Abstract
BackgroundThe aim of the study was to explore Fc mutations of a humanised anti-Lewis-Y antibody (IgG1) hu3S193 as a strategy to improve therapeutic ratios for therapeutic payload delivery.MethodsFour hu3S193 variants (I253A, H310A, H435A and I253A/H310A) were generated via site-directed mutagenesis and radiolabelled with diagnostic isotopes iodine-125 or indium-111. Biodistribution studies in Lewis-Y-positive tumour-bearing mice were used to calculate the dose in tumours and organs for therapeutic isotopes (iodine-131, yttrium-90 and lutetium-177).Results111In-labelled I253A and H435A showed similar slow kinetics (t1/2β, 63.2 and 62.2 h, respectively) and a maximum tumour uptake of 33.11 ± 4.05 and 33.69 ± 3.77 percentage injected dose per gramme (%ID/g), respectively. 111In-labelled I253A/H310A cleared fastest (t1/2β, 9.1 h) with the lowest maximum tumour uptake (23.72 ± 0.85 %ID/g). The highest increase in tumour-to-blood area under the curve (AUC) ratio was observed with the metal-labelled mutants (90Y and 177Lu). 177Lu-CHX-A" DTPA-hu3S193 I253A/H310A (6:1) showed the highest tumour-to-blood AUC ratio compared to wild type (3:1) and other variants and doubling of calculated dose to tumour based on red marrow dose constraints.ConclusionsThese results suggest that hu3S193 Fc can be engineered with improved therapeutic ratios for 90Y- and 177Lu-based therapy, with the best candidate being hu3S193 I253A/H310A for 177Lu-based therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0180-0) contains supplementary material, which is available to authorized users.
Highlights
The aim of the study was to explore Fc mutations of a humanised anti-Lewis-Y antibody (IgG1) hu3S193 as a strategy to improve therapeutic ratios for therapeutic payload delivery
The hu3S193 heavy chain (HC) was ligated into the pEE6.4 mammalian expression vector (Lonza Biologics, Slough, UK) via a HindIII and EcoRI double digest. pEE6.4/hu3S193 HC was used as a template for site-directed mutagenesis (GeneTailorTM Site-Directed Mutagenesis System (Invitrogen) or QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene) to introduce the following substitutions in the CH2 and CH3 domains: I253A, H310A, H435A and I253A/H310A [25]
Production of hu3S193 variants with short elimination half-lives To investigate whether hu3S193 antibodies with reduced serum persistence would benefit payload delivery, three hu3S193 single variants (I253A, H310A, H435A) and one double variant (I253A/H310A) were generated
Summary
The aim of the study was to explore Fc mutations of a humanised anti-Lewis-Y antibody (IgG1) hu3S193 as a strategy to improve therapeutic ratios for therapeutic payload delivery. Our laboratory has previously evaluated smaller forms such as diabody, F(ab′) and tetrameric scFv constructs without an Fc fragment These constructs showed a rapid elimination phase compared to wild-type IgG1, more rapid tumour targeting with the maximum tumour uptake observed at an earlier time point but strongly reduced maximal tumour uptake [12,13,14]. Another approach studied in preclinical studies to improve therapeutic ratio is pretargeted RIT. The multistep targeting approach allows the antibody to clear first before administrating the therapeutic radionuclide, and this approach has shown high increases in therapeutic ratio [1, 15]
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