Rat-mouse hybrid IgG

Rat-mouse hybrid IgG is a monoclonal antibody with binding sites for two different antigens, typically CD3 and a tumor antigen, making it a type of bispecific monoclonal antibody. In addition, its intact Fc-part can bind to an fc receptor on accessory cells like conventional monospecific antibodies. The net effect is that this type of drug links T cells (via CD3) and monocytes/macrophages, natural killer cells, dendritic cells or other Fc receptor expressing cells to the tumor cells, leading to their destruction. At first, monoclonal antibodies against the desired antigens are produced using mouse CHO cells. Simultaneously, monoclonal antibodies against another antigen are produced using rat CHO cells. These two CHO cells are hybridized to produce hybrid-CHOs or quadromas of hybrid (trifunctional) antibody. The trifunctional antibody is extracted and purified using protein A chromatography. Trifunctional antibodies eliminate tumor cells by means of antibody-dependent cell-mediated cytoxicity and cytotoxic T cell responses with emphasis on CD8 T cells. These trifunctional antibodies also elicit individual anti-tumor immune responses in cancer patients. The principle of trifunctional antibody activity is shown in Fig. 1. Trifunctional antibody activates T cells, and release TNF and IFN-γ cytokines. In addition, FcγR-positive cells are sent to the tumor foci, where they actively produce proinflammatory cytokines IL-6, IL-12, GM-CSF, and DC-CK1. The interaction of T cells and CD14-positive monocytes, activated via Fc/FcR, leads to increased expression of CD83, CD86 and CD40, providing T cells with an additional costimulatory signal from CD40/CD40L or CD80- CD86/CD28, which enhances the therapeutic effect of the antibodies. In such case tumor cells are effectively destroyed not only by direct attack of T cells, but through other mechanisms such as antibody-dependent cellular cytotoxicity, phagocytosis, perforin/granzyme mediated lysis, and induction of apoptosis by activated immune system accessory cells. The resulting necrotic and apoptotic tumor particles are phagocytized, processed, and presented by professional antigen-presenting cells (macrophages, dendritic cells), which is a prerequisite for long-term anticancer immunization.

Fig. 1. Principle of Triomab® antibody action. TAA: tumor-associated antigen; ADCC: antibody-dependent cellular cytotoxicity. (Adopted from: Vasilenko, E., Mokhonov, V., Gorshkova, E., Astrakhantseva, I. (2018) Bispecific Antibodies: Formats and Areas of Application. Molecular Biology. 52. 323-334.)

Formats of bispecific antibodies (BsAbs)

Many formats have been developed for BsAb generation as listed in the following table.