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Genomic Antibody Technology

Antibodies are important research tools, diagnosticreagents and therapeutic drugs. As reagents they areused in a variety of different immunoassay formats

including western blot, immunohistochemistry (IHC),ELISA, sandwich immunoassay, immunoprecipitation,ow cytometry, and functional assays. The way in

which an antibody is made determines the form ofantigen it binds, the site of binding, the strength ofbinding, crossreactivity, and the type of assay in whichthe antibody will work. Therefore, it is necessary that

antibodies be developed and selected in a mannerconsistent with the intended purpose.

Conormational Protein Epitopes

Proteins have specic functions that are dependent on

their native 3-dimensional conformation. It has beenknown for over 30 years that the majority of antibodiesmade to intact native proteins do not react to thesame protein sequence in a denatured form (1,2). The

traditional method of making antibodies is to immunizeanimals with full-length puried native or recombinant

proteins. A signicant obstacle to this approach is the

development and purication of protein antigens in

sufcient quantities and qualities for immunization and

subsequent antibody purication or selection.

The preparation of native protein antigens is so onerous

that it led researchers to search for less costly, lesstime-consuming alternatives to full-length proteinimmunization. Since the 1980s, the most commonmethod by which antibodies to proteins are made isto immunize animals with short linear peptides andhope that the resultant anti-peptide antibodies cross-react with native protein. Peptide immunogens madeup of 10-20 amino acids are highly exible and adopt

many different 3-D conformations, of which only a verylimited number will elicit an antibody capable of cross-reacting with the native protein. While anti-peptide

antibodies may have high titer to the immunizingpeptide, typically only a small percentage of theantibodies made using this method are capable ofbinding native protein - resulting in very low specic

activity or, as is often the case, no binding activity atall. Indeed, a crucial test of peptides capacity to elicitantibodies that bind native protein structures is thegeneration of protective antibodies when administeredas synthetic vaccines. Unfortunately, since 1990 over100 synthetic peptides have entered clinical trials asvaccines and none have been approved (3).

Discontinuous Protein Epitopes

In addition to having a specic 3-dimensional shape,

the site on an antigen recognized by an antibody, theepitope, is made up of amino acid residues that arenot all adjacent to one another in the linear amino acidsequence, i.e., they are located in a discontinuousmanner along the protein sequence (Figure 1). It is wellestablished that the vast majority of epitopes on nativeprotein surfaces are comprised of discontinuous aminoacid residues that only come into close spatial proximity

when the protein is properly folded (4).

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As a research program develops thereis usually a need for antibodies that

bind native protein, and it is frequentlythe case that the rst antibodies thatwere useful in western blot and IHCare not useful in assays requiring

recognition of native 3-dimensionalprotein conformation.

Figure 1. Discontinuous proten epitope. Antigenamino acids contacted by antibody (yellow) are notadjacent in linear sequence. Contact residues arecontained within 218aa linear protein sequence. Ashort peptide can not form this epitope.

X-ray Crystal Structure PDB 3G04

TSHR antigen

Linear amino acid sequence of TSHR

38 255218 aa


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Figure 2 shows the actual sequence positions of theamino acids comprising the epitopes of 5 highlycharacterized inuenza hemagglutinin neutralizing

monoclonal antibodies. These antibodies demonstrate

that epitopes recognized by real-world functional, i.e.,neutralizing, antibodies are discontinuous in nature.

Failed Peptide Antibodies

Decades of research has led to the conclusion thatpeptides are poor mimics of discontinuous epitopes(5), and that peptide immunizations are not a practicalstrategy for targeting discontinuous epitopes (6).Given that the vast majority of protein epitopes areconformationally-determined and discontinuous, andthat the most common method of developing antibodiesis using small, linear, unstructured peptides, it is nowonder that a common complaint among antibodyusers is that most antibodies dont work.

The Inuence o Assay Type

One of the most important considerations whenattempting to develop an antibody is how it will beused. Both western blot and IHC subject the proteinto signicant chemical and physical processes that

alter 3-dimensional protein conformation, and it iscommon that antibodies that work in these assaysdo not work in other assay formats (e.g., sandwichELISA with minimally processed serum samples or ow

cytometry with live cells) and vice versa. This effect is

more pronounced with monoclonal antibodies becausethey recognize only a single epitope conformation.Polyclonal antibodies made to native proteins comprise

a population recognizing both native and denaturedepitopes and generally are useful in a wider variety ofassay applications.

Figure 3shows the reactivity of two polyclonalantibodies, GA3177 and GA3179, to EGFR protein inwestern blot and sandwich ELISA. The data

demonstrate that GA3177 binds EGFR in sandwich

ELISA but not western blot, while GA3179 binds inboth assays. In the western blot, EGFR is denatured

by boiling in detergent and reducing agent while inthe ELISA it is maintained in physiologic buffer. These

results demonstrate a critical aspect of antibody

technology, i.e., a single antibody rarely works inall types of immunoassays, and successful antibodydevelopment is dependent on understanding theintended application and designing antibody reactivityin a way that is consistent with how the antibody will beused.

Early in a research program, it is frequently a rst

objective to establish the presence or absence of aprotein in a specic sample type or biological process.

Western blot and IHC are ideally suited for theseactivities and polyclonal antibodies are cost-effectivereagents in these cases. As a research program

develops there is usually a need for antibodies that bindnative protein, and it is frequently the case that therst antibodies that were useful in western blot and IHC

are not useful in assays requiring recognition of native3-dimensional protein conformation. This is especially

true when the rst reagents were made using peptide

immunogens.

Figure 2. Discontiuousepitopes of 5 inuenzahemagglutinin neutralizingmonoclonal antibodies. CRS= Contact Residue Span.

In most critical antibody applications, it is not sufcient for an antibody to simplybind anywhere on the protein antigen, rather, it is necessary that the antibody

bind to a specic site or region that correlates with biological signicance.

PDB Mab1-50 51-100 101-150 151-200 201-250 251-300

CR CRS

20 94

17 66

18 124

19 227

12 38

86 256

3SM5 CH65

2VIS IgG1, Lambda

3LZF 2D1

1QFU Neutralizing Fab

3GBN Cr6261

SUM ALL

Contact Residues (CR)

Infuenza HA Ammino Acid Sequence

3 3.5 4

Purifed hEGFRSandwich ELISA with Full-Length

Purifed Human EGFR (hEGFR)

4

3

2

1

010.1 10 100

EGFR Concentration, nM

OD650nm

GA3179

GA3177

GA3177 Capture

GA3179 Detector

Figure 3. The type of assay inuences antibody reactivity.Polyclonal antibody GA3177 binds native epitopes in ELISAbut not the same epitopes when they are denatured inwestern blot. GA3179 binds epitopes in both assays.


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In a mature project it is often the case that routineassays are being used that have dened performance

specications and large amounts of antibody are

required. In these applications, long-term, consistentsupply of antibody is critical, and monoclonal antibodiesare preferred reagents. While the uniform consistencyof monoclonal antibodies is a preferred characteristic ofreagents in all applications, the time and cost to developthem is signicantly greater than polyclonal antibodies,

and because they only recognize a single epitope theyare less likely to react in a variety of assay types.

A common antibody development scenario entailsrelatively low cost polyclonal reagents for earlydiscovery activities followed in later stages byadditional antibody development, includingmonoclonals, made in such a way that they will reactto the form of the antigen as it exists in the new

intended applications.

Immunodominant Protein EpitopesThe gold standard approach for developing antibodies

to native proteins is to immunize animals with full-length native or recombinant proteins. While scientistsbelieve that antibodies to virtually every region of aprotein exist within the complete antibody repertoire

of an animal, the majority of antibodies made tofull-length protein antigens are restricted to a few

immunodominant regions of the protein (7,8). Inmost critical antibody applications, it is not sufcient

for an antibody to simply bind anywhere on the proteinantigen, rather, it is necessary that the antibody bind to

a specic site or region that correlates with biologicalsignicance. In cases where the site of interest is

non-immunodominant, it is very difcult to develop

antibodies with the required performance specications

using full-length protein antigens.

Thus, there is a critical need for a means of directing

antibody development to predened, biologically-

relevant, 3-dimensional epitopes of native proteins.

SDIX Genomic Antibody Technology

SDIX Genomic Antibody Technology (GAT) was

developed to produce antibodies that have performancecharacteristics superior to conventional antibodies madeusing peptide and full-length protein immunization.GAT is designed to produce antibodies to predened

native protein epitopes for critical research, biomarkerdiscovery, diagnostic and therapeutic applications. GAT

includes immunization of rabbits and mice with DNAvectors encoding relatively large (approximately 100aa)

portions of proteins. Frequently, these sequences aredesigned to code for independent protein domains. The

3-dimensional conformation of an individual domainis relatively independent of other parts of a proteinand there is a signicantly greater probability that a

domain will fold into a native conformation and elicitconformation-specic antibodies than a short linear

peptide.

A critical aspect of GAT is immunization with DNA

rather than protein. Even highly puried preparations ofprotein contain contaminants that antibodies are madeto in immunized animals. DNA immunization encodesonly the target of interest, absent of contaminants,resulting in high specic activity antibody responses.

In addition, in vitro expression and puricationprocesses subject proteins to physical stress that canalter native 3-D conformation. A signicant advantage

of DNA immunization is that the protein is made in vivowithin 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 toproperly folded 3-D conformations. Evidence that GAT

results in conformation-specic antibodies can be seen

in rabbit polyclonal Genomic Antibody GA3177 (Figure3), which reacts with EGFR in physiologic buffer but not

when it is denatured in western blot.

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.


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Site-Directed Specifcity

A signicant advantage of immunizing with explicitly

designed protein fragments is the ability to targetregions of biological signicance rather than unspecied

immunodominant regions of full-length proteins. This

site-directed specicity is a unique advantage of GAT.

Examples of biological sites that can be targeted in

this way include extracellular or intracellular regionsof membrane proteins, alternative splice sites, proteininteraction sites, enzyme active sites, sites of post-translational modications and even regions harboring

single amino acid substitutions. Another importantapplication of GAT site-directed specicity is the

ability to design and develop antibody reagent pairsto different regions of a protein for use in sandwichimmunoassays (Figure 4).

Polyclonal and Monoclonal GenomicAntibodies

Both rabbits and mice can be immunized using GenomicAntibody Technology. Rabbit polyclonal antibodies are

puried by afnity chromatography using recombinant

polypeptides representing the immunizing sequence.Monoclonal antibodies (mAbs) are derived from

immunized mice using hybridoma technology andselected using the same recombinant polypeptideantigens, and/or other antigens that may be availableand appropriate to select mabs for a specic purpose.

Figure 5 shows an example of a ow cytometry

screening assay of hybridoma supernatants derivedfrom mice immunized with extracellular regions of the

membrane protein EGFR using GAT. In this example,

multiple mabs were detected that specically 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 inthis manner bind native epitopes.

Comparing SDIX Genomic and PeptideAntibody Perormance

In a recent systematic study (9), it was demonstratedthat polyclonal antibodies generated using GAT

performed much better than peptide antibodiesin sandwich immunoassays with native proteins,western blot and IHC. In this study, three leadingpeptide manufacturers designed and produced three

peptides each for ten well-characterized, immunogenic,commercially-available protein targets. Similarly, threeGAT DNA constructs were designed for each protein.

Two rabbits were immunized with each peptide-

conjugate and GAT immunogen. Full-length protein

(FLP) immunizations were used as positive controls.Antibodies were afnity-puried on cognate antigens.

In antigen-coated microtiter plate ELISAs, GAT

antibodies recognized 7 out of 10 full-length nativeproteins with signicantly higher specic activity than

did peptide antibodies (Figure 6). In sandwich ELISAs,GAT antibodies recognized 8 out of 10 full-length

native proteins with signicantly greater sensitivity

than peptide antibodies when paired with antibody tofull-length protein (Figure 7), and the likihood that atleast 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 peptideantibodies (Figure 8a).

The most striking performance differential was in

sandwich ELISAs where pairs of GAT antibodies were

compared with peptide antibody pairs. Forty percentof the proteins at 1 nM, 30% at 100 pM, and 20%

Figure 5. Flow cytometry screening of hybridoma supernatantsfrom mice immunized with EGFR using GAT. Red = A431,Black = HEK293, Blue = CHO, Turquoise = Jurkat.

Figure 4. Development of optimized antibody pairs forsandwich immunoassays.

0

35

70

105

140

A

B

C

D

E

F

G

H

Genomic Antibody Sequences

mAb supn 1 mAb supn 2

mAb supn 3 GR01 positive control


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at 10 pM, could be detected by at least one pair ofGAT antibodies, while no combinations of peptide

antibodies detected protein at any concentrationtested (Figure 8b).

In western blotting, 90% and 93% of GAT antibodies

recognized the cognate protein at 100 ng/ml or 1000ng/ml antibody probing concentration, respectively.

In contrast, only 47% and 57% of peptide antibodieswere found to be immunoreactive.

Both DNA and peptide immunization strategies yieldedantibodies highly suitable for IHC, although in termsof sensitivity and specicity, the GAT antibodies were

judged to be superior.

Success in one assay didnt guarantee success inanother. GAT antibodies consistently performed in

the broadest number of assays. Six out of the 6 GAT

antibodies positive in IHC also worked in westernblot compared to only 1 of 6 peptide antibodies.

Twenty-eight of 29 GAT antibodies worked in at leastone of the assays compared to only 19 of 30 peptideantibodies.

These ndings demonstrate that GAT generates

antibodies to both conformational and linear epitopesthat have superior performance characteristics and areuseful in a broader range of applications than peptideantibodies.

SDIX Genomic Antibody Perormance in

IHC at The Human Protein Atlas

To further evaluate GAT antibody performance, a

single polyclonal rabbit antibody was made to eachof 552 different protein antigens and independentlyevaluated by the Human Protein Atlas (HPA) projectagainst 46 different normal human tissues, 20different cancer types and 47 different human celllines by IHC. As a result of these studies 294 GAT

antibodies (53%) were selected for inclusion in theatlas. The results from all analyses of all 294 GAT

antibodies are available for viewing at the HPA dataportal (http://www.proteinatlas.org/).

Membrane Protein Antibodies

Membrane proteins are of signicant pharmaceutical

interest as they represent critical biological switches inmolecular pathways making them obvious biomarkerand drug targets. However, this class of proteins istypically challenging to make antibodies to becausefull-length membrane proteins may not adopt theirnative conformation in the absence of the lipidbilayer in which they reside. In addition, membrane

Protein

Figure 7. Sensitivity of antibodies for antigen in

sandwich ELISA. Symbols represent minimumdetection limits of full length antigen proteins.

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

Protein

Figure 6. Specic activity of puried antibodies indirect ELISA to full length proteins. Symbolsrepresent the minimum antibody concentrationrequired to achieve an optical density of 0.5 in ELISA.


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Reerences

1. Berzofsky, J.A., G.K. Buckenmeyer, G. Hicks, F.R.N. Gurd, R.J. Feldmann, and J. Minna. 1982. Topographic antigenicdeterminants 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 dened bymonoclonal 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. Guand 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 peptidemimics 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 dened by monoclonal antibodies correspond tostructural 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 assayapplication on antibody performance - a systematic comparative evaluation. PLoS ONE 6, e28718. doi:10.1371/journal.pone.0028718

Summary

Antibodies to protein antigens are important researchtools, diagnostic reagents and therapeutic drugs.Functional proteins exist in dened 3-dimensional

conformations and antibodies bind conformationalepitopes comprised of discontinuous amino acids.Peptide immunogens are limited in their ability to elicit

antibodies that bind native protein epitopes. GenomicAntibody Technology (GAT) generates polyclonal and

monoclonal antibodies to conformationally-determined,discontinuous protein epitopes. GAT employs DNA

immunization with sequences representing proteinregions or domains. In vivo expression of relatively

large, intact protein domains results in the generationof antibodies that bind conformational epitopes onnative proteins in a wide variety of immunoassayformats. Immunization with pre-dened protein

domains enables site-directed antibody targeting ofbiologically relevant protein regions and is a meansfor overcoming undesirable antibody responses to

immunodominant epitopes of full-length proteins.GAT antibodies have been shown to have superiorperformance characteristics over peptide antibodies inwestern blot, sandwich immunoassay, and IHC, andbind to native membrane proteins on the surfaces ofliving cells in ow cytometry.


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