Synthetic Cancer‐Targeting Innate Immune Stimulators Give Insights into Avidity Effects

Abstract

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen-binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid-phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two "binder" peptides that target ephrin A2 and integrin α receptors and an "effector" peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.

Multifunctional Scaffolds for Assembling Cancer-Targeting Immune Stimulators Using Chemoselective Ligations

“…The ISEr peptide-polymer scaffold was synthesized by SPPS as described previously with a L-propargyl glycine (alkyne functionality) on the end of each PEG 27 chain (Conibear et al, 2018a). The short N-formylated “effector” peptide fMIFL (Rot et al, 1987) bore a glycine spacer and two lysine residues to which the PEG 27 chains were coupled via the side chains (Figure S1).…”

“…In addition to increasing the modularity of ISEr synthesis, we further used CuAAC ligations to generate multivalent ISErs to explore avidity and affinity effects of cancer cell binding (Conibear et al, 2018a). Using a similar strategy to that described for the bivalent ISErs above, we synthesized an effector-PEG 27 scaffold with either two L-propargyl glycine residues on the ends of each of two PEG 27 chains, or four PEG 27 chains each with one L-propargyl glycine residue (Figures S6, S7).…”

“…In both cases, we observed side-products with two or no Aoa functionalities that were challenging to purify. Nevertheless, the desired bispecific ISErs were obtained in acceptable purity for bioactivity assays, which showed that the bispecific bivalent ISEr had higher affinity for PC-3 cancer cells (K d = 13.0 ± 2.3 nM) than either of the two monospecific bivalent ISErs (K d = 30.4 ± 10.0 nM and 68.9 ± 19.0 nM) (Conibear et al, 2018a).…”

“…Whereas, the first strategy is straightforward and does not require any additional steps, it relies on the compatibility of the chosen dye or tag to SPPS conditions. The C-terminal biotin was used to determine the immune-activating and cancer cell-binding activities of a series of multivalent and multispecific ISErs generated using the CuAAC and oxime ligations described above (Conibear et al, 2018a).…”

“…We have previously reported the development of a prototype innate immune system engager (ISEr), a peptide-polymer conjugate that mimics the functions of antibodies in targeting cancer cells and eliciting an immune response (Brehs et al, 2017) ISErs are synthesized entirely by solid phase peptide synthesis (SPPS) and comprise two or more “binder” peptides that specifically target receptors overexpressed on cancer cells and are conjugated via polyethylene glycol (PEG) linkers to an “effector” peptide that stimulates an immune response (Figure 1) (Brehs et al, 2017). To explore the cancer cell specificity and immune stimulatory activity of ISErs, we have generated variants bearing two or more “binders” targeting the same or different receptors and have also used them for targeting cytotoxic drug molecules to cancer cells (Conibear et al, 2017b, 2018a). Further development and optimisation of the ISEr concept and its application to other types of cancer cells requires investigation of additional binder and effector peptides, combinations of binder and effector peptides and labeling of the ISErs to detect them in cells and tissues.…”