Humanized antibody

Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of 'humanization' is usually applied to monoclonal antibodies developed for administration to humans (for example, antibodies developed as anti-cancer drugs). Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system (such as that in mice). The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients (see also Human anti-mouse antibody). There are other types of antibodies developed. The International Nonproprietary Names of humanized antibodies end in -zumab, as in omalizumab (see Nomenclature of monoclonal antibodies).The humanization processes takes advantage of the fact that production of monoclonal antibodies can be accomplished using recombinant DNA to create constructs capable of expression in mammalian cell culture. That is, gene segments capable of producing antibodies are isolated and cloned into cells that can be grown in a bioreactor such that antibody proteins produced from the DNA of the cloned genes can be harvested en masse. The step involving recombinant DNA provides an intervention point that can be readily exploited to alter the protein sequence of the expressed antibody. The alterations to antibody structure that are achieved in the humanization process are therefore all effectuated through techniques at the DNA level. Not all methods for deriving antibodies intended for human therapy require a humanization step (e.g. phage display) but essentially all are dependent on techniques that similarly allow the 'insertion' or 'swapping-out' of portions of the antibody molecule.Humanization is usually seen as distinct from the creation of a mouse-human antibody chimera. So, although the creation of an antibody chimera is normally undertaken to achieve a more human-like antibody (by replacing constant region of the mouse antibody with that from human) simple chimeras of this type are not usually referred to as humanized. Rather, the protein sequence of a humanized antibody is essentially identical to that of a human variant, despite the non-human origin of some of its complementarity determining region (CDR) segments responsible for the ability of the antibody to bind to its target antigen.The humanization process may, however, include the creation of a mouse-human chimera in an initial step (mouse variable region spliced to human constant region). Thereafter the chimera might be further humanized by the selective alteration of the sequence of amino acids in the variable region of the molecule. The process must be 'selective' to retain the specificity for which the antibody was originally developed. That is, since the CDR portions of the variable region are essential to the ability of the antibody to bind to its intended target, the amino acids in these portions cannot be altered without the risk of undermining the purpose of the development. Aside from the CDR segments, the portions of the variable regions that differ from those in humans can be corrected by exchanging the appropriate individual amino acids. This is accomplished at the DNA level using mutagenesis.It is possible to produce a humanized antibody without creating a chimeric intermediate. 'Direct' creation of a humanized antibody can be accomplished by inserting the appropriate CDR coding segments (so-called 'donor', responsible for the desired binding properties) into a human antibody 'scaffold' (so-called 'acceptor'). As discussed above, this is achieved through recombinant DNA methods using an appropriate vector and expression in mammalian cells. That is, after an antibody is developed to have the desired properties in a mouse (or other non-human), the DNA coding for that antibody can be isolated, cloned into a vector and sequenced. The DNA sequence corresponding to the antibody CDRs can then be determined. Once the precise sequence of the desired CDRs are known, a strategy can be devised for inserting these sequences appropriately into a construct containing the DNA for a human antibody variant. The strategy may also employ synthesis of linear DNA fragments based on the reading of CDR sequences.There are technologies that completely avoid the use of mice or other non-human mammals in the process of discovering antibodies for human therapy. Examples of such systems include various 'display' methods (primarily phage display) as well as methods that exploit the elevated B-cell levels that occur during a human immune response.