Fusion have developed a range of antibody engineering services based around monoclonal antibodies:

Service 1

Monoclonal Sequencing: The sequence of a monoclonal antibody is important for patent issues and therapeutic approval. Fusion Antibodies have a rapid monoclonal antibody sequencing service.

Service 2

ScFv / Fab Production: Single chain & poly chain antibody fragments are commonly used as research reagents and are finding increased use as therapeutics. Fusion Antibodies have developed a technology, as an extension of the Monoclonal Sequencing Service, to convert full monoclonal antibodies from a hybridoma cell line into single chain antibody fragments. These can be expressed in E. coli or CHO cells.

Service 3

Antibody Humanization: The major disadvantage of using murine monoclonals as therapeutic agents is that repeated dosing leads to a human anti-murine antibody response (HAMA). Humanization of murine IgG is used to try to reduce the HAMA response. Humanized antibodies have a greater half-life in vivo meaning a longer duration of therapy. They also have the advantage of repeat administration in chronic cases and greater intensity at lower doses.

ScFv / Fab production Service

Fusion have developed an E. coli based protocol for the production of custom single chain variable fragments (scFv) and Fab recombinant antibodies. There is no other company in the world that offers scFv production as a service. There are many advantages in using scFv's or Fab's, particularly in the fields of therapeutics and diagnostics. scFv's offer several advantages over monoclonal antibodies as carrier of radionuclei and drugs to tumours, including greater tumour penetration due to their small size, low kidney uptake, rapid blood clearance, and a lower negative response by the human immune system.

scFv Construction
There are three main entry points in the construction of an scFv:
· Option I - Manufacture of scFv from synthetic DNA.
· Option II - Manufacture of scFv from monoclonal cell line.
· Option III - Manufacture of scFv from accession number.

Option 1- Manufacture of scFv from synthetic DNA
The fastest route to an scFv is starting from the actual DNA sequence of the desired scFv. In this scenario we design an artificial gene using oligonucleotides, assemble it in vitro and clone it into our in-house expression vectors. We can then express the scFv in E. coli and purify it; this has a timescale of around 8 weeks.

Option 2 - Manufacture of scFv from Monoclonal Cell Line
The second route is to create an scFv from the monoclonal cell line. The client provides the monoclonal hybridoma cell line. 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. Typical production of the purified scFv is 8 weeks.

Option 3 - Manufacture of scFv from Accession Number
The third route is starting with an accession number of the target protein. We can clone, express and purify the protein and use this as an immunogen to raise a monoclonal in mice. We can then screen for the relevant monoclonal characteristics and use this cell line as the basis for scFv. The procedure is the same as Option II from this point on. This has a timescale of 28 weeks, 20 weeks of which are dedicated to producing the monoclonal cell line. The process is described in more detail in the following paragraphs.
Following the identification of a highly specific monoclonal antibody, the hybridoma cell line producing the antibody will be used as a source of Vl and Vh fragments. Hybridoma mRNA will be isolated from the cells using standard methods and a cDNA copy prepared using the reverse transcription (RT) PCR with random oligonucleotide primers. This cDNA will then be used as a template, using specific oligonucleotide primers, for the amplification of the Vh and Vl chains.
The Vl and Vh chains will then be amplified with primers containing specific cloning sites to facilitate construction into our expression vector. The primers are also engineered to create the peptide linker sequence between the heavy and light chains.

Fab Construction
Fab fragments differ from scFv’s in that as well as containing variable domains, constant regions are included giving rise a molecule of the form VhCh1-s-s-Cl-Vl where -s-s- represents a disulphide bridge. Thus their construction and ultimate expression is somewhat more complex. The same heavy and light variable chains used for scFv construction will be used in the construction of Fab. Unlike scFv’s the Fab fragments have to be expressed as two separate chains i.e. Vh-Ch1 and Vl-Cl.
This service is useful when there is no monoclonal antibody available and the sequences of the variable regions are known. Otherwise it is more cost effective to create the Fab enzymatically using papain; creates two Fab fragments and the Fc fragment, or pepsin; creates F(ab')2 and pFc' fragments.

If you are interested in scFv or Fab production please contact Fusion and we can discuss your requirements.

 

Antibody Humanization Services

Background
Strategies have been developed to reduce the HAMA (human anti-mouse antibody) response in patients undergoing antibody therapy. Murine antibody variable domains can be recombined with human antibody constant domains to produce a chimeric antibody. The modular nature of antibodies enables researchers to convert a murine monoclonal antibody into one that has some human segments but still retains its original binding specificity. Chimeric antibodies are designed to reduce the HAMA response while maintaining a high specificity. They permit multiple dosing and increased half-life of each dose. Also the human effector function can be assessed.

Technology
Fusion Antibodies have developed a technology for the rapid production of cell lines expressing full length recombinant antibodies and fully humanized antibodies. A humanized antibody is engineered from a murine monoclonal antibody following our monoclonal sequencing procedures.

Stage I
The antibody variable domains are amplified from the clients hybridoma cell line, cloned and sequenced. By overlap extension PCR, a signal peptide is added to the variable domains and these are linked to the human or murine constant domain isotype of the clients choice, eg IgM, IgG2b, Ig3, etc.
Stage II
The antibody heavy and light chains are cloned into our bicistronic mammalian retroviral expression vector. A retroviral packaging cell line is transfected with the vector, and post-transfection the supernatant from this is used to infect our in-house CHO cell line.
Stage III
Antibody expressing cells can be selected by antibiotic resistance or FACS in some cases. The antibody produced from the CHO cells are tested by ELISA.