell lysis at low levels in vitro, administration of this bispecific format to patients is associated with significant challenges. Due to their small size, BiTE®s are rapidly cleared from the circulation and dosing of patients thus requires continuous infusion. As the dosing regimen has an overall duration of more than 2 months, this treatment has a significant impact on the quality of life of the patients.
There thus remains a need for effective full length bispecific T cell engaging IgG molecules in eradicating aberrant cells that combine a long circulatory half-life upon intravenous administration without the need for continuous infusion without being immunogenic and with only limited side effects.