Development of a Novel Engineered Antibody Format for PSMA-Targeted Radionuclide Therapy

Abstract

Prostate cancer remains a prevalent and lethal malignancy across the globe. Despite ongoing advances in therapeutic approaches, these remain ineffective, and new treatments are drastically needed. Prostate-specific membrane antigen (PSMA)-targeted radionuclide therapy is a well-developed approach for prostate cancer treatment; however, current small molecule and antibody carriers for molecular radiotherapy each have drawbacks in their biodistribution and consequent side effects as highlighted in current clinical trials. To address this, we developed an approach to bioengineer the well clinically validated antibody carrier HuJ591 to yield an engineered, full-length antibody construct that achieves the beneficial fast pharmacokinetic profile of small molecule carriers alongside the enhanced tumor targeting and reduced renal toxicity of antibody carriers. We report here a rational design process to produce a novel humanized PSMA-targeting antibody designed for the delivery of radiation with abrogated FcRn recycling that aims to reduce blood circulation time and minimize systemic exposure. We demonstrate that these IgG-based constructs retain the favorable properties of HuJ591, such as inherent protein stability, expression in systems compatible with industrial manufacture, and comparable, highly specific PSMA-binding characteristics. We then radiolabeled constructs with the diagnostic radionuclide ⁶⁴Cu as a surrogate for therapeutic radionuclide payloads and undertook a proof-of-concept preclinical imaging study to probe the resulting in vivo behaviors. This demonstrated the success of this design strategy to yield the intended in vivo and radiopharmaceutical characteristics, with the resulting construct being rapidly cleared from circulation over 3 days. Together, this study demonstrates the rational design of a novel targeting antibody platform for PSMA-expressing tumors with reduced systemic exposure. Such a platform is extremely promising for future radiotherapeutic delivery approaches, whereby effective tumor treatment can be achieved while mitigating potential hematologic toxicity observed with standard antibody delivery approaches.

Keywords: PET imaging; PSMA; antibody engineering; pharmacokinetics; prostate cancer; targeted therapy.

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Overarching scheme illustrating FcRn recycling of antibody carriers and the relationship with systemic exposure compared with the design approach taken here. In brief, native IgG antibodies in circulation undergo neonatal Fc receptor (FcRn)-mediated endosomal recycling from catabolic cells, thereby greatly extending their circulation time. When these IgG are then radiolabeled, this results in undesirable systemic exposure. The approach undertaken herein is to use antibody engineering to abrogate IgG FcRn binding and recycling, thereby producing constructs with enhanced clearance rates while maintaining the high specificity and affinity of antibody carriers.

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Diagram showing the relationship of PSMA-binding antibodies described in this work including targeted engineering of ANT4044 variants to abolish FcRn and Fc interactions to abrogate FcRn-mediated recycling and antibody-dependent cellular-cytotoxicity (ADCC) mechanisms, respectively.

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Visualization of Pepscan analysis to identify putative epitopes of PSMA bound to HuJ591 and ANT4044. For visual comparison of the epitopes bound by each antibody, identified epitopes are identified on a model of the PSMA monomer (AlphaFold: AF-Q04609-F1-v4).

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ANT4044 IgG variants binding profiles. (A) Representative multicycle sensorgram data and fitted curves demonstrating PSMA affinity of all constructs (colored lines; raw data for PSMA applied over a 2-fold dilution range from 25 nM (teal) to 1.5625 nM (red), black lines; fitted data). (B) Steady state affinity sensorgrams of the binding of constructs to FcRn at pH 6.0.

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ANT4044 IgG1 (H310A, H435Q) functionalization for preclinical ⁶⁴Cu PET imaging. (A) Schematic outline of IgG DOTA and ⁶⁴Cu radiolabeling for preclinical PET imaging. (B) LC–MS spectrum for ANT4044 IgG1-DOTA (H310A, H435Q) to determine DOTA DAR. (C) Radio-SEC chromatogram of [⁶⁴Cu]­ANT4044 IgG1-DOTA (H310A, H435Q), demonstrating the effective radiolabeling of monomeric IgG species.

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Preclinical biodistribution and PET imaging of [⁶⁴Cu]-labeled constructs in an LNCaP xenograft model. Color profiles of all plots were as per legend in (B). (A) Exemplar PET-CT image of ANT4044 IgG1 (H310A, H435Q) biodistribution at 48 h post-administration (arrow highlights tumor). (B) Ex vivo biodistribution of all ANT4044 constructs in comparison to HuJ591 at 48 h post-administration. (C) Blood profile of ANT4044 constructs in comparison to HuJ591 over 120 h post-administration. (D) Tumor:Blood ratio of ANT4044 constructs in comparison to HuJ591 at 48 h post-administration. * Denotes p < 0.05, ** Denotes p < 0.01 and *** Denotes p < 0.001 via unpaired t test. Where no significance level is indicated, differences between groups were not statistically significant.