A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity
Division of Oncology, Department of Medicine, Stanford University Medical Center, and Departments of Chemical Engineering and Bioengineering, Stanford University, Stanford, CA 94305.Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2012; 109(36):14526-31. DOI: 10.1073/pnas.1211018109
Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.
Get notified about updates to this publicationFollow publication
A vaccine directed to B cells and produced by
cell-free protein synthesis generates potent
Patrick P. Ng
, Ming Jia
, Kedar G. Patel
, Joshua D. Brody
, James R. Swartz
, Shoshana Levy
, and Ronald Levy
Division of Oncology, Department of Medicine, Stanford University Medical Center, and Departments of
Chemical Engineering and
Stanford University, Stanford, CA 94305
Contributed by Ronald Levy, July 10, 2012 (sent for review May 26, 2012)
Clinical studies of idiotype (Id) vaccination in patients with
lymphoma have established a correlation between the induced
anti-Id antibody responses and favorable clinical outcomes. To
streamline the production of an Id vaccine, we engineered a small
diabody (Db) molecule containing both a B-cell–targeting moiety
(anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was
designed to penetrate lymph nodes and bind to noncognate B cells
to form an antigen presentation array. Indeed, the αCD19-Id mol-
ecule accumulated on B cells in vivo after s.c. administration. These
noncognate B cells, decorated with the diabody, could then stim-
ulate the more rare Id-speciﬁc B cells. Peptide epitopes present in
the diabody linker augmented the response by activating CD4
helper T cells. Consequently, the αCD19-Id molecule induced a ro-
bust Id-speciﬁc antibody response and protected animals from tu-
mor challenge. Such diabodies are produced in a cell-free protein
expression system within hours of ampliﬁcation of the speciﬁcIg
genes from the B-cell tumor. This customized product can now be
available to vaccinate patients before they receive other, poten-
tially immunosuppressive, therapies.
bispeciﬁc antibody fragments
diotype (Id), the unique Ig molecule of each lymphoma tumor,
is a good target for the immune system. Passively administered
monoclonal antibodies (mAbs) against this target are effective in
therapy (1). Furthermore, studies of Id vaccination had sug-
gested a correlation between induced anti-Id antibody responses
and progression-free survival and overall survival of patients (2 –
4). Despite these encouraging results, phase III trials have not
established a clinical beneﬁt from Id vaccination, except for
a possible subset of patients who have prolonged remissions after
initial chemotherapy (5–7). One possible problem may have been
the chemical conjugation of Id to the carrier protein, keyhole
limpet hemocyanin (KLH). Antigenic determinants on the Id
could have been damaged in this process (8). Recombinant vac-
cines that do not require chemical conjugation may lead to im-
proved immunogenicity and clinical outcomes.
Recent studies on antigen (Ag) acquisition by B cells have
provided new insights for vaccine design. The majority of B cells
reside in follicles within secondary lymphoid organs. Foreign Ags
in the form of immune complexes are transported into lymph
node follicles by subcapsular sinus macrophages (9–11), and into
spleen follicles by marginal zone B cells (12). In the follicles,
nonspeciﬁc B cells retain immune complexes on their cell sur-
faces. Some complexes are transferred to follicular dendritic cells
(9–11), whereas others may directly cross-link the Ag-speciﬁc
receptors (BCRs) on cognate B cells (10, 11). These roles played
by noncognate B cells in the generation of speciﬁc antibody
responses were previously not appreciated. In addition to forming
immune complexes that facilitate entering the follicles and pre-
senting on the cell surface, foreign Ags may also be endocytosed,
processed, and presented as peptides that activate CD4
which in turn, provide costimulation to cognate B cells. These
attributes argue for the use of foreign carrier proteins such as
KLH to help stimulate antibody responses against self-Ags that
do not form immune complexes. However, chemical conjugation
has been shown to reduce vaccine potency (8). Recombinant Id
vaccines may offer distinct advantages because they can be pro-
duced with built-in carrier moieties.
It was recently discovered that small molecules (<70 kDa) can
enter follicles more efﬁciently through specialized conduits (13,
14). We therefore designed a recombinant vaccine below this
size limit. To provide cell surface anchorage for Ag retention and
presentation, we delivered the vaccine to noncognate B cells
within the follicle by targeting to CD19, a B-cell–speciﬁc mole-
cule (15). We created a bispeciﬁc diabody (Db), containing the
variable regions of a rat anti-mouse CD19 mAb and those of the
38C13 mouse B-cell lymphoma Id [αCD19-Id, molecular weight:
52 kDa]. We envisioned that the αCD19-Id would form a “lawn”
of Ags on the surface of follicular B cells, where they could cross-
link the BCR of the rare Ag-speciﬁc B cell among them. Fur-
thermore, coligation of the BCR with CD19 could result in
synergistic activation of the speciﬁc B cells (15). Nonsyngeneic
sequences, such as the rat variable regions in the Db, might help
by activating CD4
T cells (Fig. 1).
We used an in vitro (cell-free) protein synthesis (CFPS) system
for mammalian proteins that can assemble intrachain disulﬁde
bonds (16, 17). The reaction contains the DNA template for
each polypeptide chain, an energy source, substrates, and cellular
machinery from Escherichia coli that can carry out both tran-
scription and translation. A small reaction can produce protein
sufﬁcient for vaccination in a matter of hours, as opposed to the
usual methods of mammalian cell protein production that take
several weeks. We produced and screened several structural
variants of αCD19-Id. The most active form was then used for in
Diabody Design, Production, and Initial Characterizations. αCD19-Id
is a heterodimer of noncovalently associated polypeptides con-
taining the variable regions of 38C13 and anti-CD19, separated
Ser linkers (Fig. 2A). We produced four different αCD19-
Ids with the respective variable domains in different orientations
(Fig. 2A and Fig. S1). The only polypeptides that incorporate
a radiolabeled amino acid are those encoded by the supplied
templates. This labeling allows quantiﬁcation and SDS/PAGE
autoradiography without puriﬁcation, thus expediting screening
Author contributions: P.P.N., J.R.S., S.L., and R.L. designed research; P.P.N., M.J., K.G.P.,
and J.D.B. performed research; P.P.N., J.R.S., S.L., and R.L. analyzed data; and P.P.N., J.R.S.,
S.L., and R.L. wrote the paper.
The authors declare no conﬂict of interest.
Present address: Division of Hematology and Medical Oncology, Department of Medi-
cine, Mount Sinai School of Medicine, New York, NY 10029.
To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
September 4, 2012
no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1211018109
of various constructs. The “open” feature of CFPS also allowed
us to adjust the relative amounts of the two template plasmids to
ensure a 1:1 chain ratio in each Db heterodimer. The Db pro-
teins were screened by ﬂow cytometry for appropriate binding
activities (Fig. 2B). Bispeciﬁc binding was determined using
a target cell (A20) that expresses surface CD19 and a detector
consisting of an anti-38C13 Id mAb. As a negative control cell we
used a subclone of the A20 cell line that had lost cell surface
expression of CD19 (A20/CD19
) (18). Among these four
products, α CD19-Id-1 showed the best bispeciﬁc binding activity
(Fig. 2B). We made a negative control Db that had the variable
regions of a rat mAb of irrelevant speciﬁcity (RatFv-Id) (Fig.
S1). Both Dbs were conﬁrmed to be single species heterodimers
by SDS/PAGE and size-exclusion (SE)-HPLC (Fig. S2 A and B),
and were used for subsequent studies.
αCD19-Id Localized to B Cells in Vivo. We injected mice in-
tradermally (i.d.) with ﬂuorophore-labeled Dbs and analyzed
cells from the draining lymph nodes by ﬂow cytometry. αCD19-
Id, but not RatFv-Id, was retained speciﬁcally on B cells (B220
population) but not on T cells (CD3
population) (Fig. 3A), and
this occurred as early as 2.5 h after injection (Fig. S3 A). This
rapid accumulation is similar to a report of a small Ag (turkey
egg lysozyme, 14 kDa) that traveled through conduits into fol-
licles (14). The efﬁciency of B-cell targeting was even more ap-
parent when cells from the spleen and blood were analyzed 2 h
after i.v. injection of αCD19-Id. Again, we found binding of the
speciﬁc Db to B cells, but not to T cells (CD3
macrophages, or granulocytes (CD11b
) (Fig. 3B
and Fig. S3 B and C).
Id-Speciﬁc BCR Activation by αCD19-Id–Decorated B Cells. For this
test we constructed an Id-speciﬁc B cell (A20/α38BCR) by
transfecting the A20 cell line to express a membrane-anchored
form of the anti-Id antibody (Fig. S4). We demonstrated that
splenic B cells recovered from animals injected with αCD19-Id
(Fig. 4A), or A20 cells decorated with αCD19-Id in vitro (Fig.
S5A), could trigger the phosphorylation of intracellular BCR
pathway signaling molecules in Id-speciﬁc B cells. These signals
peaked at 20 min and declined gradually over 30–60 min after
the stimulator and responder cells came in contact (Fig. 4 A and
B). This stimulation did not occur in the negative control cell
line, native A20, that lacked the speciﬁc anti-Id BCR. We also
found that A20 cells decorated with αCD19-Id induced a stron-
ger activation signal than that induced by an equal amount of
free αCD19-Id, and reached a level to that induced by the
pentameric 38C13 IgM protein (Fig. S5B). Another way αCD19-
Id could stimulate an Id-speciﬁc B cell is by cross-linking its BCR
to its CD19 surface molecule. In fact, αCD19-Id induced phos-
phorylation of phosphatidylinositol 3-kinase (PI3K), a signaling
molecule directly downstream of CD19 (15), as well as the ex-
tracellular signal-regulated protein kinase (ERK) (Fig. 4C).
Neither the negative control RatFv-Id (Fig. 4C) nor an anti-
CD19 mAb induced such phosphorylation.
Id-Speciﬁc B Cells Captured αCD19-Id from Db-Decorated B Cells and
Internalized the Vaccine Molecule.
Ag-speciﬁc B cells need to in-
ternalize their cognate Ag for processing and presentation
to receive CD4
T-cell help. Splenic B cells from mice injected
with ﬂuorophore-labeled αCD19-Id (Fig. 5A), or A20 cells dec-
orated with ﬂuorophore-labeled αCD19-Id in vitro (Fig. S6),
could transfer the Db to A20/α38BCR cells, but not to A20
cells lacking the speciﬁc BCR. We also conﬁrmed that αCD19-Id
was internalized by A20/α38BCR cells using confocal micros-
copy (Fig. 5B).
αCD19-Id Induced Both an Id-Speciﬁc Antibody Response and a Db-
Speciﬁc T-Cell Response.
αCD19-Id induced a robust Id-speciﬁc
IgG response, comparable to that induced by 38C13-KLH. By
contrast, immunization with RatFv-Id, αCD19 + 38C13, or
αCD19-Av-38C13 failed to induce a signiﬁcant response (Fig.
6A). Whereas both groups of antibody responding mice made
predominantly anti-Id IgG1, αCD19-Id induced a slightly higher
percentage of anti-Id IgG2 than that induced by 38C13-KLH
(33.8 ± 6.2% vs. 22.4 ± 2.8%, as mean ± SEM).
The anti-Id antibody response induced by 38C13-KLH requires
T cells (8). That was also the case for αCD19-Id. Depletion
T cells from animals before vaccination with the Db
dramatically reduced the anti-Id IgG responses (from 77, 50, and
20 μg/mL to 6, 0, and 0 μg/mL serum). Lymphocytes from animals
B cell receptor
T cell receptor
Fig. 1. Proposed model: Id-speciﬁc B cells are stimulated by αCD19-Id tar-
geted to CD19 on B cells in lymphoid follicles.
Fig. 2. Design and characterization of αCD19-Id. (A) Design of expression
plasmids and schematic of one of four αCD19-Ids. Coexpression of both
plasmids in the same CFPS reaction produces two polypeptides that assemble
into a noncovalent heterodimeric Db. Locations of heavy chain variable
domains (α19 V
and 38 V
), and light chain variable domai ns (α19 V
) of anti-CD19 and 38C13, respectively, T7 promoters (T7), ribosomal
binding sites (rbs), (Gly)
Ser linkers (L), hexahistidine tag (H
), and stop
codons (stop) on the expression plasmids are indicated, as are the 38C13 Id
and the binding site for CD19 on the Db. (B) Flow cytometry analysis of
αCD19-Id bispeciﬁc binding. A20 cells were incubated with CFPS products
containing 5 μg of each αCD19-Id variant (—) or with mock CFPS product
cells incubated with the same CFPS product (---)
served as a negative target cell control. Cells were then washed and stained
with Alexa Fluor 488-conjugated anti-38C13 mAb.
Ng et al. PNAS
September 4, 2012
vaccinated with RatFv-Id and αCD19-Id proliferated (Fig. S7)and
secreted gamma IFN (IFN-γ)(Fig.6B)toboththespeciﬁcand
nonspeciﬁc Dbs. There was no response to anti-CD19 mAb or to
rat IgG (Fig. 6B). These results indicate that it was a component
other than the Ig variable regions (i.e., the linker) shared by both
Dbs, that provided T-cell responses and help to Id-speciﬁc B cells.
Indeed, peptides most likely to bind the major histocompatibility
complex II (MHCII) expressed by C3H/HeN mice (http://imed.
med.ucm.es/Tools/rankpep.html) fall within the V-domain linker
junction (FDYWGQGTTLTVSSGGGGSDIVMTQS) shared by
both Dbs. It is suprising that animals immunized with the vaccine
containing a complex with anti-CD19 and avidin did not have
activated T cells speciﬁc for these xenogeneic Ags. However, the
lack of such helper T cells may explain the poor anti-Id antibody
response induced by this complex.
Vaccination with αCD19-Id Protected Mice from a Systemic Tumor
Vaccinated mice were challenged with lethal doses
of the aggressive 38C13 lymphoma. Mice vaccinated with RatFv-
Id showed no protection compared with unvaccinated mice. In
contrast, mice that received αCD19-Id were protected to a simi-
lar degree as those vaccinated with 38C13-KLH (Fig. 7). Tumors
from animals vaccinated with αCD19-Id or 38C13-KLH still
bound to immune sera generated by these vaccines. Therefore,
the lack of protection for these animals could not be explained
by the expansion of tumor cells expressing Id variants.
Patients with follicular lymphoma can be induced to make anti-
Id antibodies against their tumors. Those who make such a re-
sponse have improved overall survival compared with those who
do not (2, 3). However, randomized controlled trials have failed
to prove a clinical beneﬁt from Id vaccination (5–7). An expla-
nation for this discrepancy may be that the ability to make anti-Id
antibody is simply an indicator for which the patient is destined
to survive longer. An alternative explanation is that anti-Id
antibodies are protective against tumor growth, but only if the
response is robust. In one phase III trial, all of the patients
produced antibodies against the KLH carrier protein, indicating
a certain level of general immune competence, but more than
half of them failed to generate anti-Id antibodies (5). One pos-
sible problem may have been the chemical conjugation to KLH,
a process that is difﬁcult to control, especially by the glutaral-
dehyde method that was used. It has been established that glu-
taraldehyde can damage antigenic determinants of an Id and
abrogate tumor protection in that animal model (8). For patients
Fig. 3. αCD19-Id targeted speciﬁcally to B cells in vivo. Mice were injected
with αCD19-Id, RatFv-Id, or 38C13 IgM, each conjugated to A lexa Fluor
488, or with buffer. (A) Draining lymph nodes (LN) were harvested 8 h after
i.d. injections. (B) Spleens (SP) and peripheral blood (PB) were harvested
2 h after i.v. injections. Leukocytes from these organs were stained with
ﬂuorophore-conjugated mAbs speciﬁc for B220, CD3, CD11b, and F4/80,
and analyzed by ﬂow cytometry. The percentages of gated total leuko-
cytes in the Upper Right quadrants are indicated. One of two experiments
Fig. 4. Id-speciﬁc BCR activation by αCD19-Id and αCD19-Id –decorated B
cells. (A) Splenic B cells recovered from mice 2 h after i.v. injection with
αCD19-Id (B-Db) or with PBS (B) were incubated for the indicated times with
responder cells, either A20 or A20/ α38BCR that were prelabeled with Cell-
Trace Violet dye. Cells were ﬁxed, permeabilized, stained with PE-conju-
gated antibodies speciﬁc for the phosphorylated forms of PLC-γ2 and Syk,
and analyzed by ﬂow cytometry. Responses of gated responder cells are
shown. (B) Kinetics of BCR signaling induced by αCD19-Id –decorated A20
cells (A20-Db). The percentages of A20/α38BCR responder (●) and A20
negative control responder (▲) cells are shown. Data are pooled from three
experiments (an example is shown in Fig. S5A). The percentage of BCR sig-
naling cells for each incubation time was calculated from the corresponding
histograms: [% PE
cells in response to A20-Db (red line)] − [% PE
in response to A20 (black line)]. (C) A20/α38BCR cells were stimulated for
10 min at 37 °C with Dbs, 38C13, or control IgM. Cell lysates were analyzed
by Western blotting using antibodies speciﬁc for the phosphorylated forms
of ERK and the p55 subunit of PI3K, and for total ERK and actin. Repre-
sentative results of three experiments are shown.
www.pnas.org/cgi/doi/10.1073/pnas.1211018109 Ng et al.
where each Id is unique, the conjugation chemistry may affect
each product to a different degree. Therefore, new vaccines that
do not require chemical conjugation may lead to improved im-
munogenicity and clinical outcomes. To achieve this goal, we
and others have tested various forms of recombinant Id vaccines
(17, 19–23). A common approach is to produce fusions of Id
sequences to targeting moieties that direct the construct to cy-
tokine receptors or to other activating receptors on dendritic
cells, macrophages, and other antigen-presenting cells (APCs)
(19, 22, 23). The peptides derived from Id proteins would then
be presented to T cells (24, 25).
Herein, we report an alternative strategy designed to activate
Id-speciﬁc B cells. This approach targets Id to the surface of non-
cognate B cells where they can be presented as intact molecules
to cognate B cells. Vaccines targeted to the complement receptor
2 (CD21) expressed on a variety of immune cells, including B cells,
have been constructed by several groups. Some showed enhance-
ment of Ag-speciﬁc immunity (26, 27), whereas others reported
unexpected suppression of antibody responses (28, 29). We chose
to target Id to the CD19 molecule expressed exclusively on B
cells. There is no competing ligand for CD19 as there is for CD21.
Importantly, it is known that the majority of CD19-antibody com-
plexes remain on or recycle to the surface of B cells even after
extended periods (30, 31). Furthermore, coligation of CD19 to the
BCR lowers the activation threshold of B cells (15).
Syngeneic Ig are poor immunogens. However, Id mixed with
complete Freud’s adjuvant can generate anti-Id antibody to
protective levels in several tumor models (32, 33). Interestingly,
no anti-Id antibody can be induced this way in the 38C13 model
(34). The unusually poor immunogenicity of this Id may be due
to the lack of somatic mutation in its V
resulting in a paucity of CD4
T-cell epitopes. The 38C13 Id can
be made immunogenic by coupling it to KLH. KLH binds to
natural antibodies and complement (36), and has been shown to
be transported into the follicles of lymph nodes (14). KLH also
contains peptide epitopes that activate CD4
T cells. These
properties make KLH an effective carrier.
We show that a robust anti-Id antibody response can also be
induced by fusing 38C13 Id to anti-CD19. Although different
from Id-KLH, αCD19-Id may achieve similar immune-stimula-
tory functions by alternative strategies. Being small, Db can enter
follicles through conduits, as inferred from the speed that
αCD19-Id reached B cells in the lymph node (Fig. S3A). Similar
conduit systems have been found to channel small molecules into
the T-cell areas of a lymph node, and into the white pulp of the
spleen (37). αCD19-Id may also be actively transported into
spleen follicles by marginal zone B cells expressing CD19.
αCD19-Id anchored to CD19 on abundant noncognate B cells
provided cross-linking of BCRs on the cognate B cell. Indeed,
αCD19-Id bound to B cells induced a stronger BCR signal than
free αCD19-Id, and reached the level induced by the pentameric
38C13 IgM (Fig. 4 A and B and Fig. S5).
T cells were required for the anti-Id response gener-
ated by αCD19-Id. The rat variable regions of anti-CD19 might
have been expected to be the source of CD4
However, instead, our data indicate that the nonnatural
Ser linker p rovid ed such epito pes (Fig. 6B and Fig. S7).
The potential to generate immune-stimulatory epitopes is an-
other advantage of recombinant Id vaccines over native Ig Id
vaccines, in addition to avoiding the regulatory T-cell epitopes
found on Ig constant regions (38). Ding et al. repo rted that B
cellstargetedbyananti–CD19-Ag conjugate could prime
T cells (39). We have no evidence for th is because the
nontargeting RatFv-Id was as effective as αCD19-Idinacti-
vating T cells. It is l ikel y t hat some molecules of both Dbs were
internalized and presented to T cells by macrophages or den-
dritic cells. However, in addition, some αCD19-Id targeted to
noncognate B cells where they formed an array to present the Id
to cognate B cells. By contrast, the nontargeting RatFv-Id in-
duced no anti-Id antibody response, nor did the 38C13 IgM,
a good cross-linker of Id-speciﬁc BCR but lacking T-cell epitopes.
Together, these results underscore the importance of vaccines such
as αCD19-Id that are designed to activate both cognate B cells and
Rituximab is now a part of the standard therapy for follicular
lymphoma, therefore, therapeutic vaccine strategies for lym-
phoma will need to be used in conjunction with this mAb that
depletes normal B cells. Rituximab can blunt antibody responses
to new Ags but it does not ablate an existing response once it is
established by prior vaccination (40, 41). Id vaccines produced
rapidly by cell-free protein synthesis, as tested here, can be
available before rituximab is used. This strategy may have the
additional beneﬁt of delaying the use of rituximab, and there-
fore, the development of rituximab resistance.
Materials and Methods
Plasmids. To construct expression plasmids for Dbs, RNAs were extracted from
hybridomas producing the anti-CD19 rat IgG2a/κ (1D3) (18) and a rat IgG2a/κ
of irrelevant speciﬁcity (H22-15-5) (RNeasy; Qiagen). The V
sequences were isolated using the SMART RACE kit (Clontech) and primers
speciﬁc to rat IgG2a constant region 1 (5′-ggaaatagcccttgaccaggcatcc-3′)and
Fig. 5. Id-spe ciﬁ c B cells captured and internalized αCD19-Id. (A) Mice were
injected i.v. with PBS (B) or with αCD19-Id conjugated to Alexa Fluor 488
(B-Db). Splenic B cells recovered after 2 h were incubated for 1 h at 37 °C
with A20 or A20/α38BCR cells prelabeled with Violet dye, then ﬁxed and
analyzed by ﬂow cytometry. One of two experiments is presented. (B)
A20/α38BCR cells were incubated at 0 °C or 37 °C for 30 min with Alexa Fluor
488-conjugated αCD19-Id. Cells were washed, ﬁxed, and analyzed by con-
focal microscopy. Representative images of cells are shown with a z-section
thickness of 2.4 μm. (Scale bar, 10 μm.)
Ng et al. PNAS
September 4, 2012
κ constant region (5′-gactgaggcacctccagttgctaactg-3′). These sequences and
those of the 38C13 cells (35) were codon optimized for expression in E. coli
with the online resource, DNAworks. The pY71 expression vector (42) con-
tains T7 promoter and termination sequences. The coding region, ﬂanked by
the 5′ NdeI and 3′ SalI sites, contains two V sequences separated by a linker.
An analysis of potential secondary structures in the upstream 58 nucleotides
and the codons of the ﬁrst nine amino acids was performed using the online
resource, Mfold. Silent codon changes were made to eliminate G:C pairings
that stabilize secondary structures, which may impede translation. Over-
lapping oligonucleotides of the coding regions were designed (DNAworks),
purchased (IDT), assembled by PCR, and cloned into pY71. The plasmid
expressing a membrane-bound anti-38C13 IgM, created for the present
work, has been described (42).
Flow Cytometry. Db in vitro binding assay. Cells (10
) were incubated with CFPS
products for 1 h on ice, then with 1 μg Alexa Fluor 488-conjugated S1C5 for
30 min. Cells were washed after each incubation, ﬁxed with 2% (wt/vol)
paraformaldehyde, and analyzed on a FACScalibur (Becton Dickinson).
Db in vivo trafﬁcking studies. Mice were injected i.d. on the abdomen or i.v.
in the tail with 10 μg AlexaFluor 488 conjugates. Cells isolated from
the indicated body compartments were incubated with Fc blocker, stained
with ﬂuorophore-conjugated mAbs, washed, ﬁxed, and analyzed as
described above. The animal study protocol was approved by the Stanford
University Institutional Animal Care and Use Committee.
Db transfer studies. A20 or A20/ α38BCRcellsat10
cells/mL in PBS were in-
cubatedwith5μM CellTrace Violet dye (Invitrogen) for 20 min at 37 °C, washed,
and cultured overnight in full media before use. Splenocytes were harvested
from mice injected i.v. with buffer or 20 μg Alexa Fluor-488 conjugated αCD19-
Id. A total of 3 × 10
splenic B cells were mixed with 3 × 10
cells, centrifuged for 30 s, and incubated for 1 h at 37 °C. Cells were washed,
ﬁxed, and analyzed on an LSR II cytometer (Becton Dickinson).
Signal transduction assays. Splenic B cells and Violet dye-labeled cells were
mixed, incubated at 37 °C for 15 s before adding 3.3 mM hydrogen peroxide.
The cells were immediately vortexed, centrifuged for 30 s, and incubated
for the indicated time. Cells were washed with cold PBS, ﬁxed for 30 min
in BD CytoFix/CytoPerm solution, washed with BD Perm/Wash buffer, and
incubated for 30 min with PE-conjugated mAbs. After washes, cells were
ﬁxed and analyzed.
ELISAs. Se rum anti-Id IgGs were quantiﬁed as described previously (17).
To quantify IFN-γ, splenocytes were seeded (5 × 10
cells per well) in
96-well U-bottom plates in 100 μL media (5% FBS, 100 μg/mL gentamy-
cin). A ﬁnal concentration of 50 μg/mL of 38C13 IgM, anti-CD19, re-
spective isotype control antibodies, 10 μg/mL of KLH, avidin, or 2 μg/mL
of Dbs was added. Culture supernatants were tested with an IFN-γ ELISA
kit (Thermo Scientiﬁc).
ACKNOWLEDGMENTS. We thank D. Czerwinski and R. Rajapaksa for
technical expertise in ﬂow cytometry; A. Virrueta for technical assistance;
C.-C. Kuo for advice on molecular biology; and R. Houot and H. Kohrt for
producing the anti-CD4 mAb. This work was supported by a Leukemia and
αCD19 + 38C13
αCD19 + 38C13
Fig. 6. Immune responses to αCD19-Id. (A) αCD19-Id induced a robust Id-speciﬁc antibody response. Mice received four biweekly i.d. vaccinations given twice
on consecutive days. Vaccines consisted either of 6 μg Dbs or an Id molar equivalent of 38C13 IgM, either chemically conjugated to KLH (38C13-KLH), mixed
with anti -CD19 mAb (αCD19 + 38C13), or conjugated to anti-CD19 mAb by avidin ( αCD19-Av-38C13). Sera collected a week after the last immunization were
tested by ELISA for antibodies against the 38C13 Id. Data were combined from four studies. The number of animals in each group is indicated. Each bar on the
graph represents the mean serum anti-Id IgG concentration ± SEM of each group. None of the sera reacted with a mouse IgM/κ isotype control. (B) IFN-γ
production by splenocytes from immunized mice. Mice (two to three per group) were vaccinated as in A. Spleens from each group were harvested and pooled
a week later. Splenocytes were cultured for 4 d with Ags listed in the legend in hexaplicate wells each. IFN-γ in culture supernata nts was measured by ELISA.
n. s. (P = 0.70)
0 20 40 60 80 100
s since tumor challen
n. s. (P = 0.27)
P = 0.015
n. s. (P = 0.91)
0 20 40 60 80 100
Days since tumor challenge
Fig. 7. Vaccination with αCD19-Id protected mice from tumor. (A) Mice (10
per group) received αCD19-Id, RatFv-Id, 38C13-KLH, or buffer as described in
Fig. 6A. Ten days later, mice were challenged with 100 38C13 cells by i.v.
injection. (B) Mice (10 per group) were vaccinated with αCD19-Id, 38C13-
KLH, or buffer and challenged with 400 cells. Survival was analyzed by the
Kaplan–Meier method and the log-rank statistical test.
www.pnas.org/cgi/doi/10.1073/pnas.1211018109 Ng et al.
Lymphoma Society Specialized Center of Research (SCOR) program grant,
Ruth L. Kirschstein Grant 5 T32 AI07290 (to P.P.N.), and a Lymphoma
Research Foundation fellowship (to P.P.N.). R.L. is an American Cancer Soci-
ety clinical research professor.
1. Davis TA, Maloney DG, Czerwinski DK, Liles TM, Levy R (1998) Anti-idiotype antibodies
can induce long-term complete remissions in non-Hodgkin’s lymphoma without
eradicating the malignant clone. Blood 92:1184–1190.
2. Weng WK, Czerwinski D, Timmerman J, Hsu FJ, Levy R (2004) Clinical outcome of
lymphoma patients after idiotype vaccination is correlated with humoral immune
response and immunoglobulin G Fc receptor genotype. J Clin Oncol 22:4717–4724.
3. Ai WZ, Tibshirani R, Taidi B, Czerwinski D, Levy R (2009) Anti-idiotype antibody re-
sponse after vaccination correlates with better overall survival in follicular lymphoma.
4. Navarrete MA, et al. (2011) Upfront immunization with autologous recombinant
idiotype Fab fragment without prior cytoreduction in indolent B-cell lymphoma.
5. Levy R, Robertson M, Leonard J, Vose J, Danney D (2008) Results of a phase 3 trial
evaluating safety and efﬁcacy of speciﬁc immunotherapy, recombinant idiotype (Id)
conjugated to KLH (Id-KLH) with GM-CSF, compared to non-speciﬁc immunotherapy,
KLH with GM-CSF, in patients with follicular non Hodgkin’s lymphoma (FNHL). Ann
Oncol 19(suppl 4):101–102.
6. Freedman A, et al. (2009) Placebo-controlled phase III trial of patient-speciﬁc immu-
notherapy with mitumprotimut-T and granulocyte-macrophage colony-stimulating
factor after rituximab in patients with follicular lymphoma. J Clin Oncol 27:
7. Schuster SJ, et al. (2011) Vaccination with patient-speciﬁc tumor-derived antigen in
ﬁrst remission improves disease-free survival in follicular lymphoma. J Clin Oncol 29:
8. Betting DJ, Kaﬁ K, Abdollahi-Fard A, Hurvitz SA, Timmerman JM (2008) Sulfhydryl-
based tumor antigen-carrier protein conjugates stimulate superior antitumor immu-
nity against B cell lymphomas. J Immunol 181:4131–4140.
9. Carrasco YR, Batista FD (2007) B cells acquire particulate antigen in a macrophage-rich
area at the boundary between the follicle and the subcapsular sinus of the lymph
node. Immunity 27:160–171.
10. Phan TG, Grigorova I, Okada T, Cyster JG (2007) Subcapsular encounter and com-
plement-dependent transport of immune complexes by lymph node B cells. Nat Im-
11. Phan TG, Green JA, Gray EE, Xu Y, Cyster JG (2009) Immune complex relay by sub-
capsular sinus macrophages and noncognate B cells drives antibody afﬁnity matu-
ration. Nat Immunol 10:786–793.
12. Ferguson AR, Youd ME, Corley RB (2004) Marginal zone B cells transport and deposit
IgM-containing immune complexes onto follicular dendritic cells. Int Immunol 16:
13. Gretz JE, Norbury CC, Anderson AO, Proudfoot AE, Shaw S (2000) Lymph-borne
chemokines and other low molecular weight molecules reach high endothelial ven-
ules via specialized conduits while a functional barrier limits access to the lymphocyte
microenvironments in lymph node cortex. J Exp Med 192:1425–1440.
14. Roozendaal R, et al. (2009) Conduits mediate transport of low-molecular-weight
antigen to lymph node follicles. Immunity 30:264–276.
15. Fearon DT, Carroll MC (2000) Regulation of B lymphocyte responses to foreign and
self-antigens by the CD19/CD21 complex. Annu Rev Immunol 18:393–422.
16. Goerke AR, Swartz JR (2008) Development of cell-free protein synthesis platforms for
disulﬁde bonded proteins. Biotechnol Bioeng 99:351–367.
17. Kanter G, et al. (2007) Cell-free production of scFv fusion proteins: an efﬁcient ap-
proach for personalized lymphoma vaccines. Blood 109:3393–3399.
18. Shoham T, et al. (2003) The tetraspanin CD81 regulates the expression of CD19 during
B cell development in a postendoplasmic reticulum compartment. J Immunol 171:
19. Tao MH, Levy R (1993) Idiotype/granulocyte-macrophage colony-stimulating factor
fusion protein as a vaccine for B-cell lymphoma. Nature 362:755–758.
20. Syrengelas AD, Chen TT, Levy R (1996) DNA immunization induces protective im-
munity against B-cell lymphoma. Nat Med 2:1038–1041.
21. King CA, et al. (1998) DNA vaccines with single-chain Fv fused to fragment C of tet-
anus toxin induce protective immunity against lymphoma and myeloma. Nat Med 4:
22. Biragyn A, Tani K, Grimm MC, Weeks S, Kwak LW (1999) Genetic fusion of chemokines
to a self tumor antigen induces protective, T-cell dependent antitumor immunity. Nat
23. Fredriksen AB, Bogen B (2007) Chemokine-idiotype fusion DNA vaccines are poten-
tiated by bivalency and xenogeneic sequences. Blood 110:1797–1805.
24. Biragyn A, et al. (2004) Chemokine receptor-mediated delivery directs self-tumor
antigen efﬁciently into the class II processing pathway in vitro and induces protective
immunity in vivo. Blood 104:1961–1969.
25. Schiavo R, et al. (2006) Chemokine receptor targeting efﬁciently directs antigens to
MHC class I pathways and elicits antigen-speciﬁc CD8+ T-cell responses. Blood 107:
26. Dempsey PW, Allison ME, Akkaraju S, Goodnow CC, Fearon DT (1996) C3d of com-
plement as a molecular adjuvant: Bridging innate and acquired immunity. Science
27. Lou D, Kohler H (1998) Enhanced molecular mimicry of CEA using photoaf
crosslinked C3d peptide. Nat Biotechnol 16:458–462.
28. Suradhat S, et al. (2001) Fusion of C3d molecule with bovine rotavirus VP7 or bovine
herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immu-
nization. Vet Immunol Immunopathol 83:79–92.
29. Gor DO, Ding X, Li Q, Greenspan NS (2006) Genetic fusion of three tandem copies of
murine C3d sequences to diphtheria toxin fragment B elicits a decreased fragment B-
speciﬁc antibody response. Immunol Lett 102:38–49.
30. Pulczynski S, Boesen AM, Jensen OM (1993) Antibody-induced modulation and in-
tracellular transport of CD10 and CD19 antigens in human B-cell lines: An immuno-
ﬂuorescence and immunoelectron microscopy study. Blood 81:1549–1557.
31. Du X, Beers R, Fitzgerald DJ, Pastan I (2008) Differential cellular internalization of
anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity. Cancer Res
32. Lynch RG, Graff RJ, Sirisinha S, Simms ES, Eisen HN (1972) Myeloma proteins as tumor-
speciﬁc transplantation antigens. Proc Natl Acad Sci USA 69:1540–1544.
33. George AJ, Tutt AL, Stevenson FK (1987) Anti-idiotypic mechanisms involved in sup-
pression of a mouse B cell lymphoma, BCL1. J Immunol 138:628–634.
34. Kaminski MS, Kitamura K, Maloney DG, Levy R (1987) Idiotype vaccination against
murine B cell lymphoma. Inhibition of tumor immunity by free idiotype protein.
J Immunol 138:1289–1296.
35. Carroll WL, Starnes CO, Levy R, Levy S (1988) Alternative V kappa gene rearrange-
ments in a murine B cell lymphoma. An explantation for idiotypic heterogeneity.
J Exp Med 168:1607–1620.
36. Thornton BP, Vĕtvicka V, Ross GD (1994) Natural antibody and complement-mediated
antigen processing and presentation by B lymphocytes. J Immunol 152:1727–1737.
37. Sixt M, et al. (2005) The conduit system transports soluble antigens from the afferent
lymph to resident dendritic cells in the T cell area of the lymph node. Immunity 22:19–29.
38. De Groot AS, et al. (2008) Activation of natural regulatory T cells by IgG Fc-derived
peptide “Tregitopes”. Blood 112:3303–3311.
39. Ding C, Wang L, Marroquin J, Yan J (2008) Targeting of antigens to B cells augments
antigen-speciﬁc T-cell responses and breaks immune tolerance to tumor-associated
antigen MUC1. Blood 112:2817–2825.
40. DiLillo DJ, et al. (2008) Maintenance of long-lived plasma cells and serological
memory despite mature and memory B cell depletion during CD20 immunotherapy in
mice. J Immunol 180:361–
41. Takata T, et al. (2009) Attenuated antibody reaction for the primary antigen but not
for the recall antigen of inﬂuenza vaccination in patients with non-Hodgkin B-cell
lymphoma after the administration of rituximab-CHOP. J Clin Exp Hematop 49:9–13.
42. Patel KG, et al. (2009) Cell-free production of Gaussia princeps luciferase—antibody
fragment bioconjugates for ex vivo detection of tumor cells. Biochem Biophys
Res Commun 390:971–976.
Ng et al. PNAS
September 4, 2012
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.
[Show abstract] [Hide abstract] ABSTRACT: Tumor immunotherapy holds great promise in controlling multiple myeloma (MM) and may provide an alternative treatment modality to conventional chemotherapy for MM patients. For this reason, a major area of investigation is the development of cancer vaccines to generate myeloma-specific immunity. Several antigens that are able to induce specific T-cell responses are involved in different critical mechanisms for cell differentiation, inhibition of apoptosis, demethylation and proliferation. Strategies under development include infusion of vaccine-primed and ex vivo expanded/costimulated autologous T cells after high-dose melphalan, genetic engineering of autologous T cells with receptors for myeloma-specific epitopes, administration of dendritic cell/plasma cell fusions and administration expanded marrow-infiltrating lymphocytes. In addition, novel immunomodulatory drugs may synergize with immunotherapies. The task ahead is to evaluate these approaches in appropriate clinical settings, and to couple them with strategies to overcome mechanisms of immunoparesis as a means to induce more robust clinically significant immune responses. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
- "In an APC-targeting approach, but using protein rather than DNA, Id + scFv was fused with scFv specific for CD19 in a diabody format. Targeting of CD19 on B cells increased Id-specific responses . ONCH-1998; No. of Pages 16 "
[Show abstract] [Hide abstract] ABSTRACT: To circumvent pathology caused by infectious microbes and tumor growth, the host immune system must constantly clear harmful microorganisms and potentially malignant transformed cells. This task is accomplished in part by T-cells, which can directly kill infected or tumorigenic cells. A crucial event determining the recognition and elimination of detrimental cells is antigen recognition by the T cell receptor (TCR) expressed on the surface of T cells. Upon binding of the TCR to cognate peptide-MHC complexes presented on the surface of antigen presenting cells (APCs), a specialized supramolecular structure known as the immunological synapse (IS) assembles at the T cell-APC interface. Such a structure involves massive redistribution of membrane proteins, including TCR/pMHC complexes, modulatory receptor pairs, and adhesion molecules. Furthermore, assembly of the immunological synapse leads to intracellular events that modulate and define the magnitude and characteristics of the T cell response. Here, we discuss recent literature on the regulation and assembly of IS and the mechanisms evolved by tumors to modulate its function to escape T cell cytotoxicity, as well as novel strategies targeting the IS for therapy.
- "T and B cells from adaptive immunity have been shown to play key roles in tumor immunity; these cells can be engaged to prevent and control tumorogenesis     . Although, antibodies against tumor antigens and immune-modulatory molecules have been shown to be helpful in tumor treatment   , T cells are often involved in this process and have been shown to play significant roles in the control of proliferative malignant cells    . T cells recognize cognate antigens as small peptides bound to self-MHC molecules (pMHC complexes, Figure 1) [13, 15–19, 52, 53]. "
[Show abstract] [Hide abstract] ABSTRACT: A basic tenet of antibody-based immunity is their specificity to antigenic determinates from foreign pathogen products to abnormal cellular components such as in cancer. However, an antibody has the potential to bind to more than one determinate, be it an antigen or another antibody. These observations led to the idiotype network theory (INT) to explain immune regulation, which has wax and waned in enthusiasm over the years. A truer measure of the impact of the INT is in terms of the ideas that now form the mainstay of immunological research and whose roots are spawned from the promise of the anti-idiotype concept. Among the applications of the INT is understanding the structural implications of the antibody-mediated network that has the potential for innovation in terms of rational design of reagents with biological, chemical, and pharmaceutical applications that underlies concepts of reverse immunology which is highlighted herein.
- "Despite that pathogen-associated animal models were often used to validate vaccination with anti-Ids, anti-Id vaccination has made it to the clinic for cancer. A number of monoclonal antibodies that mimic distinct human tumor-associated antigens as well as Id vaccines have demonstrated encouraging results in clinical studies for solid tumors (Bhattachary-Chatterjee et al., 2000; Bhattacharya-Chatterjee et al., 2001; Maruyama et al., 2000; Ruffini et al., 2005; Lee et al., 2007; Neninger et al., 2007; Fernandez et al., 2010; Hernandez et al., 2011; Ng et al., 2012). While the theoretical hypothesis is sound, trials have been limited and have not been tested prospectively. "