Article

Non-classical binding of a polyreactive α-type anti-idiotypic antibody to B cells

Immunobiology Division, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba.
Molecular Immunology (Impact Factor: 2.97). 10/2010; 48(1-3):98-108. DOI: 10.1016/j.molimm.2010.09.006
Source: PubMed
ABSTRACT
Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the "HCDR4", located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies.

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Molecular Immunology 48 (2010) 98–108
Contents lists available at ScienceDirect
Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm
Non-classical binding of a polyreactive -type anti-idiotypic antibody to B cells
Tays Hernández
a
, Cristina Mateo de Acosta
a,
, Alejandro López-Requena
a
, Ernesto Moreno
b
,
Ruby Alonso
c
, Yuniel Fernández-Marrero
a
, Rolando Pérez
d
a
Immunobiology Division, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba
b
Tumor Biology Division, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba
c
System Biology Division, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba
d
Research and Development Direction, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba
article info
Article history:
Received 29 June 2010
Received in revised form
10 September 2010
Accepted 14 September 2010
Available online 16 October 2010
Keywords:
Alpha-type anti-idiotypic antibodies
FcRIIb
Antibody binding site
B-CLL
abstract
Detailed information on the immunological relevance of -type anti-idiotypic antibodies is lacking after
more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-
human chimeric version of the B7 MAb, a polyreactive -type anti-idiotypic antibody, generated against
an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispeci-
ficity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as
well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this anti-
body might have immunoregulatory properties, and therefore we investigated the possible interaction
of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition
assays using flow cytometry indicated that this antibody is capable of binding the Fc receptor II (FcRII).
The recognition of FcRII-expressing K562, Raji and Daudi human cell lines, together with the capability
of inhibiting the binding of an anti-human FcRII antibody to these cells, suggest that B7Y33 interacts
with both the FcRIIa and FcRIIb isoforms. We evaluated the contribution to the binding of different
surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the con-
struction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the “HCDR4”,
located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by per-
forming key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by
combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispeci-
ficity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that
this phenomenon occurs in a non-classical way with the participation of both the variable and constant
regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to
two FcRIIb molecules through the Fc and Fv regions, or simultaneously to FcRIIb and another unknown
antigen on B cells. The FcRIIb has recently received great attention as an attractive target for therapies
directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lympho-
cytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell
malignancies.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
With the postulation of the Idiotypic Network Theory by Jerne
in 1974 (Jerne, 1974), the knowledge about the function and
Abbreviations: Ab, antibody; B-CLL, B-cell chronic lymphocytic leukemia; BCR, B
cell receptor; CH, heavy chain constant domain; ELISA, enzyme linked immunosor-
bent assay; Fc, crystallizable fragment; FITC, fluorescein isothiocyanate; Fv, variable
fragment; HCDR, heavy chain complementarity determining region; HFR, heavy
chain framework; IVIg, intravenous immunoglobulin; MAb, monoclonal antibody;
PBMC, peripheral blood mononuclear cells; PE, phycoerithryn; PBS, phosphate
buffer saline; VH, heavy chain variable region; V, light chain variable region.
Corresponding author. Tel.: +53 7 2143160; fax: +53 7 2720644.
E-mail address: cristina@cim.sld.cu (C.M. de Acosta).
structure of antibodies moved from the interaction with their anti-
gens, to the complex relationships they can establish between
them, of potential relevance for the immune system regula-
tion (Winkler et al., 1979; Shoenfeld, 2004). The existence of
-type anti-idiotypic antibodies, having the ability of inhibiting
the interaction of the Ab1 antibody with the nominal antigen
and able to mimic the latter molecule, has been largely exploited
for the design of vaccines (Poskitt et al., 1991; Betáková et al.,
1998). However, the -type anti-idiotypic antibodies, which do
not impair the Ab1-antigen binding, have not been extensively
studied.
Gangliosides are normal components of the plasma membranes
of most mammalian cell types, and have been also associated with
malignant transformation (Marquina et al., 1996; Malykh et al.,
0161-5890/$ see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2010.09.006
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T. Hernández et al. / Molecular Immunology 48 (2010) 98–108 99
2001; Kannagi et al., 2008). Diverse strategies have been reported
to elicit antibodies specific for these low immunogenic molecules
(Helling et al., 1994, 1995; Livingston and Ragupathi, 1997). Our
group has obtained several ganglioside-specific antibodies (Alfonso
et al., 1995; Vázquez et al., 1995), some of which, in different formu-
lations, have been able to induce a strong anti-idiotypic response
in mice (Vázquez et al., 1998). We have thus isolated some mon-
oclonal antibodies (MAbs) that behaved as -or-type (those
that inhibit the interaction of the Ab1 antibody with the nomi-
nal antigen but do not mimic it) anti-idiotypes depending on the
species in which they were used as immunogens for the obten-
tion of Ab3 antibodies (Vázquez et al., 1998; Hernández et al.,
2005). Moreover, we have also generated, immunizing mice with
the anti-ganglioside antibodies mentioned above, non-paratopic
specific or -type anti-idiotypic MAbs. One of them, the polyre-
active B7 MAb, was obtained by immunizing BALB/c mice with
the anti-NeuAc-GM2 ganglioside E1 MAb (Macías et al., 1999). The
B7 MAb has shown an anti-tumor effect in a murine model of
melanoma. Furthermore, it also exhibits in vitro some properties
that resemble those of the intravenous immunoglobulin prepara-
tion (IVIg), such as the inhibition of the proliferation of human B
and T cell lines and of human normal lymphocytes activated with
different mitogens (Macías et al., 1999). These findings suggested
that B7 MAb might play a similar immunoregulatory role as pro-
posed for the IVIg pool, which has been successfully used in the
treatment of autoimmune and inflammatory diseases and lym-
phoproliferative disorders (Kazatchkine and Kaveri, 2001; Krause
and Shoenfeld, 2005). The mechanisms of action of this prepara-
tion rely on both the variable and constant regions of the IgG.
The Fv region is responsible for the recognition of soluble and
membrane-associated self-molecules, as well as idiotypes of sol-
uble immunoglobulins and B cell receptors (BCR). On the other
hand, the Fc portion contributes to IVIg effects through interac-
tions with Fc receptors (FcR), modulation of FcRIIb expression
and saturation of FcRn (Negi et al., 2007).
Though the reactivity of B7 MAb with antibodies and non-
immunoglobulin antigens has been well documented (Macías et al.,
1999), the nature of the interaction of this antibody with different
cells of the immune system has not been yet elucidated.
The present work demonstrates the existence of a high avidity
interaction of B7Y33, a mutated chimeric version of B7 MAb, with B
lineage cells. The interaction with the low affinity receptor FcRIIb
proved to be critical for the recognition of this cell type. Our results
point out a non-classical interaction of B7Y33 with B cells, which
involves both the variable and constant regions of the antibody. The
recognition of peripheral B lymphocytes from B cell chronic lym-
phocytic leukemia (B-CLL) patients by B7Y33 supports its potential
application for the treatment of B cell malignancies.
2. Materials and methods
2.1. Cells
K562 (human erythroleukemia), Raji (Burkitt’s lymphoma),
Daudi (Burkitt’s lymphoma), NS0 (murine myeloma), MB16F0
(non-metastatic C57Bl/6 murine melanoma) and F3II (murine
breast cancer) cell lines as well as chimeric antibodies-expressing
NS0 transfectomas, were cultured at 37
C, 5% CO
2
, in Dulbecco’s
modified Eagles medium (DMEM) supplemented with 10% heat
inactivated fetal calf serum (FCS), antibiotic mixtures of penicillin
(100 U/mL) and streptomycin (100 g/mL), and 2 mM l-glutamine.
For the selection of whole antibody-producing transfectomas,
DMEM-F12 containing 10% FCS and histidinol at 10 mM was used
as selective medium.
2.2. Monoclonal antibodies (MAbs)
The E1 (Alfonso et al., 1995), P3 (Vázquez et al., 1998), and F6
(Alfonso et al., 1995) IgM MAbs were purified from mouse ascitic
fluid by gel filtration chromatography using a Sephacryl S-300 high
resolution column (Pharmacia, Uppsala, Sweden) equilibrated with
phosphate-buffered saline (PBS) containing 0.5 M NaCl.
The following chimeric antibodies were used: P3 (López-
Requena et al., 2003), two of its mutants (with arginine-to-serine
replacements at HCDR1 Kabat position 31 and HCDR3 Kabat posi-
tions 98 and 100a, respectively) (López-Requena et al., 2007a), 1E10
(López-Requena et al., 2003), C5 (Roque-Navarro et al., 2003) and
14F7 (Roque-Navarro et al., 2008) (all human IgG1, ). The chimeric
antibodies, including those obtained during this work, were puri-
fied from transfectoma culture supernatants by Protein-A Affinity
Chromatography (Pharmacia, Uppsala, Sweden) and analyzed by
SDS-PAGE under reducing conditions.
The murine FcRII/III-specific 2.4G2 antibody (rat, IgG2a) and
the murine MHC-II-specific M5/114.15.2 antibody (rat, IgG2b) were
purified from 2.4G2 and M5/114.15.2 hybridoma supernatants,
respectively, by Protein-G Affinity Chromatography.
The specificity of the purified antibodies was confirmed by
enzyme-linked immunosorbent assay (ELISA).
2.3. Vectors
The pAH4604 and pAG4622 vectors, containing the human 1
and constant regions, respectively, have been described in detail
(Coloma et al., 1992) and were kindly provided by Dr. Sherrie L.
Morrison, Department of Microbiology and Molecular Genetics,
UCLA, USA.
The pAH4604 (Ala/Ala) vector is a mutated version of pAH4604,
where the leucine residues at positions 234 and 235 of the human
1 CH2 region were replaced by alanines (Hinojosa et al., 2010). It
was used for the expression of the B7Y33LALA mutant.
2.4. Variable region genes
The genes of the B7Y33 heavy chain variable region (VH) and its
mutants were chemically synthesized (Geneart GmbH, Regensburg,
Germany). The B7Y33 VH gene was designed from the published
B7 VH MAb coding sequence (Hernández et al., 2007), with the
substitution of the threonine residue at Kabat position 33 by a tyro-
sine. The changes contained in each mutant are detailed in Table 1.
The synthetic genes were digested HincII/NheI (NEB, New England
Biolabs, Ipswich, MA) from pGA4 Geneart vector and cloned into
pAH4604 or pAH4604 (Ala/Ala), previously digested EcoRV/NheI
(NEB).
The B7 MAb light chain variable region (V) gene (Hernández
et al., 2007) was digested EcoRV/SalI (NEB) and inserted into the
equally digested pAG4622.
2.5. Chimeric antibody expression
NS0 cells were transfected by electroporation with 10 g of both
pAG4622 containing chimeric B7 MAb light chain, and pAH4604 or
pAH4604 (Ala/Ala) bearing chimeric B7Y33 MAb heavy chain or its
mutants, all linearized through PvuI (NEB) digestion.
NS0 transfectoma cells expressing chimeric P3 light chain or
chimeric 1E10 light chain (López-Requena et al., 2003) were trans-
fected as above with pAH4604 bearing chimeric B7Y33 MAb heavy
chain.
The transfection method and the selection of transfectomas
secreting chimeric IgG have been previously described (López-
Requena et al., 2007a).
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100 T. Hernández et al. / Molecular Immunology 48 (2010) 98–108
Table 1
Constructed mutants of the B7Y33 VH. Numbering is according to Kabat et al. (1991).
Positions
Variants 28 31 53 54 57 73 75 98 99 100
B7Y33 Thr Glu Asn Asn Ala Lys Ser Glu Ala Thr
H1 Arg Arg
H2 Tyr Tyr Thr
H3 Lys Ser Gin
H4 Thr Gin
2.6. MAb-biotin conjugation
The antibodies (1 mg/mL) were extensively dialyzed with borate
buffer 0,2 M, pH 8,5 and then incubated with 100 mg/mL of biotin
N-hydroxysuccinimide ester (Sigma, Saint Louis, MO) for 4 h with
gentle agitation at room temperature. Finally, they were dialyzed
against PBS (Bayer and Wilchek, 1980).
2.7. Antibody binding assays
Binding of chimeric B7Y33, its mutants and hybrid molecules
(VHB7Y33/VE10 and VHB7Y33/VP3) to anti-ganglioside IgMs
(E1, P3 and F6 MAbs) was determined using an ELISA assay pre-
viously described (López-Requena et al., 2007a). Briefly, microtiter
plates (Microlon
®
600, high binding, Greiner Bio One, Germany)
were coated overnight at 4
C with 10 g/mL of the anti-ganglioside
antibodies and blocked for 1 h at 37
C with PBS-T-BSA (phosphate-
buffered saline containing 0.05% Tween 20, pH 7.5, and 1% bovine
serum albumin). After an incubation of 2 h at 37
C with the
chimeric antibodies, the plates were washed and an alkaline
phosphatase-conjugated goat anti-human IgG (-chain-specific)
antibody (Sigma) was added. After 1 h at 37
C the reaction was
developed with p-nitrophenylphosphate substrate solution and
absorbance monitored at 405 nm (Organon Teknika Inc., reader,
Salzburg, Austria). A similar experiment was used to test binding
of B7Y33 to chimeric P3 antibody and two of its mutants. The lat-
ter antibodies were used for coating the plates, B7Y33 was used
biotinylated and reactivity was detected using alkaline-conjugated
streptavidin (Jackson Immunoresearch, West Grove, PA).
Three samples of each experiment were tested and the coef-
ficient of variation was <10% for all assays. Background values of
absorbance were less than 0.1.
2.8. Flow cytometry analysis
2.8.1. Isolation of spleen cells
Spleen cells from naïve BALB/c mice were obtained by mechan-
ical dissociation, and erythrocytes were lysed by hypotonic shock
in ammonium chloride solution 0.15 M. The cells were washed in
cold PBS containing 2% BSA (PBS–BSA 2%).
2.8.2. Isolation of PBMC
Peripheral blood mononuclear cells (PBMC) from a healthy
human donor and B-CLL patients were isolated by density gradient
centrifugation using Ficoll-Paque
TM
PLUS (Amersham Pharmacia
Biotech AB, Uppsala, Sweden). PBMC were resuspended in PBS–BSA
2%.
2.8.3. Recognition of B lymphocytes and cell lines
10
6
BALB/c mouse spleen cells or NS0, Raji, Daudi, MB16F0 and
F3II cells were incubated with chimeric antibodies on ice for 30 min,
and washed with PBS–BSA 2%. The binding of chimeric antibodies
was detected by incubation with a FITC-conjugated rabbit anti-
human IgG F(ab
)
2
(Dako, Denmark) for 20 min on ice. In case of
double labeling, the samples were incubated with a PE-conjugated
anti-B220 antibody (BD Biosciences Pharmingen, NJ).
10
6
PBMC from a healthy human donor and B-CLL patients
and K562 cells were incubated with biotinylated chimeric anti-
bodies on ice for 30 min. After washing with PBS–BSA 2%, the
binding was detected with PE-conjugated streptavidin (BD Bio-
sciences Pharmingen). For the identification of the human B cell
population, the samples were labeled with a FITC-conjugated anti-
CD19 antibody (AbD Serotec, Germany). Chimeric C5, 1E10 and P3
antibodies were used as isotype-matched controls.
2.8.4. FcRII expression in murine cell lines
For the evaluation of theexpressionof FcRII in three murine cell
lines (NS0, MB16F0 and F3II), 10
6
cells were incubated with purified
2.4G2 MAb. For the detection of 2.4G2 binding, a biotinylated goat
anti-rat immunoglobulin antibody (BD Biosciences Pharmingen)
and FITC-conjugated streptavidin (BD Biosciences Pharmingen)
were used.
2.8.5. Competition assays
The competition experiments were performed by incubating
10
6
BALB/c mouse spleen cells with increasing concentrations of
a donkey anti-mouse IgM ( chain-specific) antibody (Jackson
Immunoresearch) for 30 min on ice. After washing with PBS–BSA
2%, the cells were incubated with 2.5 g/ml of biotinylated B7Y33
for 30 min at 4
C. Reactivity was measured using FITC-conjugated
streptavidin (BD Biosciences Pharmingen). A PE-conjugated anti-
B220 antibody (BD Biosciences Pharmingen) was used to identify
the B cell population.
The inhibition assay with an anti-mouse FcRII antibody was
done by incubating BALB/c mouse spleen cells or NS0 cells
with purified 2.4G2 antibody for 30 min on ice. Then, cells
were incubated with biotinylated B7Y33 at 2.5 g/ml. Reactivity
was measured using PE-conjugated streptavidin. The inhibi-
tion percentage was calculated as: % inhibition = [(% binding
without inhibitor % binding with inhibitor)/% binding without
inhibitor]*100. In the experiment with spleen cells, an anti-MHC-
II antibody was used as control inhibitor. For the NS0 cells, the
biotinylated chimeric anti-NeuGc-GM3 ganglioside 14F7 antibody
was used as negative control.
Human cells K562 and Raji were first incubated with B7Y33 at
15 g/mL (K562) or 5 g/mL (Raji), and then with PE-conjugated
anti-human FcRII AT10 antibody (AbD Serotec) for 30 min on
ice. Binding of AT10 antibody conjugate was also assessed in the
absence of B7Y33.
2.8.6. “Molecular complementarity” binding experiments
H3 or H4 mutants were mixed with B7Y33LALA mutant at differ-
ent ratios (1:1, 1:10 and 10:1; 1 stands for 2.5 g/ml and 10 stands
for 25 g/ml) and incubated at 37
C for 30 min. Mixes were added
to spleen cells from BALB/c mice. Reactivity was measured adding
a FITC-conjugated rabbit anti-human IgG F(ab
)
2
(Dako) for 20 min
on ice. The samples were incubated with a PE-conjugated anti-B220
antibody (BD Biosciences Pharmingen).
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T. Hernández et al. / Molecular Immunology 48 (2010) 98–108 101
In all cytometry assays the mean fluorescence intensity (MFI)
and percentage of stained cells were determined with a FACScan
instrument (Becton Dickinson, NJ). The WinMDI 2.8 program was
used to analyze a total of 10
4
cells acquired on every FACS experi-
ment. All the assays were performed at least twice.
3. Results
3.1. The B7Y33 mutant chimeric antibody
The B7 MAb is an -type anti-idiotypic antibody with multi-
specificity and self recognition properties (Macías et al., 1999).
The study of its biological properties required a constant source
of antibody that was unavailable due to the instability of the
secreting hybridoma (Macías A., personal communication). For
this reason, we decided to construct a chimeric version of the
molecule (human 1, constant regions), also considering the
envisaged therapeutic potential and the practical benefit of avoid-
ing the traces of host IgG contaminants coming from the murine
ascites. However, the attempts to obtain a transfectoma with
appropriate antibody productivity levels were ineffective, some-
how in agreement with the B7 hybridoma instability. Afterwards,
we obtained several variants of the recombinant antibody by
introducing single point mutations in the heavy chain variable
region (VH) (our unpublished data). One of these mutants, named
B7Y33, where the threonine residue at position 33 (Kabat num-
bering) of the VH was substituted by a tyrosine, was efficiently
expressed. Hence, we continued working with this mutant, tak-
ing into account that it kept the B7 MAb immunochemical
properties.
3.2. B7Y33 binds to unrelated immunoglobulins via a
non-classical binding site
B7Y33, as reported before for the B7 MAb (Macías et al.,
1999), is highly polyreactive, being able to interact with several
anti-ganglioside IgM antibodies as well as non-immunoglobulin
antigens (Supplementary Fig. 1). In order to study the molecular
features of its variable region that support this multispecificity, we
constructed chimeric hybrid molecules by combining the B7Y33
heavy chain with unrelated light chains. We selected the light
chains of an anti-NeuGc-ganglioside MAb, named P3 (Vázquez et
al., 1995) and its anti-idiotype, named 1E10 (Vázquez et al., 1998).
In both cases, the mutants exhibited higher reactivity, as compared
with B7Y33, with two of the IgM antibodies that were tested, while
showing almost identical reactivity towards the rest (Fig. 1A). This
result suggests that although the light chain variable region (V)
modulates the interaction of B7Y33 with these antibodies, the VH
domain is the main responsible for its binding properties.
Based upon the sequence reported for B7 VH (Hernández et al.,
2007) we constructed and expressed mutants with substitutions
in each of the three HCDRs, as well as in the so-called HCDR4,
located in the framework 3 (HFR3) (Franklin et al., 2004; Bond et
al., 2005). We called these mutants H1, H2, H3 and H4, respectively
(Table 1). As shown in Fig. 1B, and although with some differences
with respect to the wild antibody, none of the mutants lost the
capability of recognizing the murine IgMs. Indeed, both H1 and H2
mutations modified similarly the recognition of E1 and F6 antibod-
ies by B7Y33, while did not have impact on the reactivity against P3.
On the other hand, H3 mutations strongly influenced the reactivity
against P3, unlike the E1 and F6 IgMs. These results point out the
Fig. 1. Reactivity of B7Y33, its H1 (HCDR1), H2 (HCDR2), H3 (HCDR3) and H4 (HFR3) mutants and the hybrid VHB7Y33/VP3 and VHB7Y33/V1E10 antibodies, with anti-
ganglioside antibodies. (A and B) Microtiter plates were coated with 10 g/ml of murine IgM E1, P3 and F6 mAbs. Purified B7Y33, its mutants and the hybrid antibodies were
added unconjugated at 10 g/ml. Reactivity was measured using an alkaline phosphatase-conjugated anti-human IgG ( chain-specific) antibody. Light grey bars represent
the reactivity of B7Y33, black bars of its mutants or the hybrid antibodies, and dark grey bars of the isotype-matched chimeric C5 antibody. (C) Microtiter plates were coated
with 10 g/ml of chimeric P3 antibody (circles) and its mutants P3S31 (HCDR1, squares) and P3S98;100a (HCDR3, triangles). Biotinylated B7Y33 was added at different
concentrations and the reactivity was measured using alkaline phosphatase-conjugated streptavidin. Black curves represent the reactivity of biotinylated B7Y33 and grey
curves of isotype-matched biotinylated chimeric 14F7 antibody.
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102 T. Hernández et al. / Molecular Immunology 48 (2010) 98–108
Fig. 2. Self-binding assay of B7Y33 and its variable and constant region mutants. Microtiter plates were coated with 10 g/mL of B7Y33 (A), its variable region mutants H1
(HCDR1) (B), H2 (HCDR2) (C), H3 (HCDR3) (D) and H4 (HFR3) (E), and its CH2 mutant B7Y33LALA (F). Rituximab (Rx) was used as isotype-matched control (G). Different
concentrations of biotinylated antibodies were added to the plates coated with the same non-biotinylated molecules. Reactivity was measured using alkaline phosphatase-
conjugated streptavidin.
existence of at least one non-classical binding site other than the
conventional one on B7Y33 that explains its polyreactivity. Taking
into account that E1 and F6 MAbs belong to the same V
H
family, Q52
(López-Requena et al., 2007b), being almost identical (only one con-
servative change at framework 1), the reactivity profile of H1 and
H2 mutants might indicate the recognition of a region that does
not include the HCDR3 of these IgMs. In addition, we tested the
binding of B7Y33 to two previously described mutants of P3 MAb,
which lost the ganglioside binding capability. One of the mutants
(unable also to bind to P3-specific anti-idiotypes) has a substitu-
tion in HCDR1, while the other one has two substitutions in HCDR3
(López-Requena et al., 2007a). Again, the interaction of B7Y33 was
unaffected (Fig. 1C). It is thus interesting to notice that B7Y33 does
not interact with P3 throughthedemonstrated classical binding site
of this latter antibody. P3 MAb V
H
gene also belongs to the Q52 fam-
ily, but it displays several differences at HCDRs 1 and 2 with respect
to E1 and F6 MAbs (López-Requena et al., 2007b). Collectively, these
results demonstrate that B7Y33 is an anti-idiotypic antibody that
recognizes non-paratopic regions on different antibodies.
An unusual property of the B7 MAb is its ability for self-binding
(our unpublished data). As for other immunochemical properties of
B7 MAb, B7Y33 also displayed a homophilic recognition capability.
Interestingly, this property was not affected in any of the evalu-
ated mutants which were able of recognizing themselves (Fig. 2)
and the wild type antibody (data not shown). This last result is an
additional indication of immunoglobulin interactions not involving
the classical binding site.
3.3. FcRIIb is critical for the binding of B7Y33 to B cells
The previous observations prompted us to test whether B7Y33
recognizes immunoglobulins not only in soluble forms, but also
as surface BCRs. We first assessed the binding of B7Y33 to B lym-
phocytes of both murine and human origin. B7Y33 recognized a
large percentage of spleen B cells from naïve BALB/c mice (Fig. 3A
and C). In addition, it interacted extensively with peripheral B lym-
phocytes from a healthy human donor and B-CLL patients (Fig. 3B
and D). In all cases, binding was demonstrated at concentrations at
which the interaction of the isotype-matched control antibody was
negative.
We further tested whether B7Y33 interacts directly with the
BCR, using an inhibition assay. Preincubation of cells with increas-
ing concentrations of an anti-IgM antiserum did not affect the
binding of B7Y33 to murine B lymphocytes (Fig. 4A). This result
is not conclusive, considering the possibility of a non-complete
blocking by the antiserum of all the potential binding sites on
the BCR. For that reason, we evaluated the recognition of a non-
immunoglobulin-expressing murine myeloma (NS0) by B7Y33,
which resulted to be positive (Fig. 4B). Taken together, these results
indicate that B7Y33 recognizes an antigen other than the BCR on B
cells.
Then, we evaluated the possible interaction of B7Y33 with other
pan-B molecules. We first focused on the CD20 molecule, whose
murine and human versions share 73% similarity (Tedder et al.,
1988). For that purpose, we compared the capability of B7Y33 to
recognize human B cells with that of the commercial anti-CD20
antibody (Rituximab). We used PBMC from the same three B-CLL
patients, as in the previous experiments. Rituximab recognized
clearly only two out of the three tested samples, indicating a het-
erogeneity in the expression of CD20 by these malignant B cells
(Fig. 3D). In contrast, B7Y33 was able to interact also with the lym-
phocytes of the sample that was negative to Rituximab binding
(Fig. 3D). These results rule out the CD20 molecule as a target of
B7Y33 on human B lymphocytes.
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T. Hernández et al. / Molecular Immunology 48 (2010) 98–108 103
Fig. 3. Binding of B7Y33 to murine and human B lymphocytes. (A and C) Spleen cells from naïve BALB/c mice; (B) PBMC from a healthy human donor; (D) PBMC from B-CLL
patients. Cells were incubated with 5 g/mL of unconjugated (A) or biotinylated (B and D) B7Y33 and Rituximab, or with different concentrations of unconjugated B7Y33
(C). The binding was measured using a FITC-conjugated anti-human IgG antibody (A and C) or PE-conjugated streptavidin (B and D). Chimeric C5 antibody was used as
isotype-matched control. Results are representative of two performed assays.
The second pan-B molecule we chose was FcRIIb, considering it
is conserved in mice and humans (Brooks et al., 1989; Nimmerjahn
and Ravetch, 2008). This molecule belongs to a family of receptors
involved in the interaction of immunoglobulins with cells, and is
the only member of this family that is expressed on B lymphocytes
(Amigorena et al., 1989; Nimmerjahn and Ravetch, 2008). Preincu-
bation of cells with the murine FcRII/III-specific 2.4G2 antibody
led to a marked reduction of B7Y33 binding to murine spleen B
Fig. 4. B7Y33 recognizes an antigen other than the BCR on B cells. (A) Inhibition assay of B7Y33 binding to murine B cells using an anti-mouse IgM antiserum. Spleen cells from
BALB/c mice were first incubated with different concentrations of the inhibitor antiserum, and then with biotinylated B7Y33 at a non-saturating concentration (2.5 g/mL).
Reactivity was measured using FITC-conjugated streptavidin. A commercial PE-conjugated anti-B220 antibody was used to identify the B cell population. (B) Binding of B7Y33
to murine myeloma NS0 cell line. Cells were incubated with 10 g/mL of the antibody. Binding was detected with a FITC-conjugated anti-human IgG antibody. Light grey
curve represents the background fluorescence, black curve the binding of B7Y33 and dark grey curve the binding of the isotype-matched chimeric C5 antibody. Results are
representative of two performed assays.
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104 T. Hernández et al. / Molecular Immunology 48 (2010) 98–108
cells and to the NS0 cell line (FcRIIb-positive, supplementary Fig.
2; Gillet et al., 2007). This effect was not observed either with an
anti-MHC-class II antibody or with the anti-NeuGc-GM3 ganglio-
side 14F7 MAb (Carr et al., 2000), which binds to these murine cells
(Fig. 5).
Afterwards, we extended the study to other FcRII isoforms, by
evaluating the recognition of B7Y33 against a panel of murine and
human cell lines with different expression patterns of this receptor.
Consistently with the above finding, B7Y33 bound to the human
K562 (FcRIIa-positive; Sibéril et al., 2006), Raji (FcRIIb-positive;
Mackay et al., 2006) and Daudi (FcRIIb-positive; Mackay et al.,
2006) cell lines (Supplementary Fig. 3). Nointeraction was observed
(Supplementary Fig. 3) with the murine MB16F0 and F3II cell lines
(FcRII-negative, supplementary Fig. 2).
The inhibition by B7Y33 of the binding of a commercial
anti-human FcRII antibody to the K562 and Raji cells further cor-
roborated the interaction of our antibody with FcRII. The fact that
no binding was detected for the isotype-matched control antibody
(Fig. 5) indicates that binding of B7Y33 to these cells was not simply
a result of the interaction of its Fc region with the FcRII.
3.4. Involvement of B7Y33 constant and variable regions in its
interaction with B lymphocytes
In order to demonstrate the role of the B7Y33 Fc fragment
in the recognition of B cells, we constructed a heavy chain con-
stant region mutant with impaired FcR binding. We selected
the double leucine 234 and 235-to alanine mutation in the CH2
domain (Kabat numbering), which is well known to abolish the
IgG-FcRII interaction (Huizinga et al., 1989; Sarmay et al., 1992;
Xu et al., 2000; Hinojosa et al., 2010). The resulting antibody,
called B7Y33LALA, retained, as expected, the recognition of E1 MAb
(Supplementary Fig. 4) as well as the self-binding activity (Fig. 2F),
but showed no recognition of murine B lymphocytes (Fig. 6A) and
NS0 myeloma cells (Fig. 6B). This result confirms the contribu-
tion of the Fc–FcRII interaction in the binding of B7Y33 to these
cells.
We used two approaches to test the contribution of the B7Y33
variable region to the interaction with B lineage cells. First, we
tested the previously described hybrid antibodies. As shown in
Fig. 7, the VHB7Y33/VP3 antibody did not recognize the cells,
while the VHB7Y33/VE10 hybrid molecule did bind, although
weaker than the wild antibody. We then assessed whether the
H1–4 mutants were also able to bind to B and NS0 cells. The
H1 and H2 mutants behaved as the wild antibody (Fig. 6A). In
contrast, the interaction was affected for the H3 and H4 vari-
ants (Fig. 6A). Taken together, these results further support the
conclusion that the interaction of B7Y33 with B cells does not
depend on its capability of recognizing different immunoglobulins,
and that both the variable and constant regions participate in the
binding.
Finally, we tried to demonstrate a possible way of interaction
of B7Y33 with B cells based on both Fc and Fv direct contacts
with FcRII and the subsequent stabilization of these interactions
through the homophilic and heterophilic binding activity displayed
by this antibody and its H3 and H4 mutants. However, we did not
detect any B cell binding when we mixed the H3 or H4 mutants
with B7Y33LALA (data not shown) (see Fig. 8
I, II).
Fig. 5. B7Y33 binds to the FcRII. (A) Spleen cells from BALB/c mice were preincubated with different concentrations of the anti-murine FcRII/III 2.4G2 antibody (left panel)
or an anti-MHCII antibody (control inhibitor) (right panel); (B) murine myeloma NS0 cells were preincubated with the 2.4G2 antibody at 2 g/mL. Then, cells were incubated
with biotinylated B7Y33 at 2.5 g/ml. Reactivity was measured using PE-conjugated streptavidin. In (B), biotinylated chimeric anti-NeuGc-GM3 ganglioside 14F7 antibody
was used as negative control inhibitor. (C) Human K562 (left panel) and Raji (right panel) cells were preincubated with B7Y33 at 15 g/mL (K562) or 5 g/mL (Raji). Then, cells
were labeled with the PE-conjugated anti-human FcRII AT10 antibody at 2 g/mL. Chimeric 1E10 antibody was used as isotype-matched control. The mean fluorescence
intensity (MFI) and the percentage of labeled cells are indicated. Results are representative of two performed assays.
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T. Hernández et al. / Molecular Immunology 48 (2010) 98–108 105
Fig. 6. Binding of B7Y33 mutants to B lineage cells. Spleen cells from BALB/c mice (A) and NS0 cells (B) were incubated with 20 g/ml of B7Y33 and its mutants: H1 (HCDR1),
H2 (HCDR2), H3 (HCDR3), H4 (HFR3) and B7Y33LALA (CH2), followed by labeling with a FITC-conjugated anti-human IgG antibody. A commercial PE-conjugated anti-B220
antibody was used to identify the B cell population. Chimeric C5 antibody was used as isotype-matched control. Results are representative of two performed assays. Percentage
of labeled cells is indicated.
Fig. 7. Binding of VHB7Y33/unrelated V hybrid antibodies to B lineage cells. Spleen cells from BALB/c mice (A) and murine myeloma NS0 cells (B) were incubated with
20 g/mL of B7Y33, VHB7Y33/VP3, VHB7Y33/V1E10 and isotype-matched chimeric P3, 1E10 and C5 antibodies. Reactivity was measured using a FITC-conjugated anti-
human IgG antibody. Results are representative of two performed assays.
4. Discussion
The B7 MAb is an anti-idiotypic antibody (Macías et al., 1999)
generated by immunization of BALB/c mice with the NeuAc-GM2
ganglioside-specific E1 MAb (Alfonso et al., 1995). Following tra-
ditional criteria, B7 is an -type anti-idiotypic antibody, because it
does not inhibit the binding of E1 MAb to the ganglioside. Addition-
ally, it exhibits a wide-spectrum reactivity that includes antibodies
of different isotypes and specificities, and non-immunoglobulin
antigens (Macías et al., 1999). More importantly, it has also shown
anti-tumor properties (Macías et al., 1999). Therefore, it is impor-
tant to study the molecular basis of the immunochemical behavior
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106 T. Hernández et al. / Molecular Immunology 48 (2010) 98–108
Fig. 8. Schematic representation of the models of binding of B7Y33 to B lineage cells. (I) B7Y33 binds to FcRIIb through its Fc. B7Y33-FcRIIb interaction is stabilized by
inter-Fv homophilic binding. (II) Two molecules of B7Y33 bound to two molecules of FcRIIb, one through its Fc and the other through its variable region, interact by inter-Fv
homophilic binding. (III) B7Y33 binds to one molecule of FcRIIb through its Fc and to another molecule of FcRIIb through its variable region. (IV) B7Y33 binds to FcRIIb
through its Fc, and to another unknown surface antigen through its variable region.
of this antibody and identify its targets on immune system cells,
in order to understand the mechanisms underlying its potential
therapeutic effects.
E1 antibody, used to generate B7 MAb, is an IgM of germ line ori-
gin (López-Requena et al., 2007b), as is frequently found among the
anti-ganglioside antibodies of the natural repertoire (Vollmers and
Brandlein, 2007; López-Requena et al., 2007b). As demonstrated
in this work, B7 MAb interacts with E1 MAb and other unrelated
antigens through a non-conventional binding site. Furthermore, it
also binds to B cells in a non-classical way. It is likely that, when
using as antigen an antibody with the characteristics displayed by
the E1 MAb, the anti-idiotypic antibodies generated may include
specificities and other features of the natural repertoire, some of
them with immunoregulatory properties. In that sense, we are cur-
rently studying the possible in vivo effect of B7 MAb on the antibody
response against non-immunogenic self immunoglobulins, when
administered together to mice.
B7Y33 recognizes unrelated soluble immunoglobulins, but this
observation does not explain its binding to B cells. We demon-
strated that B7Y33 also binds to a myeloma cell devoid of surface
immunoglobulin, which, in spite of indicating the relevance of an
antigen other than BCR in the recognition, does not exclude a pos-
sible low affinity interaction with this receptor.
Our results indicate that FcRIIb, which is highly conserved in
mice and humans (Brooks et al., 1989), is the major target of B7Y33
on B lymphocytes from both species. Though this receptor belongs
to a family of molecules whose main function is associated to the
binding of antibodies through their Fc, the data presented here
suggest a particular type of interaction. The binding of chimeric
B7Y33 antibody to the murine FcRIIb, a low affinity receptor for
monomeric IgG and the only FcR expressed on B cells (Amigorena
et al., 1989; Nimmerjahn and Ravetch, 2008), seems to be unique, as
compared with other antibodies with the same human 1 isotype.
Moreover, the recognition of different human cell lines express-
ing both FcRIIa and FcRIIb is coherent with the high similarity
of the extracellular domains of these two isoforms (Brooks et al.,
1989).
The construction of VH and Fc mutants of B7Y33 allowed us to
elucidate the contribution of these two regions to the binding to
B cells. Our results evidenced the relevance of the idiotype, which
has no participation in conventional Fc-FcR interactions.
We proposed four possible models of interaction of B7Y33 with
B cells (Fig. 8). The four possibilities were designed and evaluated
using all the evidences gathered in this work, as well as the pre-
viously known polyreactive nature of B7 MAb (Macías et al., 1999;
Hernández et al., 2007).
Self-binding is one of the features that characterize some of the
so-called “superantibodies” (Kohler and Paul, 1998; Kohler, 2000).
This property consists of interactions between two identical anti-
body molecules through a region different from the conventional
binding site. It provides a mechanism for amplifying the binding
of an antibody to a cell surface antigen (Yan et al., 1996; Zhao et
al., 2002). Following this concept, we proposed a first model based
on the binding of B7Y33 to FcRIIb through its Fc and stabilization
of the complex by Fv homophilic interactions (Fig. 8-I). This model
can be rejected because of the intact self binding activity of non-B
cell binding VH mutants (H3 and H4).
The second model suggests that self-binding through the Fv
region stabilizes both the Fc- and Fv-mediated direct interaction
of B7Y33 with FcRIIb (Fig. 8-II). From such a model of interaction,
one could expect a “molecular complementarity” of the H3/H4 and
B7Y33LALA mutants for the binding to these cells: the lack of recog-
nition of H3/H4 through the idiotype, and the impaired interaction
of B7Y33LALA through the Fc, would be compensated by the inter-
action with FcRII of the B7Y33LALA idiotype and the H3/H4 Fc,
respectively. As mentioned above, we demonstrated that this type
of interaction does not occur.
Considering the polyspecific nature of B7 MAb (Macías et al.,
1999), one antibody molecule could in principle bind to two FcRIIb
molecules through two different sites: one on the Fv and the other
on the Fc (Fig. 8-III). Another very similar model would consist of
the binding of this antibody to a surface antigen other than FcRIIb,
while having its Fc engaged by this receptor. We think that this is
the most probable model (Fig. 8-IV), because it would be consis-
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T. Hernández et al. / Molecular Immunology 48 (2010) 98–108 107
tent also with at least one report in which an antibody required
stabilization by interaction with FcRIIb for optimal binding to its
antigen (CD22) on the surface of B cells (Walker and Smith, 2008).
Nevertheless, with our current data we are unable to rule out the
third possibility (Fig. 8-III). More experiments are needed to deter-
mine which model of interaction is the correct one.
FcRIIb plays an important role in B cell homeostasis and the
regulation of the immune response (Rahman et al., 2007; Xiang
et al., 2007; Smith and Clatworthy, 2010). It is overexpressed in
lymphomas of B cell origin such as follicular lymphoma (Callanan
et al., 2000). Other B cell malignancies like mantle cell, splenic
marginal zone and small lymphocytic lymphomas also express this
molecule (Camilleri-Broët et al., 2004; Rankin et al., 2006). These
findings make the FcRIIb an interesting target for treating those
kinds of B cell disorders. The recognition of B lymphocytes from
B-CLL patients by B7Y33 turns this antibody into a potential ther-
apeutic tool. The well known anti-CD20 Rituximab is currently a
powerful weapon against Non-Hodgkin lymphoma with outstand-
ing results in the clinics (Rothe et al., 2004). Nonetheless, its use is
limited in patients with low or no expression of the CD20 molecule
on their malignant B cells. Here, we demonstrated that such cells
are still recognized by B7Y33; therefore, it would be interesting to
investigate the effect of our antibody on them.
In summary, this work reports the high avidity binding of B7Y33,
a polyreactive -type anti-idiotypic antibody, to human and mouse
B cells. Our data indicate the participation of both the variable
and constant regions of B7Y33 in this binding, where the inter-
action with the FcRIIb molecule on the surface of B cells seems to
play a crucial role. The recognition of peripheral B lymphocytes
from B-CLL patients by B7Y33 suggests its potential application
in the treatment of B cell malignancies. The interaction of B7Y33
with FcRIIb merits further studies in order to evaluate its possi-
ble effects on the regulation of the immune system as well as the
therapeutic potential of this antibody.
Conflict of interest
The authors have no financial conflict of interest.
Acknowledgements
This work was supported by the Center of Molecular Immunol-
ogy. We thank Katya Sosa for her helpful technical assistance.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molimm.2010.09.006.
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    • "Mouse T lymphoma cell line CTLL-2 was used as mouse CD137-expressing cells (Fig. 7A), and human B lymphoma cell line Raji was used as human FcγRII-positive cells (Hernández et al., 2010). T-cell activating agonistic activity of anti-CD137 antibody was measured with the production of mouse IFN-γ production of CTLL-2 co-cultured with Raji cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIa(R131) allotype to a similar degree because FcγRIIb and FcγRIIa(R131) are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131). This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIa(R131) and FcγRIIa(H131) is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the CH2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.
    Full-text · Article · Jun 2013 · Protein Engineering Design and Selection
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    [Show abstract] [Hide abstract] ABSTRACT: Multispecificity is not a well-understood property of some antibodies. Different functions have been attributed to multispecific natural antibodies, commonly associated with the neutralization and clearance of antigens. Much less is known about the role of antibodies like these, based on their idiotypic connectivity. B7Y33 is a chimeric IgG1 version of a polyreactive α anti-idiotype antibody that is able to interact with different immunoglobulin and non-immunoglobulin antigens. Here we report the capacity of this antibody to enhance the immunogenicity of several autologous IgMs in adjuvant-free conditions. Our results suggest that the formation of immune complexes seems to be necessary, but not sufficient, to this activity. The potential involvement of the interaction of B7Y33 with the FcγRIIb is discussed.
    Full-text · Article · May 2012 · mAbs
  • [Show abstract] [Hide abstract] ABSTRACT: An antibody specific for the variable regions, or idiotype, of another antibody (Ab1) is termed anti-idiotypic antibody (Ab2). The Idiotypic Network is one of the most polemic theories in the field of Immunology. Nevertheless, naturally occurring anti-idiotypic antibodies have been associated with the progression or control of some pathological conditions. The most exploited property of anti-idiotypic antibodies is their ability to mimic antigens of diverse chemical nature. Thus, immunization with an Ab2 generated against an Ab1 specific for a given antigen may elicit an immune response specific for the antigen. Vaccine formulations based on anti-idiotypic antibodies have been assayed for the treatment of cancer and other diseases.
    No preview · Chapter · Aug 2014