Ever since the first mAb drug, antibody engineering technologies have drastically evolved. Each successive generation of mAb therapeutics was intended to enhance the clinical benefits and minimize toxicities. The main areas of focus in therapeutic mAb design are high target specificity, better systemic retention, and low immunogenicity. The following are the different generations of antibodies that have made an impact.
The murine antibodies were the first antibodies to be developed at lab scale using the hybridoma technology in rodents. But because of the differences between the human and rodent immune system, usage of murine antibodies resulted in cytotoxicity. Thus their continuous administration often resulted in allergic reactions and anaphylactic shock, termed as human anti-mouse or anti-murine antibody (HAMA) response. Anti-CD3 mAb of murine origin (OKT-3), the first therapeutic mAb, approved for treatment of transplantation rejection was discontinued primarily due to severe HAMA response in patients. Nevertheless, murine antibodies serve as frameworks for antibody development. Also due to the improved and optimized protocols for antibody generation from hybridoma mouse cell lines, this technology is considered popular and economical to produce new antibodies.
In order to minimize the HAMA response, chimeric antibodies were manufactured. These antibodies consist of 65% human genetic component by recombination of human constant regions and mouse variable regions in a suitable expression system. There are some chimeric antibodies approved by the FDA for use in human therapy and research, for eg., Infliximab, Rituximab, Abciximab. Presently even though there is a declining interest in developing chimeric antibodies for clinical applications, chimeric molecules can be widely used in the initial stage of antibody humanization strategies and also serve as controls and calibrators for specific research needs or in diagnostics applications.
The urgency to overcome the obstacles presented by low complementarity of the mouse line developed antibodies to the patient’s immune system, gave way to the creation of humanized antibodies. The humanized antibodies are close to 95% in human origin, with only the complementarity determining regions (CDRs) of the variable regions having mouse-sequence origin. However, these Abs sometimes present lower affinity than the parent murine mAbs with respect to binding with antigens. So as to increase the antibody-antigen binding affinity, techniques such as chain-shuffling randomization can be employed to introduce some transformations into the CDR. Daclizumab is the first FDA-approved humanized antibody, used in the treatment of multiple sclerosis.
Fully human sequence derived antibodies have no murine sequence, and are largely produced via two sources: phage display technologies and transgenic mice. The first fully human sequence-derived antibody to be approved for therapeutic use was adalimumab (Humira), a fully human IgG1 antibody specific for TNFalpha that was selected via phage display of human VH and VL sequences.
The two main technology platforms that are currently used for manufacturing fully human mAbs are:
This method is used to produce diverse libraries of phage displayed antibody variable regions (scFv or Fab) thus providing quick access of target specific mAbs, without creating a library of hybrid clones. Through biopanning, an in-vitro screening technique using specific antigens, the phages expressing antibody fragments exhibiting better affinities are identified, sequenced and used for development of fully human mAbs.
Transchromosomal engineering process has aided the introduction of transgenes encrypting human immunoglobulin heavy and light chains, into mice germline. This has led to successful development of mAb drugs from human-antibody producing mouse. Some of the advantages of this technology include direct generation of full-length IgG, more diversity and in-vivo affinity maturation. However, this method is not preferred for toxic antigens.