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Simple Summary Bispecific antibodies are a very effective immunotherapy against different types of cancer since they activate T cells in the presence of tumor cells. However, they can cause severe side effects, such as a systemic inflammation called cytokine release syndrome. We aimed to clarify an important mechanism that causes cytokine release syndrome. In cocultures of T cells with endothelial cells or lymphoid cells, application of bispecific antibodies can induce T cell activation and cytokine release in the absence of tumor cells. By blocking the adhesion molecule CD18, this interaction is interrupted and the unwanted T cell activation is diminished. CD18 blockade, however, does not interfere with T cell activation when tumor cells are present. Therefore, CD18 blockade could prevent side effects of bispecific antibodies without decreasing the anti-tumor effect. Abstract T cell-recruiting bispecific antibodies (bsAbs) are successfully used for the treatment of cancer. However, effective treatment with bsAbs is so far hampered by severe side effects, i.e., potentially life-threatening cytokine release syndrome. Off-target T cell activation due to binding of bispecific CD3 antibodies to T cells in the absence of target cells may contribute to excessive cytokine release. We report here, in an in vitro setting, that off-target T cell activation is induced by bsAbs with high CD3 binding affinity and increased by endothelial- or lymphoid cells that act as stimulating bystander cells. Blocking antibodies directed against the adhesion molecules CD18/CD54 or CD2/CD58 markedly reduced this type of off-target T cell activation. CD18 blockade—in contrast to CD2—did not affect the therapeutic activity of various bsAbs. Since CD18 antibodies have been shown to be safely applicable in patients, blockade of this integrin holds promise as a potential target for the prevention of unwanted off-target T cell activation and allows the application of truly effective bsAb doses.

The introduction of monoclonal antibodies (mAbs) has largely improved treatment options for cancer patients. The ability of antitumor mAbs to elicit antibody-dependent cellular cytotoxicity (ADCC) contributes to a large extent to their therapeutic efficacy. Many efforts accordingly aim to improve this important function by engineering mAbs with Fc parts that display enhanced affinity to the Fc receptor CD16 expressed, e.g., on natural killer (NK) cells. Here we characterized the CD133 mAb 293C3-SDIE that contains an engineered Fc part modified by the amino acid exchanges S239D/I332E—that reportedly increase the affinity to CD16—with regard to its ability to induce NK reactivity against colorectal cancer (CRC). 293C3-SDIE was found to be a stable protein with favorable binding characteristics achieving saturating binding to CRC cells at concentrations of approximately 1 µg/mL. While not directly affecting CRC cell growth and viability, 293C3-SDIE potently induced NK cell activation, degranulation, secretion of Interferon-γ, as well as ADCC resulting in potent lysis of CRC cell lines. Based on the preclinical characterization presented in this study and the available data indicating that CD133 is broadly expressed in CRC and represents a negative prognostic marker, we conclude that 293C3-SDIE constitutes a promising therapeutic agent for the treatment of CRC and thus warrants clinical evaluation.