High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

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.

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models

“…All the selected scFvs (GH2-20, GH2-61 and GH2-75) were among the most potent targeting modules in a large set of non-covalently linked immunotoxins, with IC 50 for scFv-AL2-PE38KDEL < 0.01 nM and IC 50 for scFv-AL1-PE38KDEL < 0.1 nM, as measured in previous work. 9 The epitopes of GH2-61 and GH2-75 overlap with that of the positive control antibody H32, for which the epitope has been identified on domain I of HER2-ECD as determined with negative stain electron microscopy. 9 The epitope of GH2-20 does not overlap with that of H32, and indirect evidence suggests that its epitope is situated on domain IV of HER2-ECD, but does not overlap with trastuzumab's epitope, 8 which is also situated on domain IV of HER2-ECD as determined with x-ray crystallography (PDB code: 1N8Z).…”

“…9 The epitopes of GH2-61 and GH2-75 overlap with that of the positive control antibody H32, for which the epitope has been identified on domain I of HER2-ECD as determined with negative stain electron microscopy. 9 The epitope of GH2-20 does not overlap with that of H32, and indirect evidence suggests that its epitope is situated on domain IV of HER2-ECD, but does not overlap with trastuzumab's epitope, 8 which is also situated on domain IV of HER2-ECD as determined with x-ray crystallography (PDB code: 1N8Z). 18 GH2-61, GH2-20, H32 and trastuzumab IgG1 antibodies have similar on/off rates and nano-molar monovalent dissociation constants binding to HER2-ECD (measured with surface plasmon resonance (SPR) shown in Supplementary Table S1); the affinity of GH2-75 IgG1 to HER2-ECD is slightly inferior in terms of the monovalent dissociation constant and the off-rate binding to HER2-ECD ( Supplementary Table S1).…”

“…2 Three HER2-ECD-positive single-chain variable fragment (scFv) candidates (GH2-20, GH2-61 and GH2-75) from the synthetic antibody libraries 7,8 were selected through the high throughput antibody-based immunoconjugate discovery platform. 9 Those with the highest potencies as targeting modules toward N87 cells were reformatted into human IgG1 antibodies and tested together with two positive control humanized IgG1 antibodies (trastuzumab and H32, which are humanized murine anti-HER2-ECD antibodies 9 ) and one isotype negative control IgG antibody (nc). All these IgG1 antibodies were constructed with the same human IgG1 framework.…”

“…The efficacies of these IgG1 antibodies as the targeting modules for delivering cytotoxic payloads to N87 xenograft tumor models in vivo and to cultured N87 cells in vitro were scrutinized with three forms of immunoconjugates in this work: 1) ADC in the form of IgG1-vc-MMAE (monomethyl auristatin E linked to the IgG1 via valine-citrulline dipeptide cathepsin-cleavable linker); 10 2) immunotoxin of a single polypeptide chain in the form of scFv-PE38KDEL fusion protein, where PE38KDEL is a truncated form of Pseudomonas exotoxin (PE) A subunit toxin,- 11,12 as pioneered by Pastan and colleagues; 13 and 3) immunotoxin in the form of IgG1-AL1-PE38KDEL, where the IgG1 is non-covalently linked to PE38KDEL through the adaptor-toxin fusion protein AL1-PE38KDEL. 9 The AL1 fragment is composed of consecutive Protein A and Protein L separated by a polypeptide linker that enables Protein A and Protein L binding to the framework regions of the IgG1 simultaneously with nano-molar affinity. 9 Since the IgG1 candidates were reformatted from the scFvs derived from the synthetic antibody libraries, which were designed using antibody-protein interaction principles from computational and experimental analyses 8,[14][15][16][17] and built on a single human variable domain antibody germline framework (IGKV1-NL1*01/IGHV3-23*04), [7][8][9] the IgG1s bind to Protein A and Protein L through the variable domain framework heavy and light chain regions, respectively, without affecting the antigen binding sites of the antibodies.…”

“…9 The AL1 fragment is composed of consecutive Protein A and Protein L separated by a polypeptide linker that enables Protein A and Protein L binding to the framework regions of the IgG1 simultaneously with nano-molar affinity. 9 Since the IgG1 candidates were reformatted from the scFvs derived from the synthetic antibody libraries, which were designed using antibody-protein interaction principles from computational and experimental analyses 8,[14][15][16][17] and built on a single human variable domain antibody germline framework (IGKV1-NL1*01/IGHV3-23*04), [7][8][9] the IgG1s bind to Protein A and Protein L through the variable domain framework heavy and light chain regions, respectively, without affecting the antigen binding sites of the antibodies. [7][8][9] The differences in the cytotoxic payloads and linkers among the three forms of immunoconjugate were anticipated to result in different effects on the efficacies of the antibodies as the immunoconjugates' targeting modules, and hence provide information for ADC candidate selection and further development.…”