Abstract
Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.
Highlights
Through interacting with a specific cell surface target has relied on screening of large number of candidate antibodies20–26—the principles governing the efficiencies for the internalization of the immunoconjugates and the delivery of the toxin payloads remain limitedly understood
Because immunotoxin construction rate is limited by the low-throughput recombinant protein production and purification procedures, we developed an adaptor-toxin fusion proteins AL1-PE38KDEL and AL2-PE38KDEL for high throughput screening of the GH2 antibodies as targeting modules for delivering PE38KDEL, which is a truncated form of Pseudomonas Exotoxin A (PE) A subunit toxin[17,32]
To enable a high throughput screening platform for the soluble scFvs in culture media without purification of the scFvs (Fig. 1), we constructed the adaptor-toxin fusion proteins: AL1-PE38KDEL (Fig. 2) and AL2-PE38KDEL (Fig. 3), according to the structure of Protein A and Protein L in complex with a scFv of IGKV1-NL1*01/IGHV3-23*04 framework (PDB code: 4HKZ)[33] (Figs 2A and 3A). These adaptor-toxin fusion proteins binds to recombinant scFv or IgG with the IGKV1-NL1*01/IGHV3-23*04 framework, self-assembling into non-covalently-linked immunotoxin complexes in crude mixture of culture media, allowing the self-assembled immunotoxin complexes to be tested on cells cultures in micro-titer plates for cytotoxicity assay in high throughput format without the rate-limiting processes of subcloning and purification (Fig. 1)
Summary
Through interacting with a specific cell surface target has relied on screening of large number of candidate antibodies20–26—the principles governing the efficiencies for the internalization of the immunoconjugates and the delivery of the toxin payloads remain limitedly understood. Antibodies used as targeting modules in immunoconjugates are more likely to result in optimally functional therapeutics by satisfying the following criteria: adequate affinity and specificity to the target receptor; capable of inducing receptor-mediated endocytosis; capable of delivering the toxin payload to subcellular locations for optimal cytotoxicity; of human origin to reduce immunogenicity; easy to manufacture with high expression efficiency and protein stability To this end, we have constructed a phage-displayed synthetic antibody library (GH2) with a single human variable domain antibody germline framework: IGKV1-NL1*01/ IGHV3-23*0427, on which the antibody libraries were designed based on the antibody-protein interaction principles derived from computational and experimental analyses[27,28,29,30,31]. Combining the synthetic antibody libraries with the high throughput screening platform developed in this work enables better engineering capabilities for developing optimally functional antibody-based targeting modules in immunoconjugates
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