HYBRIDOMA AND HYBRIDOMICS
Volume 22, Number 1, 2003
© Mary Ann Liebert, Inc.
Rapid Monoclonal Antibody Generation Via Dendritic
Cell Targeting In Vivo
JODY D. BERRY,*1 ALEXEI LICEA,*1,2 MIKHAIL POPKOV, 1 XOCHITL CORTEZ,2 ROBERTA FULLER,1
MARIKKA ELIA,1 LISA KERWIN, 1 DIANE KUBITZ,1 and CARLOS F. BARBAS, III1
ABSTRACT
Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. Previous studies have
demonstrated that targeting foreign antigens to DC leads to enhanced antigen (Ag)-specific responses in vivo.
However, the utility of this strategy for the generation of MAbs has not been investigated. To address this
question we immunized mice with IgG-peptide conjugates prepared with the hamster anti-murine CD11c MAb
N418. Synthetic peptides corresponding to two different exposed regions of DC-specific ICAM-3 grabbing
nonintegrin (DC-SIGN), a human C-type lectin, were conjugated to N418 using thiol-based chemistry. The
N418 MAb served as the targeting molecule and synthetic peptides as the Ag (MAb-Ag). A rapid and peptide
specific serum IgG response was produced by Day 7 when the synthetic peptides were linked to the N418
MAb, compared to peptide co-delivered with the N418 without linkage. Spleen cells from N418-peptide immunized mice were fused on Day 10, and three IgG1/k monoclonal antibodies (MAbs) were selected to one of
the peptide epitopes (MID-peptide). One of the MAbs, Novik 2, bound to two forms of recombinant DC-SIGN
protein in enzyme-linked immunosorbent assay (ELISA), and was specifically inhibited by the MID-peptide
in solution. Two of these MAbs show specific binding to DC-SIGN expressed by cultured human primary DC.
We conclude that in vivo DC targeting enhances the immunogenicity of synthetic peptides and is an effective
method for the rapid generation of MAbs to predetermined epitopes.
INTRODUCTION
ing cells via major histocompatibility complex (MHC) class II
MAbs,(10) to B cells via surface IgD(11) or immunoglobulin Fc
receptors,(12,13) and to dendritic cells (DC) via MAb-peptides
Ags.(14,15) In general, these MAbs-Ags produce enhanced antibody responses. However, most studies of serum antibody responses examined secondary responses in which high titer affinity-matured IgG is produced. The utility of these approaches
for facilitating the production of MAbs has not previously been
investigated.
The immunogenicity of a vaccine candidate antigen is the
ability to engender antibodies that recognize the corresponding
native epitopes. Ideally, a subunit vaccine is comprised of an
antigen that is not only immunogenic but also engenders protective antibodies. Research on synthetic peptide vaccines originated from early observations of immune responses to chemically synthesized linear peptides corresponding to protective
A
are one of the main effector arms of the specific
immune system, and are the only one capable of completely preventing an infection.(1) The exquisite specificity of
antibodies to individual epitopes makes them desirable as therapeutics and as targeting domains to host proteins. Many methods for improving the immunogenicity of synthetic and recombinant vaccine candidates have been evaluated. Some of
these methods include: Tandem co-delivery of T-cell epitopes(2); changing epitope multiplicity, orientation, or polarity(3–5) ; conformationally optimized peptides(6) ; route of
immunization(7); and MAP-peptide multimer technique.(8) Another method is to target antigens directly to cells of the immune system through the use of MAb-Ags or other molecules.(9)
A variety of MAb-Ags have been targeted to antigen presentNTIBODIES
1 The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, BCC-515, 10550 North
Torrey Pines Road, La Jolla, CA 92126.
2 Centro de Investigacion Cientifica y de Educacion Superior de Ensenada. Departamento de Acuacultura, Biotecnologia Marina, Tijuana, Mexico.
*These authors contributed equally to this work.
23
24
BERRY ET AL.
epitopes of polio and foot and mouth disease viruses.(16,17) In
these cases the protective peptides were identified by epitope
mapping with neutralizing serum or MAbs. Unfortunately, the
peptide vaccine field in general suffers from a serious general
drawback—low immunogenicity. While peptide-carrier combinations can be antigenic (stimulate anti-peptide antibodies)
these antibodies tend to recognize the native epitopes poorly.
Low immunogenicity has been attributed to many factors including conformational flexibility of the native antigen, and the
low amount of native structural information portrayed in small
synthetic peptides. Alternatively, studies with a highly characterized model peptide epitope in various conformational constraints reveals that the MAb on-rates, rather than conformation, correlates with affinity of the MAbs raised to these
structures.(18) While this may be a characteristic limited to
MAb-peptide binding studies, even these model antigenic determinants required co-delivery with adjuvants and multiple
boosters in order to produce high titer IgG responses. Because
concentration may be more important than absolute affinity for
antibody-mediated protection,(19) the ability of a vaccine to
rapidly engender a high titer, secondary-like, response may be
more important than the absolute affinity of the antibody produced.
From an applied perspective, the generation of rapid high
titer IgG responses by in vivo DC targeting in mice should facilitate MAb production. DC are sentinels of the immune system and are capable of stimulating powerful antigen-specific
immune responses. DC have become the focus of many new
vaccine strategies. Importantly, targeting of MAb-Ags to DC
stimulates primary antibody responses by B cells.(20,21) Similar
strategies may induce protective primary antibody responses
against infectious or toxic threats if given just before or soon
after exposure.(15) The isolation of monoclonal antigen specific
antibodies from MAb-Ag immunized animals would also serve
as an unequivocal confirmation of the MAb-Ag targeting technique. In this paper we sought to extend the findings of Wang
et al.(15) by evaluating DC targeting of synthetic peptide MAbAgs as an in vivo method for the rapid production of MAbs.
MATERIALS AND METHODS
Targeting antibody–antigen preparation
N418 hybridoma [American Type Culture Collection (ATCC)]
was grown in the Integra CELLine CL1000 with BD-Cell-MAb
media. The CL1000 was inoculated with 1 3 108 cells in 15
mL of BD-Cell, 20% FCS. Purified antibody was dialyzed twice
against PBS and stored frozen at 280°C until used further.
Synthetic peptides were commercially synthesized and HPLC
purified (United Biochemical Research Inc., Seattle, WA). Two
synthetic peptides were used; one, termed the C-terminal
peptide (H2N-CSRDEEQFLSPAPATPNPPPA— COOH) corresponds to the unique C-terminal residues of the DC-SIGN
protein(22); and the second is called the MID-peptide (H2NCPEKSKLQEIYQELTQLKAAVGEL— COOH) and is a consensus peptide corresponding to a domain repeated eight times
between the membrane and the C-terminal lectin and was previously to raise antiserum to DC-SIGN.(23) This sequence is
also common to the recently discovered DC-SIGNR protein.(24)
The peptides were conjugated to the purified N418 IgG MAb
pretreated with SMCC linker (Pierce, Rockford, IL) through the
designed free N-terminal cysteine residue. MALDI mass-spectroscopy was used to evaluate linkage of the synthetic peptides
to the N418 molecule.
Mice were immunized with 2 mg of these conjugates, half
to intraperitoneal (i.p.) and to intradermal (i.d.) locations simultaneously. Following 7 days the animals were bled from the
tail vein, the serum was treated at 37°C for 1 h, then 4°C
overnight, and clots removed. Sera was serially diluted in 96well V-bottom plates using 2%-bovine serum albumin (BSA)
phosphate-buffered saline (PBS) as diluents. Standard enzymelinked immunosorbent assay (ELISA) was performed using the
same peptides linked to BSA at 100 ng/well. ELISA assays
were stained with a commercial goat anti-mouse IgG—horseradish peroxidase (HRP) conjugated antibody (Southern
Biotechnology Associates, Birmingham, AL) at 1/1000 dilution
in 2% BSA-PBS. The ELISA was developed with standard 2,29azino-bis(3-ethylbenzo thiasoline-6-sulfonic acid) (ABTS) substrate in citrate buffer (pH 4.4) and 0.2% H2O2. The plates were
read at 405 nm after 1 h. The mice display reciprocal reactivity confirming peptide specific immune responses and not reactivity to the common SMCC-linker group used for both BSA
and IgG linkage of peptides. The serum dilution used in all
ELISA assays was 1/250 in 2% BSA-PBS, unless otherwise
noted.
Recombinant DC-SIGN protein construction and
purification (pmalA9/pgexA9)
Recombinant fragment of the human DC-SIGN protein containing the external repeat domains from residues 62 to 260 in
the mature protein (termed A9 fragment) was cloned. The domain is found between (and excluding) the putative transmembrane domain and the C-terminal C-type lectin. Total RNA was
harvested with an RNEasy kit (Qiagen, Valencia, CA) on
.2.5 3 106 human primary DC (BioWhittaker, Walkersville,
MD). Complementary DNA was produced using the Superscript
RT system (Invitrogen, Carlsbad, CA) and the cDNA was amplified using specific primers and high-fidelity polymerase (Invitrogen). Electrocompetent XL-1 blue (Stratagene, La Jolla,
CA) E. coli colonies were screened for inserts using polymerase
chain reaction (PCR) and the sequence of the insert confirmed
by cycle sequencing. The DC-SIGN A9 fragment was subcloned
using the same conditions into the Sfi1 precut vector pGex4T
(Amersham Biosciences, Piscataway, NJ) modified to contain
Sfi1 sites. Colonies determined to be insert positive using vector specific primers and were sequenced to confirm identity.
Clones with correct DC-SIGN fragment sequences in pMalp
and pGex4T were expressed as fusion proteins (pMal-A9 and
pGex-A9, respectively). Both pMal-A9 and pGex4T-A9 were expressed in XL-1 blue E. coli. Protein was purified from sonicated cell lysates using amylose resin columns (New England
Biolabs, Beverly, MA). For pGex4T-A9, expression was carried out in two 500-mL super broth cultures. Each culture was
induced for 6 h with isopropyl-beta D -thiogalactopyranoside
(IPTG) (0.3 mM) when the optical density (OD) was between
0.5 and 2.0. Purification from sonicated cell lysates was
achieved using glutathione sepharose-4B slurry (Amersham
Biosciences). The eluted samples were concentrated and
25
IN VIVO DELIVERY OF PEPTIDE EPITOPES
brought into PBS using Centriprep 210 sec (10,000 MW cutoff) (Millipore, San Clemente, CA). Protein size was confirmed
by Coomassie staining of protein gels run against prestained
MW marker (BioWhittaker) upon premade 15% sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAGE) (Biorad, San
Diego, CA).
Fusion reagents and hybridoma production
Splenocytes were harvested 10 days postimmunization, and
fused to the P3X63Ag8.653 myeloma in log phase according
essentially to the protocol of Lane.(25) Briefly, the PEG1500
(Roche Molecular Biochemicals, Mannheim, Germany) was
added drop-wise over 1 min while gently tapping the tube containing the myeloma-splenocyte pellet on the flow hood surface. The cells were swirled for an additional minute. The polyethylene glycol (PEG) was diluted by addition of 8 mL of
serum-free RPMI media (RPMI-0; Biowhittaker) over 1 min.
The cells were washed twice in RPMI-0, and resuspended in
incomplete RPMI with 10% fetal calf serum FCS, and 4% hybridoma cloning factor (IGEN International Inc., Gaithersburg,
MD) and hypoxanthine, aminopterine, and thymidine (HAT)
additive (Invitrogen). Cells were plated out to 10 96-well tissue culture plates (BD Biosciences; San Diego, CA) and cultured at 37°C and 5% CO2 overlay. Tissue culture supernatants
were assayed for binding to peptide-bovine serum albumin
(BSA) conjugates in ELISA when foci were visible to the eye.
The Costar 3690 96-well ELISA plates (Corning, Acton, MA)
were coated with respective peptide-BSA conjugates (100
ng/well) in PBS overnight at 4°C and then blocked with 4%
BSA, 1% Ovalbumin in PBS, for 2 h at 37°C. Supernatant (100
mL/well) was incubated neat for 1 h at 37°C. The ELISA plates
were washed ten times with dH2O and patted dry on a paper
towel. A pan-goat anti-mouse IgG-HRP antibody (Southern
Biotechnology Associates; Birmingham, AL) was used at
1:2000 in 2% BSA in PBS for 45 min at 37°C and washed as
above. Binding was detected with ABTS. The OD was read at
405nm at 1 h of development, or as indicated. Fusion efficiency
was monitored in parallel by testing the supernatants for IgG
production using enzyme immunoassay (EIA) plates coated
with 200 ng/well of unconjugated, pooled, goat anti-mouse
kappa and lambda light-chain antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for IgG capture. Wells
corresponding to positive signals for a single peptide-BSA conjugate were retested and subcloned at limiting dilution twice.
MAb supernatants were isotyped at the 6 well-stage with a
mouse-HRP clonotyping kit (Southern Biotechnology Associates) with optimized working dilutions and developed as above.
Flow cytometry on normal human cells
Normal human dendritic cells (NHDC, blood-derived
CD142 ) and human umbilical-vein endothelial cells (HUVEC)
were purchased from Biowhittaker. Culturing of NHDC was
performed in LGM-3 growth media (Biowhittaker) complete
with albumin, insulin, transferrin, gentamicin, GM-CSF (500
U/mL), IL-4 (1000 U/mL), and 5% of human plasma. HUVEC
were maintained in EGM growth media (Biowhittaker) complete with hEGF, hydrocortisone, insulin, gentamycin, amphotericin-B, and 2% fetal bovine serum (FBS). The EGM media
was supplemented with Bovine Brain Extract before use
(Biowhittaker). After 4 days of culture, NHDC and HUVEC
were washed once with N-z-hydroxyethylpiperazine-Nz ethanesulphonic acid (HEPES) Buffered Saline Solution (HBSS;
Biowhittaker) and harvested by mild trypsinization with 0.025%
trypsin, 0.01% ethylene-diaminetetraacetic acid (EDTA) in
HBSS (Biowhittaker). NHDC found in suspension (non-adherent) were also collected by extensive washing with PBS, and
stained separately from the remaining adherent NHDC. NHDC
were washed once with PBS, resuspended in 1 mL of LGM-3,
and incubated for 1 h at 37°C in the presence of 1 mg/mL of
human IgG to prevent binding through Fc portion of the Abs.
Cells were washed once with PBS and resuspended at 106
cells/mL of FACS buffer (1% BSA, 0.03% NaN3, 25 mM
HEPES in PBS, pH 7.4 sterile filtered). Aliquots of 100 mL
containing about 105 cells were distributed into wells of V-bottom serocluster plates (BD Biosciences). One hundred microliters of purified from hybridoma cultures MAb (5 mg/mL) were
then added to the cells and incubated for 30 min at room temperature (RT). Cells were washed once (spun down at 1800 rpm
for 2–3 min) with 200 mL of FACS buffer and incubated with
100 mL of fluorescein isothiocyanate (FITC)-conjugated affinity-purified donkey anti-mouse IgG (H1L) antibody (Jackson
ImmunoResearch Laboratories), diluted to 1:100 in FACS
buffer for 30 min at RT. Cells were washed twice as above, resuspended in 200 mL of FACS buffer, and transferred to FACStube for analysis in a FACS scan flow cytometer with CellQuest
software (Becton Dickinson, San Jose, CA).
RESULTS
Conjugation of synthetic peptides to N418
(anti-CD11c)
The purified hamster IgG MAb N418 was used as the carrier moiety and coupled to either the MID- or C-terminal peptides. Coupling was mediated using commercial reagents and
the terminal Cys residues instituted by design. Coupling efficiency was evaluated by a shift in molecular weight of the IgG
molecules on SDS-PAGE gels (data not shown) and confirmed
using mass spectroscopy. The average molecular weight of the
IgG with coupling groups was about 159,078 kDa as predicted,
and the size shifted to 175,931 kDa with the MID-peptides
groups attached (Fig. 1). The MID peptide is 2200 kDa therefore we estimate that the average valence of peptide attachment
is approximately 7 peptides per N418 hamster IgG molecule.
Serological responses to MAb-Ag conjugates
Groups of mice were immunized with MAb-peptide Ag conjugates and tested for homologous and heterologous serum IgG
reactivity on Day 7. The specific serum IgG was tested on the
same synthetic peptides conjugated to BSA as antigen. Groups
of mice (n 5 4–6) were immunized with N418 conjugated with
the C-terminal peptide, or the MID-peptide, or C-terminal peptide and N418 unconjugated. A fourth group of mice was immunized with N418 MAb but with only half of the molecules
conjugated to the C-terminal peptide. Mice immunized with the
C-terminal peptide MAb-Ag have a serum IgG end-point titer
of 1/6000 for the homologous peptide-BSA conjugate and no
reactivity with the MID-peptide BSA conjugate. Conversely,
26
BERRY ET AL.
FIG. 1. Overlay of the MAb–Ag conjugates analyzed in mass spectroscopy. The upper line represents the hamster MAb N418
conjugated to the MID-peptide. The lower line represents the same purified IgG with coupling reagent but without peptide attachment.
mice immunized with the MID-peptide MAb-Ag have a serum
IgG end point titer of 1/1200 for the homologous peptide-BSA
conjugate, and no reactivity for the C-terminal peptide BSA
conjugate (data not shown). The clear reciprocal reactivity of
the serum indicated that the rapid IgG response is specific for
the peptide moieties, and not the common linker groups (Fig.
2). Mice co-immunized with naked N418 and C-terminal peptide conjugated N418 show a reduced serum IgG reactivity
for the homologous C-terminal peptide-BSA conjugate (Fig.
2). This may reflect a dose-dependent or competitive effect
for CD11c on the DC. Co-immunization of mice with N418
MAb and free C-terminal peptide does not produce measurable serum IgG to either peptide-BSA conjugates by Day 7.
Similarly, mice immunized with free MID-peptide and N418
do not produce measurable serum IgG by Day 7 to either peptide-BSA conjugate. These data show that conjugation of peptides to the anti-CD11c antibody are required to elicit rapid
IgG responses.
FIG. 2. Serum IgG reactivity for peptides. Serum was collected 7 days after a single immunization of the MAb-Ag conjugates
without adjuvant. The analysis shown was done at a 1/250 dilution of serum. White bars represent C-terminal peptide IgG reactivity; Black bars represent MID-peptide IgG reactivity. Each column represents the averaged value of the indicated number of
mice. The standard errors are shown.
27
IN VIVO DELIVERY OF PEPTIDE EPITOPES
MAbs from fusion at Day 10
The splenocytes from a single mouse immunized with N418MID-peptide and N418-C-terminal peptide were harvested on
Day 10. Three hybridomas were identified as MID-peptide reactive from 250 IgG-producing wells and were selected and
grown for further studies. All three of these MAbs are of the
IgG1/kappa isotypes. This is consistent with the observations
of Wang et al.(15) as IgG1 was the predominant IgG class observed in the rapid responses to their model antigens. These
MAbs react with the MID-peptide BSA conjugate specifically
and not with the C-terminal peptide nor with the common linker
group (Fig. 3). Fusion efficiency was far lower for the C-terminal peptide immunized mouse, as there were no C-terminal
peptide specific clones identified from the 80 IgG producing
wells. This is despite the fact that the serological response of
the C-terminal peptide immunized mice were 5 times higher,
and the splenic inflammation was far greater for the C-terminal immunized mouse compared with the MID-peptide immunized mouse. This shows that fusion efficiency still cannot always be predicted based upon phenotypic markers.
Specificity of anti-DC-SIGN MAbs
The MID-peptide MAbs were tested for reactivity with recombinant DC-SIGN fragments. Two recombinant DC-SIGN
fragments spanning the repeat-domain from which the MIDpeptide corresponds, were tested for binding in ELISA. One of
the clones, Novik2, reacted with recombinant DC-SIGN proteins (Fig. 3). Novik2 does not react with irrelevant pGex and
pMaL fusion proteins and is the only MAb to react with whole
DC-SIGN expressed in cell lysates in western blot (data not
shown). Thus, our recombinant DC-SIGN proteins are antigenic
for only one of three linear peptide epitope specific MAbs. The
binding of Novik2 to both pMaL-A9 and pGex-A9 was specifically inhibited by soluble MID-peptide, but not by the C-terminal-peptide (Figs. 4A and B, respectively). This shows that
FIG. 4. Peptide inhibition of MAb binding. Novik2 MAb
binding on pMaL-A9 fragment (A) and pGex-A9 fragment (B).
The black squares represent MAb binding in the presence of Cterminal peptide; Black circles represent binding in the presence of MID-peptide; The open circle represents binding in the
absence of peptide inhibitors. The average of three different assays is shown.
the binding of Novik2 was specific for the MID-peptide epitope in the recombinant DC-SIGN A9 fragments.
DC-SIGN expression on NHDC
FIG. 3. Reactivity of MID-peptide MAbs on recombinant human DC-SIGN A9 fragment and the corresponding DC-SIGN
peptides. Binding of three MID-peptide-specific MAbs identified by hybridoma technique is shown. The average of three
different assays is shown.
Finally, we tested the two MAbs, Novik2 and 3, for reactivity with native DC-SIGN on NHDC in flow cytometry. We
found that two MID-peptide specific MAbs reacted with a reproducibly small percentage (8–11%) of nonadherent NHDC
subpopulations, which had high levels of DC-SIGN expression,
compared with isotype matched control (Figs. 5E and F). Bloodderived DC are known in the literature to express DC-SIGN in
between 4–14% of their population.(26) Thus, our anti-DCSIGN MAbs that bind fit precisely into this range. In contrast,
only Novik2 MAb was able to detect DC-SIGN, which was
weakly expressed by adherent NHDC subpopulation (Figs. 5C
and D; large peaks shifts to right for Novik2). Novik3 was unable to detect the lower level of DC-SIGN expression. The small
bright fluorescent peak of cells are most likely due to remaining loosely adherent DC, which contaminate the adherent cell
population after our imperfect separation of DC subpopulations
28
BERRY ET AL.
FIG. 5. Flow cytometry on primary human cells. HUVEC (A, B), adherent NHDC (C, D), and nonadherent NHDC (E, F) were
treated with MAb Novik2 (bold line on left panel) or MAb Novik3 (bold line on right panel) and stained with FITC-conjugated
donkey anti-mouse IgG antibody. The dashed line corresponds to irrelevant isotype-matched control mouse MAb. Numbers in
the figure represent the percentage of bright stained cells in the corresponding subpopulations of NHDC. MFI, mean fluorescence
intensity.
based upon gross phenotype of being adherent or nonadherent
NHDC in tissue culture. HUVEC were uniformly negative for
DC-SIGN (Figs. 5A and B). This shows that the synthetic peptides are immunogenic for the native DC-SIGN protein and suggests that the peptide epitopes in the A9 region are more accessible on DC cells than in recombinant fusion proteins.
Overall, these findings indicate that the MAb-Ag peptide DC
targeting strategy is useful for the rapid generation of MAbs to
predetermined epitopes.
DISCUSSION
The DC-SIGN-specific MAbs described herein are the first
set of MAbs produced using the immunotargeting MAb-Ag
technique. They are specific to a peptide-epitope from DC-
SIGN. DC-SIGN is a type II membrane protein with a C-terminal lectin-binding domain, and is involved in DC interaction
with other immune system cells.(22) DC-SIGN was initially
cloned from the cDNA of human placenta(23) and it was subsequently shown to be highly expressed on DC and to bind
ICAM-3.(27,28) Recently, a closely related homolog to DCSIGN, termed DC-SIGNR was described, that exhibits 77%
amino acid identity with DC-SIGN.(24) The DC-SIGNR expression was documented on endothelial cells in human placenta, lymph node sinuses, and hepatic sinusoids, but not on
peripheral blood-derived DC.(28) Using the MAb-Ag technique
with DC-SIGN peptides borne on a DC targeting MAb-Ags, we
obtained specific hybridomas in 8 weeks. This marked reduction in the 2–5 months normally needed to immunize and boost
an animal prior to the fusion and subcloning process will directly translate into savings of time and money.
29
IN VIVO DELIVERY OF PEPTIDE EPITOPES
In the present study, we demonstrate that the N418 MAb-Ag
system is useful for the rapid generation of antigen specific
MAbs. Moreover they show that the IgG1 class of antibodies
is generated within days of a single-step immunization by targeting CD11c1 cells, including DC, of the mouse. This reduces
the amount of time needed to house and immunize animals compared to normal priming and boosting strategies. Indeed a single immunization of just 2 mg of antigen allowed us to produce
peptide specific, recombinant protein reactive, and native cell
expressed protein reactive MAb in just 8 weeks. The time required for the in vitro cloning can be reduced further through
the use of direct picking of individual foci from in soft-agar(29)
the conditions for which have been greatly redefined (R.P.
Beecroft and T.W. Pearson, University of Victoria, Canada,
personal communication). Thus, the production of MAbs via
hybridoma fusion from immune mice has a secure future in antibody discovery in particular in light of new technologies
such as the generation xenomice expressing nonrodent Vgenes.(30–32) The rapidity of the IgG response is intriguing from
an immunological standpoint. A rapid serum IgG response is
engendered to the synthetic peptide conjugates by Day 7, the
magnitude of these IgG responses is more modest with respect
to those seen by Wang et al.(15) Recently, Wang et al.(15) showed
that targeting model antigens to the mainly DC borne antigenCD11c via a hamster anti-mouse CD11c MAb N418(33) enhanced the serum IgG antibody levels in the primary response.
A single low-dose immunization of MAb-Ag, in this case a hapten or goat anti-hamster antibody, produced high titer IgG antibody in only 7 days without adjuvant.(15) The mice had serum
IgG titers as high as 1:100,000 to the goat moiety as early as
Day 7. Remarkably, this was in naive mice without the use of
any classical adjuvants and an irrelevant hamster MAb conjugated to the same antigen produced little measurable serum IgG
until Day 20. Clearly, this type of response will vary depending on individual antigens and on the parameters of this type
of response remain to be determined. We thereby extend these
results by demonstrating the use of in vivo DC targeting to accelerate MAb discovery. The predominant IgG class recovered
by Wang et al.(15) was IgG1, which is consistent with the isotype of the MAbs we recovered in our fusion.
The molecular basis for this enhanced response remains to
be determined. Similar rapid and potent IgG responses are seen
in some viral antigens. Viral particle with repeating antigenic
structures on cytopathic viruses elicit rapid antibody responses.(34) While the basis for this rapid response is also not
clear, some general findings are relevant. Sequencing of many
VSV MAbs from early and late in the response showed that
multiple V-region genes are recruited early on in the response.(35) Remarkably, the MAbs selected early to VSV on
Day 7 had the same high affinity as those from late in the immune response. It is likely that the repeat structures of viruses
such as VSV may bind directly to DC receptors triggering a
rapid B-cell response and generation of antibody. Affinity studies on DC targeted MAb-Ags at different time points will test
the generality of this effect. The influence of DC in the recruitment of B-cell clonotypes and studies of V-gene usage and
mutation may clarify the T-cell dependence of B cells producing these rapid responses,(36,37) respectively. For example, DC
are capable of eliciting B-cell responses directly to live cytopathic virus in a CD40 independent(38,39) and T-cell independent manner(38) they tend to concomitantly stimulate T-cell
help. The structure of the antigen is apparently important as Tcell-independent antiviral antibody responses were seen in the
VSV glycoprotein when expressed as a repetitive antigen but
not as an irregular cell surface protein.(19) Indeed, the more
rigidly arrayed the VSV glycoprotein, the less B-cell primary
and secondary responses are T-cell dependent.(34) Evidence
suggests that the DC are intimately involved in the rapidly protective antibody response to live but not UV inactivated VSV
virus.(39) We speculate that the targeting of MAb-conjugated
antigens onto the surface of murine DC cells may similarly
serve to localize Ag into repeat-like structures on the surface
of the murine DC. If this strategy translates to protective epitopes in other systems it would be valuable as a biomedical
countermeasure in the event of an intentional release of a pathogen or its products for postexposure immunization, or for a rapid
boost to immunity of individuals who may be exposed to a
pathogen in short order.(15) Currently we do not know the
longevity or the protective capacity of this type of antibody response.
These data confirm the general utility of the immunotargeting approach(9) for producing rapid antibody responses in mice.
The enhanced immunogenicity of synthetic peptides delivered
as MAb-Ags to DC in vivo may overcome past problems of
limited immunogenicity that subdued the initial promise of peptide vaccines.(16,17,40) In support, synthetic peptide vaccines delivered to the mucosa of the nose and to the skin (areas rich in
DC cells) have efficient systemic and mucosal immune responses.(41) We would also predict that the MAb-Ag technique
would work as a mucosal immunization strategy in mice as
these tissues are rich in DC. Clearly synthetic peptide-based
vaccines will not be protective in all cases,(42) however, the in
vivo DC targeting strategy can work for whole proteins.(15)
Clearly, further studies are warranted to determine if such rapid
IgG responses can be engendered against whole microorganisms or toxins, and if these antibodies provide in vivo protection against challenge with the live organisms. It will also be
interesting to see if the same MAb-Ag strategy can translate to
larger animals and humans. In humans, we speculate that DCSIGN targeting antibodies, like the ones prepared here, might
serve as appropriate DC-targeting MAbs for the MAb-Ag approach described here. This technique uses extremely well-defined and characterized subunit immunogens. In this respect,
the MAb-Ag method is particularly well suited to accommodate antibody discovery, via hybridoma or phage display, in the
rapid field of genomics. The pace at which new target proteins
are being identified has greatly exceeded the ability of immunologists to produce quality MAb reagents. Clearly, the development of techniques that reduce the amount of time cost and
effort are critical for the efficient production of MAbs and the
development of new vaccines based on such proteomic experiments. In summary, by using in vivo DC targeting with MAbAgs, we have demonstrated here the rapid generation of MAbs
specific for a peptide epitope of human DC-SIGN.
ACKNOWLEDGMENTS
The authors thank Mike Kubitz for excellent technical assistance on the hybridoma production. Our thanks also to the
Scripps Research Institute Department of Animal Services and
Department of Mass Spectrometry. JDB is supported by a Cana-
30
BERRY ET AL.
dian Institutes for Health Research (CIHR) fellowship (former
Medical Research Council/NHRDP HIV/AIDS fellowship of
Canada). Work on this article was supported by grants from the
National Institute of Health 5R01AI37470-06 to C.F.B. III.
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Address reprint requests to:
Carlos F. Barbas, III, Ph.D.
The Skaggs Institute and Department of Molecular Biology
The Scripps Research Institute, BCC-526
10550 North Torrey Pines Road
La Jolla, CA 92037
E-mail: carlos@scripps.edu
Received for publication September 13, 2002. Accepted for
publication November 20, 2002.