Gene Therapy (2001) 8, 1555–1561  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Anti-idiotypic induced by genetic immunisation are directed exclusively against combined VL/VH determinants

F Benvenuti1,2 and OR Burrone1 1International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy

DNA vaccines encoding the idiotype of immunoglobulins of brane-displayed idiotypes revealed that DNA immunisation tumour B cells were shown to induce protection in several induced a polyclonal response restricted to confor- mouse lymphoma models. The mechanism of rejection of mational combined formed by the parental VL/VH tumour cells has not been fully understood, but there is association. Immune sera raised by scFv DNA vaccination strong evidence suggesting that engagement of the idiotype did not show any detectable reactivity towards chimeric by anti-idiotypic antibodies may directly result in inhibition of scFvs containing only one of the two immunising V regions, tumour growth. In this study, we have investigated the struc- indicating that the response against combined VL/VH deter- tural basis of the idiotypic/anti-idiotypic interaction following minants is highly dominant. Remarkably, the same immun- immunisation with DNA vaccines. scFvs containing only one ogen, delivered as scFv protein, induced antibodies also of the two tumour-derived V regions recombined to an irrel- directed against chain-specific determinants. These findings evant V region partner were generated. These constructs indicate that presentation of properly folded idiotypes results encoding a secretory form of the scFv were used as immun- in a highly specific antibody response directed exclusively to ogens to induce anti-Id antibodies. The same scFvs were private idiotypic determinants of the VL/VH combination of expressed as membrane-bound molecules on the surface of the . Gene Therapy (2001) 8, 1555–1561. mammalian cells. Analysis of immune sera on the mem-

Keywords: anti-idiotypic antibodies; DNA vaccination; lymphoma immunotherapy

Introduction murine BCL1 lymphoma model, genetic vaccination with a construct containing the IdBCL1 in a scFv format, fused Immunoglobulins bind through the variable (V) to the CH3 exon of the human IgG1 constant region was regions of the two-polypeptide components, the light (L) able to induce high titres of anti-Id antibodies and resist- and the heavy (H) chains. Sequence variability of ance to tumour challenge.7 We showed that both tumour- immunoglobulin V regions is generated by differential V derived V regions were essential for the induction of region genes usage, genetic recombination and somatic protective . hypermutation. The particular combination of the two V Recent studies indicated the importance of direct regu- regions define the idiotype (Id), a unique antigenic struc- latory effects of anti-Id antibodies on tumour growth.8,9 tural feature that can be specifically detected by anti-Id This raises the question of the binding specificity of anti- antibodies. Since idiotypes reflect the uniqueness of each idiotypic antibodies. Although several strategies to immunoglobulin molecule, and each B induce high titres of anti-lymphoma idiotype antibodies expresses a single arrangement of light and heavy chain using scFv DNA vaccines have been described, little is V regions, idiotypes represent clonal signatures of indi- + known on the structural basis of the idiotype/anti- vidual B cells. In the case of surface Ig malig- idiotype interaction following DNA vaccination. The con- nancies, such as B cell lymphomas, idiotypes are there- tribution of VH and VL variable domains to the idiotype fore ideal tumour-associated antigens that can be used as structure has been investigated in other systems. Most of targets for immunotherapy. Active immunisation using the studies involved the use of mAbs, yielding different 1–3 idiotypic protein and more recently, genetic immunis- results. There are reports in which the reactivity of mAbs ation with plasmid DNA encoding tumour-derived Ig was shown to depend on the presence of both parental 4–7 variable regions was found to be effective in inducing V regions,10–12 as well as cases in which the binding was anti-Id antibodies and protection against tumour chal- 13–15 mapped on the VH, regardless of the associated VL. lenge. Our laboratory has demonstrated that, in the Analysis of the polyclonal anti-Id responses of sera raised against the major Id (CRIA) of anti-p-azobenzenearsonate revealed that 20 to 35% of anti-Id antibodies reacted with Correspondence: OR Burrone 2Present address: U520, Institute Curie, 12 rue Lhomond, 75005 Paris, high affinity with H chains recombined with non-CRIA 16 France related L chains. Received 16 March 2001; accepted 12 July 2001 So far, no data have been reported regarding the speci- Specificity of anti-Id antibodies induced by scFv DNA immunisation F Benvenuti and OR Burrone 1556 ficity of anti-Id responses following DNA immunisation. Specificity of anti-Id antibodies for VL and VH combined In this study, we explored the contribution of the two determinants tumour-derived V regions in the induction of the anti-Id We investigated the overall polyclonal anti-Id antibody immune response by scFv DNA vaccination, as well as response raised by pBCL1, in terms of reactivity against

the specificity of the polyclonal anti-Id antibodies for specificVL,VH, or combined VL/VH determinants combined VL/VH versus not combined VL or VH determi- expressed in the context of folded idiotypes. To address nants. We show that genetic immunisation with plasmids this issue we developed a genetic strategy to display encoding scFv idiotypes induced a polyclonal immune idiotypes arising from different V region pairings on the response that is exclusively directed against determinants surface of transfected cells. We designed a membrane depending on the immunising VL/VH association. On the version of SIP (⑀-mSIP), based on the human membrane contrary, sera raised by immunisation with purified scFv IgE . These molecules contain a scFv fused to the BCL1 BCL1 protein reacted with VL and VH determinants C-terminal region of the human membrane IgE H chain, associated with irrelevant V region partners. from ⑀CH4 down to the cytoplamic tail.19 ⑀-mSIP are efficiently expressed as membrane proteins20 allowing Results flow cytometry visualisation of anti-Id Abs reacting with the displayed idiotypic determinants. DNA cassettes Induction of anti-Id antibodies by scFv DNA encoding scFvs of BCL1, 6C6 and the two BCL1/6C6 immunisation chimera were cloned into the ⑀-mSIP vector (Figure 1a) We have previously demonstrated that genetic immunis- and transfected into the Ig−, non-secreting mouse myel- ation with a scFv idiotype construct was able to induce oma cell line Sp2/0. Positive clones showing comparable a protective response against the BCL1 lymphoma.7 The expression levels for each construct were selected gene construct contained the two BCL1 variable regions (Figure 2b). in the VL-VH orientation, separated by an 18-amino acid Proper display of the IdBCL1 on Sp2/0 cells transfected linker that ensures optimal folding of the scFv molecules. with ⑀-mBCL1 was demonstrated by flow cytometry 4 In agreement with studies in other lymphoma models, using two specific reagents: an anti-IdBCL1 mAb the fusion of a xenogeneic domain (the CH3 domain of (10/4A12)21 and the anti-Id serum raised by pBCL1 the human IgG1 constant region) to the idiotype-enco- (Figure 2c). In addition, since mAb 10/4A12 binds to VH ding sequence, proved to be essential for the induction 21 6C6 BCL1 of BCL1 we tested it on the VL /VH chimera. As of anti-Id antibodies (data not shown), indicating the shown in Figure 2c, mAb 10/4A12 stained SP2/0 trans- need to break tolerance. In addition, this scFv- BCL1 17 fected cells, indicating that VH is also properly dis- huCH3 design (called SIP) leads to efficient expression played in the context of a non-parental Id. No cross-reac- and secretion of the encoded protein. tivity was observed with sera induced by p6C6 on cells In order to evaluate the relative contribution of displaying the BCL1 idiotype, thus excluding the pres- tumour-derived VL and VH in the induction of the anti-Id ence of antibodies directed against the 18 aa linker antibody response, we generated two different constructs between V and V , identical for all the soluble and (pV BCL1/V 6C6 and pV 6C6/V BCL1) containing a scFv L H L H L H membrane scFvs (see below). with either V or V of BCL1 paired, respectively, to V L H H We next examined the reactivity of sera induced by the or V of an irrelevant murine , mAb L protective pBCL1 construct7 towards the chimeric 6C6.18 The 6C6 antibody was chosen because its V regions idiotypes displayed on the surface of Sp2/0 cells. Strik- have low homology to those of BCL1. While mAb 6C6 ␬ ingly, polyclonal anti-Id immune sera raised by pBCL1 has a light chain and a VH corresponding to family VI, ␭ did not react to either of the two BCL1/6C6 Id chimeras BCL1 light chain is (family I) and the VH corresponds BCL1 6C6 6C6 BCL1 to family XXIV. As a control, we also constructed a com- (VL /VH and VL /VH , indicating lack of anti- 6C6 Id antibodies directed against determinants on folded plete scFv -huCH3 (p6C6) (Figure 1a). All these con- BCL1 BCL1 structs showed similar levels of expression in transfected VL or VH in the context of non-parental pairings Sp2/0 cells. (Figure 3). The ability of each construct to induce an antibody To extend the analysis to other cases, we performed response against the BCL1 idiotype (IdBCL1) and against crossed binding analysis of the displayed idiotypes with BCL1 6C6 6C6 BCL1 the human ␥1-CH3 portion was analysed by ELISA on sera raised by pVL /VH ,pVL /VH and p6C6. sera of animals receiving three shots of DNA-coated gold Flow cytometry data shown in Figure 4 clearly demon- particles. While serum levels of antibodies directed strated that each membrane-displayed idiotype was only against the hu-CH3 domain were comparable for all four recognised by antibodies induced by the corresponding BCL1 6C6 6C6 BCL1 soluble scFv. Noteworthy, no detectable cross-reactivity constructs, both chimeric pVL /VH ,pVL /VH as well as the control p6C6 failed to evoke detectable lev- was observed by any of the immune sera raised with els of anti-IdBCL1 antibodies (Figure 1b). Immune sera scFvs containing only one of the two V regions. All four were also tested by flow cytometry for their ability to cases presented provided strong evidence that the anti- bind the Id expressed on the surface of BCL1 lymphoma Id immune response induced by our scFv-huCH3 genetic cells (Figure 1c). Independently from the dilution tested, vaccination was strictly confined to conformational only sera from animals immunised with pBCL1 were able VL/VH combined determinants with no reactivity against to stain BCL1 cells, thus confirming the inability of either determinants displayed on either VL or VH, indepen- of the two tumour-derived V regions associated with an dently from the other V region partner. The inability of irrelevant partner to elicit antibodies against the parental sera raised with pBCL1 to recognise purified BCL1 IgM idiotype. Hereby, the pairing of both parental VL and VH protein in Western blotting confirmed that DNA vacci- in a scFv was essential to induce antibodies against the nation induced exclusively conformational anti-Id Ab IdBCL1. (data not shown; see also Figure 5).

Gene Therapy Specificity of anti-Id antibodies induced by scFv DNA immunisation F Benvenuti and OR Burrone 1557

Figure 1 (a) Schematic representation of constructs used for genetic vaccination. (b) Ab responses induced by DNA immunisation. Antibody levels were measured by ELISA. Mean OD values (1:400) for each vaccinated group, corresponding to anti-IdBCL1 and anti-huCH3, are indicated as solid bars. (c) Flow cytometry analysis of antibodies induced by DNA immunisation on BCL1 lymphoma cells. Tumour cells were incubated with dilutions of immune sera from the indicated groups. The figure shows a representative experiment at 1:200 dilution.

Reactivity against chain-specific determinants Discussion The lack of reactivity against chain-specific determinants suggested that the response against epitopes deriving Active immunotherapy of B cell malignancies using from the VL/VH association was highly dominant. This idiotypic protein and DNA vaccination has been success- implies presentation to the of a confor- ful in murine lymphoma models. Although several mational idiotype, as the one expressed on the native reports indicated that anti-Id antibodies play an essential immunoglobulin. This is the case of our immunising sol- role in the rejection of tumour cells.6,8,9,22,23 the binding uble scFv molecules, which are efficiently secreted in of anti-Id antibodies to the idiotype on the surface of lym- tissue culture supernatants of transfected cells main- phoma cells has not been mapped. In the present study taining the -binding capacity.17 It was then inter- we explored the structural basis of the Id/anti-Id interac- esting to examine whether the observed reactivity was tion upon immunisation with scFv plasmid DNA. Start- peculiar to genetic vaccination. To analyse with the same ing from our BCL1 model system we generated four dif- system the reactivity of sera induced by the classical pro- ferent Id genetic constructs, two parental (BCL1 and 6C6) tocol of protein immunisation, we immunised mice with and two chimeric (mismatched BCL1/6C6 V region the scFvBCL1-huCH3 protein purified from SP2/0 cells associations) that were used both as in a transfected with pBCL1 (Figure 1a). We chose for the soluble SIP version (scFv-huCH3), as well as displayed analysis protein-induced sera yielding ELISA titres of target idiotypes in their membrane-bound forms anti hu-CH3 antibodies identical to that induced by (⑀-mSIP). pBCL1 DNA. Interestingly, flow cytometry (Figure 5a) Our strategy is particularly relevant for the analysis of revealed that anti-Id antibodies induced by protein the Id/anti-Id interaction in native-like conditions. In immunisation, as opposed to those induced by DNA flow cytometry, as opposed to ELISA tests, Ids are dis- immunisation, reacted with the two chimeric displayed played on the surface of living cells rather than purified BCL1 6C6 6C6 BCL1 idiotypes (VL /VH and VL /VH ). Thus, protein and coated on a plastic support. In addition, each V immunisation induced an anti-Id response containing region can be analysed independently from its own part- BCL1 BCL1 antibodies that also recognise VL and VH chain- ner, but folded as it would be in the original idiotype. specific determinants, in the context of a different Moreover, the idiotype protein displayed in the scFv for- idiotype. In addition, the protein-induced serum, but not mat is identical to the one used to elicit the immune the DNA-induced serum, was able to recognise linear response. chain-specific idiotypic epitopes as revealed by their reac- We demonstrated that the parental V regions associ- tivity on Western blots. Only the protein-induced serum ation in our DNA vaccine is an absolute requirement to recognised the three different denatured idiotypes that induce anti-Id antibodies against the BCL1 IgM. The anti- BCL1 6C6 BCL1 contain BCL1 V regions, namely BCL1, VL /VH , and Id antibodies induced by our scFv DNA vaccination 6C6 BCL1 VL /VH (Figure 5b). These results demonstrate that were previously shown to induce protection against the specificity of the anti-Id antibody response depends tumour challenge.7 In this report, we show that these on the way in which the same protein antigen is antibodies are directed exclusively against confor- presented to the immune system. mational combined VL/VH determinants. Although it is

Gene Therapy Specificity of anti-Id antibodies induced by scFv DNA immunisation F Benvenuti and OR Burrone 1558

Figure 2 (a) Scheme of the ⑀-mSIP construct used for membrane display of idiotypes in Sp2/0 cells. (b) Membrane expression of ⑀-mSIPs by Sp2/0 cells transfected with the four ⑀-membrane scFv constructs. Sp2/0 wild-type (control) or transfected cells (as indicated) were incubated with FITC- conjugated anti-human IgE and analysed by flow cytometry. (c) Idiotype display on transfected Sp2/0 cells. Expression of the IdBCL1 on cells transfected ⑀ BCL1 with the -mBCL1 construct was assessed using mAb 10/4A12 and immune sera raised by pBCL1. Display of VH on cells expressing the chimeric 6C6 BCL1 Id VL /VH was revealed with mAb 10/4A12.

likely that most anti-Id antibodies recognise epitopes for- med by combined VL/VH sequences, it cannot be excluded that some recognise determinants on a single V region, whose conformations are strictly dependent on the other V region partner. We extended the analysis to three additional idiotypes: one containing V regions from an unrelated murine mAb (6C6) and two chimeric Id generated by mismatching BCL1 and 6C6 V regions. Crossed analysis of the four different immune sera on the four membrane displayed idiotypes showed that, in all cases, the anti-Id immune response was directed exclusively at the original immunising VL/VH combination, with complete absence of antibodies recognising determinants in any of the sin- gle V regions displayed in the context of a different idiotype. These data indicated that epitopes formed by the VL/VH association are highly dominant, inducing an immune response in which antibodies against them become the major component. However, epitopes dis- played on single V regions exist and are immunogenic, Figure 3 Reactivity of anti-Id antibodies induced by pBCL1. Flow cyto- BCL1 metry of Sp2/0 cells displaying parental or chimeric idiotypes following as demonstrated by the reactivity of scFv -huCH3 pro- ⑀ ⑀ BCL1 6C6 ⑀ 6C6 BCL1 ⑀ transfection with: -mBCL1, -mVL /VH -m VL /VH and - tein-induced sera. As expected, the protein induced m6C6, reacted with different dilutions of sera induced by genetic immuni- response was also protective upon BCL1 tumour cell zation with pBCL1. A representative experiment at 1:200 dilution is challenge (data not shown). shown.

Gene Therapy Specificity of anti-Id antibodies induced by scFv DNA immunisation F Benvenuti and OR Burrone 1559

Figure 4 Cross analysis of immune sera on chimeric idiotypes. Sera induced by the indicated plasmids were incubated with transfected Sp2/0 cells (as in Figure 3) displaying the different idiotypes and analysed by flow cytometry. A representative experiment at 1:200 dilution is shown.

Figure 5 (a) Comparative reactivity of DNA-induced versus protein-induced sera. Transfected Sp2/0 cells displaying IdBCL1 or chimeric Ids were incu- bated with sera induced by pBCL1 (anti-Id DNA) or by scFvBCL1-huCH3 protein (anti-Id prot) and analysed by flow cytometry. (b) Reactivity in Western immunoblot of protein-induced or DNA-induced anti-Id sera on the indicated denatured idiotypes.

The phenomenon of dominance might be due to the For this reason, it is likely that following genetic vacci- higher avidity or higher representation of nation, a high percentage of properly folded molecules reacting with antigenic structures peculiar to VL/VH are secreted and rendered available for processing by the associations, thus resulting in sequestration of the avail- immune system. In protein immunisation, ex-vivo treat- able antigen by these specific lymphocytes. In the case of ments such as purification and mixing with adjuvant, protein immunisation, that involves presentation to the may affect folding of a fraction of the protein disclosing immune system of larger amounts of antigen than DNA otherwise hidden epitopes. However, we did not observe immunisation, the higher availability of antigen would any differences in the specificity of the anti-IdBCL1 suffice to stimulate a broader range of idiotype-specific response induced by protein immunisations with or lymphocytes, including those (presumably less reactive) without Freund’s adjuvant. In both cases the sera reacted against chain-specific epitopes. On the contrary, genetic with the IdBCL1, as well as with the two chimeric Ids (data immunisation relies on small amounts of antigen pro- not shown). duced and assembled by cells of the host. In mammalian The data presented indicate that our scFv DNA vacci- cells, a protein directed to the secretory pathway encoun- nation design results in a highly specific anti-idiotypic ters the endoplasmic reticulum quality control machinery immune response that strictly depends on the quaternary that promotes the release of correctly folded proteins.24 structure of the antigen. Although we analysed this

Gene Therapy Specificity of anti-Id antibodies induced by scFv DNA immunisation F Benvenuti and OR Burrone 1560 phenomenon only in the case of VL/VH domains, the vated Sepharose 4B resin (Pharmacia Biotech, Uppsala, indication that DNA vaccination induces conformational Sweden) conjugated to goat anti-human IgG Ab (Dako, antibodies can also be of interest for other antigens. Glostrup, Denmark). The purified protein in PBS was It has been suggested that binding of anti-Id antibodies injected subcutaneously three times at 2-week intervals, to the idiotype on lymphoma cells can result in direct using 25 ␮g of protein per mouse in a total volume of killing of tumour cells without recruitment of other effec- 100 ␮l. For immunisations with adjuvant, 25 ␮g/mouse tor mechanism.8,9 The degree of growth inhibition of protein in CFA were injected subcutaneously three mediated by different antibodies directed against the times at 2-week intervals. Sera were collected via retro- lymphoma immunoglobulin was shown to depend on the orbital puncture 2 weeks after the last boost. extent of crosslinking.25 This may in turn depend on the orientation of the target epitopes,26 as well as on the ELISA and FACS analysis 27 affinity of the crosslinker. In this setting, it is worth Anti-idiotypic antibody levels in immune sera were mea- mentioning that DNA vaccination was shown to be as sured by ELISA on plates coated with 3 ␮g/ml of BCL1 efficient as protein immunisation in inducing protection, IgM protein immunopurified from the 123bcl1 although total levels of anti-Id antibodies against the lym- hybridoma supernatant. Reactivity against the human 4 phoma immunoglobulin were much lower. CH3 portion was determined using plates coated with 3 Altogether these considerations are important for the ␮g/ml of human IgG (Sigma). Immune sera were serially design of proper antigenic immunogens in anti-Id diluted and bound Abs were detected with (HRPO)-con- immunotherapies and for the correct analysis and follow- jugated goat anti mouse IgG (Kirkegaard and Perry, Gai- up of anti-Id antibody responses in clinical trials. thersburg, MD, USA). To detect the expression of the ⑀- m SIPs, Sp2/0 trasfected cells were stained with FITC Materials and methods conjugated goat anti-human IgE (1:200). Analysis of immune sera on BCL1 or Sp2/0 transfected cell were Mice, cell lines and transfections always performed using samples yielding comparable ELISA values against the hu-CH3 portion. BCL1 lym- Ten to 12-week-old Balb/c mice were obtained from Har- phoma cells or Sp2/0 transfected cells were incubated at lan (Milan, Italy) and housed at the ICGEB animal house. 4°C with different dilutions of immune sera in 3% BCL1 is a spontaneous B cell leukemia/lymphoma of BSA/PBS containing 0.02% NaN . Bound antibodies were BALB/c origin that expresses high levels of surface 3 detected with FITC conjugated goat anti-mouse IgG IgM/␭.28 The hybridoma 123bcl1 secreting BCL1 IgM was (Kirkegaard and Perry). mAb 10/4A12 is a rat mAb spe- kindly provided by Dr FK Stevenson (Tenovus Labora- cific for V of BCL1.21 Staining with mAb 10/4A12 was tory, Southampton University Hospital Trust, Sou- H − performed by incubation for 1 h (1:200) followed by a thampton, UK). The Ig non-secreting mouse myeloma FITC-conjugated goat anti-rat IgG (Kirkegaard and Sp2/0 cell line used for transfection experiments was Perry). Fluorescence analysis was performed with a purchased from the American Type Culture Collection FACScalibur (Becton Dickinson, San Jose, CA, USA). (Rockville, MD, USA). Transfections were performed by electroporation as described elsewhere,29 using 400 ␮g/ml of G418 (Geneticin; Life Technologies, Inc., Western blotting × 7 Gaithersburg, MD, USA) for selection of Sp2/0 clones. Membrane extracts were obtained from 4 10 cells that were resuspended at a concentration of about 107/ml in Idiotypic constructs hypotonic buffer (20% PBS containing 0.25 m sucrose, 20 Cloning of pBCL1 and p6C6 (EMBL accession number mm NEM and protease inhibitors) and lysed by three X985337 and X98538) has been described in detail pre- freeze/thawing cycles. The suspension was centrifuged 7,15 BCL1 6C6 10 min at 700 g to separate nuclei and unlysed cells, and viously. To construct plasmid pVL /VH the frag- 6C6 the supernatant was ultracentrifuged for 1 h at 100000 g. ment PstI/BspEI encoding VH was inserted into the BCL1 The membrane pellet was resuspended in 400 ␮l of TNN pBCL1 vector in place of the VH . Similarly, plasmid 6C6 BCL1 buffer (50 mm Tris HCl pH 8.0, 250 mm NaCl, 0.5% NP40) pVL /VH was generated by substitution of the BCL1 containing 20 mm NEM and protease inhibitors (1 mm ApaLI/SpeI fragment encoding the VL with the one 6C6 phenyl-methyl-sulfonyl-fluoride, 0.8 ␮m aprotinin, 40 ␮m encoding VL . The membrane bound version of each ␮ ␮ idiotype was obtained by subcloning the HindIII-BspEI bestatin, 22 m leupeptin and 15 m pepstatin A, all from Sigma). 30 ␮l of this membrane extract was separated in fragment (signal peptide-VL-linker-VH) of pBCL1, BCL1 6C6 6C6 BCL1 ⑀ SDS polyacrylamide gel electrophoresis in reducing con- pVL /VH ,pVL /VH and p6C6 into the -mSIP 20 ditions, transferred to PVDF membrane, and probed with vector that contains the C-terminal region of the short BCL1 isoform of human membrane IgE ⑀H chain (⑀CH4, either protein-induced or DNA-induced anti-Id EMPD, transmembrane and cytoplamic domains). serum, followed by peroxidase-conjugated rabbit anti- mouse IgG (Dako) and detected with the ECL chemilumi- Immunisations nescent reagent (Amersham-Pharmacia Biotech). For DNA vaccination, mice were immunised three times intradermally using the BioRad gene delivery device Acknowledgements (BioRad, Hercules, CA, USA). The abdominal area of mice was shaved, and 1 ␮m gold particles carrying 1–3 F Benvenuti was supported by a SISSA predoctoral fel- ␮g DNA were shot at 400 psi, every 2 weeks lowship. We are grateful to Mauro Sturnega and Dario For protein immunisation, the BCL1 scFv protein was Pippan for excellent technical assistance. We thank Pro- affinity-purified from tissue culture supernantants of fessor Freda Stevenson for providing the BCL1 specific Sp2/0 cells transfected with pBCL1 using a CNBr-acti- mAb 10/4A12 and the 123bcl1 hybridoma cell line. We

Gene Therapy Specificity of anti-Id antibodies induced by scFv DNA immunisation F Benvenuti and OR Burrone 1561 also thank Facundo Batista and Luca Vangelista for help- 14 Weissenhorn W et al. VH-related idiotopes detected by site ful comments on the manuscript. directed mutagenesis. J Immunol 1992; 149: 1237–1241. 15 Potter KN et al. Molecular characterisation of a cross-reactive idiotope on human immunoglobulin utilizing the VH4–21 gene References segment. J Exp Med 1993; 178: 1419–1428. 1 Campbell M et al. Idiotype vaccination against murine B cell 16 Haba S, Lascombe MB, Poljak RJ, Nisonoff A. Structure of lymphoma: humoral and cellular responses elicited by tumour idiotypes associated with antiphenylarsonate antibodies derived IgM and its molecular subunits. J Immunol 1987; 139: expressing an intrastrain crossreactive idiotype. J Exp Med 1989; 2825–2833. 170: 1075–1790. 2 Kaminski MS, Kitamura K, Maloney DG, Levy R. Idiotype vacci- 17 Li E et al. 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