Soluble ENPP1 Polypeptides Without SMB Domain

Solution Overview

Problem

Current ENPP1-containing fusion proteins exhibit reduced stability and increased immunogenicity due to the Somatomedin B (SMB) domain, leading to challenges in preventing unwanted calcification in soft tissues.

Innovation Solution

Development of variant ENPP1 polypeptides lacking the SMB domain, with specific amino acid modifications to enhance stability and reduce homodimerization, proteolytic resistance, and immunogenicity, while maintaining enzymatic activity, such as those with substitutions at positions 816, 817, or 818, and fusion with heterologous proteins like the Fc domain for increased circulating half-life.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If ENPP1-containing fusion proteins with SMB domain are used, then therapeutic activity for preventing calcification is achieved, but stability is reduced and immunogenicity is increased

Engineering Contradiction:

Improvetherapeutic activityVSAvoidprotein stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent removes the SMB domain from the ENPP1 fusion protein structure. Specifically, the invention uses ENPP1 variants that lack the SMB domain (amino acids 104-189), thereby eliminating the source of instability and immunogenicity while retaining the catalytic domain functionality for preventing ectopic calcification.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces specific amino acid substitutions at positions 816, 817, or 818 in the ENPP1 sequence to modify protein properties. These parameter changes enhance proteolytic resistance and stability without compromising therapeutic activity, directly addressing the stability issue caused by the original SMB domain-containing structure.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If ENPP1-containing fusion proteins with SMB domain are used, then therapeutic activity for preventing calcification is achieved, but immunogenicity is increased

Engineering Contradiction:

Improvetherapeutic activityVSAvoidimmunogenicity

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent removes the SMB domain from the ENPP1 fusion protein structure. Specifically, the invention uses ENPP1 variants that lack the SMB domain (amino acids 104-189), thereby eliminating the source of instability and immunogenicity while retaining the catalytic domain functionality for preventing ectopic calcification.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces specific amino acid substitutions at positions 816, 817, or 818 in the ENPP1 sequence to modify protein properties. These parameter changes enhance proteolytic resistance and stability without compromising therapeutic activity, directly addressing the stability issue caused by the original SMB domain-containing structure.

Inventive Principle:

Principle #35Parameter changes

3Stability of the object's composition

If variant ENPP1 polypeptides with amino acid substitutions are used, then stability and proteolytic resistance are enhanced, but structural complexity increases

Engineering Contradiction:

Improveprotein stabilityVSAvoidprotein structure complexity

Core Design Contradiction:

Stability of the object's compositionVSDevice complexity

Solution Approach 1:

The patent applies localized modifications rather than global structural changes. Specifically, only three specific amino acid positions (816, 817, or 818) are substituted, while the rest of the protein structure remains relatively simple and unchanged. This localized approach enhances stability without significantly increasing overall structural complexity.

Inventive Principle:

Principle #3Local quality

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 variant ENPP1 polypeptides demonstrate enhanced stability, reduced immunogenicity, and increased proteolytic resistance, resulting in a homogenous composition with improved therapeutic efficacy in preventing ectopic calcification, including soft tissue calcification and arterial calcification.

Implementation Method 1

ENPP1 (also known as PC-1) and ENPP3 are type 2 extracellular membrane-bound glycoproteins located on the mineral-depositing matrix vesicles of osteoblasts and chondrocytes, and hydrolyze extracellular nucleotides (principally ATP) into adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi).

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Implementation Method 2

Tissue Non-specific Alkaline Phosphatase (TNAP) removes PPi via direct hydrolysis of PPi into Pi.

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Data Source

PatentUS20230313158A1Soluble ENPP1 proteins and uses thereof

Publication Date: 2023.10.05 BIOMARIN PHARMACEUTICAL INC

AI summary

In certain aspects, the present invention provides novel soluble ENPP1 or ENPP3 polypeptides, as well as compositions and methods for using those variants to treat an indication associated an ENPP1 or ENPP3 deficiency. The compositions and methods provided herein are useful in treating diseases associated with an ENPP1 or ENPP3 deficiency such as pathological calcification or pathological ossification.