Camelized Human Heavy Chain Variable Domain via Gene Conversion

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Solution Overview

Problem

Efficient production of heavy chain-only antibodies using the immune system of a host animal is challenging due to the requirement for autonomous heavy chain variable domains that can fold and bind to antigens without aggregating.

Innovation Solution

A transgenic animal is developed using gene conversion, where the endogenous immunoglobulin heavy chain locus comprises a functional human heavy chain gene and operably linked pseudogenes that donate nucleotide sequences to the human heavy chain variable domain, incorporating camelizing amino acid substitutions to enable autonomous binding.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If heavy chain-only antibodies are produced using conventional immunoglobulin genes, then the antibodies can be expressed autonomously, but the variable domains lack the stability and solubility characteristics of camelid antibodies

Engineering Contradiction:

Improveautonomous expression capabilityVSAvoidvariable domain stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent applies parameter changes by introducing specific amino acid substitutions (camelizing mutations) into the human heavy chain variable domain sequence. These substitutions modify the chemical and physical parameters of the variable domain, conferring camelid-like stability and solubility properties while maintaining autonomous expression capability.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates a composite antibody structure by combining human immunoglobulin framework regions with camelid variable domain characteristics through gene conversion. This hybrid approach integrates the autonomous expression advantage of human antibodies with the enhanced stability of camelid antibodies.

Inventive Principle:

Principle #40Composite materials

2Adaptability or versatility

If gene conversion is used to diversify the antibody repertoire, then the number of different antibodies increases, but the process complexity increases

Engineering Contradiction:

Improveantibody repertoire diversityVSAvoidgene locus structure

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent segments the immunoglobulin heavy chain locus into functional components: a functional heavy chain gene and multiple pseudogenes. The pseudogenes serve as reservoirs of camelizing mutations that can be transferred to the functional gene through gene conversion, enabling diversity generation without complicating the overall locus architecture.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent uses pseudogenes as intermediary elements that facilitate gene conversion events. These pseudogenes contain pre-evolved camelizing mutations and act as donors that can transfer beneficial sequences to the functional heavy chain gene, simplifying the diversification process by providing ready-made mutational variants.

Inventive Principle:

Principle #24Intermediary (Mediator)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach allows for the production of camelized human immunoglobulin heavy chains capable of autonomous antigen binding, enhancing stability and solubility, and expanding the antibody repertoire through gene conversion events.

Implementation Method 1

a transgenic animal that uses gene conversion for antibody diversification comprising B cells in which the endogenous immunoglobulin heavy chain locus comprises: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a human heavy chain variable domain; and (b) a plurality of pseudogenes that are operably linked to the functional immunoglobulin heavy chain gene and that donate, by gene conversion, nucleotide sequence to the nucleic acid encoding the human heavy chain variable domain

Methodology Applied

Scientific EffectGene conversion:

Data Source

PatentUS12102070B2Camelization of a human variable domain by gene conversion

Publication Date: 2024.10.01 CRYSTAL BIOSCIENCE INC

AI summary

This disclosure provides, among other things, a transgenic animal that uses gene conversion for antibody diversification comprising B cells in which the endogenous immunoglobulin heavy chain locus comprises: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a human heavy chain variable domain; and (b) a plurality of pseudogenes that are operably linked to the functional immunoglobulin heavy chain gene and that donate, by gene conversion, nucleotide sequence to the nucleic acid encoding the human heavy chain variable domain of (a), wherein the pseudogenes are upstream or downstream of the functional immunoglobulin heavy chain gene and encode variable domains that have camelizing amino acid substitutions.