Sortase A Enzymatic Conjugation for Multispecific Polypeptides

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Solution Overview

Problem

Current methods for delivering therapeutic molecules, such as antibodies and toxins, lack specificity and efficiency, leading to systemic toxicity and reduced efficacy due to non-targeted distribution within the body.

Innovation Solution

The use of Ca2+-dependent sortase A from Staphylococcus aureus to enzymatically conjugate polypeptide domains with endoplasmic reticulum retention signals, enabling the production of multispecific binders and tailored therapeutic molecules that can be specifically targeted to disease-specific cells by combining binding entities with improved retention and delivery properties.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional delivery methods are used, then therapeutic molecules can be administered systemically, but specificity and efficiency are reduced leading to systemic toxicity

Engineering Contradiction:

ImprovespecificityVSAvoidsystemic toxicity

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The therapeutic molecule is divided into separate polypeptide domains (first and second domains) that are independently expressed and then conjugated together in vivo through sortase A-mediated ligation. This segmentation allows each domain to be optimized for specific functions (targeting, effector activity) and enables controlled assembly at the target site, improving specificity while reducing systemic exposure and toxicity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Sortase A serves as an enzymatic intermediary that facilitates the conjugation of polypeptide domains containing specific motifs (LPXTG and oligoglycine). This intermediary enables precise, controlled assembly of therapeutic molecules in vivo, ensuring that the complete therapeutic agent is formed only when needed, thereby reducing premature systemic exposure and associated toxicity.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If conventional delivery methods are used, then therapeutic molecules can be distributed throughout the body, but efficiency is reduced due to non-targeted distribution

Engineering Contradiction:

ImproveefficacyVSAvoidoff-target distribution

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

Different polypeptide domains are designed with specific local qualities: one domain contains a sortase motif for enzymatic conjugation, another contains an oligoglycine motif for ligation, and additional domains provide targeting specificity or effector functions. This local differentiation ensures that each part of the molecule performs its designated function, improving overall efficacy while minimizing off-target distribution through precise molecular design.

Inventive Principle:

Principle #3Local quality

3Reliability

If sortase A is used for enzymatic conjugation, then specific and efficient targeting is achieved, but the process requires multiple polypeptide domains and motifs

Engineering Contradiction:

Improvetargeting precisionVSAvoidmolecular structure

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The sortase A recognition motifs (LPXTG and oligoglycine) serve as universal interfaces that can be incorporated into various polypeptide domains regardless of their specific function (targeting, effector activity, structural). This universality allows the same conjugation mechanism to be applied across different therapeutic molecule designs, simplifying the overall approach despite the presence of multiple domains, as the conjugation interface remains consistent and predictable.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 creation of highly specific, tailor-made therapeutic molecules with enhanced targeting and reduced off-target binding, achieving higher therapeutic efficacy and minimizing side effects by ensuring precise delivery to disease sites.

Implementation Method 1

The use of Ca2+-dependent sortase A from Staphylococcus aureus to enzymatically conjugate polypeptide domains

Methodology Applied

Scientific EffectEnzymatic catalysis: Enzyme

Implementation Method 2

producing and selecting molecules formed by the combination of two different entities... by using a transpeptidase, such as sortase A

Methodology Applied

Scientific EffectTranspeptidation: Chemical Bonding

Implementation Method 3

by adding an endoplasmic reticulum retention signal to the sortase and to one of the entities... one of the polypeptide domains and the soluble sortase A enzyme contain an endoplasmic reticulum retention signal sequence

Methodology Applied

Scientific EffectProtein targeting and retention:

Data Source

PatentUS9862779B2Method for the production and selection of molecules comprising at least two different entities and uses thereof

Publication Date: 2018.01.09 F HOFFMANN LA ROCHE INC

AI summary

Herein is reported a method for producing a polypeptide comprising at least two polypeptide domains comprising the step of cultivating a cell comprising (a) a nucleic acid encoding a soluble S. aureus sortase A with a C-terminal endoplasmic reticulum retention signal, (b) a nucleic acid encoding a first polypeptide domain comprising at its C-terminus a sortase motif followed by an endoplasmic reticulum retention signal, and (c) a nucleic acid encoding a second polypeptide domain comprising at its N-terminus at least a diglycine, whereby the cell secretes the sortase A conjugate of the first polypeptide domain and the second polypeptide domain, thereby producing a polypeptide comprising at least two polypeptide domains.