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Prepublished online as a Blood First Edition Paper on October 31, 2002; DOI 10.1182/blood-2002-03-0831.
IMMUNOBIOLOGY
From the Department of Medical Zoology, Jichi Medical
School, Tochigi, Japan; and the Department of Tropical
Medicine and Medical Microbiology, John A. Burns School of Medicine,
Leahi Hospital, University of Hawaii at Manoa, Honolulu, HI.
A novel bispecific single-chain antibody fragment (biscFv) has been
constructed to address the possibility of a new approach to malaria
therapeutic drug development. The biscFv consists of 2 different
single-chain antibody fragments linked by a flexible peptide linker
(Gly4-Ser)3. Of the 2 scFv fragments,
one is directed against a conserved epitope of the 19-kDa C-terminal
fragment of the major surface protein of human malignant malaria
parasite, Plasmodium falciparum, and the other is directed
against the CD3 antigen of human T cells. The biscFv expressed
by a recombinant baculovirus retained the antigen-binding properties of
the corresponding univalent single-chain antibody fragments and
formed a bridge between P falciparum and T cells.
In cooperation with T cells, the biscFv specifically induced not only
interferon Malaria kills between 1.5 and 2.7 million people
each year, and between 300 and 500 million others fall ill from it,
often severely.1 The greatest cause of mobility and
mortality is caused by infection with Plasmodium falciparum.
The emergence and spread of drug resistance to common antimalarial
drugs and the current unavailability of vaccines against any form of
malaria has increased the urgency of new drug development efforts for the control of P falciparum malaria.
Bispecific single-chain antibody fragments (biscFvs) are proteins that
have 2 different binding specificities: the first binding site is
specific for an antigen present on the target cell, and the second
binding site recognizes an antigen on immune effector T cells involved
in signal transduction and T-cell activation. It is thought
that biscFvs bridge target cells and effector cells, resulting in
target-cell destruction. The potential use of biscFv for immunotherapy
has been reported in several models. For instance, anti-CD3 biscFvs
with antitumor scFv have been shown to activate T cells and mediate
redirected cellular cytotoxicity in vitro and in vivo, resulting in
tumor lysis.2-6 Several clinical trials have supported the
clinical potential of anti-CD3 biscFvs with antitumor
scFv.7-9 In addition, a biscFv composed of CD4 and anti-CD3 also has been reported to retarget T cells against
gp120-expressing HIV-infected cells.10-12 Thus,
application of biscFvs appears to be a promising avenue for malaria
immunotherapy because biscFvs do not rely on the induction of antibody
production but rather entail the stimulation and targeting of
cell-mediated mechanisms. Moreover, unlike antibodies, which are often
strain specific, biscFvs would act upon P falciparum strains
of diverse genetic makeup.
Conserved epitopes expressed on the surface of parasite-infected
erythrocytes or the merozoite surface could serve as potential targets
for selective immunotherapeutic molecules. PfMSP-1 is a prominent
antigen in malaria vaccine development and has been found to induce
antibodies that bind to the merozoite surface, inhibit parasite growth
in vitro, and induce a protective immune response against malaria
infection in nonhuman primates in vivo.13-16 Although
portions of the PfMSP-1 gene undergo allelic variation, 19-kDa
C-terminal processing product of PfMSP-1 (PfMSP-119), which appears to be an important target of the protective immune response, is
highly conserved and may serve as a potential target of immunotherapy. Taken together, the bridging between blood-stage parasites and T cells
via anti-CD3 biscFv with anti-PfMSP-119 scFv would be expected to trigger cellular activation and the release of the cytokines responsible for macrophage or neutrophil activation, resulting in phagocytosis and killing of blood-stage malaria parasites. To our knowledge, no biscFv has been exploited to date as an
immunotherapeutic drug intervention in the field of developing new
drugs for malaria.
In this paper we describe a genetically engineered biscFv consisting of
anti-CD3 scFv and anti-PfMSP-119 scFv. This 5.2-OKT3 biscFv was expressed in insect cells using a recombinant baculovirus expression system. The 5.2-OKT3 biscFv antigen-binding characteristics, in vitro merozoite targeting properties, and in vitro blood-stage parasite killing potential of 5.2-OKT3 biscFv-activated effector cells
are presented. The potential usefulness of this biscFv approach for
activation of T-cell-mediated antimalarial mechanisms is discussed.
Parasites, cell lines, and antibodies
Cloning and sequencing of the 5.2 and OKT3
V genes
For the cloning of the OKT3 VH and VL genes, the first-stranded cDNA were similarly synthesized from mRNA extracted from OKT3 hybridoma cells. Based on the sequence information of the OKT3 VH and VL genes,19 the OKT3 VH and VL genes were amplified from the cDNAs with specific sets of primers, pOKT3VH-1/pOKT3VH-2 and pOKT3VL-1/pOKT3VL-2, respectively (Table 1). After amplification of the VH and VL genes, overlapping PCR was performed to construct the OKT3 scFv gene under the same conditions, but with the pOKT3VH-1 and pOKT3VL-2 primers. The OKT3 scFv DNA was digested with BamHI and SfiI, and inserted into BamHI/SfiI-digested pBACgus-13.1scFv. The resulting transfer vector was designated pBACgus-OKT3 scFv. Thus, the 5.2 scFv and OKT3 scFv genes are nonhuman genes obtained from mouse hybridoma cells. For the construction of a biscFv, the OKT3 gene with (Gly4Ser)3 linker sequence at the 5' end was amplified from pBACgus-OKT3 scFv with pOKT3VH-GC and pOKT3VL-2 primers (Table 1). The PCR product was digested with BglI, and inserted into SfiI-digested pBACgus-5.2 scFv. The resulting transfer vector was designated pBACgus-5.2-OKT3 biscFv. Expression and purification of scFvs Recombinant baculoviruses were generated by cotransfection of Sf9 cells with pBACgus-5.2 scFv, pBACgus-OKT3 scFv, or pBACgus-5.2-OKT3 biscFv and linearized baculovirus DNA BacVector-3000 (Novagen) according to the manufacturer's protocol. For protein production, High Five cells were grown to a density of 1.5 × 106 cells/mL in Ex-Cell 405 medium (JRH Bioscience, Lenexa, KS). Cells were infected with recombinant baculovirus at a multiplicity of infection of 3 (MOI = 3) and incubated at 27°C for 3 days. All scFv antibodies were purified from culture supernatants by affinity chromatography on a Ni-NTA Superflow column (Qiagen GmbH, Hilden, Germany) following chromatography on a Q-Sepharose anion exchange column (HiPrep 16/10 QXL, Pharmacia Biotech). The purity of the proteins was checked by silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein yield was estimated by comparison with bovine serum albumin (BSA) standards.Immunoblotting Preparation of purified native gp195 and 42-kDa PfMSP-1 C-terminal polypeptide (rPfMSP-142) expressed by recombinant baculovirus was described previously.20 The protein samples were separated on SDS-PAGE and transferred to nitrocellulose membrane. Following blocking with BSA, the membranes were incubated with the purified scFvs at a concentration of 50 µg/mL. Bound scFvs were detected with biotinylated mouse anti-FLAG M2 mAb (Eastman Kodak, Rochester, NY) followed by development with 5-bromo-4-chloro-3-indolylphosphate p-toluidin salt/nitroblue tetrazolium chloride substrate (Life Technologies, Rockville, MD) as described previously.17Preparation of lymphocytes Human peripheral blood mononuclear cells (PBMCs) were obtained from heparinized peripheral blood of a single donor with no previous exposure to malaria by centrifugation on Lymphoprep (Nycomed, Oslo, Norway). The cells were washed and resuspended in malaria complete medium. PBMC preparations were used for flow cytometry, intercellular bridge formation, cytokine, growth inhibition, and phagocytosis assays.Flow cytometry analysis T cells were isolated from PBMCs by nonadherence to a nylon wool column (WAKO Pure Chemical Industries, Tokyo, Japan) as described previously.21 Aliquots of 2 × 106 T cells in 200 µL RPMI 1640 were mixed with protein samples or parent mAbs at a final concentration of 50 µg/mL. Binding of scFvs to T cells was determined by flow cytometry as described previously.19 In all experiments, freshly prepared PBMCs were used without any pretreatment.Intercellular bridge formation CD8+ T cells were isolated from PMBCs by positive sorting using anti-CD8 magnetic beads (Dynal AS, Oslo, Norway). More than 90% of the resulting cells were positive for CD8. For preparation of merozoites, P falciparum culture was treated with 10 µM E64 (Sigma Aldrich, St Louis, MO) to enrich schizont-stage parasites using the procedure described by Salmon et al.22 After washing free of E64, the parasites were incubated for 3 to 6 hours in the presence of 50 µg/mL 5.2-OKT3 biscFv. CD8+ T cells were added to the malaria culture when release of merozoites was detected. After incubation for one hour, cells were fixed with cold acetone/ethanol (6:4) on slide glass. After blocking with phosphate-buffered saline (PBS) containing 5% normal goat serum, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-FLAG M2 mAb for one hour. The cells were washed 3 times with PBS, mounted with Vectashield (Vector Laboratories, Burlingame, CA) containing 4'6'-diamidino-2-phenylindole (DAPI), and observed by fluorescence microscopy.Cytokine assay The PfMSP-119 gene was cloned from P falciparum MAD20 strain, and inserted into the pBACgus-1 vector (Novagen) to construct a baculovirus transfer vector. Recombinant baculovirus expressing the PfMSP-119 gene, designated recBac-PfMSP-119, was constructed as routine procedure. Because recombinant PfMSP-119 protein (rPfMSP-119) was found to express a conformation-dependent epitope that is recognized by 5.2 mAb, the protein was purified by affinity chromatography on a 5.2 mAb column. Microtiter plates coated with 1 µg/mL purified rPfMSP-119 were incubated with scFv samples (50 µg/mL) for one hour at room temperature. Plates were washed with PBS, and a total of 2 × 105 PBMCs in 200 µL RPMI 1640 medium containing 10% fetal calf serum was added and incubated at 37°C under 5% CO2. After 24 hours, supernatants of cultures were collected, and the levels of interleukin-2 (IL-2), interferon (IFN-), and tumor necrosis
factor  (TNF-) were measured by an enzyme-linked immunosorbent
assay (ELISA; Genzyme, Boston, MA).
Inhibition and phagocytosis assay ScFv samples were dialyzed against RPMI 1640 and then adjusted to a concentration of 200 µg/mL. Synchronization of blood-stage parasites was achieved after repeated sorbitol treatment.23 Schizont-stage parasites were diluted with unparasitized red blood cells (RBCs) to give a final parasitemia of 1.0% at a 4% hematocrit. Then 50 µL scFv, 2 × 105 freshly prepared PBMCs in 50 µL complete media, and 100 µL parasitized RBCs were added in triplicate to wells of a 96-well plate. Each well contained 10 µg scFv, 2 × 105 PBMCs and a 2% hematocrit of RBCs at a 0.5% parasitemia. The experiment comprised the following: (1) parasite culture alone; (2) parasite culture with PBMCs; (3) parasite culture with scFv sample; and (4) parasite culture with scFv sample and PBMCs. All cultures were incubated at 37°C in 5% CO2 for 24 or 72 hours. Supernatants from the cultures were collected and the levels of IL-2, IFN-, and TNF- were measured by an ELISA
(Genzyme). Growth inhibition was assessed after 72 hours of culture by
microscopic examination of Giemsa-stained thin smears from each well,
and percentage of parasitized RBCs was estimated by the counting of at
least 1000 RBCs per smear. The mean ± SD parasitemia for each treatment was calculated from 3 replicate blood smears in each experiment. The specific growth inhibition (SGI) index was calculated according to Bouharoun-Tayoun et al24 by the following
equation: SGI of scFv alone = 100 × [1 ({%
parasitemia with scFv sample}/{% parasitemia with medium
alone})]; SGI of PBMCs = 100 × [1({% parasitemia with
PBMCs}/{% parasitemia with medium alone})]; SGI of scFv plus
PBMCs = 100 × [1({% parasitemia with PBMCs and scFv sample}/{% parasitemia with scFv sample})/({% parasitemia
with PBMCs}/{% parasitemia with medium alone})].
Phagocytosis by monocytes was studied morphologically. After 24 or 72 hours of incubation of parasites, PBMCs, and scFv samples, the cells were resuspended with a pipettor to dislodge them from each well and cytocentrifuged thin smears were prepared using the total pellet, stained with Giemsa, and examined microscopically (× 1000). The phagocytosis rate was evaluated by counting the number of monocytes that had ingested one or more P falciparum merozoites per 500 monocytes in 2 separate experiments in triplicate. Differences between cultures were assessed by Student t test.
Construction and expression of scFvs We selected the anti-PfMSP-119 5.2 mAb, derived originally against the Uganda Palo Alto strain of P falciparum (FUP), as a means of targeting blood-stage parasites because it recognizes an epitope within the 19-kDa C-terminal region of MSP-1 that is highly conserved in different P falciparum field isolates.20,25 The 5.2 mAb itself has no direct effect on merozoite invasion or intraerythrocytic growth of the parasite in vitro. The VH and VL genes encoding 5.2 mAb were cloned from 5.2 hybridoma cells by RT-PCR. The 5.2 scFv construct was inserted into the phage display vector pCANTAB5E. After transformation of Escherichia coli HB2151, the resultant phage pool (4 × 104) was screened with rPfMSP-119. Finally, we obtained one phage which had the capability of binding to rPfMSP-119 (data not shown). The VH and VL genes encoding 5.2 mAb were sequenced from this positive phage. The VH and VL genes encoding OKT3 mAb were cloned from OKT3 hybridoma cells by RT-PCR with primers designed from previous publication by Arakawa et al19 The 5.2 scFv and OKT3 scFv genes were connected through a (Gly4Ser)3 linker to construct the 5.2-OKT3 biscFv gene. Figure 1 shows the nucleotide sequence and the predicted amino acid sequence of the 5.2-OKT3 biscFv gene.
To facilitate high-level expression, 3 recombinant baculoviruses
were generated harboring the following genes: (1) 5.2 scFv, specific for P falciparum MSP-1; (2) OKT3 scFv,
specific for human CD3; and (3) 5.2-OKT3 biscFv,
encoding a bispecific molecule consisting of 5.2 scFv fused to OKT3
scFv (Figure 2). The 5.2 and OKT3 scFv constructs each contain an intra-scFv
(Gly4-Ser)3 linker connecting the C-terminal
VH segment with the N-terminal VL segment. The 5.2-OKT3 biscFv construct contains an additional intra-scFv
(Gly4-Ser)3 linker connecting the C-terminal
5.2 VL segment with the N-terminal OKT3 VH
segment. All 3 constructs contain the melittin signal leader peptide
followed by the FLAG epitope tag at the N terminus and a histidine tag
at the C terminus.
All constructs were expressed as secreted soluble proteins and purified by Ni-NTA chromatography from culture supernatants of High Five cells infected with recombinant baculoviruses. Each purified preparation appeared as a single band of the appropriate molecular size by SDS-PAGE (Figure 2A). As expected from the predicted amino acid sequences of scFvs, the apparent molecular weight of 5.2-OKT3 biscFv (77 kDa) was double that of 5.2 scFv (34 kDa) or OKT3 scFv (33 kDa). Binding properties of 5.2-OKT3 The binding properties of 5.2-OKT3 biscFv to PfMSP-1 and CD3 were shown by immunoblot and fluorescent-activated cell sorting (FACS) analysis, respectively. As shown in Figure 2B, 5.2-OKT3 biscFv specifically reacted with 19-kDa and 42-kDa purified, parasite PfMSP-1 (lane 2) of the P falciparum FUP strain as well as 42-kDa rPfMSP-142 (lane 4). The specificity of 5.2-OKT3 biscFv was indistinguishable from that of the parent 5.2 mAb (lanes 1,3). Figure 3 shows that OKT3 scFv (panel C) and 5.2-OKT3 biscFv (panel D) were comparable to OKT3 mAb (panel A) in binding to human T cells, as shown by their increased fluorescence intensity compared with 5.2 mAb (panel B) or 5.2 scFv (panel E). These results clearly demonstrate that 5.2-OKT3 biscFv retains the binding properties of both parental antibodies.
We examined whether 5.2-OKT3 biscFv can form a bridge between merozoites and T cells. To facilitate the observation of bridge formation, CD8+ T cells were isolated from PBMCs as effector cells capable of binding to OKT3. At the moment that schizont-stage parasites rapidly rupture to release merozoites from their parasitophorous vacuole following removal of E64, human CD8+ T cells were added to the culture in the presence of 5.2-OKT3. DAPI staining showed merozoites were agglutinated on the surface of CD8+ T cells. The binding sites exhibited intense fluorescence, whereas no positive signal was detected in schizont-stage parasites. This result indicates that CD8+ T cells can bind to free merozoites through 5.2-OKT3 biscFv. Immunologic characteristics of 5.2-OKT3 scFv To investigate whether the bridge formation shown in Figure 4 could activate T cells, we examined cytokine production following bridge formation between rPbMSP-119 and PBMCs through 5.2-OKT3 biscFv. rPbMSP-119 was immobilized to concentrate the binding complex of rPbMSP-119 and 5.2-OKT3 biscFv in a well. Unbound scFvs were washed away to confirm the specific binding of 5.2-OKT3 biscFv and rPbMSP-119. PBMCs were added and cultured in the plate for 24 hours. The production of IL-2, IFN-,
and TNF- in culture supernatants was measured by an ELISA
assay. Figure 5A shows that production of these cytokines was specifically induced by 5.2-OKT3 biscFv. Regardless of the absence or presence of soluble
rPfMSP-119, both OKT3 scFv and 5.2-OKT3 biscFv
could activate PBMCs to induce approximately 10% to 30% of cytokine
(TNF- but not IFN-) produced by bridge formation through 5.2-OKT3
biscFv (data not shown). Presumably, this low level of activation is
due to direct binding of 5.2-OKT3 biscFv or OKT3 scFv to T cells. This
result is consistent with previous reports that cross-linking of OKT3
is more effective to activate T cells compared with the soluble
form.26,27
Functional activity of 5.2-OKT3 biscFv To assess the potential therapeutic value of 5.2-OKT3 biscFv against P falciparum blood-stage parasites, we measured cytokine production, P falciparum growth inhibition and parasite/merozoite phagocytosis in cocultures of PBMCs, and P falciparum blood stages in the presence of 5.2-OKT3 biscFv. Similar to the result in Figure 5A, significant levels of IFN- and TNF- production were induced in the cocultures when
5.2-OKT3 biscFv was present (Figure 5B). In contrast, high levels of
TNF- production were induced by PBMCs even in the absence of any
biscFv, consistent with previous work in which high levels of TNF-
activity were detected when monocytes were cocultured with P
falciparum schizont-stage parasitized erythrocytes that
subsequently ruptured.28 No IL-2 production was detected in this experiment (data not shown).
As shown in Table 2, 5.2-OKT3 biscFv in
cocultures of P falciparum and PBMCs caused
inhibition of parasite growth at an SGI of 36.8%, whereas 5.2 scFv and
OKT3 scFv cocultured with PBMCs enhanced parasite growth as shown by
negative SGIs of
In the present study, we constructed 5.2-OKT3 biscFv to design a
potentially immunotherapeutic ligand for blood-stage malaria parasites.
We found that 5.2-OKT3 biscFv possessed specific binding activities for PfMSP-119 on the surface of merozoites and
for CD3 on human T cells. Binding of 5.2-OKT3 biscFv to human T cells induced an increase in production of IFN- Based on our data and on mechanisms proposed by others for biscFv
biologic activity, we propose that bridge formation between parasites and T cells through 5.2-OKT3 biscFv provides the
initial trigger for the parasite clearance (Figure
7). T cells are stimulated with binding
of 5.2-OKT3 biscFv to CD3 and release soluble mediators like IL-2,
IFN-
Druilhe and coworkers24,32 found that human IgG of protected hyperimmune subjects inhibits parasite growth (SGIs = 60, approximately 90%) in the presence of monocytes (2 × 105/well) via antibody-dependent cellular inhibition (ADCI), whereas antibodies alone either had no effect or enhanced parasite growth.24,32 Parasite growth inhibition (SGI = 36.8%) observed in the presence of 5.2-OKT3 biscFv and PBMCs may or may not occur by similar mechanisms as ADCI. Because the main purpose of the present study was to address the potential usefulness of biscFvs in malaria patients, we used freshly prepared PBMCs (2 × 105/well) rather than purified T cells and monocytes to simulate naive culture conditions without prestimulation. Consequently, the number of monocytes responsible for phagocytosis might be insufficient to completely inhibit parasite growth inhibition. It is also possible that T cells and/or monocytes used in the present studies may have not been fully activated under the current culture conditions. Additional signals may be required to achieve efficient killing of ingested parasites. Studies using an antitumor scFv have identified a requirement for a B7-mediated costimulatory signal along with TCR-mediated binding for optimal T-cell activation.33 More extensive studies are required to define the requirements for optimal interactions between T cells, mononuclear effector cells, and 5.2-OKT3 biscFv. BiscFv-mediated selective immunotherapy has been used to retarget T-cell cytotoxicity against tumor cells, and some of these molecules are now being evaluated in clinical trials.7-9 An application of this mechanism for the control of infectious diseases is the use of a biscFv composed of OKT3 scFv and CD4 to target and kill HIV-infected cells.10,11 These immune-activating properties of biscFv may provide a means for enhancing biscFv therapy via several distinct mechanisms. In addition to the current example of 5.2-OKT3 biscFv as an immunotherapeutic drug, we have recently constructed an immunotoxin composed of 5.2 scFv and an antimalaria toxin. The potential parasitocidal effect of this immunotoxin on parasite growth inhibition is now under investigation. The present study demonstrates that 5.2-OKT3 biscFv offers
distinct advantages as an antimalarial therapeutic: (1) the
PfMSP-119 epitope recognized by 5.2-OKT3 biscFv is highly
conserved and thus is suitable as a target molecule; (2) 5.2-OKT3
biscFv cross-linking to T cells and malaria parasites results in the
production of IFN-
We would like to thank Dr Okazaki for advice for FACS analysis and Dr Kasahara for critical reading of the manuscript.
Submitted March 18, 2002; accepted October 28, 2002.
Prepublished online as Blood First Edition Paper, October 31, 2002; DOI 10.1182/blood-2002-03-0831.
Supported in part by a grant-in-aid for Scientific Research on Priority Areas (08281104) and by a grant-in-aid for Scientific Research (C) (13670253) from the Ministry of Education, Science, Culture, and Sports, Japan, and also by a grant for aid from the Ministry of Health, Welfare, and Labour for International Medical Cooperation, Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Shigeto Yoshida, Department of Medical Zoology, Jichi Medical School, 3311-1 Yakushiji Minamikawachimachi, Tochigi 329-0498, Japan; e-mail: shigeto{at}jichi.ac.jp.
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© 2003 by The American Society of Hematology.
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  S. Yoshida, T. Sudo, M. Niimi, L. Tao, B. Sun, J. Kambayashi, H. Watanabe, E. Luo, and H. Matsuoka Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito Blood, February 15, 2008; 111(4): 2007 - 2014. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
