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Genomic Antibody Technology

Antibodies are important research tools, diagnostic reagents and therapeutic drugs. As reagents they are used in a variety of different immunoassay formats including western blot, immunohistochemistry (IHC), ELISA, sandwich immunoassay, immunoprecipitation, flow cytometry, and functional assays. The way in which an antibody is made determines the form of antigen it binds, the site of binding, the strength of binding, crossreactivity, and the type of assay in which the antibody will work. Therefore, it is necessary that antibodies be developed and selected in a manner consistent with the intended purpose.

Discontinuous Protein Epitopes

In addition to having a specific 3-dimensional shape, the site on an antigen recognized by an antibody, the epitope, is made up of amino acid residues that are not all adjacent to one another in the linear amino acid sequence, i.e., they are located in a discontinuous manner along the protein sequence (Figure 1). It is well established that the vast majority of epitopes on native protein surfaces are comprised of discontinuous amino acid residues that only come into close spatial proximity when the protein is properly folded (4).

Conformational Protein Epitopes

Proteins have specific functions that are dependent on their native 3-dimensional conformation. It has been known for over 30 years that the majority of antibodies made to intact native proteins do not react to the same protein sequence in a denatured form (1,2). The traditional method of making antibodies is to immunize animals with full-length purified native or recombinant proteins. A significant obstacle to this approach is the development and purification of protein antigens in sufficient quantities and qualities for immunization and subsequent antibody purification or selection. The preparation of native protein antigens is so onerous that it led researchers to search for less costly, less time-consuming alternatives to full-length protein immunization. Since the 1980s, the most common method by which antibodies to proteins are made is to immunize animals with short linear peptides and hope that the resultant anti-peptide antibodies crossreact with native protein. Peptide immunogens made up of 10-20 amino acids are highly flexible and adopt many different 3-D conformations, of which only a very limited number will elicit an antibody capable of crossreacting with the native protein. While anti-peptide antibodies may have high titer to the immunizing peptide, typically only a small percentage of the antibodies made using this method are capable of binding native protein - resulting in very low specific activity or, as is often the case, no binding activity at all. Indeed, a crucial test of peptides capacity to elicit antibodies that bind native protein structures is the generation of protective antibodies when administered as synthetic vaccines. Unfortunately, since 1990 over 100 synthetic peptides have entered clinical trials as vaccines and none have been approved (3).

X-ray Crystal Structure PDB 3G04

TSHR antigen

Linear amino acid sequence of TSHR 38 255

218 aa

Figure 1. Discontinuous proten epitope. Antigen amino acids contacted by antibody (yellow) are not adjacent in linear sequence. Contact residues are contained within 218aa linear protein sequence. A short peptide can not form this epitope.

As a research program develops there is usually a need for antibodies that bind native protein, and it is frequently the case that the first antibodies that were useful in western blot and IHC are not useful in assays requiring recognition of native 3-dimensional protein conformation.
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Contact Residues (CR)

PDB 3SM5 2VIS 3LZF Mab CH65
IgG1, Lambda

3.5

4 CR 20 17 18 19 12 86 CRS 94 66 124 227 38 256

Influenza HA Ammino Acid Sequence 1-50 51-100 101-150 151-200 201-250 251-300

2D1

Figure 2. Discontiuous epitopes of 5 influenza hemagglutinin neutralizing monoclonal antibodies. CRS = Contact Residue Span.

1QFU Neutralizing Fab 3GBN SUM Cr6261 ALL Purified hEGFR

GA3177 GA3179

OD 650 nm

Figure 2 shows the actual sequence positions of the amino acids comprising the epitopes of 5 highly characterized influenza hemagglutinin neutralizing monoclonal antibodies. These antibodies demonstrate that epitopes recognized by real-world functional, i.e., neutralizing, antibodies are discontinuous in nature.

Sandwich ELISA with Full-Length Purified Human EGFR (hEGFR) 4 3 2 1 0 0.1 1 10 100
GA3177 Capture GA3179 Detector

Failed Peptide Antibodies

Decades of research has led to the conclusion that peptides are poor mimics of discontinuous epitopes (5), and that peptide immunizations are not a practical strategy for targeting discontinuous epitopes (6). Given that the vast majority of protein epitopes are conformationally-determined and discontinuous, and that the most common method of developing antibodies is using small, linear, unstructured peptides, it is no wonder that a common complaint among antibody users is that most antibodies dont work.

EGFR Concentration, nM

Figure 3. The type of assay influences antibody reactivity. Polyclonal antibody GA3177 binds native epitopes in ELISA but not the same epitopes when they are denatured in western blot. GA3179 binds epitopes in both assays.

The Influence of Assay Type

One of the most important considerations when attempting to develop an antibody is how it will be used. Both western blot and IHC subject the protein to significant chemical and physical processes that alter 3-dimensional protein conformation, and it is common that antibodies that work in these assays do not work in other assay formats (e.g., sandwich ELISA with minimally processed serum samples or flow cytometry with live cells) and vice versa. This effect is more pronounced with monoclonal antibodies because they recognize only a single epitope conformation. Polyclonal antibodies made to native proteins comprise a population recognizing both native and denatured epitopes and generally are useful in a wider variety of assay applications. Figure 3 shows the reactivity of two polyclonal antibodies, GA3177 and GA3179, to EGFR protein in western blot and sandwich ELISA. The data

demonstrate that GA3177 binds EGFR in sandwich ELISA but not western blot, while GA3179 binds in both assays. In the western blot, EGFR is denatured by boiling in detergent and reducing agent while in the ELISA it is maintained in physiologic buffer. These results demonstrate a critical aspect of antibody technology, i.e., a single antibody rarely works in all types of immunoassays, and successful antibody development is dependent on understanding the intended application and designing antibody reactivity in a way that is consistent with how the antibody will be used. Early in a research program, it is frequently a first objective to establish the presence or absence of a protein in a specific sample type or biological process. Western blot and IHC are ideally suited for these activities and polyclonal antibodies are cost-effective reagents in these cases. As a research program develops there is usually a need for antibodies that bind native protein, and it is frequently the case that the first antibodies that were useful in western blot and IHC are not useful in assays requiring recognition of native 3-dimensional protein conformation. This is especially true when the first reagents were made using peptide immunogens.

In most critical antibody applications, it is not sufficient for an antibody to simply bind anywhere on the protein antigen, rather, it is necessary that the antibody bind to a specific site or region that correlates with biological significance.
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In a mature project it is often the case that routine assays are being used that have defined performance specifications and large amounts of antibody are required. In these applications, long-term, consistent supply of antibody is critical, and monoclonal antibodies are preferred reagents. While the uniform consistency of monoclonal antibodies is a preferred characteristic of reagents in all applications, the time and cost to develop them is significantly greater than polyclonal antibodies, and because they only recognize a single epitope they are less likely to react in a variety of assay types. A common antibody development scenario entails relatively low cost polyclonal reagents for early discovery activities followed in later stages by additional antibody development, including monoclonals, made in such a way that they will react to the form of the antigen as it exists in the new intended applications.

SDIX Genomic Antibody Technology

SDIX Genomic Antibody Technology (GAT) was developed to produce antibodies that have performance characteristics superior to conventional antibodies made using peptide and full-length protein immunization. GAT is designed to produce antibodies to predefined native protein epitopes for critical research, biomarker discovery, diagnostic and therapeutic applications. GAT includes immunization of rabbits and mice with DNA vectors encoding relatively large (approximately 100aa) portions of proteins. Frequently, these sequences are designed to code for independent protein domains. The 3-dimensional conformation of an individual domain is relatively independent of other parts of a protein and there is a significantly greater probability that a domain will fold into a native conformation and elicit conformation-specific antibodies than a short linear peptide. A critical aspect of GAT is immunization with DNA rather than protein. Even highly purified preparations of protein contain contaminants that antibodies are made to in immunized animals. DNA immunization encodes only the target of interest, absent of contaminants, resulting in high specific activity antibody responses. In addition, in vitro expression and purification processes subject proteins to physical stress that can alter native 3-D conformation. A significant advantage of DNA immunization is that the protein is made in vivo within cells of the host animal. Thus, the protein is a mammalian expression product and undergoes normal cellular processing that destroys misfolded protein, increasing the probability that antibodies are made to properly folded 3-D conformations. Evidence that GAT results in conformation-specific antibodies can be seen in rabbit polyclonal Genomic Antibody GA3177 (Figure 3), which reacts with EGFR in physiologic buffer but not when it is denatured in western blot.

Immunodominant Protein Epitopes

The gold standard approach for developing antibodies to native proteins is to immunize animals with fulllength native or recombinant proteins. While scientists believe that antibodies to virtually every region of a protein exist within the complete antibody repertoire of an animal, the majority of antibodies made to full-length protein antigens are restricted to a few immunodominant regions of the protein (7,8). In most critical antibody applications, it is not sufficient for an antibody to simply bind anywhere on the protein antigen, rather, it is necessary that the antibody bind to a specific site or region that correlates with biological significance. In cases where the site of interest is non-immunodominant, it is very difficult to develop antibodies with the required performance specifications using full-length protein antigens. Thus, there is a critical need for a means of directing antibody development to predefined, biologicallyrelevant, 3-dimensional epitopes of native proteins.

A common antibody development scenario entails relatively low cost polyclonal reagents for early discovery activities followed in later stages by additional antibody development, including monoclonals, made in such a way that they will react to the form of the antigen as it exists in the new intended applications.

Site-Directed Specificity
A significant advantage of immunizing with explicitly designed protein fragments is the ability to target regions of biological significance rather than unspecified immunodominant regions of full-length proteins. This site-directed specificity is a unique advantage of GAT. Examples of biological sites that can be targeted in this way include extracellular or intracellular regions of membrane proteins, alternative splice sites, protein interaction sites, enzyme active sites, sites of posttranslational modifications and even regions harboring single amino acid substitutions. Another important application of GAT site-directed specificity is the ability to design and develop antibody reagent pairs to different regions of a protein for use in sandwich immunoassays (Figure 4).

Polyclonal and Monoclonal Genomic Antibodies

Both rabbits and mice can be immunized using Genomic Antibody Technology. Rabbit polyclonal antibodies are purified by affinity chromatography using recombinant polypeptides representing the immunizing sequence. Monoclonal antibodies (mAbs) are derived from immunized mice using hybridoma technology and selected using the same recombinant polypeptide antigens, and/or other antigens that may be available and appropriate to select mabs for a specific purpose. Figure 5 shows an example of a flow cytometry screening assay of hybridoma supernatants derived from mice immunized with extracellular regions of the membrane protein EGFR using GAT. In this example, multiple mabs were detected that specifically bound full-length EGFR on A431 cells with unique patterns of reactivity. Detection of EGFR on the surface of intact, living cells is further evidence that antibodies made in this manner bind native epitopes.

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Genomic Antibody Sequences

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Comparing SDIX Genomic and Peptide Antibody Performance

In a recent systematic study (9), it was demonstrated that polyclonal antibodies generated using GAT performed much better than peptide antibodies in sandwich immunoassays with native proteins, western blot and IHC. In this study, three leading peptide manufacturers designed and produced three peptides each for ten well-characterized, immunogenic, commercially-available protein targets. Similarly, three GAT DNA constructs were designed for each protein. Two rabbits were immunized with each peptideconjugate and GAT immunogen. Full-length protein (FLP) immunizations were used as positive controls. Antibodies were affinity-purified on cognate antigens. In antigen-coated microtiter plate ELISAs, GAT antibodies recognized 7 out of 10 full-length native proteins with significantly higher specific activity than did peptide antibodies (Figure 6). In sandwich ELISAs, GAT antibodies recognized 8 out of 10 full-length native proteins with significantly greater sensitivity than peptide antibodies when paired with antibody to full-length protein (Figure 7), and the likihood that at least one of the 3 GAT antibodies would detect 1000, 100 and 10 pM of antigen was 100, 80 and 40% respectively, compared to 40, 10 and 0% for peptide antibodies (Figure 8a).

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SDIX Genomic Antibodies Figure 4. Development of optimized antibody pairs for EGFR Monoclonal Antibodies
sandwich immunoassays.

mAb supn 1

mAb supn 2

mAb supn 3

GR01 positive control

The most striking performance differential was in sandwich ELISAs where pairs of GAT antibodies were Figure 5. Flow cytometry screening of hybridoma supernatants compared with peptide antibody pairs. Forty percent from mice immunized with EGFR using GAT. Red = A431, of the proteins at 1 nM, 30% at 100 pM, and 20%
Black = HEK293, Blue = CHO, Turquoise = Jurkat. 4

Figure 6. Specific activity of purified antibodies in direct ELISA to full length proteins. Symbols represent the minimum antibody concentration required to achieve an optical density of 0.5 in ELISA.

at 10 pM, could be detected by at least one pair of GAT antibodies, while no combinations of peptide antibodies detected protein at any concentration tested (Figure 8b). In western blotting, 90% and 93% of GAT antibodies recognized the cognate protein at 100 ng/ml or 1000 ng/ml antibody probing concentration, respectively. In contrast, only 47% and 57% of peptide antibodies were found to be immunoreactive. Both DNA and peptide immunization strategies yielded antibodies highly suitable for IHC, although in terms of sensitivity and specificity, the GAT antibodies were judged to be superior. Success in one assay didnt guarantee success in another. GAT antibodies consistently performed in the broadest number of assays. Six out of the 6 GAT antibodies positive in IHC also worked in western blot compared to only 1 of 6 peptide antibodies. Twenty-eight of 29 GAT antibodies worked in at least one of the assays compared to only 19 of 30 peptide antibodies. These findings demonstrate that GAT generates antibodies to both conformational and linear epitopes that have superior performance characteristics and are useful in a broader range of applications than peptide antibodies.

Protein

Figure 7. Sensitivity of antibodies for antigen in sandwich ELISA. Symbols represent minimum detection limits of full length antigen proteins.

SDIX Genomic Antibody Performance in IHC at The Human Protein Atlas

Protein

Figure 8. Success rate of antibodies at detecting the indicated concentrations of full length protein antigens.

To further evaluate GAT antibody performance, a single polyclonal rabbit antibody was made to each of 552 different protein antigens and independently evaluated by the Human Protein Atlas (HPA) project against 46 different normal human tissues, 20 different cancer types and 47 different human cell lines by IHC. As a result of these studies 294 GAT antibodies (53%) were selected for inclusion in the atlas. The results from all analyses of all 294 GAT antibodies are available for viewing at the HPA data portal (http://www.proteinatlas.org/).

Membrane Protein Antibodies

Membrane proteins are of significant pharmaceutical interest as they represent critical biological switches in molecular pathways making them obvious biomarker and drug targets. However, this class of proteins is typically challenging to make antibodies to because full-length membrane proteins may not adopt their native conformation in the absence of the lipid bilayer in which they reside. In addition, membrane
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proteins may contain significant posttranslational modifications such as disulfide bonds or glycosylation that can be additional determinants of epitope structure.

In vivo expression of GAT membrane protein domains allows them to be presented as appropriately folded and modified immunogens. Using the technology, antibodies have been generated to a number of membranebound proteins such as EGFR, CD4, CD44, CD22 and CD33 which have been well characterized in whole-cell flow cytometry assays (Figure 9). Figure 10 shows binding of a mouse monoclonal antibody generated using GAT to the highly glycosylated, singlepass membrane protein CD33 on peripheral blood mononuclear cells (PBMCs).

Figure 9. Detection of membrane proteins in flow cytometry using polyclonal rabbit GAT antibodies.

Figure 10. Flow cytometry evaluation of CD33 monoclonal antibodies.

Summary
Antibodies to protein antigens are important research tools, diagnostic reagents and therapeutic drugs. Functional proteins exist in defined 3-dimensional conformations and antibodies bind conformational epitopes comprised of discontinuous amino acids. Peptide immunogens are limited in their ability to elicit antibodies that bind native protein epitopes. Genomic Antibody Technology (GAT) generates polyclonal and monoclonal antibodies to conformationally-determined, discontinuous protein epitopes. GAT employs DNA immunization with sequences representing protein regions or domains. In vivo expression of relatively

large, intact protein domains results in the generation of antibodies that bind conformational epitopes on native proteins in a wide variety of immunoassay formats. Immunization with pre-defined protein domains enables site-directed antibody targeting of biologically relevant protein regions and is a means for overcoming undesirable antibody responses to immunodominant epitopes of full-length proteins. GAT antibodies have been shown to have superior performance characteristics over peptide antibodies in western blot, sandwich immunoassay, and IHC, and bind to native membrane proteins on the surfaces of living cells in flow cytometry.

References
1. Berzofsky, J.A., G.K. Buckenmeyer, G. Hicks, F.R.N. Gurd, R.J. Feldmann, and J. Minna. 1982. Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin. J. Biol. Chem., 256, 3189. 2. Stave, J.W., J.L. Card, D.O. Morgn, and V.N. Vakharia. 1988. Neutralization of type O1 foot-and-mouth disease virus defined by monoclonal antibodies and neutralization-escape virus variants. Virol., 162, 21. 3. Hans, D., P.R. Young, and D.P. Fairlie. Current status of short synthetic peptides as vaccines. Med. Chem., 2, 627. 4. van Regenmortel, M.H.V. 2009. What is a B-Cell Epitope? In Methods in Molecular Biology, Epitope Mapping Protocols. U. Reineke and M. Schutkowski, eds. Humana Press. p. 3. 5. Ponomarenko, J.V., and M.H.V. van Regenmortel. 2009. B-Cell Epitope Prediction. In Structural Bioinformatics, 2nd ed. J. Gu and P.E. Bourne, eds. John Wiley & Sons. p. 849. 6. Irving, M.B., L. Craig, A. Menendez, B.P. Gangadhar, M. Montero, N.E. van Houten, and J.K. Scott. 2010. Exploring peptide mimics for the production of antibodies against discontinuous protein epitopes. Mol. Immunol. 47, 1137. 7. Jemmerson, R. 1987. Multiple overlapping epitopes in the three antigenic regions of horse cytochrome C. J. Immunol., 138, 21. 8. Smith-Gill, S.J., T.B. Lavoie, and C.R. Mainhart. 1984. Antigenic regions defined by monoclonal antibodies correspond to structural domains of avian lysozyme. J. Immunol., 133, 384. 9. Brown M.C., T.R. Joaquim, R. Chambers, D.V. Onisk, F. Yin, et al. 2011. Impact of immunization technology and assay application on antibody performance - a systematic comparative evaluation. PLoS ONE 6, e28718. doi:10.1371/journal. pone.0028718 7

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