Tumor-specific idiotype vaccines. II. Analysis of the tumor-related network response induced by the tumor and by internal image antigens (Ab2 beta)

In this study the tumor-specific immuneresponse induced by irradiated tumor cells (L1210/GZL) and by anti-idiotype antibodies was analyzed. The anti-idiotype antibodies (Ab2) were made against the paratope of a monoclonal antitumor antibody (11C1) that recognizes a tumor-associated antigen which cross-reacts with the mouse mammary tumor virus-encoded envelope glycoprotein 52. Two Ab2, 2F10 and 3A4, induced idiotypes expressed by the monoclonal antitumor antibodies 11C1 and 2B2. Cytotoxic T cells, generated by immunization with irradiated tumor cells, lyse 2F10 and 3A4 hybridoma cells. Furthermore, immunization with Ab2 induces tumor-specific cytotoxic T lymphocytes. The frequency of tumor-reactive cytotoxic T lymphocyte was found to be similar in mice immunized with Ab2 or irradiated tumor cells when examined at the precursor level. However, only 2F10 induces protective immunity against the growth of L1210/GZL tumor cells. The depletion of a L3T4+ T cell population from 2F10 immune mice was found to increase the effectiveness of transferred T cells to induce inhibition of tumor growth. The inability of 3A4 to induce antitumor immunity could be correlated with the presence of a population of Lyt2+ regulatory T cells. Collectively, these results demonstrate the existence of a regulatory network controlling the expression of effective tumor immunity. Our results demonstrate that selection of binding site-related Ab2 may not be a sufficient criteria for the development of an idiotype vaccine. A better understanding of the regulatory interactions induced by anti-idiotypes is needed for the design of effective antitumor immunotherapy.     

Tumor idiotype vaccines

In this study, the contribution of idiotype-positive antitumor antibodies (anti-Id) in protective tumor immunity was investigated. We have previously shown that among various anti-Id generated and typed as the internal-image Ab2 of the tumor-associated antibody (TAA) gp52, only 2F10 antibody induces protective immunity. Increase of the 2F10 idiotope in sera of tumor-bearing mice correlated with long-term survival, while in mice with short survival the circulating 2F10 idiotype decreased. 2F10⁺ Ig were purified from sera of tumor-bearing mice with longterm survival and the amount of 2F10⁺ anti-TAA antibodies was determined. Only about 3% of 2F10⁺ antibodies are 2F10⁺ anti-TAA⁺. Hybridomas were generated from a 2F10 high mouse with spontaneous tumor regression. Only 2 out of 52 tumor-specific hybridomas were 2F10⁺. These results suggest that the protective effect induced by 2F10 vaccination may not be directly mediated by 2F10⁺ antibodies but indirectly through the stimulation of a 2F10-specific cellular immune response.Supported in part by a grant from N.I.H. no. CA51434     

VH-related idiotopes detected by site-directed mutagenesis. A study induced by the failure to find CD4 anti-idiotypic antibodies mimicking the cellular receptor of HIV.

The function of the CD4 cell surface protein as coreceptor on T helper lymphocytes and as receptor for HIV makes this glycoprotein a prime target for an immune intervention with mAb. A detailed understanding of the structural determinants on the therapeutic CD4 mAb that are involved in Ag binding or are recognized by anti-idiotypic mAb (anti-Id) may be important for designing antibodies with optimal therapeutic efficacy. Seven anti-Id raised against the CD4 mAb M-T310 were selected from a large panel with the intention to obtain CD4 mimicking structures with specificity for HIV gp120. The selected anti-Id did not react with other CD4-specific mAb cross-blocking M-T310. Among these, mAb M-T404, although having the same L chain as M-T310 and a VH region sequence differing only at 14 amino acid positions, was not recognized by the anti-Id. M-T310 H chain complexed with the J558L L chain reacted with all anti-Id, thus demonstrating that the recognized idiotopes are located within the VH region. To identify the idiotopes of M-T310 seen by the anti-Id, variants of M-T404 containing one or more of the M-T310-derived substitutions were generated by oligonucleotide-directed mutagenesis. The reactivity pattern of the mutant proteins with the anti-Id demonstrated that the idiotopes reside within the complementarity determining region (CDR) 2 and CDR3 loops of the VH region. A major idiotope was defined by a single amino acid in CDR2 that was recognized by three anti-Id, whereas the four other anti-Id reacted with determinants of CDR3. Although the performed amino acid substitutions did influence the Id recognition, Ag binding was not significantly affected, suggesting that none of the anti-Id can be considered as a mimicry of the CD4 Ag.     

Tumor-specific idiotype vaccines. III. Induction of T helper cells by anti-idiotype and tumor cells

In a previous report, we have demonstrated the induction of tumor-specific immunity by monoclonal anti-idiotype antibodies generated against a monoclonal anti-tumor antibody, 11C1, that also cross-reacts with mouse mammary tumor virus envelope glycoprotein gp52. Also, we showed that whereas one anti-idiotype antibody, 2F10, could induce protective immunity, another anti-idiotype antibody, 3A4, induced nonprotective immunity. Here we demonstrated the existence of T helper cells which recognize anti-idiotypes that exert differential controls on tumor growth. The qualitative nature of idiotype recognizing T cells generated in response to 2F10, 3A4, irradiated tumor, and progressively growing tumor was compared. The reactivity pattern of idiotype recognizing T cells obtained from 2F10 and irradiated tumor immunized mice were similar in nature in the sense that Lyt-2- T cells obtained from these immunized mice responded to both 2F10 and 3A4 as antigen, although T cells from tumor immunized mice responded better to 3A4 antigen. On the other hand, the idiotype-recognizing T cells obtained from 3A4-immunized mice showed a similar reactivity pattern to T cells isolated from mice during the early phase of tumor growth (within day 4 to 5 after the inoculation of 10(4) live tumor cells). Lyt-2- T cells isolated from mice immunized with 3A4 or during the early phase of tumor growth responded only to 3A4 antigen. The inability of Lyt-2- T cells, isolated from 4- to 5-day-old tumor in mice, to cooperate with 2F10-TNP is not due to the absence of 2F10 idiotype recognizing T cells as 2F10 id recognizing T cells are present when examined at the precursor level. These data on the idiotype specificity of T helper cells show a correlation with the presence of anti-tumor immunity. This information will help in the design and application of idiotype vaccine in tumor immunotherapy.     

Tumor-specific idiotype vaccines. I. Generation and characterization of internal image tumor antigen

The concept of idiotype vaccines against tumor-associated antigens (TAA) was tested in the DBA/2 L1210 lymphoma subline, L1210/GZL. Monoclonal antibodies against a TAA that cross-reacts with the envelope glycoprotein gp52 of the mammary tumor virus were used to make hybridoma anti-idiotype antibodies (Ab2). In this report we describe the characterization of monoclonal anti-idiotypic antibodies against the combining site of 11C1 (Ab1), which recognizes a shared determinant of gp52 of mouse mammary tumor virus (MMTV) and the TAA of L1210/GZL. Hybridomas expressing the internal image of gp52 were screened by an idiotype inhibition assay. Mice sensitized with radiated L1210/GZL cells produced specific delayed type hypersensitivity (DTH) against the Ab2 hybridoma. Five Ab2 hybridomas were selected and were used to immunize DBA/2 mice. Such immunized animals showed specific DTH reaction against a challenge with the L1210/GZL tumor cells. Similar results were obtained in mice immunized with purified Ab2. Fluorescence-activated cell sorter analysis demonstrated that fluorescence staining of L1210/GZL cells by 11C1 can be completely inhibited with preabsorption on Ab2 hybridoma cells. Mice immunized with 2F10 and 3A4 coupled to keyhole limpet hemocyanin (KLH) contained antibodies binding to MMTV. But only in mice immunized with 2F10-KLH was significant inhibition of L1210/GZL tumor growth observed. Collectively, these results indicate that certain anti-idiotypic antibodies can mimic the MMTV gp52 antigen, as well as the gp52-like epitope expressed on the L1210/GZL tumor cells. These properties of anti-idiotypic antibodies mimicking TAA could be exploited for making idiotype vaccines against tumors.     

Murine monoclonal anti-idiotype antibody as a potential network antigen for human carcinoembryonic antigen.

Carcinomas of the gastrointestinal tract are not curable by standard therapies. Thus, new therapeutic approaches for this disease are needed. This study proposes the use of anti-Id mAb as Ag substitutes to induce anti-tumor immunity in gastrointestinal cancer patients. Recently, we have generated and characterized one monoclonal anti-Id antibody, designated 3H1 (Ab2), which mimics biologically and antigenically a distinct and specific epitope of the 180,000 m.w. carcinoembryonic antigen (CEA) primarily expressed in high density by human pancreatic and colonic tumor cells. This epitope is unique to CEA and not present on other CEA-related lower m.w. members of the Ag family also found on normal tissues. The antigenic determinant as defined by the mAb 8019 (Ab1) against which the Ab2, 3H1 was raised, is absent on normal adult tissues by immunoperoxidase staining and haematopoietic cells including granulocytes by flow cytometry analysis. Anti-Id (Ab2) 3H1 induced CEA-specific antibodies in mice and rabbits. The immune sera from both mice and rabbits competed with Ab1 for binding to the colon carcinoma cell line LS174T and inhibited the binding of radioiodinated Ab1 to Ab2. This indicates that anti-anti-Id (Ab3) in mice and rabbits share idiotopes with Ab1 (8019). Furthermore, monoclonal Ab3 that bind to CEA have been generated from mice immunized with 3H1. The Ab3 (both polyclonal as well as monoclonal) immunoprecipitated the same 180,000 m.w. CEA as Ab1 (8019) by Western blotting analysis and showed almost identical immuno-staining patterns as Ab1 on colonic adenocarcinoma tissue sections from several patients. Collectively these data suggest that Ab2 3H1 could potentially be used clinically as a network Ag for immunotherapy of patients with CEA positive tumors.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

A "network antigen" for human CD4. A murine monoclonal anti-idiotype to Leu-3a induces an anti-CD4 response in naive mice.

Previous studies have evaluated anti-CD4 mAb as idiotypic models of the HIV gp120-binding site for CD4. The success of this strategy depends upon the concept of internal image, whereby the binding paratope of the anti-CD4 structurally mimics the equivalent binding surface on HIV gp120. To test this concept of internal image, anti-idiotypic antibodies were raised against the anti-CD4, Leu-3a. If any of these anti-Id detect the paratopic idiotope on the anti-CD4 antibody, their own respective paratopes should structurally model the corresponding binding epitope on CD4 bound by Leu-3a. Consequently, the immunization of naive mice with the selected anti-Id should induce an anti-CD4 response which reflects the binding specificities of Leu-3a. Four anti-Id to Leu-3a were characterized and tested for their ability to induce anti-CD4 responses in naive animals. Although one anti-Id induced an anti-CD4 response in mice, no such response could be detected in other species. Thus the failure to raise anti-Id with internal image characteristics may provide an explanation for the lack of anti-gp120 activity reported in anti-Id antisera raised to multiple anti-CD4 antibodies.     

Conducting polymer based fluorescence quenching as a new approach to increase the selectivity of immunosensors

Polypyrrole (Ppy) has been shown to be a superior matrix for fluorescence detection based immunosensors: (i) the fluorescence of polypyrrole and polypyrrole modified by entrapped proteins was almost not detectable when this polymer was excited by near UV 325 nm light; (ii) polypyrrole quenched the fluorescence of such fluorescence agents as fluoresceine 5(6)-isothiocyanate, rhodamine B and enzyme-horseradish peroxidase (HRP) by almost 100% if they were deposited in the solution as a drop at the Ppy surface followed by evaporation of the solvent. According to our knowledge, this work is first application of Ppy in the design of a fluorescence-based immunosensor, where low Ppy fluorescence background and Ppy induced fluorescence quenching were exploited. These sensors were devoted to the detection of antibodies against bovine leukemia virus (BLV) protein gp51 (anti-gp51-Ab). A biological recognition system of this fluorescence immunosensor model was based on polypyrrole with entrapped BLV proteins gp51 (gp51/Ppy). This gp51/Ppy layer was applied for the detection of anti-gp51-Ab. Secondary antibodies against anti-gp51-Ab labeled with HRP (Ab*) were applied as fluorescence-detectable labels that are able to recognize specifically and interact with the complex of gp51 proteins and anti-gp51-Ab antibodies (gp51/anti-gp51-Ab). It was demonstrated that fluorescence of non-specifically adsorbed Ab* was almost completely quenched by the Ppy substrate. In addition, enzymatic activity of HRP was exploited as a traditional reference method for verification of the formation of the immune complex gp51/anti-gp51-Ab/Ab*.     

Immunisation of macaques with SIV env recombinants: Specificity of T cell and antibody responses and evaluation of protective efficacy

Macaques were immunised with lentil lectin purified recombinant SIVmac (BK28) derived gp160 (rgp160) with or without live vaccinia (vac)-env (BK28) priming, followed by a final boost with solid matrix antibody antigen (SMAA)-gp160 (J5) complexes and challenged with the SIVmac molecularly cloned virus J5M. Rgp160 and vac-env plus gp160 induced strong Ab responses against the homologous virus. Live vac-env did not enhance or prolong the antibody response, however, T cell responses were stronger. Analysis of the specificity of the immune response demonstrated that sequence variation within SIVmac viruses can affect antibody and T cell recognition. A single booster immunisation with the heterologous SIVmac J5 env recombinant protein was not sufficient to protect against the molecularly cloned virus J5M. These findings further illustrate the difficulty of generating protective immunity with immunogens based on single sequence recombinants.     

Immune response to human immunodeficiency virus. In vivo administration of anti-idiotype induces an anti-gp160 response specific for a synthetic peptide

Anti-idiotypic antibodies (anti-Id) to chimpanzee antibodies directed against a synthetic peptide corresponding to a native epitope associated with gp41 of human immunodeficiency virus (HIV) envelope glycoprotein were produced in rabbits. The peptide was analogous to amino acid sequences 735 to 752 from the human T cell leukemia virus-IIIB isolate of HIV. Characteristics of the anti-Id preparation included: 1) detection of a shared determinant present on a second chimpanzee and one of three rabbit antibody preparations directed against the synthetic peptide, 2) failure to recognize an idiotype (Id) in BALB/c mouse antisera to the peptide, and 3) partial inhibition of the homologous chimpanzee Id preparation from binding either peptide or a recombinant HIV gp160 preparation. Immunization of BALB/c mice with the anti-Id induced an antipeptide response which bound a recombinant gp160 preparation without subsequent peptide or gp160 exposure. The anti-gp160 containing sera from mice immunized with anti-Id were able to inhibit the Id-anti-Id reaction indicating that an Id-positive antibody response was induced. This Id is not normally expressed in the murine anti-gp 160 immune response to the synthetic peptide and suggests that this anti-Id may activate normally silent clones. This study indicates that Id networks may be operational during the immune response to HIV epitopes. Alternatively, anti-Id may be useful in altering the serologic characteristics of an antibody response to HIV and may offer potential for modulating the immune response in this viral infection.     

Functional analysis of a T cell line specific for antiidiotypic antibodies to a Schistosoma mansoni protective epitope. II. Induction of protective immunity in experimental rat schistosomiasis.

In our previous work on the idiotypic network in the rat model of schistosomiasis we showed that immunization with an IgE mAb specific for 26/56-kDa parasitic Ag resulted in the production of anti-anti-Id antibodies of both the IgG and IgE classes. Further studies demonstrated that anti-Ab2 T cell lines, obtained by immunization with Ab2 antibodies, functioned as conventional Th cells; they were MHC-restricted and required APC to proliferate in the presence of the native schistosomula Ag and the Ab2 antibodies. We report the involvement of these anti-Ab2 cells in the regulation of protective immunity. The transfer of long term culture anti-Ab2 T cell lines into LOU/M rats, followed by a challenge infection by Schistosoma mansoni 1 day after the cell transfer led to a slight increase in the worm burden. On the contrary, the transfer of anti-Ab2 T cells 90 days before S. mansoni infection induced a significant reduction of the worm burden (up to 57%). T cells recovered from the protected rats were stimulated by the native schistosomula Ag as well as by tryptic fragments of IgG isolated from the Ab2 sera, in the presence of irradiated thymic cells as APC. We also analyzed the humoral response developed by the rats after transfer with the anti-Ab2 T cell lines. The sera induced various inflammatory cells into cytotoxic effectors against the larvae of S. mansoni, arguing for the presence of functional IgE in the sera. Moreover, when these sera were passively transferred into rats infected 1 day later, a significant reduction of the worm burden was observed. However, antibody-dependent cytotoxic mechanisms efficient 10 days after the anti-Ab2 T cell transfer did not correlate with the protective immunity which required a 90-day delay to be established. These data suggest that the protective immunity induced by the anti-Ab2 cells is supported both by the cellular and humoral components and that in a future vaccinating strategy the idiotypic network may play a crucial role.     

Prevention of experimental autoimmune thyroiditis through the anti-idiotypic network.

The central goal in the therapy of autoimmune diseases is to develop potent tools able to exert specific control of the immune response to self Ag. Anti-Id may provide such specific immunodulators because the relevance of the idiotypic network in autoimmunity is well documented. We now describe the protective immunity against experimental autoimmune thyroiditis induced exclusively by a thyroglobulin (Tg)-specific cytotoxic T cell clone and show that this down-regulation occurs through the generation of anti-Id antibodies (Ab) (Ab2Beta) which recognize the paratope of a anti-Tg mAb (Ab1) specific to the pathogenic epitope of the Tg molecule. We further analyze the various steps of the Ab responses (Ab1, Ab2, and Ab3) in terms of poly-, mono-, and autospecificities for the pathogenic epitope of the Tg molecule and for the idiotope of the related Ab.     

Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.     

Structure and binding properties of a monoclonal anti-idiotypic autoantibody to anti-DNA with epibody activity.

We report the isolation and characterization of a mouse autoanti-idiotypic mAb (D7.4 IgG2a), which is directed against a major public Id (A52 IgG2b) in the murine and human autoimmune response to DNA. The natural anti-Id mAb has been produced in the course of the SLE-like disease in a female NZB/NZW F1 mouse and showed a dual specificity (epibody activity) for the public Id (A52) and for the autoantigen (DNA). The two binding activities were shown to reside in the Fab portion of the epibody and were highly specific for their respective Ag. A complete nucleotide sequence analysis of the D7.4 H and L chain V-region genes combined with computer comparisons to available Ig sequences may suggest a charge interaction between the H chain CDR3 segments of the Id and anti-Id antibodies. The D7.4 epibody may be a component of the self-binding, idiotypically connected network of natural antibodies. Alternatively, it could be elicited against the potentially pathogenic, DNA-containing immune complexes in order to facilitate their removal from the circulation of diseased individuals.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Anti-tumor immunity against CT26 colon tumor in mice immunized with plasmid DNA encoding beta-galactosidase fused to an envelope protein of endogenous retrovirus

Endogenous retroviral gene products have been recognized as being expressed in human cancerous tissues. However, these products have not been shown to be antigenic targets for T-cells, possibly due to immune tolerance. Since carcinogen-induced colon tumor CT26 expresses an envelope protein, gp70, of an endogenous ecotropic murine leukemia virus that is comparable to human tumor-associated antigens, we examined whether a DNA vaccine containing the gp70 gene induces protective immunity against CT26 cells. Injection of mice with plasmid DNA (pDNA) encoding gp70 alone failed to induce anti-gp70 antibody (Ab) or anti-CT26 cytotoxic T lymphocyte (CTL) responses. However, immunization with pDNA encoding the beta-galactosidase (beta-gal)/gp70 fusion protein induced anti-gp70 Ab and anti-CT26 CTL responses and conferred protective immunity against CT26 cells. These results indicate that beta-gal acts as an immunogenic carrier protein that helps in the induction of immune responses against the poorly immunogenic gp70. Considering these results, it is possible that potential tolerance to the endogenous retroviral gene products expressed by human tumors may be overcome by DNA vaccines that contain an endogenous retroviral gene fused to genes encoding immunogenic carrier proteins.     

Monoclonal anti-idiotypic antibody mimicking the principal neutralization site in HIV-1 GP120 induces HIV-1 neutralizing antibodies in rabbits

A murine mAb BAT123 (Ab1) directing to the principal neutralization site of human T cell leukemia virus (HTLV)-IIIB gp120 (amino acid residue 308-322) was used to generate syngeneic anti-Id mAb (Ab2). Among the Ab2, a mAb AB19-4 was characterized by both serologic and biologic methods to be paratope-specific (Ab2 beta), bearing the internal image of the neutralization site. AB19-4 was found to bind specifically to BAT123 and also to its mouse-human chimeric form in ELISA. The binding of AB19-4 to BAT123 was specifically inhibited by HTLV-IIIB gp120 and the synthetic epitope peptides of HTLV-IIIB and HTLV-IIIMN defined by BAT123. AB19-4 also inhibited the binding of BAT123 to HTLV-IIIB-infected H9 cells in flow cytometric studies. Polyclonal goat and sheep antisera against HTLV-IIIB gp120 reacted specifically with AB19-4, suggesting that AB19-4 may recognize cross-species idiotopes. Rabbits immunized with purified AB19-4 generated anti-anti-Id antibodies (Ab3) that reacted specifically with HTLV-IIIB gp120 and the BAT123-binding epitope peptides of HTLV-IIIB and HTLV-IIIMN. The Ab3 bound to H9 cells infected by HTLV-IIIB or HTLV-IIIMN and inhibited the infection of CEM cells by HTLV-IIIB or HTLV-IIIMN, whereas BAT123 also bound H9 cells infected by HTLV-IIIB or HTLV-IIIMN but neutralized only HTLV-IIIB. Our data suggest that AB19-4 mimics the neutralization site on HIV-1 gp120 defined by BAT123. The induction of immunity to HIV using internal-image Ab2 to HIV-neutralizing antibodies may provide a viable approach for developing effective vaccines for AIDS.     

DNA fusion vaccines against B-cell tumors

DNA vaccination is currently being explored as a potential strategy for combatting cancer. However, tumor antigens are often weak and the immune system of patients may be compromised. For B-cell tumors, immunoglobulin idiotypic antigens provide defined targets but are poorly immunogenic. Fusion of a sequence derived from tetanus toxin to the genes encoding idiotypic determinants has proved highly effective in activating protective anti-tumor immunity. DNA fusion vaccines containing immuno-enhancing sequences can augment and direct immune attack on a range of target antigens. Gene-based fusion vaccines offer ease of manipulation and flexible design to activate effective attack on cancer.     

Tumor idiotype vaccines. VI. Synergistic anti-tumor effects with combined "internal image" anti-idiotypes and chemotherapy

Anti-Id antibodies that have biologic activity as stimulators of specific immunity have been used in experimental vaccines and tumor protection models. However, very little is known about the therapeutic potential of anti-Id antibodies in animals and men. In this study we explored the combination of anti-Id and chemotherapy in a murine tumor system for which we had previously generated protective anti-Id mAb. First, we investigated various protocols by using a protective anti-Id in active immunization. Mice preimmunized before tumor transfer and challenged again after tumor survived significantly longer. Next, we explored the use of soluble anti-Id as immunostimulator in tumor-bearing mice. Although this treatment did not induce long-term survival, it significantly increased survival time. Interestingly, this anti-Id effect was dose dependent, whereby large and small doses had no effect. Finally, stimulatory anti-Id therapy and cyclophosphamide (Cy) treatment was combined. Tumor bearing mice were given a single dose of Cy followed by different doses of soluble anti-Id. The optimal effect on tumor growth and survival was achieved with 80 mg/kg Cy and 10 micrograms/mouse of anti-Id, where 80% of mice survived longer than 100 days. These results provide guidelines for developing clinical protocols for cancer patients by using combination therapy of anti-Id and chemotherapy.