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Construction and characterization of DNA encoding the single-chain variable fragment of the anti-idiotype 1A7 mimicking the tumor-associated disialoganglioside GD2 Hasan E. Zeytin,1 Pulak K. Tripathi,2 Malaya Bhattacharya-Chatterjee,2 Kenneth A. Foon,2 and Sunil K. Chatterjee2

1Department of Microbiology and Immunology, University of Kentucky Medical Center, Lexington, Kentucky 40536; and 2Department of Internal Medicine, Division of Hematology Oncology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267.

Anti-idiotype antibody, 1A7, functionally mimics the tumor-associated antigen disialoganglioside GD2, which is overexpressed on the surface of a number of neuroectodermal tumors such as melanoma, neuroblastoma, soft tissue sarcoma, and small cell carcinoma of the . of mice with 1A7 generated the production of anti-GD2 . In a phase I , immunization of patients with 1A7, mixed with the adjuvant QS21, demonstrated that 1A7 could act as a surrogate antigen for GD2 and induce strong humoral immune responses in advanced stage melanoma patients. DNA vaccines have recently been shown to invoke humoral as well as cellular responses in injected hosts against the transgene product. To evaluate the efficiency of DNA vaccines encoding anti-idiotype antibodies, we constructed expression encoding the variable heavy (VH) and variable light (VL) chains of 1A7. The plasmids were made in two configurations, expressing either the VH (pc1A7VHLnVL) or the VL (pc1A7VLLnVH) chain of 1A7 at the amino terminus, linked together by a 15- linker (Ln). In vitro / assays and of CHO-K1 cells with the plasmids demonstrated that a 30-kDa protein was expressed by both configurations of the single-chain variable fragment. This protein can be specifically precipitated by monoclonal anti-GD2 antibody, 14G2a. Following intramuscular injection in mice, the plasmids were detectable in the injected tissues for at least 3 months and the injected plasmids actively transcribed the single-chain variable fragment 1A7 at the injected site. A single, intramuscular immunization of a group of C57BL/6 mice with pc1A7VLLnVH in phosphate-buffered saline induced humoral immune responses against 1A7 as well as GD2, the nominal antigen. Multiple , however, were required to elicit stronger immune responses. Cancer (2000) 7, 1426±1436

Key words: DNA ; anti-idiotype antibody; ganglioside GD2; melanoma.

ncreased and aberrant membrane expression of different the covalent linkage of ganglioside to keyhole limpet Igangliosides, such as GD2, on tumors of neuroectodermal hemocyanine, co-injection of a strong adjuvant, and, most origin including malignant melanoma, neuroblastoma, soft important of all, the complex purification steps of these tissue sarcoma, and small cell carcinoma of lung tumor has glycospingolipids. been reported.1 GD2 is absent in most normal tissues, except As an alternate to purified ganglioside, we have chosen for low levels in brain and peripheral nerve. Thus, GD2 is an to use surrogate molecules. Our immunization strategy is excellent target for immunotherapy of tumors of neuroecto- based on the immune network concept of Jerne.7 Accord- dermal origin, such as melanoma (reviewed in Ref. 2). ing to this concept, immunization with an antibody (Ab1) Although of cancer patients with different against an antigen can generate several types of anti- gangliosides has been tested by a number of investigators,3,4 idiotype antibodies (Ab2) in the injected host. One set of GD2 is weakly immunogenic in humans.5,6 Other limitations these anti-idiotype antibodies, designated Ab2 , acts as the of using GD2 as a include the requirement of internal image of the antigen and mimics its molecular features. Thus, injection of Ab2 to the new host will elicit a new set of antibodies, which, besides binding to the Ab2 molecule (Ab3 response), will also bind to the Received March 1, 2000; accepted July 8, 2000. 0 Address correspondence and reprint requests to Dr. Sunil K. Chatterjee, nominal antigen (Ab1 response). The efficacy of anti- PhD, Department of Internal Medicine, University of Cincinnati, The Vontz idiotype antibodies as cancer vaccines has been demon- Center for Molecular Studies, Room No. 1314, Cincinnati, OH 45267- strated in a number of preclinical and clinical studies 8 0509. (reviewed in Ref. ).

1426 Cancer Gene Therapy, Vol 7, No 11, 2000: pp 1426±1436 ZEYTIN, TRIPATHI, BHATTACHARYA-CHATTERJEE, ET AL: DNA VACCINE ENCODING SCFV OF ANTI-IDIOTYPE ANTIBODY 1427

We have generated a number of anti-idiotype antibodies radish peroxidase±conjugated goat antimouse IgG, Fc- for active, specific immunotherapy of different cancer specific alkaline phosphatase±conjugated goat antimouse patients.9±12 One of our anti-idiotype antibodies, designated IgG, and all glycolipids were purchased from Sigma (St. 1A7,12 mimics GD2. Immunization of small animals and Louis, MO). All restriction enzymes were obtained from 35 nonhuman with 1A7 induced strong anti-1A7 as Promega (Madison, WI) and [ S] L -methionine was from well as anti-GD2 humoral responses in the injected hosts.12 DuPont NEN (Boston, MA). 1A7 mixed with the adjuvant QS-21 has also been used to immunize advanced stage melanoma patients.13 All patients Generation of anti-idiotype antibody 1A7 in this study demonstrated strong anti-1A7 antibody The murine monoclonal IgG2a antiganglioside GD2 anti- responses. Isotype analysis showed that these antibodies body 14G2a (Ab1) was used to immunize syngeneic were mostly IgG and specifically bound to 1A7 (Ab3 BALB/c mice. Hybridoma fusion, cloning, and selection response) as well as disialoganglioside GD2 (Ab1 0 of the monoclonal anti-idiotype 1A7 (Ab2 ), as well as the response). These results demonstrated that a significant production of ascites in bulk quantities in mice, were against GD2 can be mounted and performed as described previously.10 1A7 was purified from immunological tolerance against GD2 can be broken in ascites by affinity chromatography on protein A-CL melanoma patients by vaccination with an anti-idiotype Sepharose 4B column, followed by diethylaminoethyl antibody. (DEAE) ion exchange chromatography. The purity of the Although the injection of 1A7 invoked a strong humoral isolated immunoglobulin (>99%) was determined by immune response against GD2, the presumed cellular sodium dodecyl sulfate polyacrylamide gel electrophoresis immune responses could not be detected. Various (SDS-PAGE), high-pressure liquid chromatography, and constructs have been reported to invoke combined humoral isoelectric focusing. and cellular immune responses against the encoded (reviewed in Ref. 14 ). To explore the feasibility of using DNA vaccines encoding anti-idiotype antibodies, we Molecular cloning of 1A7 and the determination of the developed plasmids expressing the 1A7 single-chain vari- sequence of cDNA encoding the VH and VL domains able fragment (scFv) as a new generation of anti-idiotype Total RNA was isolated by using the single-step procedure17 vaccines. from 1Â107 1A7 hybridoma cells. First strand cDNA was ScFv consisting of a single variable heavy (VH) and synthesized using a SuperScript Preamplification kit (Life variable light (VL) chain domain is the smallest antibody Technologies). DNA fragments encoding the VH of 1A7 fragment capable of binding to an antigen.15,16 In this were amplified by polymerase chain reaction (PCR) using communication, we report the construction of two anti- the mixed oligonucleotide forward primer 50 -ACTAGTC- idiotype DNA vaccines, designated pc1A7VHLnVL and GACATGGCTGTCYTRGSGCTRCTCTTCTGC and the pc1A7VLLnVH, encoding two different configurations of reverse primer 50 -CCCAAGCTTCCAGGGRCCARKGGA- the scFv of 1A7. We present results demonstrating that scFvs TARACIGRTGG corresponding to sequences of the leader of 1A7 encoded by these plasmids are expressed in vitro and (signal ) region amino acids À12 to À20 and the in vivo and the expressed protein can bind to the monoclonal constant region amino acids 119±126 of IgG. For the VL Ab1 antibody, 14G2a. Moreover, our results demonstrate cDNA, the mixed oligonucleotide forward primer used was that a single intramuscular injection of these DNA vaccines, 50-ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTG- without any adjuvant or , induced anti-1A7 as well GTGCTG and the reverse primer was 50 -CCCAAGCTT- as anti-GD2 antibodies in the injected mice. However, ACTGGATGGTGGGAAGATGGA, corresponding respec- multiple immunizations were required for induction of tively to À10 to À19 amino acids of the VL leader sequence stronger immune responses. and the 116±122 amino acids of the mouse  constant region (I=inosine, R=A or G, Y=C or T, S=C or G, K=G or T). The amplified fragments of cDNA were subcloned into MATERIALS AND METHODS pT7Blue(R) plasmid to obtain the two starting plasmids, pB1A7VH and pB1A7VL, respectively encoding the VH Materials and VL of 1A7. Nova blue±competent cells were trans- Monoclonal anti-GD2 antibody 14G2a (IgG2a, ) was formed with pB1A7VH and pB1A7VL using the protocol kindly provided by Dr. Reisfeld (The Scripps Research supplied by the manufacturer (Novagen). Plasmids from Institute, La Jolla, CA). Plasmid pT7Blue(R) and Nova selected clones were prepared by the miniprep procedure and blue±competent cells were purchased from Novagen the DNA sequence of the double-stranded plasmid was (Madison, WI). pcDNA3 expression vector was obtained determined by Sequenase, version 2.0 kit from United States from Invitrogen (Carlsbad, CA). Columns for the purifica- Biochemical (Cleveland, OH). The DNA sequence was tion of plasmids were from Qiagen (Valencia, CA). CHO- determined from both orientations using T7 primer KI cells were obtained from the American Type Culture (50 -TAATACGACTCACTATAGGG) and U19 primer (5 0 - Collection (ATCC, Rockville, MD). DH5 -competent cells GTTTTCCCAGTCACGACGT). To determine the entire were purchased from Life Technologies (Gaithersburg, sequence of the amplified cDNA fragments, oligonucleo- MD). Production of the control anti-idiotype antibody, tides were synthesized corresponding to the sequences of the 3H1, was described previously.10 Antimouse IgG  50 and 30 ends of the sequence obtained in the first round and (negative control monoclonal antibody), biotinylated horse- were used for a second round of sequencing.

Cancer Gene Therapy, Vol 7, No 11, 2000 1428 ZEYTIN, TRIPATHI, BHATTACHARYA-CHATTERJEE, ET AL: DNA VACCINE ENCODING SCFV OF ANTI-IDIOTYPE ANTIBODY

Verification of the cDNA clone by amino acid sequence CGGATCCCAGGTGCAGGTGAAGGAGTCA and the 0 For the amino acid sequence determination of 1A7 variable reverse primer (P8) was 5 -TGACTCCTTCACCTGCAC regions, 50 g of purified 1A7 was diluted with sample CTGGGATCCGCCGCCACCCGAGCCGCCACCGCCC- loading buffer (50 mM Tris±HCl, pH 6.8, 1% SDS, 1% GAGCCACCTCCCCCTTTGATTTCCAGCTTGGTGC- glycerol, 0.1% -mercaptoethanol) and heated to 1008C for C. The two sets of primers, P5 and P6, were boiled for 10 3 minutes. The denatured protein was loaded onto a 7.5% minutes, cooled to room temperature, and subjected to PCR polyacrylamide gel (Bio-Rad Miniprotean II Dual Slab with VH and VL as templates. To obtain the plasmid of VH± Cell) containing SDS and subjected to electrophoresis at 200 VL configuration, P1 and P4 were also added to the PCR V for 1 hour. Proteins in the gel were transferred to reaction mixture. Similarly, for the VL±VH configuration, polyvinylidene difluoride membranes at 30 mA overnight. primers P3 and P2 were used. The PCR products 800 bp The transfer buffer contained 25 mM Tris±192 mM glycine were purified with QIAquick PCR purification Kit (Qia- and 20% (vol/vol) methanol. The membranes were stained gen). by quick dipping in 0.1% Coommassie Brilliant Blue Construction of 1A7 scFv expression vectors (Sigma) in 50% methanol/50% acetic acid, followed by washing in a solution containing 40% methanol plus 10% PCR products of VHLnVL and VLLnVH of 1A7 and acetic acid. After drying the membranes at room tempera- pcDNA3 were digested with EcoRV and XbaI and were run ture, the stained VH and VL chain bands were excised with a on the 0.8% low melting point agarose gel at 10 mA for 4 clean razor blade. Proteins on the membrane slices were hours. Expected bands were cut out from the gel with a razor subjected to N-terminal microsequencing by automated and DNA fragments were purified using the GELase Edman degradation using an Applied Biosystem Model Agarose Gel Digestion Preparation according to the protocol 477A protein sequencer using pulsed liquid chemistry and of the manufacturer (Epicentre, Madison, WI). The EcoRV- online phenyl-ethiohydantoin amino acid identification. and XbaI-digested pcDNA3 and VHLnVL or VLLnVH of Each protein was subjected to 10±15 degradative cycles scFv were then ligated with T4 DNA Ligase (Promega) by and converted cleavage products from each cycle were incubating at 168C overnight. The recombinant plasmids, analyzed by reverse-phase high-pressure liquid chromato- pc1A7VHLnVL and pc1A7VLLnVH, were used for the graphy. transformation of DH5 -competent cells (Life Technolo- gies). Small- and large-scale plasmid purifications were Construction of 1A7 scFv expression plasmids in two performed by using appropriate Qiagen purification kits. configurations In vitro transcription and translation of pc1A7VHLnVL and cDNAs of VH and VL chains of 1A7 were amplified from pc1A7VLLnVH pB1A7VH and pB1A7VL, respectively, by PCR. For the VH chain, the forward primer (P1) was 5 0 -GCCGATATCAC- pc1A7VHLnVL and pc1A7VLLnVH (1 g each) were CATGGCTGTCTTGGGGCTGCTC and the reverse primer incubated with 40 L of TNT T7 Quick Master Mix (TNT 0 T7 Quick-Coupled Transcription/Translation System, Pro- (P2) was 5 -CATCTCTAGATTATGAGGAGACGGTGAC- 35 TGAGGT. EcoRV and XbaI restriction sites were incorpo- mega) and with 10 Ci of [ S] L -methionine for 1 hour at rated into those primers. The start codon at the 50 end of the 308C. Five-microliter aliquots from the reaction mixture forward primer (bold) and the stop codon at the 3 0 end of the were mixed with 10 L of sample loading buffer (50 mM reverse primer (bold) were also included. For VL chain, the Tris±HCl, pH 6.8, 1% SDS, 1% glycerol) and were run on forward primer (P3) was 5 0 -GCCGATATCACCATG- the 12% SDS-PAGE gel for 1 hour at 30 mA. After fixing GAGTTGCCTGTTAGGCTG and the reverse primer (P4) the gel in 30% methanol/10% acetic acid solution for 30 was 50-CATCTCTAGATTATTTGATTTCCAGCTTGGTG- minutes, the gel was rinsed with water for 30 minutes and CC. EcoRV and XbaI restriction sites (italics) and start and dried in a gel drier (Bio-Rad, model 583) at 808Cfor2 stop codons were also incorporated in these two primers. hours. Dried gels were exposed to KODAK X-OMAT AR cDNAs for VH and VL were linked together by a 15-amino film (Eastman Kodak, Rochester, NY) for autoradiography. acid linker (Ln), (Gly 4Ser) 3. In order to get two different configurations of scFv, two different sets of Ln primers were Transfection of CHO-K1 cells with pc1A7VHLnVL and synthesized. The forward primer for VH±VL configuration pc1A7VLLnVH (P5) was 5 0-TCAGTCACCGTCTCCTCAGGGGGAGG- CHO-K1 cells (2Â105) were plated on six-well Falcon TGGCTCGGGCGGTGGCGGCTCG GGTGGCGGCGG- plates overnight in 1 mL Dulbecco's Modified Eagle ATCCGATGTTTTGATGACCCAA. The reverse primer Medium containing 10% fetal calf serum. The cells were (P6) was 5 0 -TTGGGTCATCAAAACATCGGATCCGC- transfected with 2 g of plasmid plus 20 L Lipofectamine CGCCACCCGAGCCGCCACCGCCCGAGCCACCTCC- (Life Technologies) according to the manufacturer's proto- CCCTGAGGAGACGGTGACTGA. The 50 end of the col. After 36 hours at 378C in a tissue culture incubator, 20 0 35 forward primer contained nucleotides corresponding to the 3 Ci of [ S] L -methionine was added to the media. The end of the VH chain and the 30 end of the Ln contained supernatant was collected after 5 hours and a 300-L aliquot nucleotides corresponding to the 50 end of the VL chain was incubated with 2 g of either 14G2a, 3H1 (negative (bold). The forward primer for VL±VH configurations control anti-idiotype antibody), or antimouse IgG  (P7) was 5 0 -GGCACCAAGCTGGAAATCAAAGGGG- (negative control monoclonal antibody) for 4 hours. GAGGTGGCTCGGGCGGTGGCGGCTCGGGTGGCGG- Antigen±antibody complexes were precipitated by incuba-

Cancer Gene Therapy, Vol 7, No 11, 2000 ZEYTIN, TRIPATHI, BHATTACHARYA-CHATTERJEE, ET AL: DNA VACCINE ENCODING SCFV OF ANTI-IDIOTYPE ANTIBODY 1429 tion with Protein A Sepharose 4 Fast Flow gel (Pharmacia The specimens were sliced into 4-m sections. Following Biotech, Piscataway, NJ) for 4 hours at 48C followed by fixation with xylene for 15 minutes, the sample was thorough wash with phosphate-buffered saline (PBS). The rehydrated by treatment with ethanol/methanol. The endo- precipitates were finally resuspended in 10 L sample genous peroxidase activity was quenched by treatment with loading buffer (50 mM Tris±HCl, pH 6.8, 1% SDS, 1% 0.3% hydrogen peroxide for 30 minutes. The tissue was glycerol) for gel electrophoresis. All samples were run on treated with 10% goat serum in PBS for 30 minutes to block the 12% SDS-PAGE for 1 hour at 30 mA and the gel was nonspecific antibody binding. After five washes with PBS, processed and autoradiographed as described above for in the tissue was incubated with the primary antibodies (14G2a, vitro transcription translation assay. 3H1 in PBS) or 1% bovine serum albumin (BSA) in PBS for 90 min. The slides were washed with PBS (five times) Transcription of pc1A7VLLnVH in mice and incubated with biotinylated goat antimouse IgG for 60 Fifty micrograms of pc1A7VLLnVH was injected into the minutes. After thorough washing with PBS, the tissue was leg quadriceps of C57BL/6 mice. At different intervals, the treated with peroxidase-conjugated streptavidin for 30 mice were sacrificed and muscle tissues removed. Total minutes at room temperature. The slides were developed RNA from the muscle was obtained by Trizol reagent (Life by incubation with AEC substrate kit for horseradish Technologies) according to the manufacturer's protocol. The peroxidase (Vector Laboratories, Burlingame, CA) and RNA was incubated with RNase-free bovine pancreatic counterstained with hematoxylin for 10 minutes. DNase I (Amersham Pharmacia Biotech, Piscataway, NJ), followed by reverse transcription (RT) PCR using a kit Immunization of mice with DNA vaccines (TITAN One Tube RT-PCR System, Boehringer-Man- Four groups of 4±6-week-old C57BL/6 female mice were nheim, Indianapolis, IN). The forward primer was 50 - injected with 100 g of purified plasmids into two leg GCCGATATCACCATGGAG TTGCCTGTTAGGCTG and quadriceps (50 g/leg). Group 1 was injected only once the reverse primer was 50 -CATCTCTAGATTATGAGGA- with pc1A7VLLnVH. The other three groups were immu- GACGGTGACTGAGGT. Ten microliters of RT-PCR nized every other week for four times with: group 2, pcDNA; products was digested with KpnI and electrophoresed on a group 3, pc1A7VLLnVH; and group 4, pc1A7VHLnVL. 1% agarose gel for 1 hour at 10 mA. Following electrophor- Sera were drawn before each immunization and stored at esis, the gel was depurinated with 0.25 N HCl, denatured with À208C for enzyme-linked immunosorbent assay (ELISA). 0.5 N NaOH±1.5 M NaCl, and neutralized with 1.5 M NaCl, 0.1 M Tris pH 7.5. DNA from the gel was transferred to Pepsin digestion of anti-idiotype antibody 1A7 into F(ab) 2 nitrocellulose membrane using Strategene (La Jolla, CA) and Fc fragments PosiBlot Pressure Blotter and Pressure Control Station in 20Â SSC as transfer buffer (3 M NaCl±0.3 M sodium citrate, pH, Ten milligrams of 1A7 was digested with pepsin and F(ab) 2 7.0). The transfer was carried out for 1 hour at 75 mm Hg fragment was purified from Sepharose A column by using pressure and DNA on the membrane was cross-linked by UV. ImmunePure F(ab) 2 Preparation Kit (Pierce Chemicals, The probe, 32P-labeled pc1A7VLLnVH, was prepared by Rockford, IL) according to the manufacturer's protocol. using Random Primer DNA Labeling Kit according to the manufacturer's (Life Technologies) protocol. The mem- Assay for induction of anti-1A7 (Ab3) response brane was incubated with prehybridization solution (6Â The 96-well Falcon plates were coated with 50 Lof5g/ SSC, 5Â Denhardt's reagent, 0.5% SDS, 100 g denatured, mL of 1A7 F(ab) 2 in carbonate±bicarbonate buffer, pH 9.6, fragmented salmon sperm DNA, 50% formamide) at 428C overnight. After blocking with 3% BSA in PBS, the plates for 4 hours in a hybridization oven.18 The prehybridization were incubated for 16 hours with 50 L of 1:10 diluted solution was discarded and the membrane was incubated mouse sera. Plates were washed seven times with PBS with prewarmed hybridization solution (6Â SSC, 5Â containing 0.05% Tween-20 and incubated with 50 Lof Denhardt's reagent, 0.5% SDS, 100 g denatured, fragmen- diluted Fc-specific alkaline phosphatase±conjugated goat ted salmon sperm DNA, 50% formamide containing the antimouse IgG for 2 hours. After washing, the color was 32P-labeled pc1A7VLLnVH probe at 428C for 16 hours in developed with alkaline phosphatase substrate and the OD the hybridization oven. The membrane was washed with was read at 405 nm. increasing stringency of SSC±SDS, as follows; 2Â SSC± 0.5% SDS, 15 min; 2Â SSC±0.1% SDS, 15 min; 0.1Â Assay of induction of anti-GD2 antibodies (Ab10 response) SSC±0.5% SDS, 30 min; and 0.1Â SSC for 5 min. The wet membrane was wrapped with saran wrap and exposed to X- Microtiter plates (96-well) were coated with 100 ng of dif- ray film (KODAK-XAR-2, Eastman Kodak) for auto- ferent gangliosides: GD2 [GalNac 1!4(NeuAc2!8Neu- radiography. Ac 2!3)Gal 1!4Glc!cer],GD3,[NeuAc 2!8NeuAc- 2!3Gal 1!4Glc!cer], GM1, [Gal 1!3GalNac 1! 4(NeuAc 2!3)Gal 1!4Glc!cer], GM2, [GalNac 1! Immunohistochemistry of muscle tissues after injection of 4(NeuAc 2!3)Gal 1!4Glc!cer], GM3, [NeuAc 2! DNA vaccines 3Gal 1!4Glc!cer], and GT1b, [NeuAc 2!3Gal 1! Fifty micrograms of pc1A7VLLnVH was injected into leg 3GalNac 1!4(NeuAc 2!8NeuAc 2!3)-Gal 1!4Glc quadriceps of C57BL/6 mice. After 7 days, the muscle tissue !cer]. The wells were blocked with 1% BSA in PBS. Sera was removed, fixed in formalin, and embedded in paraffin. from three mice from each group were pooled. The test

Cancer Gene Therapy, Vol 7, No 11, 2000 1430 ZEYTIN, TRIPATHI, BHATTACHARYA-CHATTERJEE, ET AL: DNA VACCINE ENCODING SCFV OF ANTI-IDIOTYPE ANTIBODY samples and Ab1 monoclonal antibody, 14G2a, were diluted RESULTS 1:10 with PBS±BSA and 100 L was added in triplicate wells. Cloning of cDNA of 1A7 and the determination of the After 4 hours of incubation at room temperature with shaking, nucleotide sequence the plates were washed with PBS. Bound antibodies were detected by treatment with alkaline phosphatase±conjugated Total RNA from 1A7 hybridoma cells was used to obtain goat antimouse immunoglobulin, followed by the color cDNA encoding the VH and VL chain variable domains of reaction. Readings were taken at 405 nm in an ELISA reader. 1A7 by RT-PCR using random mouse IgG primers in a kit from Novagen. Amplified VH and VL DNA fragments were incorporated into pT7Blue(R) plasmids to obtain T-cell proliferation assays pB1A7VH and pB1A7VL (Fig 1). DNA sequences of the Mice (three in each group) were immunized with plasmids were determined as described in Materials and pc1A7VHLnVL or pc1A7VLLnVH as described above. Methods. Due to the error-prone nature of Taq polymerase were isolated from the mice and pooled in PBS under used in these experiments, at least 10 pB1A7VH and sterile conditions. After smashing the in PBS, the cell pB1A7VL clones were selected for sequencing. Out of 10 suspension was washed three times with 10 mL of RPMI clones, seven showed the same sequence, which was 1640 medium, containing 10% fetal calf serum, by centrifu- accepted as the genuine sequence for 1A7. Amino acid gation at 1000Âg for 10 minutes. In each well of a 96-well sequences for VH and VL of 1A7 were translated from the Falcon plate, 2Â105 spleen cells were added and mixed gently corresponding nucleotide sequences and shown in Figure 2. with the stimulants (10 ng, 500 ng, 1 g, 1.5 g, or 2 g/ The predicted amino acid sequences were compared with the well). Stimulants tested were anti-idiotype antibody 1A7, N-terminal amino acid sequences of purified 1A7 VH and control anti-idiotype antibody 3H1,10 and the mitogen VL chains to rule out the possibility that the clones concanavalin A. After 4 days of incubation in a tissue culture originated from other mRNAs present in the 1A7 hybridoma incubator, the cells were pulse-chased with 1 Ci [ 3H]thy- cells. The predicted amino acid sequence showed exact ]thymidine. Each assay was performed at least in triplicate, homology as the purified amino acid sequence of the 1A7 and the mean was calculated from samples with SD less than VH and VL chains. The leader, framework, and comple- 10%. Stimulation indices were obtained by dividing the mentarity determining regions were identified by checking mean counts per minute of each sample by the counts per the homologies in both the nonredundant Protein Database minute obtained using the medium without stimulant. and the Kabat database19 in the National Center for

Figure 1. Molecular cloning of 1A7 heavy and light chains and the construction of scFv. Starting plasmids,pB1A7VH and pB1A7VL,were constructed by incorporation of the VH and VL chain fragments of 1A7,respectively,into pT7Blue(R) plasmid. VH and VL were joined together using the 15-amino acid linker as described in Materials and Methods.

Cancer Gene Therapy, Vol 7, No 11, 2000 ZEYTIN, TRIPATHI, BHATTACHARYA-CHATTERJEE, ET AL: DNA VACCINE ENCODING SCFV OF ANTI-IDIOTYPE ANTIBODY 1431

Figure 2. Nucleotide sequences and predicted amino acid sequences of the variable chains of anti -idiotype anti- body,1A7. DNA sequences of the VH (A) and VL (B) chains of 1A7 were determined as described in Materials and Methods. Various frameworks, complementarity determining regions, and the leader sequences were deter- mined according to Kabat et al.19

Biotechnology Information Databank using the Blastp domains may distort native conformation of the antigen- program.20 Various domains of the VH and VL chains are binding site of scFv. We have previously shown that for the shown in Figure 2. The 1A7 heavy chain belongs to mouse best antigen mimicry of scFv derived from 3H1 Ð an anti- VH subgroup IB and the light chain belongs to  II. idiotype antibody mimicking carcinoembryonic antigen Ð the VH chain should be in the amino terminus of the scFv; VH and VL should be linked by a 15-amino acid Ln.21 To Construction of plasmid vectors encoding single-chain explore the possibility that different configurations of scFv variable regions of 1A7 of 1A7 might have different in vitro or in vivo effects, two ScFv consisting of a single VH and VL chain domain is the expression vectors were constructed. In the first one, the VH smallest antibody fragment capable of binding an anti- chain was placed at the amino terminal in the construct gen.15,16 In scFv constructs, the carboxy terminus is joined to (VHLnVL) and in the other, it was placed at the carboxy the amino terminus of variable domains by a Ln of terminal in the construct (VLLnVH). The strategy for the appropriate length and flexibility. Therefore, the scFv can construction of VHLnVL is presented in Figure 1. The be constructed at least in two different configurations, either VLLnVH construct was made by a similar strategy. The VHLnVL or VLLnVH.15 These two different orientations of expression vectors, pc1A7VHLnVL and pc1A7VLLnVH,

Cancer Gene Therapy, Vol 7, No 11, 2000 1432 ZEYTIN, TRIPATHI, BHATTACHARYA-CHATTERJEE, ET AL: DNA VACCINE ENCODING SCFV OF ANTI-IDIOTYPE ANTIBODY were constructed by incorporation of the scFv cDNAs as described under Materials and Methods and schematically presented in Figure 3. The plasmids were prepared on a large scale following transformation of DH5 -competent cells. To confirm the sequences and orientations of both DNA vaccines, several sets of restriction digestion experiments were conducted. As shown in Figure 4 (lanes 2 and 3), digestion of both pc1A7VHLnVL and pc1A7VLLnVH by EcoRV plus XbaI generated a small fragment 800 bp, which was absent in a similar digest of the empty vector, pcDNA (lane 1). One KpnI restriction site was present in the VL chain and a second one in the multiple cloning site of pcDNA3 (Fig 3). As expected, KpnI digestion of pc1A7VLLnVL generated two fragments of 250 and 5900 bp sizes (lane 6), whereas the pc1A7VHLnVL digest had two bands of sizes 600 and 5600 bp (lane 5). We concluded from the results of restriction digestion experiments that the plasmids have the desired orientation. Figure 4. Confirmation of the orientations of the expression plasmids by restriction digestion. Plasmids,pcDNA (lanes 1,4), In vitro expression of 1A7 scFv protein by the plasmid pc1A7VHLnVL (lanes 2,5),and pc1A7VLLnVH (lanes 3,6) were vectors digested with EcoRV plus XbaI (lanes 1±3) or KpnI (lanes 4±6). The products were analyzed by electrophoresis in a 1% agarose gel. For the DNA vaccines to be effective, it is necessary that 1A7 The molecular weight standards are shown in the right margin. scFv be transcribed and translated by the eukaryotic machinery. We used the TNT T7 Quick-Coupled Transcrip- tion/Translation System to demonstrate the translation of the plasmids in vitro into scFv. SDS-PAGE electrophoresis of metabolically labeled translation products is presented in Figure 5. A single protein band of 30 kDa was translated using either pc1A7VHLnVL (lane 4) or pc1A7VLLnVH (lane 5); however, no radioactive band could be detected using the empty vector, pcDNA3, as the template (lane 1). Positive control DNA expressing -galactosidase (lane 2) and luciferase (lane 3) was translated into proteins of expected sizes (118 and 61 kDa, respectively). The biological activity of expressed 1A7 scFv was evaluated by transient transfection of CHO-KI cells with the plasmids. CHO-KI cells were transfected separately with pc1A7VHLnVL, pc1A7VLLnVH, or pcDNA separately, as described in Materials and Methods. Because leader sequences were incorporated in the 50 ends of these plasmids, the scFv products synthesized are expected to be released into the culture medium. Transfected cells were cultured in 35 the presence of [ S] L -methionine to label the proteins for 5 hours. The culture supernatants were treated with various antibodies and the labeled products were analyzed by SDS- PAGE electrophoresis as described in Materials and Methods. The results of these experiments are presented in Figure 6. Treatment with the monoclonal Ab1 antibody, 14G2a, of the supernatant from both pc1A7VHLnVL- and pc1A7VLLnVH-transfected CHO-KI cells resulted in the precipitation of 30-kDa bands (lanes 1 and 7), whereas no such product was present in the supernatant from pcDNA- transfected CHO-KI cells (lane 4). No protein band was present when the same supernatant was treated with either a control, isotype-matched murine monoclonal antibody (lanes 3 and 9), or the monoclonal antibody, 8019 (lanes Figure 3. Construction of the plasmids expressing 1A7 scFv in two 2 and 8), specific for an unrelated anti-idiotype antibody, 10 orientations. The expression plasmids were made in two orientations, 3H1. No labeled protein could be detected by treatment of VHLnVL and VLLnVH,as described in Materials and Methods. the culture supernatant from pcDNA-transfected CHO-KI

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translated into scFv in vivo. Fifty micrograms of pc1A7VLLnVH was injected into the leg quadriceps of C57BL/6 mice. On different days after injection, the injection site was surgically removed, total RNA from the muscle was extracted, and RT-PCR was performed. RT- PCR products in all cases were of the expected size, 800 bp (Fig 7A). To exclude the possibility of plasmid DNA contamination, all RNA samples were treated with DNase I prior to amplification. Moreover, no DNA was detected by direct PCR using 50 -VL and 30 -VH primers excluding the possibility of DNA contamination. To further confirm that RT-PCR products represent the VLLnVH DNA fragments of the 1A7 gene, the amplified products were digested with KpnI, and the products were subjected to Southern blot using randomly labeled pc1A7VLLnVH as the probe. Southern Figure 5. In vitro transcription/translation of pc1A7VLLnVH and blots of the RT-PCR products demonstrated two expected pc1A7VHLnVL. The plasmids (1 g/10 l) were mixed with 40 lof TNT mixture in the presence of [ 35S]methionine and incubated at DNA bands of sizes 600 and 200 bp (Fig 7B). Taken 308C for 1 hour. Aliquots were analyzed by SDS-PAGE as described together, these results demonstrate that pc1A7VLLnVH is in Materials and Methods. The products were: lane 1,pcDNA; lane 2, expressed in vivo and the expression lasts for at least 90 days. DNA for -galactosidase; lane 3,DNA for luciferase; lane 4, pc1A7VHLnVL; lane 5,pc1A7VLLnVH. The molecular weight Immunohistochemistry of muscle tissue following DNA standards are shown in the right margin. vaccination In vitro experiments demonstrated that scFv of 1A7 was expressed and secreted from transiently transfected CHO- K1 cells and that the resulting proteins specifically bound to cells by any of these antibodies. Taken together, these results the monoclonal antibody, 14G2a. To detect the expression of demonstrated that the 1A7 scFv translated from the plasmid 1A7 scFv in vivo,50g of pc1A7VLLnVH was injected into constructs are recognized by the specific monoclonal Ab1 the muscles of the mice. After 7 days, the muscle tissues antibody, 14G2a, and are considered biologically active. were excised and immunostained as described under Both formats of the plasmids were translated in the Materials and Methods. The immunostaining results are eukaryotic CHO-KI cells. presented in Figure 8. Whereas no staining was detected in muscles injected with pcDNA with any of the antibodies In vivo transcription of scFv of 1A7 (column II, lanes D±F), distinct staining of the muscle cells To be effective as vaccines, the host cells near the injected injected with pc1A7VLLnVH was observed following sites must take up the DNA and transcribe and translate it to incubation with the specific monoclonal antibody, 14G2a an antigen. The antigen, in turn, must invoke the desired (column I, lane C). As expected, no staining was detected in immunity. Most of the injected DNA, however, are likely to pc1A7VLLnVH-injected muscles following incubation be degraded by tissue DNases even before they are taken up with an unrelated monoclonal antibody, 8019,10 or when inside the host cells. Therefore, it is necessary to demonstrate the primary antibody was omitted (column I, lanes B and A, that the injected plasmids are indeed being taken inside the respectively). cells and genes encoding the 1A7 scFv are transcribed and

Figure 6. Transfection of CHO-KI cells with the expression plasmids. Figure 7. Expression of 1A7 scFv mRNA in vivo by the injected CHO-KI cells were transfected with plasmids,pc1A7VHLnVL (lanes, plasmids. Total RNA was extracted from the injection sites on 4±6) orpc1A7VLLnVH (lanes,7±9),as described in Materials and different days; after injection of 50 g of pc1A7VLLnVH,the RNA was Methods. Secreted labeled proteins were precipitated with indicated amplified by RT-PCR (A). The products of RT-PCR were digested antibodies,monoclonal anti-1A7/GD2 antibody,14G2a,isotype with KpnI and Southern blots were performed using labeled matched unrelated anti-idiotype antibody,8019, 10 or control isotype pc1A7VLLnVH as the probe (B). RT-PCR of the RNA samples matched unrelated antimouse antibody (anti-IgG). The products with -actin±specific primers (C). Sizes of the DNA fragments are were analyzed by SDS-PAGE on a 12% gel. shown in the right margins.

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Figure 10. Induction of anti-GD2 antibodies (Ab10 response) in the sera of mice immunized with the plasmid expressing 1A7 scFv. Falcon plates (96-well) were coated with various gangliosides (100 ng/well) and reacted with sera from mice after four immunizations Figure 8. Expression of scFv proteins in the muscles injected with the (every other week) with p1A7VHLnVL. The sera were diluted 1:10 expression plasmids. pc1A7VLLnVH (column 1) and pcDNA with PBS±BSA solution and 100 L of the diluted sera was added per (column 2) were injected into leg quadriceps of C57BL/6 mice (50 well. Details of the assays are described in Materials and Methods. g/site) and after 7 days,the muscle tissues were removed and immunostained with indicated antibodies as described in Materials and Methods. Lanes A,D,no antibody; lanes B,E unrelated isotype In order to investigate whether plasmids encoding scFv of matched anti-idiotype antibody,8019; 10 lanes C,F,1A7/GD2- 1A7 could also induce anti-1A7 antibody production in specific monoclonal antibody,14G2a. mice, we injected C57BL/6 mice with pc1A7VHLnVL, pc1A7VLLnVH, and pcDNA3 as described in Materials Anti-1A7 antibody response (Ab3 response) by and Methods. A single immunization, as well as multiple immunization of mice with DNA vaccines immunizations (every other week), was performed with the plasmids. Sera from the mice were drawn before every We previously demonstrated that injection of 1A7 anti- immunization and the amount of anti-1A7 antibodies was idiotype antibody induced the production of anti-1A7 12,22 13 assayed by ELISA as described in Materials and antibodies in animals as well as in cancer patients. Methods. The results summarized in Figure 9 demonstrate that mice injected with both pc1A7VHLnVL and pc1A7VLLnVH induced the production of anti-1A7 antibodies in the sera of mice, whereas such antibodies are not detectable in mice immunized with pcDNA. A single immunization with pc1A7VLLnVH, without any adjuvant or carrier protein, induced anti-1A7 antibody production; however, multiple immunizations augmented the immune response.

Anti-GD2 antibody response (Ab10 response) of mice immunized with p1A7VHLnVL To determine whether a clinically relevant Ab10 immune response was also induced by the DNA vaccines, groups of mice (three mice per group) were injected intramuscularly with 50 g of pc1A7VHLnVL. Preimmune sera and sera obtained after four immunizations (every other week) were separately pooled for the assay of anti-GD2 by ELISA, as described in Materials and Methods, and the results are Figure 9. Induction of anti-1A7 antibodies (Ab3 response) in the presented in Figure 10. Significant anti-GD2 antibody could sera of mice immunized with the expression plasmids. Groups of 6± be detected in the pooled sera of mice immunized with 8-week-old C57BL/6 female mice (n=8±11) were immunized with pc1A7VHLnVL compared to pooled preimmune sera. The 100 g of the plasmids once (day 0) or every other week for four times,and sera were drawn at 15-day intervals. Anti-1A7 antibodies binding of the mouse antibodies was maximum with GD2; induced in the sera were assayed by ELISA as described in Materials however, binding with other gangliosides was most likely and Methods. Each point represents the mean OD at 405 nm for each due to the presence of contaminants in the various ganglio- group. side preparations. This conclusion is supported by the fact

Cancer Gene Therapy, Vol 7, No 11, 2000 ZEYTIN, TRIPATHI, BHATTACHARYA-CHATTERJEE, ET AL: DNA VACCINE ENCODING SCFV OF ANTI-IDIOTYPE ANTIBODY 1435 that the specific monoclonal anti-GD2 antibody, 14G2a, also 30±90 minutes after injection, more than 95% of the DNA is reacted with the other gangliosides. gone.27 ± 29 However, plasmid DNA has been also shown to persist for as long as 19 months.29 We could detect the Cellular immune responses by DNA vaccines plasmid for at least 3 months after injection, and during this DNA vaccines have been shown to induce both cellular and period, the plasmids were actively transcribed (Fig 7). humoral immune responses against a variety of .14 Translation of the plasmid into protein also took place for at To determine whether the plasmids expressing 1A7 scFv least 7 days (Fig 8), a time interval adequate for invoking could induce T-cell responses, spleen proliferation assays humoral and cellular immunity in mice. were performed as described in Materials and Methods. Incorporation of genes into DNA vaccines has been demonstrated to augment the immunity induced by the Proliferation indices with 1A7 as stimulant were similar to 30 indices obtained by using 3H1, the unrelated anti-idiotype transgene. Co-injection of plasmids expressing cytokines along with DNAvaccines also appears to enhance transgene- antibody (data not shown). Stimulation indices with 31 concanavalin A as stimulant ranged from 5- to 10-fold induced immunity. Incorporation of IL-2 or GM-CSF under the assay conditions. genes into our plasmid, or co-injection of plasmids expressing these cytokines, did not increase the level of expression of 1A7 scFv in the sera of injected mice with these DISCUSSION constructs (data not shown). DNA encoding secreted proteins has been shown to induce better immune responses The immunization of mice with plasmids encoding the scFv than that encoding cell-associated proteins.32 To augment the of the anti-idiotype antibody, 1A7, induced anti-1A7 immunity of our DNA vaccines, we incorporated the antibodies in the sera of mice. More importantly, anti-GD2 immunoglobulin leader sequences for secretion of the scFv. antibodies by the DNA vaccines are clinically important, as Anti-idiotype antibody, 1A7, induced anti-1A7 and anti- adequate amounts of these antibodies with high avidity are GD2 immunity in small animals,12 nonhuman primates,22 as expected to bind to the tumor cell surface GD2 to induce cell well as humans.13 However, there are several advantages of lysis. Tumor cell lysis may include antibody-dependent DNA vaccines over protein antigens. DNA immunization has cellular cytotoxicity and complement mediated cytotoxicity. been shown to enhance dramatically the avidity of the Levels of anti-GD2 antibodies in mice induced by antibody generated compared to soluble proteins.30 No 1A7VHLnVL were 3-fold higher than the level in pre- carrier proteins or adjuvants are necessary for this type of immunized mice. This binding, however, was not restricted vaccine. In our study, delivery of the plasmids in PBS to GD2, as significant binding to other gangliosides was also invoked detectable anti-1A7 scFv antibodies in mice observed (Fig 10). This could be due to the presence of following intramuscular injection. Additional advantages of contaminants in the ganglioside preparations because DNA vaccines over protein antigens are that the plasmids are specific monoclonal anti-GD2 antibody, 14G2a, also bound stable and inexpensive to manufacture in pure form and can to other gangliosides in these experiments (Fig 10). An be transported at room temperature. Moreover, multiple alternative explanation for the binding of these antibodies to can be safely performed because double- other gangliosides could be the close structural similarities of stranded are non-immunogenic.31 Most importantly, various gangliosides. Another monoclonal anti-GM2 anti- DNA vaccines have been reported to invoke cellular immune body, 3-207, derived from the transformed lymphocytes responses in a number of studies.34 To our disappointment, from a patient vaccinated with purified GM2 also bound to the preliminary studies using T-cell proliferation assays did GD2.23 Taietal24 also obtained a series of antibodies, which not suggest the induction of cellular immune responses by showed considerable reactivity to other gangliosides follow- 1A7 anti-idiotype antibody. However, passively adminis- ing immunization of mice with purified GD2. tered antibodies as well as vaccine-induced antibodies raised ScFv is the smallest antibody fragment capable of binding against cell surface antigens, such as gangliosides, have been an antigen.16,25 The scFv can be constructed with either shown to have antitumor efficacy in animal models as well as sequence, VHLnVL or VLLnVH.25 The VHVL construct in patients (reviewed in Ref. 35 ). These antibodies are ideal typically exhibits Euclidean distances between Ln termini of for removal of circulating tumor cells or micrometastases 29±35 AÊ , whereas the VL±VH orientation generally from the bloodstream or lymphatic system, consequently exhibits distances that are 5±10 AÊ longer for the same limiting cancer spread. At present, we are evaluating the Fv.26 Thus, differential orientation of a domain may distort prophylactic and therapeutic efficacy of our DNA vaccines in the native Fv conformation. An anti-idiotype antibody acts a murine lymphoma model (EL-4), which constitutively as an antigen intended to induce Ab10 in the injected host. expresses GD2.36 Distortion of the scFv derived from an anti-idiotype antibody is likely to affect its antigen mimicry. With an anti-idiotype antibody mimicking the carcinoembryonic ACKNOWLEDGMENTS antigen, we found that only one format, VHLnVL, had biological activity.21 In the 1A7 system, both formats of scFv We thank R.A. Reisfeld (The Scripps Research Institute) for expressed by the plasmids had nearly equal biological kindly providing the monoclonal anti-GD2 antibody, 14G2a activities, assayed in vitro as well as in vivo. and Lewis Kelly for critically reading the manuscript. This Several studies have demonstrated that circular super- work was supported in part by Grants RO1 CA 72773 and coiled plasmid DNA can degrade to an extent, and within RO1 CA72018 from the National Institutes of Health.

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REFERENCES 18. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor: Cold Spring 1. Hamilton WB, Helling F, Lloyd KO, Livingston PO. Ganglio- Harbor Laboratory Press; 1989. side expression on human malignant melanoma assessed by 19. Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C. quantitative immune thin-layer chromatography. Int J Cancer. Sequences of Proteins of Immunological Interest. 5th ed. 1993;53:566±573. Bethesda: US Department of Health and Human Services, 2. Livingston P. Ganglioside vaccines with emphasis on GM2. Public Health Service, National Institutes of Health; 1991. Semin Oncol. 1998;25:636±645. 20. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic 3. Ritter G, Ritter-Boosfeld E, Adluri R, et al. Analysis of the local alignment search tool. J Mol Biol. 1990;215:403±410. antibody response to immunization with purified O-acetyl 21. Tripathi PK, Qin H, Deng S, et al. Antigen mimicry by an anti- GD3 gangliosides in patients with malignant melanoma. Int J idiotypic antibody single-chain variable fragment. Mol Im- Cancer. 1995;62:668±672. munol. 1998;35:853±863. 4. Kitamura K, Livingston PO, Fortunato SR, et al. Serological 22. Sen G, Chakraborty M, Foon KA, Reisfeld RA, Bhattacharya- response patterns of melanoma patients immunized with a Chatterjee M. Preclinical evaluation in nonhuman primates of GM2 ganglioside . Proc Natl Acad Sci USA. murine monoclonal anti-idiotype antibody that mimics the 1995;92:2805±2809. disialoganglioside GD2. Clin Cancer Res. 1997;3:1969±1976. 5. Tai T, Cahan LD, Tsuchida T, Saxton RE, Irie RF, Morton DL. 23. Yamaguchi H, Furukawa K, Fortunato SR, et al. Human Immunogenicity of melanoma-associated gangliosides in monoclonal antibody with dual GM2/GD2 specificity derived cancer patients. Int J Cancer. 1985;35:607±612. from an immunized melanoma patient. Proc Natl Acad Sci 6. Livingston PO. Approaches to augmenting the immunogenicity USA. 1990;87:3333±3337. of melanoma gangliosides: from whole melanoma cells to 24. Tai T, Kawashima I, Tada N, Ikegami S. Different reactivities ganglioside±KLH conjugate vaccines. Immunol Rev. 1995;145: of monoclonal antibodies to ganglioside lactones. Biochim 147±166. Biophys Acta. 1988;958:134±138. 7. Jerne NK. Towards a network theory of the . 25. Huston JS, Mudgett-Hunter M, Tai MS, et al. Protein Ann Immunol (Paris). 1974;125C:373±389. engineering of single-chain Fv analogs and fusion proteins. 8. Herlyn D, Somasundaram R, Li W, Maruyama H. Anti- Methods Enzymol. 1991;203:46±88. idiotype cancer vaccines: past and future. Cancer Immunol 26. Huston JS, Geoge AJT, Tai MS, et al. Single-chain Fv design Immunother. 1996;43:65±76. and production by preparative folding. In: Borrebaeck CAK, 9. Bhattacharya-Chatterjee M, Pride MW, Seon BK, Kohler H. ed. Antibody Engineering. 2nd ed. New York: Oxford Idiotype vaccines against human T cell acute lymphoblastic University Press; 1995:185±227. leukemia: I. Generation and characterization of biologically 27. Lew D, Parker SE, Latimer T, et al. Cancer gene therapy using active monoclonal anti-idiotypes. J Immunol. 1987;139:1354± plasmid DNA: pharmacokinetic study of DNA following 1360. injection in mice. Hum Gene Ther. 1995;6:553±564. 10. Bhattacharya-Chatterjee M, Mukerjee S, Biddle W, Foon KA, 28. Manthorpe M, Cornefert-Jensen F, Hartikka J, et al. Gene Kohler H. Murine monoclonal anti-idiotype antibody as a therapy by intramuscular injection of plasmid DNA: studies on potential network antigen for human carcinoembryonic anti- firefly luciferase gene expression in mice. Hum Gene Ther. gen. J Immunol. 1990;145:2758±2765. 1993;4:419±431. 11. Bhattacharya-Chatterjee M, Mrozek E, Mukerjee S, Ceriani 29. Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A. Long-term RL, Kohler H, Foon KA. Anti-idiotype antibodies as potential persistence of plasmid DNA and foreign gene expression in therapeutic agents for human breast cancer. In: Ceriani RL, ed. mouse muscle. Hum Mol Genet. 1992;1:363±369. Antigen and Antibody Molecular Engineering in Breast 30. Syrengelas AD, Chen TT, Levy R. DNA immunization induces Cancer Diagnosis and Treatment. New York: Plenum Publish- protective immunity against B-cell lymphoma. Nat Med. ing; 1994:139±148. 1996;2:1038±1041. 12. Sen G, Chakraborty M, Foon KA, Reisfeld RA, Bhattacharya- 31. Stevenson FK, Zhu D, King CA, Ashworth LJ, Kumar S, Chatterjee M. Induction of IgG antibodies by an anti-idiotype Hawkins RE. Idiotypic DNA vaccines against B-cell lympho- antibody mimicking disialoganglioside GD2. J Immunother. ma. Immunol Rev. 1995;145:211±228. 1998;21:75±83. 32. Boyle JS, Koniaras C, Lew AM. Influence of cellular location 13. Foon KA, Sen G, Hutchins L, et al. Antibody responses in of expressed antigen on the efficacy of DNA vaccination: melanoma patients immunized with an anti-idiotype anti- cytotoxic T lymphocyte and antibody responses are suboptimal body mimicking disialoganglioside GD2. Clin Cancer Res. when antigen is cytoplasmic after intramuscular DNA 1998;4:1117±1124. immunization. Int Immunol. 1997;9:1897±1906. 14. Lai WC, Bennett M. DNA vaccines. Crit Rev Immunol. 33. Boyle JS, Silva A, Brady JL, Lew AM. DNA immunization: 1998;18:449±484. induction of higher avidity antibody and effect of route on T- 15. Huston JS, Levinson D, Mudgett-Hunter M, et al. Protein cell cytotoxicity. Proc Natl Acad Sci USA. 1997;94:14626± engineering of antibody binding sites: recovery of specific 14231. activity in an anti-digoxin single-chain Fv analogue 34. Spooner RA, Deonarain MP, Epenetos AA. DNA vaccination produced in . Proc Natl Acad Sci USA. for cancer treatment. Gene Ther. 1995;2:173±180. 1988;85:5879±5883. 35. Zhang H, Zhang S, Cheung NK, Ragupathi G, Livingston PO. 16. Bird RE, Hardman KD, Jacobson JW, et al. Single-chain Antibodies against GD2 ganglioside can eradicate syngeneic antigen-binding proteins. Science. 1988;242:423±426. cancer micrometastases. Cancer Res. 1998;58:2844±2849. 17. Chomczynski P, Sacchi N. Single-step method of RNA 36. Zhao XJ, Cheung NK. GD2 oligosaccharide: target for isolation by acid guanidinium thiocyanate±phenol±chloroform cytotoxic T lymphocytes. J Exp Med. 1995;182:67±74. extraction. Anal Biochem. 1987;162:156±159.

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