Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting， with antibodies such as rituximab， to blocking of immune checkpoints to reactivate antitumor immunity. In addition， bispecific antibodies/antibody fragments are also of great interest in cancer therapy， as these constructs have the ability to redirect immune effector cells to cancer targets and， thereby， enhance therapeutic efficacy. A number of bispecific antibody formats have been reported， with the first FDA-approved bispecific antibody being blinatumomab， a so-called bispecific T cell engager (BiTE)， which redirects and potently activates T cell immune responses. Recently， we described an additional novel bispecific antibody derivative， termed RTX-CD47， which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here， we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.