Antibody Fragments Fab, scFv, VHH, DARTs and BiTE Expression and Purification

Antibody fragments represent modular building blocks for the development of larger antibody reagents and therapeutics. Increasingly, they are also directly used in therapeutic applications. Unlike human IgG monoclonals, which generally require expression in mammalian cells, human antibody fragments can be expressed in bacteria at high levels. This includes Fragment antigen-binding region (Fab region: VH-CH, VL-CL), single-chain variable fragment (scFv : VH-VL), single-domain fragments (VH, VL) and single-domain antibody (sdAb), such as V_HH fragments, the first sdAb engineered from heavy-chain antibodies found in camelids. Fusion Biolabs has both bacterial expression system and mammalian expression system for those fragments production.

Bispecific antibody (bsAb) is one kind of the next generation of antibody. The dual targeting is a particular concept linked with bsAbs, enabling them to target two different antigens on two different cells, two different antigens on the same cell, or two different epitopes on the same antigen. Chemical conjugation of two different monoclonal antibodies (mAbs), generation of a quadroma cell line (a hybrid hybridoma secreting a bsAb), and genetic engineering are the most routine methods used to produce the sAb.

There are many kinds of bsAb fragment formats. Single-chain variable fragment (scFv) based bsAb is the very common format. 

Bispecific diabody with the VH–VL configuration

Cloning and design strategy for the generation of a bispecific diabody with the VH –VL configuration is shown. In the first step, the variable fragments of binding site A and site B (V_HA or V_HB and V_LA or V_LB) are amplified separately; then the variable domains of antibody A and antibody B are fused to create the structure V_HA–V _LB and V_HB–V_LA. In the last step, the TWO fragments are introduced into one expression vector combined to generate the bispecific diabody.

Bispecific diabody with the VH–VL configuration

Bispecific single-chain diabodies (scDb) and tetravalent tandem diabody (TandAb) design

Bispecific single-chain diabodies are generated by the expression of a single fragment of the format VHA–VLB–VHB–VLA (VH-VL configuration with 10-20 amino acid length middle linker) or of the format VLA–VHB–VLB–VHA (VL-VH configuration with 10-15 amino acid length middle linker). Generally, 10 amino acid short middle linker facilitate tandem diabodies (TandAbs), whereas, long middle linker facilitate single-chain diabodies (scDbs) formation.

Bispecific single-chain diabodies (scDb) and tetravalent tandem diabody (TandAb) design

Tandem scFv fragment-based bispecific antibodies (BsAb) design

The tandem scFv fragment-based bispecific antibodies (BsAb) is constructed by two scFv (scFvA and scFvB) molecules connected through a short linker, which allows free rotation of the two separate folding and antigen-binding units, thus forming a very flexible structure (scFvA-scFvB). Since tandem scFv molecules tend to form insoluble aggregates in bacteria, variation of the domain order (e.g., the  first scFv in the VH –VL  and the second scFv in the VL–VH  orientation) or linker modifications (e.g., appropriate sequences)  can provide a solution for the generation of soluble and active protein. Alternatively, refolding protocols or mammalian expression systems can be used.

Tandem scFv fragment-based bispecific antibodies (BsAb) design

Bispecific Dual-Affinity Re-targeting molecules (DARTs): DARTs are bispecific antibodies designed to simultaneously recognize both tumor antigens and invariant epitopes of the CD3/TCR complex on T cells. By doing so, they effectively recruit T lymphocytes to tumor targets in a noncognate fashion, bypassing the MHC context required for antigen recognition by the TCR. Theoretically, this enables all T cells to be redirected against the tumor. The DART (bispecific against B-cell C19 and T-cell CD3 structure) facilitates T-cell/B-cell associations, leading to potent redirected T cell–killing of B-cell lymphoma. DARTs generally are expressed in mammalian cell system CHO or HEK293.

Bispecific antibody: Dual-affinity re-targeting molecules (DARTs) design illustration

Bispecific T cell engager (BiTE®) antibody constructs comprise tandemly-arranged scFvs. One scFv binds the TCR CD3ε subunit and the other binds a tumor-associated surface antigen (TAA).

Bispecific T-cell engaging antibodies (BiTEs) structure and design

Fusion Biolabs has developed unique genetic engineering platforms to expression and purification of DART and BiTE.

Antibody fragments offer multiple advantages over full length monoclonal antibodies as drug carriers to tumors. They are able to provide greater tumor penetration due to their small size, low kidney uptake, rapid blood clearance, and a lesser negative response by human immune system.

Our services Provides two expression platforms:

1) E. coli bacterial expression system, which is seamlessly compatible with our phage display library construction, antibody phage panning and candidate validation projects.

2) Mammalian transient expression systems in CHO or HEK293 cells.

What We Offer: 

1. Antibody Fragments (scFv, Fab, VHH, DARTs and BiTE)Produced from Synthetic DNA– This is the fastest route to construct antibody fragment. Oligonucleotides are first assembled in-vitro and then cloned into our in-house expression vectors.  The scFv is then expressed in E. coli or a mammalian cell line. The turnaround time is 1-2 weeks. 

2. Antibody Fragments (scFv, Fab, VHH, DARTs and BiTE) produced from monoclonal cell line – The customer provides the monoclonal hybridoma cells. The mRNA from the monoclonal will be cloned to create a cDNA vector from which the variable heavy (VH) and light (VL) chains are then subcloned into an expression vector.

3. Antibody Fragments (scFv, Fab, VHH, DARTs and BiTE) manufactured from accession numbers – The gene of the target protein is synthesized according to the accession number, cloned, expressed, purified.

Key Features

Process Overview

 Client provides: Antibody sequence, plasmids containing coding sequence, or hybridoma cells (>1×10⁵ cells, in liquid nitrogen or dry ice) with isotype or class information

Stage I: Variable Region (VH, VL) Sequencing or Gene Synthesis

Turnaround time: 5 days

Stage II: Bacterial expression vector or mammalian expression vector construction and sequence validation

Turnaround time: 5 days

Stage III: Transient expression or bacterial expression and affinity purification

Turnaround time: 1-2 weeks

Deliverables:

1) Purified antibody fragments (scFv, Fab, VHH, DARTs and BiTE)

2) Certificate of Analysis (COA) data sheet

Ordering Information

Antibody Fragments scFv, Fab, VHH,DARTs and BiTE Expression Services

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