重组抗体

A recombinant antibody (rAb) is generated from the cloned, in vitro-expressed heavy and light chains of a selected monoclonal antibody obtained through classical hybridoma methodology, display strategies alone or in combination, sorted single B cells from immunized animals, or by one of many other techniques. The fact that it is cloned means the rAb is defined by its primary sequence, which can be engineered to optimize affinity and be expressed in different binder formats. The numerous advantages of rAbs have been well-documented, with consistency of performance being of paramount significance.

GeneTex’s rAb protocol employs a multi-parameter FACS-based approach to isolate antigen-specific IgG+ memory B cells from an immunized animal, with subsequent cloning of the antibody variable-region genes into an IgG backbone and expression in mammalian cells (Fig. 1) (1). This protocol is very rapid and can be completed in weeks, and also affords the opportunity to identify antibodies with diverse capabilities in various applications. Importantly, it allows cloning of the heavy and light chains from the same B cell, thereby preserving natural pairing. And once cloned, the supply of a given rAb is inexhaustible with exceptional reproducibility.

Another new recombinant rabbit monoclonal reagent is GeneTex’s RAS (G12D mutant) antibody [HL10] (GTX635362) (Fig. 3). It demonstrates a robust signal and specificity for the G12D mutation in wild-type (WT) and KRAS G12D mutant pancreatic tumor tissue sections (Fig. 3A) and in established cell line lysates (Fig. 3B), respectively. This RAS (G12D mutant) antibody (GTX635362) is the first commercial recombinant version that shows exceptional specificity by IHC-P for this key tumorigenic mutant protein on sequence-verified human pancreatic tumor samples.

As mentioned above, the antigen-binding regions of a recombinant antibody can be inserted into various host IgG backbones or expressed in different binder formats (e.g., Fab fragments or scFvs). Here, GeneTex’s cloned TSG101 mouse monoclonal antibody (GTX70255) was converted to a rabbit IgG backbone (GTX635396) with preservation of performance. No cross-reaction was observed when the converted TSG101 rabbit IgG recombinant antibody was used in combination with an anti-mouse IgG secondary antibody (Fig. 4).

GeneTex’s production focus is the creation of novel recombinant rabbit monoclonal antibodies validated using enhanced protocols defined by the International Working Group for Antibody Validation (IWGAV).

Over the last decade, studies published in many of the top scientific journals have clearly described the significant impact of poorly validated antibody reagents on the reproducibility of biomedical research. The unfortunate reality is that the commercial antibody market is compromised by a substantial subset of products that have been demonstrated to be nonspecific. This means that there are two major issues that must be addressed in order to improve the overall quality of the commercial antibody market and therefore the integrity and reproducibility of the biomedical research dependent on antibodies. The first is more widespread adoption of production platforms that emphasize recombinant antibody technology (please see “Section 1: What is a recombinant antibody?”) while the second involves incorporation of improved and more coherent validation strategies. GeneTex is making major strides in both of these endeavors. 

With the goal of providing better products for its customers through innovative technology, GeneTex has now shifted all of its new production to recombinant rabbit monoclonal antibodies (please see “Section 3: Why GeneTex’s rabbit-based HL clones are a better choice.”). Fully recombinant antibodies are defined by their primary sequence, their consistent performance and supply (issues that plague traditional polyclonals and hybridoma-based monoclonals with regard to lot-to-lot variability), and their ability to be engineered (i.e., backbone switching) to meet researcher requirements. These benefits may even evoke more favorable impressions by grant review and journal editorial committees. Other outstanding advantages of recombinant rabbit antibodies stem from the favorable biological characteristics of the rabbit immune system itself.

In conjunction with its pivot to recombinant antibody production, GeneTex’s second commitment to antibody reliability is its incorporation of enhanced validation protocols, based on the IWGAV proposal mentioned above, into its manufacturing and quality assurance (QA) workflow (1). This involves evaluation of each antibody using at least one, and preferably more, of five validation “pillars” defined in the IWGAV study. GeneTex’s version of the IWGAV plan follows the five standard strategies and includes (1) Knockout/Knockdown; (2) Comparable Antibodies; (3) Immunoprecipitation followed by Mass Spectrometry (IP/MS); (4) Biological and Orthogonal Validation; and (5) Recombinant Protein Expression (Fig. 1). The company refers to this as its “5+1” Pillar Plan when referencing new recombinant antibody production, with the “+1” designation referring to the pre-validation that is inherent in the application-specific assessment that occurs with clone selection during the recombinant antibody production process (please see “New Validated Recombinant Monoclonal Antibodies from GeneTex” below).

The new recombinant rabbit monoclonal antibodies that have been generated and evaluated using the production and validation approaches described above (these products are identified by “HL” in their clone designations) are just the beginning. GeneTex is also working to replace its successful polyclonals with new recombinants. The company envisions the “HL” clones representing the highest level of GeneTex validation and manufacturing capability.

Antibody fidelity, application-dependent reliability, and performance and supply consistency are crucial for ensuring research integrity. GeneTex joins other reputable companies in leveraging recombinant antibody technology and enhanced validation to create antibodies that researchers can trust.

GeneTex’s recombinant rabbit monoclonal antibody production process is the platform for all new rabbit-based antibody generation (1). These products are distinguished by the “HL” designation in their respective clone numbers (e.g., “[HL1089]”). The rabbit, and specifically its immune system, is known to confer the following outstanding benefits for monoclonal antibody creation: broader antibody range, less complex immunoglobulin (Ig) structure, higher binding affinities, larger pool of lymphocytes to select antibodies (2). Each of these advantages is described in more detail below:

1. Broader antibody repertoire:

1. Rabbits belong to the order Lagomorpha, which is evolutionarily distinct from the order Rodentia. This means that human antigens present epitopes that are often more immunogenic in rabbits than in mice, expanding the number of generated antibodies that may in turn cross-react with murine protein orthologs (2-5).
   
2. The rabbit immune system is more successful in mounting an immune response to hapten or small molecule antigens, which is frequently not the case with the murine immune system (2-5).
   
3. Mice used to generate monoclonal antibodies are from inbred lines and offer less diversity in their immunogenic responses than do rabbits. In addition, mouse spleens are much smaller than rabbit spleens (2-5).
   
4. The rabbit immune system is different from those of mice and humans in that it utilizes somatic gene conversion (SGC) to expand its antibody range in addition to performing the same VDJ and VJ recombination and somatic hypermutation (SHM) mechanisms noted in mice and humans. Antibody diversity is further enhanced by other processes that include a kappa light chain variable gene structure distinctive for variability in the length of complementarity determining region 3 (LCDR3), as well as successive Ig development in the bone marrow, the gut-associated lymphoid tissues (GALT), and spleen and lymph node germinal centers (3, 5).

 2. Simpler Ig structure:

1. The European rabbit (Oryctolagus cuniculus) is known to have single genes for IgE and IgM, and no genes for IgD. Interestingly, its genome contains 13 IGHA genes coding for at least 10 functional IgA subclasses. Importantly, though there are allelic variants, rabbits have only one IgG gene (and thus no subclass) and therefore differ from mice and humans (each with four IgG subclasses) (4, 5).
   
   
2. In addition to the disulfide bonds found in a human or mouse IgG (linking the heavy chain pair or one heavy chain to its associated light chain), rabbit IgG antibodies demonstrate a unique intrachain disulfide bond in their K1 light chain. This is significant as perhaps 90% of IgGs in New Zealand White rabbits are IgG-k (K1), and this extra bond is thought to confer stability onto the rabbit IgG (2, 5).
   
   

3. Enhanced binding affinity:

1. Rabbit monoclonal antibodies have very high affinities with Kd values characteristically in the picomolar range, with some possessing exceptionally low picomolar Kd values (2, 5).
   
   
2. Picomolar Kds translate to increased sensitivity with consistent specificity.

4. Larger lymphocyte pool:

1. Rabbits have a longer life span and their larger size (perhaps a 100-fold weight differential between a three-month-old rabbit and a six-week-old mouse) greatly facilitates blood and tissue sampling during antibody production (5).
   
   
2. Consistent with the statements above, rabbit spleens allow isolation of 50-fold more B cells compared to murine spleens, meaning more clones recognizing a wider selection of epitopes (2-5).