Humanized Single-Domain Antibodies for Low-Immunogenic Tag Binding

Solution Overview

Problem

Existing single-domain antibodies derived from alpaca, such as NbALFA, have low sequence homology to human sequences, making them immunogenic and challenging to use in vivo applications in humans, and conventional CDR grafting methods fail to maintain high affinity and expression of humanized versions.

Innovation Solution

Retain key residues from the original camelid VHH framework, such as Arg 47, Arg 48, and Met 50, and introduce specific mutations in CDR2 and CDR3 to create humanized NbALFA antibodies with improved affinity and stability, allowing binding to the epitope tag SRLEEELRRRLTE (SEQ ID NO: 2) while facilitating SpA binding.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Object-generated harmful factors

If conventional CDR grafting methods are used to humanize NbALFA, then sequence homology to human sequences is improved, but binding affinity and expression are lost

Engineering Contradiction:

ImproveimmunogenicityVSAvoidbinding affinity

Core Design Contradiction:

Object-generated harmful factorsVSReliability

Solution Approach 1:

The patent applies local quality by making selective mutations only in specific regions (CDR2 and CDR3) while retaining the original framework regions. This allows humanization of the antibody to reduce immunogenicity while preserving the binding affinity through conservation of the functional framework structure.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent changes specific sequence parameters (amino acid residues) at defined positions in CDR2 and CDR3 to achieve humanization. By modifying these specific parameters while maintaining others, the antibody achieves human sequence homology without compromising binding affinity.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If key residues from original camelid VHH framework are retained, then binding affinity is maintained, but sequence homology to human sequences decreases

Engineering Contradiction:

Improvebinding affinityVSAvoidimmunogenicity

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent applies local quality by making selective mutations only in specific regions (CDR2 and CDR3) while retaining the original framework regions. This allows humanization of the antibody to reduce immunogenicity while preserving the binding affinity through conservation of the functional framework structure.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent changes specific sequence parameters (amino acid residues) at defined positions in CDR2 and CDR3 to achieve humanization. By modifying these specific parameters while maintaining others, the antibody achieves human sequence homology without compromising binding affinity.

Inventive Principle:

Principle #35Parameter changes

3Object-generated harmful factors

If humanized NbALFA antibodies are produced, then immunogenicity is reduced, but expression and affinity are compromised

Engineering Contradiction:

ImproveimmunogenicityVSAvoidexpression

Core Design Contradiction:

Object-generated harmful factorsVSProductivity

Solution Approach 1:

The patent applies local quality by making selective mutations only in specific regions (CDR2 and CDR3) while retaining the original framework regions. This allows humanization of the antibody to reduce immunogenicity while preserving the binding affinity through conservation of the functional framework structure.

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20260008872A1Humanized single-domain antibodies binding to epitope tags

Publication Date: 2026.01.08 NANO TAG BIOTECHNOLOGIES GMBH

AI summary

The present invention provides humanized versions of single-domain antibodies specifically binding the epitope tag of SRLEEELRRRLTE (SEQ ID NO: 2), which can be used for binding, detection, immobilization, isolation or purification a protein comprising the said epitope tag. Furthermore, the present invention relates to a nucleic acid encoding the humanized antibody and a vector comprising the nucleic acid, as well as a host cell comprising the nucleic acid or the vector described herein.