Avian B Cell Antibody Diversification via PI3Kα Inhibition

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Solution Overview

Problem

Current methods for diversifying antibody variable regions, such as animal immunization and phage display, face challenges in efficiency and specificity, and the mechanisms controlling antibody diversification, particularly in avian B cells, are not well understood.

Innovation Solution

The use of a PI3Kα-specific inhibitor, such as A66, is effective in promoting diversification of amino acid sequences in avian B cells by suppressing PI3Kα activity, enhancing the generation of antibodies with desired antigen binding capabilities.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If animal immunization methods (hybridoma method) are used to produce antibodies, then antibodies can be obtained through B cell fusion with myelomas, but time and effort are required to obtain antibodies and immune tolerance may prevent antibody acquisition

Engineering Contradiction:

Improveantibody production efficiencyVSAvoidtime required to obtain antibody

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The invention extracts the essential function of antibody production from the complex animal immunization process by using in vitro systems (phage display, cell-free systems) that separate antibody gene diversification and expression from animal physiology, thereby eliminating time-consuming animal procedures while maintaining antibody production capability

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention introduces intermediary systems such as phage particles and bacterial expression systems that mediate antibody production between genetic material and functional antibody, enabling rapid antibody generation without direct animal immunization and overcoming immune tolerance issues

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If phage display method is used to produce antibodies, then antibodies can be obtained without animal immunization, but the quality of library depends on scFv diversity and changes occur in specificity or affinity during conversion to full-length antibody

Engineering Contradiction:

Improveease of antibody productionVSAvoidantibody specificity and affinity

Core Design Contradiction:

Ease of manufactureVSManufacturing precision

Solution Approach 1:

The invention segments the antibody production process into independent modules: scFv library construction, phage display selection, and full-length antibody expression, allowing optimization of each stage separately to maintain both ease of manufacture and manufacturing precision

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention implements feedback mechanisms where selected scFv clones are characterized for affinity and specificity, and this information feeds back into library construction and selection strategies to improve full-length antibody quality while maintaining production efficiency

Inventive Principle:

Principle #23Feedback

3Productivity

If DT40 cell line system is used to produce antibodies, then full-length antibody can be obtained promptly and immune tolerance avoided, but the mechanism of variable region diversification differs from mice or humans

Engineering Contradiction:

Improveantibody production speedVSAvoidcompatibility with human antibody mechanisms

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The invention changes the genetic parameters of DT40 cells by introducing human V(D)J recombination components and human antibody gene segments, transforming the cell system to produce human-type antibodies through human-compatible diversification mechanisms while maintaining rapid production capability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention creates a composite system combining avian DT40 cell machinery with human antibody genes and recombination elements, achieving both rapid full-length antibody production and human-compatible antibody mechanisms for therapeutic applications

Inventive Principle:

Principle #40Composite materials

4Quantity of substance

If somatic mutations are induced in germinal center B cells (GANP mice) to diversify antibody variable regions, then many somatic mutations are generated, but the method is complex and requires specialized animal models

Engineering Contradiction:

Improvenumber of somatic mutationsVSAvoidcomplexity of animal model

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The invention replaces complex animal-based mechanical systems (GANP mice with engineered recombination mechanisms) with simplified in vitro biochemical systems that use enzymes and chemical reagents to induce somatic mutations directly in antibody genes, achieving high mutation numbers without animal model complexity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Data Source

PatentUS20230220049A1Method for promoting diversification of antibody variable region

Publication Date: 2023.07.13 CHIOME BIOSCIENCE INC

AI summary

A method for promoting diversification of variable regions of an antibody, particularly a method for promoting diversification of the amino acid sequences of variable regions of an antibody generated by an avian B cell population, the method including suppressing the PI3Kα activity of each avian B cell comprised in the avian B cell population expressing the antibody.