Non-N-Glycosylated CTLA-4 Proteins for Enhanced Binding

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Solution Overview

Problem

Current methods for modulating immune responses in organ transplantation, autoimmune diseases, and cancer treatment using CTLA-4 proteins face challenges due to issues with glycosylation, which affects protein function and binding affinity, particularly in yeast expression systems like Pichia pastoris, leading to suboptimal expression and purification of fully functional CTLA-4 proteins.

Innovation Solution

Development of non-N-glycosylated human CTLA-4 proteins and CTLA-4 fusion toxins, specifically designed to avoid N-linked glycosylation sites, expressed in Pichia pastoris, which maintain binding affinity and are more effectively purified and functional, including mutations at positions 113, 115, 145, and 147, and additional mutations like L141E and A66Y, to enhance their therapeutic efficacy.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If CTLA-4 proteins are expressed in yeast systems like Pichia pastoris, then production capability is improved, but N-linked glycosylation occurs at positions 113 and 145, reducing binding affinity and therapeutic efficacy

Engineering Contradiction:

Improveproduction capabilityVSAvoidbinding affinity

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent removes the problematic N-linked glycosylation sites (Asn-113 and Asn-145) from the CTLA-4 protein sequence through site-directed mutagenesis, extracting the harmful glycosylation capability while preserving the beneficial eukaryotic expression system for production

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the amino acid parameters at specific positions (113, 115, 145, 147) to prevent N-linked glycosylation, transforming the protein's chemical properties to eliminate harmful modifications while maintaining production efficiency in yeast systems

Inventive Principle:

Principle #35Parameter changes

2Ease of manufacture

If standard CTLA-4 protein sequences are used, then expression in yeast is achieved, but purification difficulty increases due to glycosylation heterogeneity

Engineering Contradiction:

Improveexpression achievementVSAvoidpurification difficulty

Core Design Contradiction:

Ease of manufactureVSDevice complexity

Solution Approach 1:

The patent extracts the source of purification complexity by removing the N-linked glycosylation sites, eliminating heterogeneity caused by variable glycosylation patterns and simplifying the purification process while maintaining yeast expression capability

Inventive Principle:

Principle #2Taking out (Extraction)

3Quantity of substance

If glycosylated CTLA-4 proteins are produced, then expression yield is improved, but therapeutic efficacy decreases due to reduced binding affinity for CD80/CD86

Engineering Contradiction:

Improveexpression yieldVSAvoidtherapeutic efficacy

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent changes the amino acid composition at critical positions (113, 115, 145, 147) to prevent N-linked glycosylation, transforming the protein to achieve both high expression yield and optimal therapeutic efficacy by eliminating glycosylation-induced affinity reduction

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS9758566B2Recombinant cytotoxic T-lymphocyte-associated protein 4 (CTLA4)

Publication Date: 2017.09.12 THE GENERAL HOSPITAL CORP

AI summary

This invention relates to recombinant CTLA-4 proteins, e.g., soluble CTLA-4 or CTLA-4 fusion toxins, and methods for making and using them.