Bispecific antibodies: design, therapy, perspectives

Sedykh, Sergey E; Prinz, Victor V; Buneva, Valentina N; Nevinsky, Georgy A

f two heavy-and two light-chain genes in one cell may result in expression of IgG-like molecules. This method allows the creation of a preparation of monoclonal bispecific anti-CD3/anti-EpCAM Abs (catumaxomab). 97 Quadroma technology implies fusion of two cell lines producing Abs, generating a hybrid hybridoma. In such cells, heavy and light chains of two different Abs combine, resulting in BsAbs with conventional IgG-like structure, which retain effector functions mediated by Fc. In cases of quadromas, BsAb constant regions of heavy and light chains can be of the same or different isotypes or even from different species (Triomab). 6 Several variants of quadroma-producing cell selection have been described. The first method uses two cell lines producing mAbs resistant to different antibiotics. In this case, hybrid cells carrying resistance markers to both antibiotics are selected. 98 One of the advantages of this method is the functional stability of fused nuclei in cell hybrids. Another method uses fluorescence-activated cell sorting: one hybridoma cell line is modified with fluorescein isothiocyanate, another with tetramethyl rhodamine isothiocyanate. 99 After fusion, hybrid cells carrying two fluorescent markers are selected. An electrofusion technique, which is innocuous and alternative to polyethylene glycol fusion, is also used. 100 The main problem of two-Ab coexpression (particularly quadromas) is the formation of up to nine variants of undesirable chimeric Abs along with the target BsAbs. This problem originates due to the homodimerization of heavy chains (instead of heterodimerization) and random binding of light chains to heavy chains. As a result, the significant disadvantage of this method is very low yield of target BsAbs. 2 The yield of heterodimers from two different heavy chains can be increased using the knob-in-hole method, in which one heavy chain with the T366W mutation ("knob": replacement with a sterically bulky amino acid) joins with