The Triomab family of trifunctional,
bispecific antibodies that maintain an
IgG-like shape are novel
tumor targeting agents. These chimeras consist of two half
antibodies, each with one light and one heavy chain, that originate from parental mouse
IgG2a and rat
IgG2b isotypes. This combination allows cost-effective
biopharmaceutical manufacturing at an industrial scale since this specific mouse/rat isotype combination favors matching of corresponding antibody halves during production by means of quadroma technology. Whereas every Triomab family member is composed of an anti-CD3 rat
IgG2b half antibody for T cell recognition, the
antigen binding site presented by the mouse
IgG2a isotype is exchangeable. Several Triomab
antibodies have been generated that bind to
tumor-associated
antigens, e.g.,
EpCAM (
catumaxomab), HER2/neu (
ertumaxomab), CD20 (FBTA05),
gangliosides GD2/GD3 (Ektomun), on appropriate
tumor target cells associated with
carcinomas,
lymphomas or
melanomas.
Catumaxomab (
Removab) was launched in Europe for treatment of malignant
ascites in April 2009. Here, we report the structural and functional characterization of this product. Mass spectrometry revealed an intact mass of 150511 Dalton (Da) and 23717 Da, 24716 Da, 51957 Da and 52019 Da of the reduced and alkylated rat light chain, mouse light chain, rat heavy chain, mouse heavy chain chains, respectively. The observed masses were in agreement with the expected masses based on the amino acid sequence obtained from
cDNA sequencing. The glycosylation profile was similar to other human
IgG consisting of biantennary
oligosaccharides with different numbers of terminal
galactose. CD spectroscopy showed mainly beta-sheets secondary structure that is typical for
IgG antibodies. Binding measurement revealed the unique trifunctional features of
catumaxomab. Other analytical tools were used to evaluate characteristics of
catumaxomab preparations, including the presence of
isoforms and aggregates.