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Electrostatic properties of human germlines and biodistribution of small biologics

Leticia Maria De Souza Cordeiro¹, Kelley Christine Atkinson¹, Argin Aivazian¹

¹Preclinical discovery, ImaginAb, Inc, Inglewood, CA, USA.

|February 9, 2024

Abstract:

Off-target biodistribution of biologics bears important toxicological consequences. Antibody fragments intended for use as vectors of cytotoxic payloads (e.g. antibody-drug conjugates, radiotherapy) can accumulate at clearance organs like kidneys and liver, where they can cause dose-limiting toxicities. Renal and hepatic uptakes are known to be affected by protein electrostatics, which promote protein internalization through pinocytosis. Using minibodies as a model of an antibody fragment lacking FcRn recycling, we compared the biodistributions of leads with different degrees of accumulation at the kidney and liver. We identified a positive electrostatic patch highly conserved in a germline family very commonly used in the humanization of approved biologics. Neutralization of this patch led to a drastic reduction in the kidney uptake, leading to a biodistribution more favorable to the delivery of highly cytotoxic payloads. Next, we conducted a high throughput study of the electrostatic properties for all combinations of VH and VL germlines. This analysis shows how different VH/VL combinations exhibit varying tendencies to create electrostatic patches, resulting in Fv variants with different isoelectric points. Our work emphasizes the importance of carefully selecting germlines for humanization with optimal electrostatic properties in order to control the unspecific tissue uptake of low molecular weight biologics.

Insights

Minimizing off-target accumulation of antibody fragments in kidneys and liver is crucial for biologic safety. Modifying electrostatic properties of antibody fragments can significantly reduce kidney uptake, improving biodistribution for targeted therapies.

Area of Science:

Biopharmaceutical development
Protein engineering
Toxicology

Background:

Off-target biodistribution of biologics can lead to toxicological issues, particularly in organs like the kidneys and liver.
Accumulation in these clearance organs is often linked to protein electrostatics, driving cellular uptake via pinocytosis.
Antibody fragments, such as minibodies, can accumulate in renal and hepatic tissues, potentially causing dose-limiting toxicities.

Purpose of the Study:

To investigate the impact of protein electrostatics on the biodistribution of antibody fragments.
To identify strategies for reducing unintended accumulation in kidney and liver tissues.
To optimize the selection of antibody germlines for improved biodistribution profiles in biologics.

Main Methods:

Utilized minibodies as a model system lacking FcRn recycling to compare biodistribution.
Identified and neutralized a conserved positive electrostatic patch on antibody fragments.
Conducted high-throughput screening of variable heavy (VH) and variable light (VL) germline combinations to assess electrostatic properties and isoelectric points.

Main Results:

Neutralization of a conserved positive electrostatic patch significantly reduced kidney uptake of antibody fragments.
This modification resulted in a more favorable biodistribution, suitable for delivering highly cytotoxic payloads.
Analysis revealed that different VH/VL germline combinations influence the formation of electrostatic patches and the resulting isoelectric points of Fv variants.

Conclusions:

Careful selection of antibody germlines with optimal electrostatic properties is essential for controlling non-specific tissue uptake of low molecular weight biologics.
Engineering electrostatic properties offers a viable strategy to mitigate off-target biodistribution and enhance the safety of antibody-based therapeutics.
Optimizing biodistribution through germline selection can improve the therapeutic index of antibody fragments used in targeted delivery systems.

Read full analysis

Keywords:

Antibody antibody drug conjugate antibody engineering biodistribution biologics electrostatics radiopharmaceutical

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