Page 82 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 5
5.1. Introduction
Bispecific antibodies, i.e. antibodies with two structurally distinct CDRs each of which binds monovalently to a different antigen, have the potential to increase binding affinity,1 improve internalization of antibody-based drugs,2, 3 or redirecting cytotoxic T-cells for increased immune response near the target cell.4, 5 Bifunctional antibodies, by comparison, are antibodies that retain the original bivalent binding fashion of a traditional antibody, however feature an additional protein module for additional functionality, for example an IL-2 fragment for T-cell activation.6 A significant amount of research regarding bispecific and bifunctional antibodies has been performed in recent years, with architectures ranging from hetero-dimerized Fab-domains to C-terminally fused scFv groups.7, 8 Recently, we reported the generation of monovalent antibody-scFv and antibody-cytokine conjugates from knob-in-hole antibodies combined with SPOCQ or sortase ligation (chapter 4).9 Alongside this work, divalent antibody-scFv and antibody- cytokine conjugates were achieved based on sortase ligation.10, 11
Trifunctional antibodies can be considered the next step for enhanced tumor targeting and/or payload delivery.8 Amongst trifunctional constructs are trispecific antibodies capable of binding to three different antigens, which have shown promising activity to combat HIV by binding multiple HIV glycoproteins.12 They can be generated via a plethora of methods such as the fusion of a single single-chain variable fragment (scFv) to a bispecific antibody (OrthoTsAb),13 or by the fusion of two unique scFv units to the light chains (LC) or heavy chains (HC) of a monospecific antibody.14 Similarly, trifunctionals in the form of bispecific ADCs can employ improved internalization of the antibody for enhanced lysosomal delivery of the toxic payload.2, 3, 15 Furthermore, trifunctionals in the form of bispecific immunocytokines16 and bispecific antibodies carrying radiolabels have been reported.17 However, the fusion of a scFv units or a cytokine to a monoclonal antibody based on recombinant technology is a strategy that is effective but rather time-consuming.
The combination of preparing a trifunctional containing both UCHT1 and IL–2 would be of particular interest. UCHT1 is an antibody targeting CD3, a T-cell co-receptor which bind to T-cells for subsequent activation.18 By introducing the scFv variant of UCHT1 on an antibody targeting acute myeloid leukemia (AML) cells, binding of both AML and T-cells was achieved, and suppressed tumor growth in mice inoculated with AML-cells.11 This effect might be further enhanced by exposing the recruited T-cells to IL-2,19 a cytokine that can activate T-cells, allowing for localized immune responses.6 By combining UCHT1 and IL-2 on tumor-targeting antibodies, we envisioned to create a system capable of efficiently recruiting and activating the immune system. Furthermore, by creating a modular approach of introducing UCHT1 and IL–2 on an antibody, the effect of varying the conjugation site can be explored, and the combination of other scFvs, cytokines, and other pharmaceutically relevant molecules can be investigated rapidly and efficiently.
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