Recombinant antibodies containing an engineered B-cell epitope capable of eliciting conformation-specific antibody responses

Vaccine,Vol.

13, No. 18,

0264-410X(95)00140-9

pp. 1770-1778,

1995 Elsevier Science Ltd Printed in Great Britain 0264-410X195 $lO+O.OO

Recombinant antibodies containing an engineered B-cell epitope capable of eliciting conformation-specific antibody responses Jeremy

Cook*

and Brian H. Barber*?

The immunogenicity of a soluble, non-self protein or peptide can be greatly enhanced by injecting this antigen coupled to an antibody spectjic for class II MHC molecules in the recipient. This adjuvant-independent immunization strategy is known as immunotargeting. We have investigated the ability of a mouse anti-class II MHC antibody to provide the three-dimensional framework for the reconstitution of a heterologous conformational B-cell epitope, spectfically the A loop epitope from the influenza virus hemagglutinin (HA). From a panel of three anti-class II MHC immunoglobulin (Ig)-A loop constructs, we found that one of these, an insertion into the FR3 region of the Ig molecule, retained its spectficity for mouse I-A”. Although mouse monoclonal antibodies spectjic for the A loop region in the HA molecule were unable to react with the Ig-A loop variants, we did find that the heavy chain CDR3 insertion construct vvas able to elicit an A loop-spec$c, HA-reactive antibody response when used as an immunogen in rabbits. These results demonstrate the potential for the Ig molecule to function successfully as a structural framework for the reconstitution and presentation of heterologous conformational B-cell epitopes. Keywords:

Recombinant

antibodies;

conformational

B-cell epitopes; influenza

In the past decade, advances in our ability to identify discrete antigenic T-cell and B-cell epitopes from any given pathogen have opened new possibilities with respect to the construction of defined subunit vaccines. Various synthetic peptides, representing defined sequences from a single antigenic protein on the surface of a virus or bacteria, have been used to generate immunity which is cross-reactive with the intact pathogen. Using this approach, one can attempt to generate antibody which are focused on key neutralizing responses epitopes, and at the same time eliminate many of the concerns associated with inactivated or attenuated whole-organism vaccines. In order for the full potential of defined subunit vaccines to be realized, however, there are several obstacles which need to be overcome. One such obstacle is the observation that, in general, synthetic peptides are poorly immunogenic’3. Thus, it is usually necessary to administer the peptides in a strong adjuvant in order to boost the level of immune response generated. However, the adjuvants currently available *Department of Immunology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada, M5S 1A8. tTo whom all correspondence should be addressed. (Received 17 April 1995; revised 5 July 1995; accepted 5 July 1995)

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hemagglutinin:

protein engineering

for use in man, such as alum, are often ineffective with respect to enhancing the immunogenicity of synthetic peptideslm3. It has been shown that the level of primary humoral immunity generated to either foreign proteins, or synthetic peptides, can be greatly enhanced by “targeting” the antigen to the surface of antigen presenting cells (APCs) in rive, without the need for adjuvant4. This has been achieved by chemically coupling the protein or peptide’ antigen to a monoclonal antibody which recognizes MHC class II molecules on the surface of the APCs. This method of immunopotentiation has been shown to be effective in several different species, includof ing mice4m~7, rabbits and ferrets’, using a number different model antigens. It remains to be determined, however, whether this adjuvant-free system would be as effective if the peptide antigen was directly fused with the targeting antibody by means of recombinant DNA technology, rather than chemically coupled to the targeting antibody. Another difficulty associated with the use of chemically synthesized peptide antigens is that many antigenic epitopes found on the surface of a native, folded protein have particular three-dimensional conformations which are not easily mimicked by small, unconstrained synthetic peptides. Therefore, the antibody response elicited

Conformation-specific

by a synthetic peptide with a particular primary amino acid sequence may not cross-react with the corresponding region of primary sequence in the native protein, due to differences in their respective conformations’*“. Because much of the outer surface of a protein is composed of loop regions of secondary structure connecting elements of P-sheet or u-helix found in the core, there have been several attempts to mimic the conformation of a given loop by constraining the corresponding synthetic peptide into a circular structure”-‘“. Although these constrained, circular synthetic peptides are sometimes better approximations of the native loop structure found in the original protein, they still suffer from the problem of being poorly immunogenic. Such peptides must therefore be coupled to a carrier protein, and injected in the context of a strong adjuvant, in order to generate loop-specific humoral immunity”*‘2. In order to address the problem of conformational constraints on peptide antigens, while at the same time moving toward the use of recombinant antibodyantigen fusions for immunotargeting, we decided to investigate the use of a targeting antibody as a threedimensional scaffold for the constraint of a conformational B-cell epitope. The targetin antibody we chose to focus on was Tib92 (10-3.6.2) & , an IgG2a murine monoclonal antibody (MAb) specific for the I-Ak molecule. The model B-cell epitope identified for study was the immunodominant A loop epitope from the influenza virus hemagglutinin molecule, a determinant previously shown to be strictly conformational’5p’9, and only by chemically-constrained pepweak1‘y mimicked tides’ ,12. Therefore, a panel of three different Ig-A loop fusion constructs were made in an attempt to create a new epitope on the surface of Tib92 which accurately reflected the conformational characteristics of the A loop as found in native HA. Two of these Ig-A loop constructs contained modifications in the heavy chain CDR3 region, one of which was a replacement of the CDR3H with the A loop, and the other an insertion of the A loop into the middle of the CDR3H. The third Ig-A loop construct was an insertion of the A loop into the middle of an external loop found in the FR3 region of the V, structure, which is not involved in the antigen binding site. It was hoped that this last construct would not only recapitulate the native A loop epitope conformation, but would retain specificity for its ligand, the class II MHC I-Ak molecule. The following report describes the construction and analysis of these Ig-A loop fusion constructs.

MATERIALS

AND METHODS

antibody responses: J. Cook and B. H. Barber

resulting 570 bp cassette was blunted and cloned into the EcoRV site of pBluescriptI1 (Stratagene). The CDR3-RA construct was made as follows: Sau3A and Ban1 sites were used to cleave out the CDR3H region of the Tib92 VH, and the A loop coding sequence was cloned into this region in the form of a double-stranded synthetic oligonucleotide cassette. The modified V, was then cloned into the genomic expression vector pSV2neo-V,315-Cy2b2’ (kindly provided by Dr M. Klein, University of Toronto) by replacing the MOPC315 V, with the modified Tib92 V, cassette to yield the pSV2neo-Vn92-CDR3-RA-Cy2b (see Figure 2~). The CDR3-IA and 74-IA constructs were constructed using oligonucleotide-directed site-specific mutagenesis (Clontech) on a 2 kb Xba I fragment of the pSV2neo-V,92-Cy2b in pBluescriptI1, followed by subcloning of the modified 2 kb Xba I fragment back into the final expression vector. All constructs were fully sequenced to confirm that the A loop coding sequence insertion was correct and in frame. Expression of the modified Tib92-IgG2b antibodies was achieved by transfecting the Sal I-linearized heavy chain vectors into the cell line 37-7, which is a heavy chain-loss variant of the parent cell line Tib92. Transfected cell lines were selected in media containing 600 ,ug ml- ’ G418 (Sigma), and stable clones were isolated by plating the cells out at 0.5 cells well - ’ in 96-well tissue culture plates. Antibody purification

Transfected cell lines were grown in RPM1 1640 media supplemented with Glutamax II (Gibco BRL), Penicillin/Streptomycin (Gibco BRL), and 10% UltraLow IgG Fetal Bovine Serum (Gibco BRL). All antibodies were purified from tissue culture supernatants on a Protein A-Sepharose fast-flow column (Pharmacia). Flow cytometry

The ability of the wildtype and modified Tib92 antibodies to bind to I-Ak was analyzed using the I-Ak expressing cell line CH12-LX22, kindly provided by Dr T. Watts, University of Toronto. Briefly, CH12-LX cells were washed twice with Dulbecco’s PBS containing 0.1% bovine serum albumin and 0.1% NaN, (“PBS/BSA”), and were then resuspended in cultured cell supernatants from modified Tib92-A loop antibodies, or control supernatants, and incubated on ice for 1 h. After two washes with PBYBSA, the cells were incubated in the presence of Goat anti-Mouse IgG2b-FITC (Caltag) for 30 min. The cells were then washed and analyzed by fluorescence-activated flow cytometry on an EPICS Profile (Coulter Electronics, Inc., Hialeah, FL).

Animals

Female New Zealand White rabbits (2.5 kg) were purchased from Rieman’s Fur Ranches Ltd (St. Agatha, ON). All animals were used in experiments within 2 months of purchase. Antibody engineering

and purification

The V, gene from Tib92 was amplified by RT-PCR using a family of V, specific 5’ primers described by Huse et ~1.” (the V2 primer successfully amplified the Tib92 V, gene), and a 3’ y2a-specific primer. The

Rabbit antisera

The purified, bromelain-cleaved portion of the hemagglutinin molecule (BHA) from the X31 strain of influenza virus was kindly provided by Dr John Skehel, National Institute for Medical Research, London, UK. Rabbits were primed subcutaneously with 100 pg of BHA in Freund’s Complete Adjuvant (FCA) (Difco), and boosted subcutaneously 3 weeks later with 50 pg of BHA in incomplete Freund’s Adjuvant (IFA) (Difco). Sera were prepared from rabbits bled 2 weeks postboost.

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antibody responses: J. Cook and B. H. Barber

The A peptide (CKRGPGSGF) was synthesized and purified to a single peak on reverse-phase HPLC by the Mobix custom peptide synthesis service of McMaster University, Ontario, Canada. The peptide was coupled to keyhole limpet hemocyanin (KLH) using bromoacetate (Boehringer N-hydroxysuccinimidyl Mannheim) to activate the carrier protein, followed by covalent bond formation with the amino-terminal cysteine of the peptide. The A peptide was also coupled to BSA, using the heterobifunctional cross-linker MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester, Pierce), according to manufacturers specifications. KLH-A peptide was injected subcutaneously (s.c.) into rabbits in FCA, and S.C.boosts were performed 3 weeks later in IFA. Anti-KLH-A peptide rabbit sera collected two weeks post-boost were analyzed for anti-peptide reactivity by ELISA using BSA-A peptide conjugate for coating.

a

G-S 1’

b \ R 1:

‘:

z

‘:

4 Y

4 S

CDR3H (residues 101-I 12) Tib92-IgG2b

d

C

/“\

HS\

B

T

%

i

N

Q

FR3H (residues 73-75) Tib924gG2b

Immunoassays

ELISA assays were performed as follows: wells were coated at 37°C for 1 h with 10 yg ml _ ’ (100 ~1 well - ‘) of antigen or capturing antibody in PBS containing 0.02% NaN, (PBSlazide), followed by three washes with PBS containing 0.05% Tween 20 (PBS/Tween), and were then blocked with 1% BSA in PBS/azide for 30 min at 37°C. One hundred microliters of conditioned supernatants or diluted sera were placed in the wells for 60 min at 37°C followed by three washes with PBS/Tween. Wells were then incubated for 1 h at 37°C with the appropriate alkaline phosphatase-conjugated secondary reagent, and once again washed three times with PBS/ Tween. Finally, 1 mg ml- I of p-nitrophenylphosphate in 10% diethanolamine was added to the wells, and the level of hydrolysis was measured by reading the O.D. at 405 nm using a Titertek Plus reader (Flow Laboratories, Canada). The presence of Kappa light chains or intact IgG2a or IgG2b immunoglobulin was detected by coating with goat anti-mouse IgG (Fab specific) to capture the antibodies or light chains from cell supernatants, followed by detection using the alkaline-phosphataseconjugated secondary antibody shown in the insert. BHA and BSA-A peptide were coated directly onto the wells, and the analysis of the Ig-A loop constructs was performed by capturing them using a goat anti-mouse IgG2b specific coating antibody. In the competition ELISA, BHA or BSA-A peptide were coated directly onto the plate. The post-immune anti-CDR3-IA rabbit No. 2 antiserum was incubated for 30 min at 37°C at a l/10 dilution, with the indicated concentrations of BHA, BSA-A peptide or no competitor. After this preincubation step, the diluted sera/competitor antigen mixtures were placed in the antigen-coated wells. Binding of the rabbit antisera to the coated antigen was detected, after three washes with PBS-Tween, using goat anti-rabbit IgG-alkaline phosphatase conjugate. All ELISAs were repeated three or more times, and data shown are from one representative experiment. Tib92-A loop antibody immunizations

Rabbits were immunized subcutaneously with 50-100 pug of recombinant antibody emulsified in FCA for the primary immunization. The rabbits were boosted 3 weeks later with 50 pugof antigen emulsified in IFA, also

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CDRJ-IA

FR3-IA

Figure 1 Tib92-A loop fusion constructs. (a) Wild-type Tib92 CDRBH amino acid sequence as found in the Tib92-lgG2b construct. (b) CDR3-RA construct, in which the wild-type CDRBA was replaced with the nine amino acid A loop sequence (the amino-terminal cysteine was replaced with a serine to avoid unwanted disulphide bond formation). (c) CDRB-IA construct, in which the A loop sequence was inserted into the tip of the wild-type CDR3H sequence. (d) Wild-tvoe Tib92-laG2b FR3 VU seauence for residues 73-75 (numbering according to Kabat ei’aL3$. (e) FR3-IA sequence, in which the A loop sequence was inserted into FR3 in place of the single serine residue at position 74

subcutaneously. Bleeds were performed 2-3 days prior to boosting to obtain pre-immune sera, and 2 weeks post-boost for the immune sera. Blood was allowed to clot at room temperature for 1 h, followed by centrifugation to recover the sera. RESULTS Construction

of Tib92-A loop constructs

Using the X-ray crystal structures of the influenza virus hemagglutinin (HA) molecule and a representative panel of murine Fab structures, we developed a strategy to create a panel of three Tib92-A loop constructs. The goal was to provide conformational constraints on the inserted HA A loop sequence which would promote the adoption of its native conformation, while at the same time retaining surface accessibility. Figure 1 outlines the details of these Tib92-A loop constructs, two of which are modifications in the CDR3H region, the other being an A loop insertion into the tip of a loop found in the FR3 region of the Tib92 V,. In developing this strategy, we considered the requirements for correct folding of the antibody, surface accessibility of the A

Conformation-specificantibody responses: J. Cook and B.H. Barber loop epitope, and appropriate conformational constraints at the base of the loop to regain the conformational characteristics exhibited by the A loop in the original hemagglutinin molecule. Given the high degree of sequence variability in CDR3H, as well as its prominent role in antigen binding, it was considered likely that antibody folding would be tolerant to alterations in this region, and that such alterations would be surface accessible on the “outside” of the antibody structure. In addition, visual analysis of a panel of Fab structures, analyzed using the program Insight11 on a Silicon Graphics workstation, revealed that the base of the A loop in BHA is similar in geometry to the base of many of the CDR3H loops found in Fab structures. For this reason, we felt that CDR3H would be a logical choice as a site in which to study the conformation of a grafted A loop. We also wanted to identify a site which would exhibit the same advantageous characteristics of CDR3H described above, but which would not abrogate the ability of the recipient antibody to bind to its class II MHC antigen, thereby enabling us to use the constructs in later targeting experiments after the addition of a T-helper epitope. Visual analysis of a variety of antibody crystal structures suggested that the /3 turn found at site 74 of the heavy chain FR3 region might fulfill these criteria. This loop is similar to the P-strand-connecting turns used to make the CDRs of the antigen binding region, but it is not involved in antigen binding because of its location, which is adjacent to, but apart from the antigen binding contact surface. It is upon these considerations and observations that we based our experimental strategy for grafting the conformational A loop, as outlined in Figure 1. Using either cassette mutagenesis (CDR3-RA) or site-specific mutagenesis oligonucleotide-directed (CDR3-IA and FR3-IA), we made the necessary modifications to the Vi, region of the Tib92 heavy chain gene, and confirmed these modifications by DNA sequencing analysis. Figure 2a shows a map of the basic vector which was modified to create the Ig-A loop expression vectors (the vector contains a genomic Tib92-IgG2b expression cassette). Following construction of the different modified heavy chain expression vectors, each Tib92-A loop construct was transfected into a heavy chain loss variant cell line of Tib92 called 37-7, and individual G418-resistant clonal lines were isolated. ELISA analysis of the supernatants of these transfected lines, as well as the wild-type Tib92-IgG2b, showed that all three constructs were expressed and secreted at similar levels (Figure Zb), indicating that the A loop-altered antibodies retained the necessary tertiary and quaternary structure to allow secretion. Analysis of Tib92-A loop binding to I-Ak Flow cytometric analysis, using the I-Ak bearing cell line CH12-LX, was used to determine whether or not the Tib92-A loop constructs retained the ability to bind to I-Ak. Figure 3 shows that, as expected, the wild-type Tib92-IgG2b antibody binds well to its cognate class II MHC antigen. The CDR3H constructs (CDR3-RA and CDR3-IA), however, no longer exhibit the ability to bind to I-Ak. This is consistent with the fact that CDR3H usually plays a central role in determining antigen specificity23. FR3-IA, by contrast, binds to I-Ak

SVZneo-VH92-Cgamma2b

a

A loop Xba

insertion

site

I

b 1.5s

n Kappa

.I 0

lgG2a

H

IgGZb

Tib9.2

37-7

nb92-lgG2b

CC+WRA

Supernatant

CDRSIA

FRSIA

sample

Figure 2 Plasmid map and analysis of expression of engineered Tib92 antibodies after heavy-chain transfection. (a) A plasmid map of the Tib92-Cg2b expression vector used for transfection and expression of the Ig-A loop constructs. (b) A heavy chain-loss variant of Tib92 (IgG2a, x), called 37-7, was transfected with various Tib92Cy2b modified heavy chain vectors. Supernatants from these G418selected clones were analyzed by a capture ELISA using goat anti-mouse IgG (Fab specific) for coating. Supernatants were incubated for 1 h at 37°C in blocking buffer, followed by goat anti-mouse Kappa, IgG2a or IgG2b specific alkaline phosphatase conjugates

unstamed S

2nd Ab only

f

E =

xb92-lgG2b

E

CWSRA

2 CDR3-IA

60 Mean

fluorescence

Figure 3 Insertion of the A loop sequence into FR3 does not affect specificity for I-Ak. Modified antibodies were analyzed for their ability to bind to class II MHC by flow cytometry as described in Materials and Methods

comparably to Tib92-IgG2b, demonstrating that this site in the FR3 is amenable to antigenic epitope insertion without affecting the original antigen specificity of the antibody.

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b q

1.2 BHA

0.8-

0.8-

0.8-

0.6-

+

BHA

+

BSA-A peptide

1

Tib92-laG2b

-

CC&A CDRSIA

---+--

FRS-IA

1.0 --+---

FRS-IA

I

0.8-1 \ 0.6-

Reciprocal

serum

dilution

Rabbit anti-BHA

Reciprocal

serum

dilution

Rabbit anti-KLH-A peptide

Figure 4 Anti BHA and anti KLH-A peptide do not recognize Ig-A loop constructs and do not cross-react with each other. (a) Rabbit anti-BHA was analyzed for binding to BHA and BSA-A peptide by direct binding ELISA as described in Materials and Methods. (b) Rabbit anti KLH-A peptide was analyzed for binding to BHA and BSA-A peptide by direct binding ELISA. (c) Rabbit anti-BHA was analyzed for binding to Fc-captured Tib92-A loop constructs by ELISA. (d) Rabbit anti KLH-A peptide was analyzed for binding to Fc-captured Tib92-A loop constructs by ELISA. The Tib92-lgG2b isotype variant lacking any HA sequence insert is used as the control antibody

Binding of rabbit antisera to Tib92-A loop constructs In order to analyze the conformational characteristics of the A loop epitope in the different Tib92-A loop constructs, we prepared an anti-BHA rabbit serum. In addition, a synthetic peptide corresponding to the A loop primary amino acid sequence was coupled to KLH and used to make an anti-A peptide serum This antiserum was used as a measure of reactivity with “unconstrained” A peptide epitope. Figure 4a shows that the rabbit anti-BHA binds well to BHA, but not to the A peptide coupled to BSA. Figure 4b shows that the rabbit anti-KLH-A peptide binds with high titer to BSA-A peptide, but exhibits no reactivity to BHA, or an unrelated peptide conjugated to BSA via the same linker (data not shown). These results confirm that the A loop is a conformationally restricted epitope which is not mimicked by the corresponding synthetic peptide’5m’9. Figure 4c and 4d show the binding of these two rabbit antisera to Fc-captured Tib92-A loop constructs.

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Compared to the Tib92-IgG2b control, rabbit anti-BHA binds slightly better to FR3-IA, but not to CDR3-RA or CDR3-IA (Figure 4~). The rabbit anti-A peptide serum binds slightly better to FR3-IA and CDR3-IA, but not to CDR3-RA. These differences are not large, but are reproducible.

Binding of anti-HA MAbs to Tib92-A loop constructs We analyzed the binding of three anti-HA MAbs, which have been shown to bind in the region of the A loop by neutralization escape mutant studies (J. Skehel, personal communication, and Smith et a1.24), to the Tib92-A loop constructs. Figure 5 shows that none of the three MAbs tested bound to the Tib92-A loop variants above the level of the negative control (Tib92IgG2b). While these data may suggest that the A loop has adopted new, unique conformations unlike either the A loop in HA, or the synthetic A peptide, it is also

Conformation-specific a

antibody responses: J. Cook and B. H. Barber

1.2

, BHA coat

0.0 ?

z p. 8

L

0.4

0.0 NRS

JCS4B

H114

CDR3lA

CDR3RA

BHA

BSA.A pep.

BHA

BSA-A pep.

Rabbit immunogen

Anti-BHA monoclonal antibody ascites (l/l 00 dilution) Figure 5 Anti A loop monoclonal Abs do not bind to the Ig-A loop constructs. Three anti-BHA monoclonal Abs (JCB4B, H114 and H159) recognizing the A site of BHA were analyzed for binding to the Fc-captured Ig-A loop Abs by ELISA. BHA and BSA-A peptide targets included for comparison were bound directly to the microtiter wells. JCB4B and H114 were detected using goat anti-mouse IgG2a specific alkaline phosphatase conjugate, and H159 was detected using goat anti-mouse IgGl alkaline phosphatase conjugate. The ELISA was performed as described in the Materials and Methods

possible that these anti-HA MAbs bind to a footprint on HA which is larger than just the A loop, and that the differences in the rest of the antigenic binding surface between HA and the Tib92-A loop constructs prevent these MAbs from efficiently recognizing the Tib92-A loop constructs in this assay. Characterization of rabbit antisera to the Tib92-A antibodies In order to examine further the conformational characteristics of the different A loop modifications, three (in the case of CDR3-RA and FR3-IA) or four (in the case of CDR3-IA) rabbits were immunized with the Tib92-A loop antibodies as described in . The sera were analyzed for reactivity to both BHA and BSA-A peptide by ELISA (Figure 6). Figure 6a shows that CDR3-RA and FR3-IA did not elicit responses which cross-reacted with BHA. However, the rabbit sera elicited by immunization with the CDR3-IA construct cross-reacted with BHA in three out of four rabbits. Figure 6b shows that two of these rabbit sera (Nos 2 and 4) also cross-reacted with BSA-A peptide. These results are markedly different from the rabbit antiKLH-A peptide sera, which do not cross-react with BHA at all despite being strongly reactive with the A that a peptide (Figure 4b). Instead they demonstrate region of the antibody molecule can provide a structural framework conducive to the folding of a conformational B-cell epitope such as the A loop, which cannot be mimicked by coupling the corresponding synthetic peptide to a carrier protein. The sera from CDR3-IA immunized rabbit No. 2, which binds to both BHA and BSA-A peptide, were analyzed in order to determine whether this observation results from a unique set of antibodies which can bind both the conformational (HA) form of the A loop, and the non-conformational A peptide, or whether this represents a mixture of two separate sets of non-cross-reactive antibodies. Figure 7 shows the results of a competition ELISA which demonstrates loop

FFtJ-IA

H159

b

1.53

BSA-A pep. coat

-I NW

CDR3lA

CCR3RA

FRB-IA

Rabbit immunogen Figure 6 The CDR3-IA construct induces a response in rabbits which cross-reacts with both BHA and BSA-A peptide. Ig-A loop constructs were injected into rabbits as described in the Materials and Methods. (a) Plates were coated with BHA, and various rabbit antisera diluted l/10 were assessed for binding. For the Ig-A loop-immunized rabbit sera, the number of each rabbit in the group is shown in the inset. (b) Plates were coated with BSA-A peptide, and various rabbit antisera diluted l/10 were assessed for binding. For the Ig-A loop-immunized rabbit sera, the number of each rabbit in the group is shown in the inset. Pre-immune normal rabbit serum (NRS) at l/10 dilution was included as a control in each case

that the anti-CDR3-IA antiserum No. 2 is blocked from binding to BHA only by BHA competition, but not by BSA-A peptide competition. Conversely, binding to BSA-A peptide is only blocked by the BSA-A peptide, but not by BHA. Thus, these data clearly demonstrate that this serum has two populations of antibodies; one which binds to the conformational A loop in BHA, and the other which reacts with the A peptide. This result is consistent with the immunogen containing at least two distinct forms of the A site, namely the configured or conformational structure found in native HA, and the unconstrained or linear form represented by the A synthetic peptide. In our view, this is most likely the result of a partial denaturation of the recombinant antibody, either as a result of handling during sample preparation, or subsequent to injection in vim, such that both intact and unfolded A loop inserts are seen by the rabbit B cells. Alternatively, the CDR3-IA construct may have a single conformation which is distinct from both the A loop as it exists in BHA and the A peptide. This unique structure might then elicit an antibody response in which some antibodies are cross-reactive with BHA, and others with the A peptide.

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BHA coat

antibody responses: J. Cook and B. l-l. Barber

BSA-A 0. coat

Coat Figure 7 CDR3-IA-immunized rabbit serum No. 2 contains a mixture of non-cross-reactive anti-BHA antibodies and anti-BSA-A peptide antibodies. The binding of CDR3-IA-immunized rabbit serum No. 2 to BHA and to BSA-A peptide was analyzed in the presence of added BHA or BSA-A peptide. The concentration of added BHA or BSA-A peptide used for blocking was determined in a titration ELISA using soluble BHA to block binding of anti-BHA to plate-bound BHA and soluble BSA-A peptide to block binding of anti-A peptide to plate-bound A peptide. The lowest concentration which gave maximal blocking was used in a blocking experiment using CDRS-IAimmunized rabbit serum No. 2 (see Figure s)

DISCUSSION Previous studies have shown that immunotargeting is an effective means of eliciting an adjuvant-independent antibody response to soluble protein or peptide antigens4-‘. In order to extend this immunization strategy towards the use of recombinant immunotargeting antibody-antigen fusion proteins, we have examined the use of the immunoglobulin molecule as a framework within which to rebuild a conformational B-cell epitope. The A loop epitope from the HA molecule of the influenza virus was chosen for our initial studies, because it represents a well-characterized25, immunodominant epitope26 which has been shown in several previous studies to be highly conformational. Specifically, synthetic peptides with the A loop sequence are not able to elicit a response which cross-reacts with BHA” 19. Two groups have reported studies of conformationally constrained, cyclic HA A loop peptides. two peptides repSchulze-Gahmen et al. ’ I synthesized resenting the HA 140-146 sequence of two influenza virus strains (X31 and X47), lengthened by two residues (Val and Thr) on each side, and cyclized with a disulphide bond. Antisera to these peptides bound to influenza virus in a solid-phase radioimmunoassay (RIA), but with much lower affinity than to the cyclic peptides themselves. Muller et al.” constructed two cyclized versions of the A loop sequence, termed D-loop and K-loop, using two different linker chemistries. Molecular modeling, competitive inhibition ELISA results and NMR analysisZ7 suggested that the D-loop adopted a compact structure which more closely mimicked the structure of the HA A loop, but anti-D-loop antisera bound to undenatured virus very poorly. However, immunizations with both the D-loop and the K-loop were able to confer significant levels of protection from viral challenge. Taken together, these studies suggest that the cyclic A loop peptides were not identical to the HA A loop structure, but were more effective at mimicking that structure than the linear synthetic A peptide.

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We wished to investigate the antibody molecule as an alternative means of constraining the A loop sequence into a structure approximating the original A loop geometry. The base of the A loop has a similar geometry to the base of the CDR3H loop in many antibody structures. We reasoned, therefore, that constraining the A loop sequence by attaching it to the CDR3H site, either at the base or at the tip of the CDR3 loop, might allow the rest of the A loop to acquire the same conformation as is found in the native HA structure. The CDR3 loop also provides the further advantage of being designed to accommodate a large variety of different sequences, which we felt would increase the chance that the overall immunoglobulin structure would not be destroyed by mutations in this region. In support of this assumption, there have been several reports of the insertion of both B-cell epitopes and T-cell epitopes into the CDR3H region of antibody structures28m’6. The reports of B-cell epitope insertions include an the NANP epitope from Plasmodium fulciparum’8, repeat epitope, and the V3 epitope from HIV-136, both of which are linear, non-conformational epitopes (as defined by cross-reactivity with the corresponding synthetic peptide). We wished to exploit the extensive knowledge of antibody structure to analyze an epitope with more rigorously conformational characteristics. In addition to inserting the A loop structure into the CDR3H region, we wished to investigate the possibility of making antigenic epitope insertions into an alternative site which did not affect the antigen specificity of the antibody. This would allow one to create an antibodyantigen immunogen which could be used in immunotargeting. Thus, in addition to the CDR3-A loop constructs, we created an A loop insertion into the fourth Vi, loop in the FR3 region of Tib92 (the other Vi, loops constitute the three CDRs), which we hoped would have the appropriate conformational characteristics while also leaving the specificity of this antibody for the I-Ak molecule unaltered. The fact that the transfectants produced antibody which could be detected in the supernatants, as well as SDS-PAGE and protein A binding characteristics of this antibody (data not shown), confirm that folding and secretion of all three Tib92-A loop constructs were unaltered. In addition, the FR3 A loop insertion was shown not to have significantly altered the specificity of this construct for the I-Ak molecule (Figure 3). This finding is consistent with a recent report by Simon and Rajewsky”, in which a randomly arising four amino acid insertion in the same FR3 site of an anti-NP antibody (Bl-8) did not affect hapten binding or the idiotypic structure. The authors suggest, however, that the insertion of 6-8 amino acids might be used to create a “CDR4” which contacts the antigen. In this study, the insertion of nine amino acids in place of the S74 residue had no effect on the ability of Tib92 to bind to I-Ak. We have also constructed other B-cell epitope insertions of up to 11 amino acids into this site, and have still retained antigen binding (data not shown). However, the details of antibody-antigen docking are not known for the association of Tib92 with I-Ak. Figure 4 shows that neither rabbit anti-BHA nor rabbit anti-A peptide antisera bind to Fc-captured A loop antibodies. In the case of the anti-BHA antiserum, it is unknown what fraction of the BHA-specific antibodies is directed to the A loop. The immunodominance of the

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A loop epitope in the case of infection with influenza virus26 may well not hold for the injection of purified BHA in FCA. In addition, it is possible that the antiBHA antibodies which recognize the A loop region may have a footprint larger than just the A loop itself, and these additional contacts would not be present in the Tib92-A loop constructs. The same can also be said of the anti-BHA MAbs analyzed in Figure 5. In the case of the rabbit anti-A peptide antiserum, however, shows that there is a high-titer response specifically to the A peptide. Thus, the fact that rabbit anti-KLH-A peptide does not bind to the Ig-A loop constructs clearly demonstrates that the A loop conformation in the Tib92-A loop constructs is significantly dissimilar to the conformation(s) adopted by the synthetic peptide. In spite of its lack of reactivity with the tested A loop-specific MAbs, we have demonstrated that the CDR3-IA construct is able to elicit an antibody response in rabbits which cross-reacts with BHA (Figure 6). In addition, Figure 7 shows that this cross-reactivity to BHA can be competitively inhibited by BHA in solution, demonstrating that the BHA does not have to undergo any local denaturation by binding to the ELISA plate in order to be recognized, as has been reported in other systems’,“. To our knowledge, this is the first report of the generation of a response to a strictly conformational viral epitope using the structural constraints provided by insertion of the epitope into the primary sequence of a “carrier” protein. This result demonstrates the feasibility of using the immunoglobulin molecule as a scaffold for defined B-cell epitopes having conformational features which are not mimicked by the corresponding synthetic peptide. In addition, we have shown that it is possible to insert an epitope sequence into the FR3 loop region of an antibody without altering the antigenic specificity of that antibody. Thus, future investigations will pursue epitope grafting into this site in an effort to combine the advantages of conformational epitope incorportion with the potential to achieve adjuvant-independent immunization via immunotargeting to class II MHC molecules. The long term goal of these studies is to develop a defined subunit vaccine based on a biosynthetically produced, recombinant immunotargeting antibody containing the required B- and T-cell epitopes to mobilize an adjuvant-independent, pathogen-neutralizing immune response.

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ACKNOWLEDGEMENTS We gratefully acknowledge the support of Connaught Laboratories Limited and the Province of Ontario Technology Fund in their funding of this research. J.C. was the recipient of a Medical Research Council of Canada Studentship. We also wish to thank Dr Farangis Saleh for her important assistance, and Drs David Burt, John Skehel and Brian Thomas for generously providing influenza reagents.

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Goodman-Snitkoff, G., Eisele, L.E., Heimer, E.P. et al. Defining minimal requirements for antibody production to peptide antigens. Vaccine 1990, 8, 257-261 Weijer, K., Pfauth, A., van Herwijnen, R. eta/. Induction of feline leukaemia virus-neutralizing antibodies by immunization with

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synthetic peptides derived from the FeLV env gene. Vaccine 1993, 11, 946-956 Steward, M.W. and Howard, CR. Synthetic peptides: a next generation of vaccines? lmmunol. Today 1987, 8, 51-58 Carayanniotis, G. and Barber, B.H. Adjuvant-free IgG responses induced with antigen coupled to antibodies against class II MHC. Nature 1987, 327, 59-61 Carayanniotis, G., Vizi, E., Parker, J.M.R., Hodges, R.S. and Barber, B.H. Delivery of synthetic peptides by anti-class II MHC monoclonal antibodies induces specific adjuvant-free IgG responses in vivo. Mol. Immunol. 1988, 25, 907-911 Carayanniotis, G., Skea, D.L., Luscher, M. and Barber, B.H. Adjuvant-independent immunization by immunotargeting antigens to MHC and non-MHC determinants in vivo. Mol. Immunol. 1991, 28, 261-267 Skea, D.L. and Barber, B.H. Studies of the adjuvantindependent antibody response to immunotargeting. J. Immuno/. 1993, 151, 3557-3568 Skea, D.L., Douglas, AR., Skehel, J.J. and Barber, B.H. The immunotargeting approach to adjuvant-independent immunization with influenza hemagglutinin. Vaccine 1993, 11, 994-l 002 Spangler, B.D. Binding to native proteins by antipeptide monoclonal antibodies. J. Immunol. 1991, 146, 1591-1595 Laver, W.G., Air, G.M., Webster, R.G. and Smith-Gill, S.J. Epitopes on protein antigens: misconceptions and realities. Cell 1990, 61, 553-556 Schulze-Gahmen, U., Klenk, H. and Beyreuther, K. Immunogenicity of loop-structured short synthetic peptides mimicking the antigenic site A of influenza virus hemagglutinin. Eur. J. Biochem. 1986, 159, 283-289 Muller, S., Plaue, S., Samama, J.P., Valette, M., Briand, J.P. and Van Regenmortel, M.H.G. Antigenic properties and protective capacity of a cyclic peptide corresponding to site A of influenza virus haemagglutinin. Vaccine 1990, 8, 308-314 Seki, J., Wang, X., Ota, A., Suzuki, Y., Sakato, N. and Fujio, H. lnducibility of protein-reactive antibodies by peptide immunization: comparison of three epitope peptides of hen egg-white lysozyme. J. Biochem. 1992, 111, 259-264 Oi, V.T., Jones, P.P., Goding, J.W., Herzenberg, L.A. and Herzenberg, L.A. Properties of monoclonal antibodies to mouse lg allotypes, H-2 and la antigens. Curr. Topics Microbial. Immunol. 1978, 81, 115-l 29 Green, N., Alexander, H., Olson, A. et a/. Immunogenic structure of the influenza virus hemagglutinin. Cell 1982, 28, 477487 Jackson, DC., Murray, J.M., White, D.O., Fagan, C.N. and Tregear, G.W. Antigenic activity of a synthetic peptide comprising the “loop” region of influenza hemagglutinin. Virology 1982, 120, 273-276 Shapira, M., Jibson, M., Muller, G. and Arnon, R. Immunity and protection against influenza virus by synthetic peptide corresponding to antigenic sites of hemagglutinin. Proc. NaN Acad. SC/. USA 1984, 81, 2461-2465 Nestorowicz, A., Tregear, G.W., Southwell, C.N. et a/. Antibodies elicited by influenza virus hemagglutinin fail to bind to synthetic peptides representing putative antigenic sites. Mol. Immunol. 1985, 22, 145-l 54 Shapira, M., Misulovin, Z. and Arnon, R. Specificity and crossreactivity of synthetic peptides derived from a major antigenic site of influenza hemagglutinin. Mol. Immunol. 1985, 22, 23-28 Huse, W.D., Sastry, L., Iverson, S.A. et a/.Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 1989, 246, 1275-l 281 Rinfret, A., Horne, C., Boux, H:, Marks, A., Dorrington, K.J. and Klein, M. lsotype modulation of idiotypic expression in recombinant isotypic variants of MOPC 315. J. Immunol. 1990, 145, 925-931 LaPan, K.E., Klapper, D.G. and Frelinger, J.A. Production and characterization of a peptide specific, anti-major histocompatibility complex class II, monoclonal antibody. Mol. lmmunol. 1991, 28, 499-504 Davies, D.R., Padlan, E.A. and Sheriff, S. Antibody-antigen complexes. A. Rev. Biochem. 1990, 59,439 Smith, C.A., Barnett, B.C., Thomas, D.B. and TemoltzinPalacios, F. Structural assignment of novel and immunodominant antigenic sites in the neutralizing antibody response of CBAICa mice to influenza hemagglutinin. J. fxp. Med. 1991, 173,953959

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Wilson, LA., Skehel, J.J. and Wiley, DC. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 angstrom resolution. Nafure 1981, 289, 366-373 Wiley, D.C., Wilson, I.A. and Skehel, J.J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 1981, 289, 373-378 Kieffer, B., Koehl, P., Plaue, S. and Lefevre, J.-F. Structural and dynamic studies of two antigenic loops from haemagglutinin: a relaxation matrix approach. J. Biomolec. NMR 1993, 3, 91-112 Billetta, R., Hollingdale, MR. and Zanetti, M. lmmunogenicity of an engineered internal image antibody. Proc. NaN Acad. Sci. USA 1991, 88,4713-4717 Zanetti, M., Filaci, G., Lee, R.H. et al. Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif. EM60 J. 1993, 12, 4375-4384 Lanza, P., Billetta, R., Svetlana, A. and Zanetti, M. Active immunity against the CD4 receptor by using an antibody antigenized with residues 41-55 of the first extracellular domain. Proc. Nat/ Acad. Sci. USA 1993, 90, 11683-l 1687 Zaghouani, H., Krystal, M., Kuzu, H. et al. Cells expressing an H chain lg gene carrying a viral T cell epitope are lysed by specific cytolytic T cells. J. Immunol. 1992, 148, 3604-3609 Zaghouani, H., Steinman, R., Nonacs, R., Shah, H., Gerhard, W. and Bona, C. Presentation of a viral T cell epitope expressed in the CDR3 of a self immunoglobulin molecule. Science 1993, 259, 224-227

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Zaghouani, H., Kuzu, Y., Kuzu, H. et al. Contrasting efficacy of presentation by major histocompatibility complex class I and class II products when peptides are administered within a common protein carrier, self immunoglobulin. Eur. J. Immunol. 1993, 23, 2746-2750 Kuzu, Y., Kuzu, H., Zaghouani, H. and Bona, C. Priming of cytotoxic T lymphocytes at various stages of ontogeny with transfectoma cells expressing a chimeric lg heavy chain gene bearing an influenza virus nucleoprotein peptide. hf. Immunol. 1993,5,1301-1307 Brumeanu, T.D., Swiggard, W.J., Steinman, R.M., Bona, C. and Zaghouani, H. Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus. J. Exp. Med. 1993, 178, 1795-1799 Zaghouani, H., Anderson, S.A., Sperber, K.E. et al. Induction of antibodies to the human immunodeficiency virus type 1 by immunization of baboons with immunoglobulin molecules carrying the principal neutralizing determinant of the envelope protein. Proc. Nat/ Acad. Sci. USA 1995, 92, 631-635 Simon, T. and Rajewsky, K. A functional antibody mutant with an insertion in the framework region 3 loop of the V, domain: implications for antibody engineering. Profein Engng 1992, 5, 229-234 Van Regenmorlel, M.H.V. Structural and functional approaches to the study of protein antigenicity. Immunol. Today 1989, 10, 266-l 71 Kabat, E.A., Wu, T.T., Reid-Miller, M., Perry, H.M. and Gottesman, KS. Sequences of proteins of immunological interest. US Department of Health and Human Services, 4th edn, 1987