Article

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
Source: PubMed

ABSTRACT

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.

Full-text

Available from: Shoshana Levy
A vaccine directed to B cells and produced by
cell-free protein synthesis generates potent
antilymphoma immunity
Patrick P. Ng
a
, Ming Jia
a
, Kedar G. Patel
b
, Joshua D. Brody
a,1
, James R. Swartz
b,c
, Shoshana Levy
a
, and Ronald Levy
a,2
a
Division of Oncology, Department of Medicine, Stanford University Medical Center, and Departments of
b
Chemical Engineering and
c
Bioengineering,
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-celltargeting 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-specic 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-specic antibody response and protected animals from tu-
mor challenge. Such diabodies are produced in a cell-free protein
expression system within hours of amplication of the specicIg
genes from the B-cell tumor. This customized product can now be
available to vaccinate patients before they receive other, poten-
tially immunosuppressive, therapies.
immunotherapy
|
tumor-specic antigen
|
bispecic antibody fragments
I
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 benet from Id vaccination, except for
a possible subset of patients who have prolonged remissions after
initial chemotherapy (57). 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 (911), and into
spleen follicles by marginal zone B cells (12). In the follicles,
nonspecic B cells retain immune complexes on their cell sur-
faces. Some complexes are transferred to follicular dendritic cells
(911), whereas others may directly cross-link the Ag-specic
receptors (BCRs) on cognate B cells (10, 11). These roles played
by noncognate B cells in the generation of specic 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
+
T cells,
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 efciently 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-cellspecic mole-
cule (15). We created a bispecic 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-specic B cell among them. Fur-
thermore, coligation of the BCR with CD19 could result in
synergistic activation of the specic 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 disulde
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
sufcient 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
vivo studies.
Results
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
by Gly
4
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 quantication and SDS/PAGE
autoradiography without purication, 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 conict of interest.
1
Present address: Division of Hematology and Medical Oncology, Department of Medi-
cine, Mount Sinai School of Medicine, New York, NY 10029.
2
To whom correspondence should be addressed. E-mail: levy@stanford.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1211018109/-/DCSupplemental.
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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 ow cytometry for appropriate binding
activities (Fig. 2B). Bispecic 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
NEG
) (18). Among these four
products, α CD19-Id-1 showed the best bispecic binding activity
(Fig. 2B). We made a negative control Db that had the variable
regions of a rat mAb of irrelevant specicity (RatFv-Id) (Fig.
S1). Both Dbs were conrmed 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 uorophore-labeled Dbs and analyzed
cells from the draining lymph nodes by ow cytometry. αCD19-
Id, but not RatFv-Id, was retained specically 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 efciency 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
specic Db to B cells, but not to T cells (CD3
+
), monocytes,
macrophages, or granulocytes (CD11b
+
and F4/80
+
) (Fig. 3B
and Fig. S3 B and C).
Id-Specic BCR Activation by αCD19-IdDecorated B Cells. For this
test we constructed an Id-specic 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-specic B cells. These signals
peaked at 20 min and declined gradually over 3060 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 specic 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-specic 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-Specic B Cells Captured αCD19-Id from Db-Decorated B Cells and
Internalized the Vaccine Molecule.
Ag-specic 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 uorophore-labeled αCD19-Id (Fig. 5A), or A20 cells dec-
orated with uorophore-labeled αCD19-Id in vitro (Fig. S6),
could transfer the Db to A20/α38BCR cells, but not to A20
cells lacking the specic BCR. We also conrmed that αCD19-Id
was internalized by A20/α38BCR cells using confocal micros-
copy (Fig. 5B).
αCD19-Id Induced Both an Id-Specic Antibody Response and a Db-
Specic T-Cell Response.
αCD19-Id induced a robust Id-specic
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 signicant 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
CD4
+
T cells (8). That was also the case for αCD19-Id. Depletion
of CD4
+
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
αCD19-Id
Large Ag
CD19
B cell receptor
T cell receptor
MHCII-peptide
CD40
CD40 ligand
CD4
+
T
B
B
B
B
Id-specific
Subcapsular sinus
Conduit
Fig. 1. Proposed model: Id-specic B cells are stimulated by αCD19-Id tar-
geted to CD19 on B cells in lymphoid follicles.
A
B
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
H
and 38 V
H
), and light chain variable domai ns (α19 V
L
and
38 V
L
) of anti-CD19 and 38C13, respectively, T7 promoters (T7), ribosomal
binding sites (rbs), (Gly)
4
Ser linkers (L), hexahistidine tag (H
6
), 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 bispecic binding. A20 cells were incubated with CFPS products
containing 5 μg of each αCD19-Id variant () or with mock CFPS product
(shaded). A20/CD19
NEG
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.
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vaccinated with RatFv-Id and αCD19-Id proliferated (Fig. S7)and
secreted gamma IFN (IFN-γ)(Fig.6B)toboththespecicand
nonspecic 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-specic 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 specic 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
Challenge.
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.
Discussion
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 benet from Id vaccination (57). 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 difcult 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
A
B
Fig. 3. αCD19-Id targeted specically 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
uorophore-conjugated mAbs specic for B220, CD3, CD11b, and F4/80,
and analyzed by ow cytometry. The percentages of gated total leuko-
cytes in the Upper Right quadrants are indicated. One of two experiments
is presented.
C
BA
Fig. 4. Id-specic 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 xed, permeabilized, stained with PE-conju-
gated antibodies specic for the phosphorylated forms of PLC-γ2 and Syk,
and analyzed by ow 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
+
cells
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 specic 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.
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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, 1923). 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-specic 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-specic 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 Freuds 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
H
and V
L
genes (35),
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).
CD4
+
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
+
T-cell epitopes.
However, instead, our data indicate that the nonnatural
Gly
4
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
cellstargetedbyanantiCD19-Ag conjugate could prime
CD4
+
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-specic 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
CD4
+
T cells.
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 benet 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 specicity (H22-15-5) (RNeasy; Qiagen). The V
H
and V
L
sequences were isolated using the SMART RACE kit (Clontech) and primers
specic to rat IgG2a constant region 1 (5-ggaaatagcccttgaccaggcatcc-3)and
A
B
Fig. 5. Id-spe cic 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 xed and
analyzed by ow 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, xed, 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.)
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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, anked 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 rst 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
6
) 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, xed with 2% (wt/vol)
paraformaldehyde, and analyzed on a FACScalibur (Becton Dickinson).
Db in vivo trafcking 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 uorophore-conjugated mAbs, washed, xed, 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
6
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
6
splenic B cells were mixed with 3 × 10
5
Violet dye-labeled
cells, centrifuged for 30 s, and incubated for 1 h at 37 °C. Cells were washed,
xed, 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, xed 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
xed and analyzed.
ELISAs. Se rum anti-Id IgGs were quantied as described previously (17).
To quantify IFN-γ, splenocytes were seeded (5 × 10
5
cells per well) in
96-well U-bottom plates in 100 μL media (5% FBS, 100 μg/mL gentamy-
cin). A nal 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 Scientic).
ACKNOWLEDGMENTS. We thank D. Czerwinski and R. Rajapaksa for
technical expertise in ow 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
B
IFN-γ (ng/mL)
Buffer
38C13-KLH
RatFv-Id
αCD19-Id
αCD19 + 38C13
αCD19-Av-38C13
70
60
50
40
30
20
10
0
Media
IgM
38C13
Rat IgG
αCD19
KLH
αCD19-Id
RatFv-Id
Avidin
μg/mL
Buffer
38C13-KLH
RatFv-Id
α
CD19-Id
αCD19 + 38C13
α
CD19-Av-38C13
n=28
n=20
n=30
n=40
n=37
n=30
A
0
30
20
10
40
50
60
Fig. 6. Immune responses to αCD19-Id. (A) αCD19-Id induced a robust Id-specic 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.
B
A
αCD19-Id
RatFv-Id
Buffer
38C13-KLH
n. s. (P = 0.70)
0 20 40 60 80 100
0
20
40
60
80
100
Da
y
s since tumor challen
g
e
Percent survival
n. s. (P = 0.27)
P = 0.015
n. s. (P = 0.91)
0 20 40 60 80 100
0
20
40
60
80
100
Days since tumor challenge
Percent survival
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
KaplanMeier method and the log-rank statistical test.
14530
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www.pnas.org/cgi/doi/10.1073/pnas.1211018109 Ng et al.
Page 5
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.
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    • "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 [90]. ONCH-1998; No. of Pages 16 "
    [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.
    No preview · Article · Jun 2015 · Critical reviews in oncology/hematology
  • Source
    • "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 [43] [44] [45] [46] [47]. Although, antibodies against tumor antigens and immune-modulatory molecules have been shown to be helpful in tumor treatment [43] [48] [49], T cells are often involved in this process and have been shown to play significant roles in the control of proliferative malignant cells [48] [49] [50] [51]. 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: 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.
    Full-text · Article · Mar 2013 · Clinical and Developmental Immunology
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    • "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. "
    [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.
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