Antibody Engineering

Since the first hybridoma monoclonal antibody has been generated, the possibility has arisen to enhance its functionality. After many years’ technology development, a certain antibody can be reconfigured into different isotypes or a variety of formats (scFv, Fab, bi-specific antibody, etc.) and other modification including affinity, immunogenicity, caninization and so on. Through modern molecular biology techniques, antibodies can be specifically modified or engineered to fit different application.

Start from the mid 1980s, antibody has became a major class of new drugs to treat cancer and autoimmune diseases. The disadvantages of murine originated antibody kept on being studied. Muromonab became the first clinical-use approved antibody therapy in 1986 and the one of only four full murine sequence. Murine antibodies have a short therapeutic half-life in human body because they are recognized by the patient immune system as foreign proteins resulting in a human anti-mouse (HAMA) response. Since then, how to humanize the antibody sequence while keeping the affinity and specificity became an important procedure in therapeutic antibody drug discovery.

Plenty of methods in in-vitro diagnostics are highly relying on a good performance antibody and the stable production considering quantity and quality. As the foundation of diagnostics, the specific antigen binding ability of antibodies is critical for a variety of purposes, such as expanding detection limits, extending pH tolerance or increasing temperature resistance. Nevertheless, it is still beneficial to sequence the valuable antibody for patent application purpose or to keep stable production as hybridoma may loss its expression during cryopreservation or subculturing.

Bio Bench is a leading service provider in antibody/protein engineering for research, therapeutic and diagnostic. Biacore T200 and Fortebio Blitz systems are both installed in-house to monitor the affinity of antibody. Biacore (Surface Plasmon Resonance, SPR technology based) and Fortebio (Bio-Layer Interferometry, BLI technology based) are two well-established techniques for detection and monitoring biomolecular interactions in real time. Used orthogonally, they can be powerful and complementary tools in basic research, drug discovery and development, and downstream bio-processing.

Antibody sequencing

It is frequently asked why my valuable antibody need to be sequenced? Here are some reasons you may take into consideration.

Patent application requires antibody sequence.
A detailed sequence for further antibody engineering.
Prevent the loss of the hybridoma cell line.
Recombinant antibody production.

Antibody sequencing process:
The mRNA will be extracted from the hybridoma clone. Then the antibody mRNA will be reverse transcript, amplified and subcloned into a vector for sequencing. At least 5 samples will be sequenced individually to confirm the final result.

Service and Time Line	Guarantee	Starting Material	Deliverables	Price
ABE120101 Antibody Sequencing	Full antibody sequence.	Hybridoma clone.	• Antibody CDR sequence. • Report.	Inquiry

Package detail:

Hybridoma clone culture and mRNA extraction.
Reverse transcrption and PCR amplification.
cDNA subcloned into vector.
Sequencing.
Sequence alignment to have the final result.
Generate detailed report.

Email to info@bio-bench.com if you have any question.

Antibody humanization and affinity maturation

Immunogenicity can cause severe problems for the patient. It can potentially cause drug efficacy reduction through antibody clearance or antibody neutralization, or toxicity due to cross-reaction with proteins in the body, which will lead to destructive consequences for the patient. Antibody humanization is the only way to reduce or remove the immunogenicity of monoclonal antibody derived from animal sources and to improve its role in the human immune system by replacing non-human originated sequences with human ones. In the past 20 years all of the approved antibody drugs are humanized antibody or full human antibody, proving that humanization is a very important step in therapeutic antibody discovery process.

Bio Bench is a one-stop service provider for antibody discovery and development, providing integrated antibody humanization and affinity maturation service. Powered either by silico CDR-grafting or by phage display technology, Bio Bench offers 2 methods to achieve humanized antibody. By applying phage display based antibody humanization, affinity maturation is combined to improve the affinity by 10 – 50 folds. Bio Bench has achieved more than 30 antibody humanization and affinity maturation projects in the past 6 years, with 1 of them in clinical phase.

3-D analysis and structuring of antibody

Fig.1 3D analysis and structuring for humanization.

Fig.2 Antibody affinity maturation strategies.

Service and Time Line	Guarantee	Starting Material	Deliverables	Price
ABE120202 In silico CDR-grafting antibody humanization 10 – 15 weeks.	>85% human sequenced antibody.	Hybridoma clone.	• Antibody humanization design. • 100μg purified humanized antibody. • Report.	Inquiry

Package detail:

Antibody sequencing (if needed).
Bio informatics analysis.
3D structure modeling and identification of back mutations.
Human germlines selection.
In silico CDR-grafting and sequence optimization.
Transient recombinant production of 9 to 18 humanized antibody variants.
Affinity test by Biacore or Fortebio (customer may discuss with Bio Bench technical support).
Detailed report.

Email info@bio-bench.com if you have any question.

Service and Time Line	Guarantee	Starting Material	Deliverables	Price
ABE120203 Phage display antibody humanization and affinity maturation Time line to be determined.	1.>90% human sequenced antibody. 2.Antibody affinity <20nM.	Hybridoma clone.	• 100μg purified humanized antibody. • Report.	Inquiry

Package detail:

Antibody sequencing (if needed).
Bio informatics analysis to confirm the FW region and CDR region.
3D structure modeling, spot mutation design and HOT-SPOT confirmation.
Phage display library construction and clone selection.
IgG subclass selected by the customer and the codon will be optimized according to expression cell line (if selected).
Transient recombinant production of humanized antibody (if selected).
Affinity test by Biacore or Fortebio (customer may discuss with Bio Bench technical support).
Detailed report.

Email info@bio-bench.com if you have any question.

Bi-Specific Antibody (BsAb)

Bi-specific Antibody (BsAb) is an artificial antibody that able to bind two different antigens or different epitopes of the same antigen. The most important application of BsAb has been to stimulate cytotoxic immune effector cells for enhanced killing of tumor cells by antibody-dependent cell-mediated cytotoxicity (ADCC) and other cytotoxic mechanisms mediated by the effector cells.

Bi-specific antibody mechanism
Each of the antibody arm binds to the biomarker on the tumor cell and CD3 on T-cell, separately. The Fc region additionally binds to an accessory cell that expresses Fc receptors, like a macrophage, NK cell or dendritic cell. This cross-binding of tumor and effector cells will sharply reduce their distance while triggering T cell killing activation, thus resulting in high tumor cell killing effect.

Bi-specific antibody structures
Developed by modern molecular biology, there are more than 50 types of bi-specific antibodies that have been synthesized with a confirmed unique function and the number is still increasing.
Going beyond bi-specific function on one antibody, multi-specific antibody is also being synthesized and studied in all of the research stages.

Antibody: Bi-specific antibody.
Antibody expression system: Mammalian 293F.
17.35mg bi-specific antibody obtained from 1L cell culture, purity>90%.

Service and Time Line	Guarantee	Starting Material	Deliverables	Price
ABE120204 Bi-specific antibody synthesis Time line to be determined.	1.An antibody binds 2 sites separately. 2.Purity >90%.	Hybridoma clones.	• 100μg purified bi-specific antibody. • Report.	Inquiry