Genetically Modified Mammals for Megabase Humanized Antibody Gene Integration
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Solution Overview
Problem
Existing methods for preparing fully human nanobody-producing mice are limited by low immune titer and antibody diversity, requiring lengthy processes like chromosome engineering and RMCE, which constrain gene modification to small fragments and result in inefficient production of humanized antibodies.
Innovation Solution
A method using the MBGE delivery system to introduce megabase-scale humanized genome fragments into non-human mammals through CRISPR-Cas9 gene editing, specifically targeting and disrupting endogenous heavy chain immunoglobulin gene loci, and introducing human IGHV, IGHD, IGHJ, IgHG2c, IgHE, and LCR regions, enabling efficient production of humanized antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chromosome engineering or RMCE technology is used to humanize heavy chain genes, then humanized antibody production is achieved, but the process takes about 5 years and requires multiple gene recombination steps
Solution Approach 1:
The patent segments the human heavy chain antibody gene cluster into multiple BAC (bacterial artificial chromosome) clones, each containing specific gene segments (IGHV, IGHD, IGHJ, IgHG2c, IgHE, IgHA, and LCR regions). These segmented BAC clones can be independently manipulated and then recombined into the mouse genome through a single transgenesis event, dramatically reducing the time required compared to traditional step-by-step gene recombination methods.
Solution Approach 2:
The patent performs preliminary actions by preparing all necessary human heavy chain antibody gene segments in advance as separate BAC clones with proper regulatory elements and structural organization. This preliminary preparation allows the entire humanized antibody system to be introduced in one step rather than requiring sequential gene modifications over multiple years.
2Ease of manufacture
If single BAC transgenesis is used, then transgene introduction is simplified, but the transgene size is constrained to about 200 kb which is insufficient for megabase-scale gene fragments
Solution Approach 1:
The patent employs a nested structure where multiple BAC clones (each containing specific human antibody gene segments) are designed to be sequentially integrated into the mouse genome. The BAC clones are organized in a nested manner where each clone contains specific gene regions that complement the others, allowing the cumulative assembly of a megabase-scale humanized antibody gene cluster through a single transgenesis event.
Solution Approach 2:
The patent creates a composite transgenic system by combining multiple BAC clones, each containing different human heavy chain antibody gene segments (IGHV, IGHD, IGHJ, IgHG2c, IgHE, IgHA, and LCR regions). This composite approach allows the system to achieve megabase-scale gene introduction capability while maintaining the ease of single-BAC transgenesis methodology.
3Adaptability or versatility
If fully human nanobody-producing mice are created, then therapeutic antibody screening is enabled, but the immune titer is lower than wild mice indicating less antibody diversity
Solution Approach 1:
The patent applies local quality by introducing only the necessary human heavy chain antibody gene segments (IGHV, IGHD, IGHJ, and IgHG2c without CH1) into the mouse genome while leaving the mouse's endogenous light chain genes intact. This localized humanization approach allows the mouse to produce fully human nanobodies with diverse sequences from the human IGHV gene segments, while maintaining the mouse's natural immune system functionality and antibody production capacity.
Solution Approach 2:
The patent copies the human heavy chain antibody gene cluster architecture into the mouse genome using BAC clones that replicate the natural human gene organization and regulatory elements. This copying approach ensures that the mouse produces authentic human nanobody sequences with full diversity, enabling direct screening of therapeutic human antibodies without requiring additional in vitro humanization steps.
4Manufacturing precision
If endogenous heavy chain immunoglobulin gene loci are disrupted, then humanized antibody production is enabled, but the immune titer and antibody diversity are reduced
Solution Approach 1:
The patent extracts and removes the mouse endogenous heavy chain immunoglobulin gene loci (specifically the IgHM, IgHA-CH1, and light chain genes) that would otherwise produce mouse antibodies. By taking out these endogenous genes, the system prevents mouse antibody production and allows exclusive production of humanized antibodies from the introduced BAC clones, ensuring full human sequence identity in the produced nanobodies.
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
The method achieves high immune titer and antibody diversity in non-human mammals, allowing for rapid production of humanized whole antibodies, single heavy chain antibodies, and nanobodies with improved efficiency and diversity.
Implementation Method 1
A method using the MBGE delivery system to introduce megabase-scale humanized genome fragments into non-human mammals through CRISPR-Cas9 gene editing, specifically targeting and disrupting endogenous heavy chain immunoglobulin gene loci
Data Source
AI summary
The present invention provides a method for preparing a genetically modified non-human mammal for producing a humanized antibody as well as use thereof, which method comprises: (1) bringing about a disruption of an endogenous heavy chain immunoglobulin gene loci in a non-human mammal; and (2) introducing a human IGHV gene, a human IGHD gene, a human IGHJ gene as well as an endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal into the non-human mammal obtained in step (1). The non-human mammal prepared according to the method of the present invention, after antigen immunization, enables efficiently producing a humanized whole antibody or single heavy chain antibody or nanobody with high immune titer.