Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.
The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment and protect the host against infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria and induces a signaling cascade that, when exaggerated, has been associated with severe sepsis. We have generated a TLR4-specific monoclonal antibody, 15C1, which neutralizes LPS-induced TLR4 activation in a dose-dependent manner. 15C1 potently blocks the effects of LPS on a panel of primary cells and cell lines in vitro. The binding of 15C1 was mapped to an epitope in the second portion of the extracellular region of TLR4, which has been shown previously to be functionally important in the recognition of LPS. Furthermore, we demonstrate a novel mechanism of inhibition, as the effects of 15C1 are partially Fc-dependent, involving the regulatory Fc␥ receptor IIA (CD32A). In addition to introducing 15C1 as a potent clinical candidate for use in the treatment of LPS-mediated indications, our work demonstrates a newly discovered pathway whose manipulation is pivotal in achieving optimal neutralizing benefit.
Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.
Immune responses to tumor -associated antigens are often dampened by a tumor -induced state of immune anergy. Previous work has attempted to overcome tumor -induced T -cell anergy by the direct injection of vectors carrying the genes encoding one of a variety of cytokines. We hypothesised that the polyclonal stimulation of T cells, preferably through the TCR complex, would result in a cascade of cytokines associated with T -cell activation and would be best able to overcome T -cell anergy. Here we use the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, either of three membrane -bound monoclonal antibodies specific for murine TCR complex. Using this system, we have expressed antibodies specific for the CD3" chain ( KT3 ), TCR / complex ( H57 -597 ), and V7 chain ( TR310 ). Tumor cells bristling with these antibodies are capable of inducing murine T -cell proliferation and cytokine production. When injected into growing tumors ( P815, RenCa, and B16F10 ), these constructs induce the activation of immune effector cells and result in the rejection of the tumor. Histological and FACS analysis of tumor -infiltrating leukocytes reveal that the injection of recombinant virus -expressing antibodies specific for the TCR complex attracts and activates ( CD25 + , CD69 + )CD4 and CD8 lymphocytes. This approach represents a novel strategy to overcome T -cell anergy in tumors and allow the stimulation of tumor -specific T cells.
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