Towards an idiotype vaccine against mammary tumors. Induction of an immune response to breast cancer-associated antigens by anti-idiotypic antibodies

Previously we have reported on the production of two sets of human monoclonal antibodies reacting with mouse mammary tumor virus (MMTV) and human mammary tumor virus (HuMTV) cross-reacting antigens. B11 is a monoclonal IgG generated by the human-human hybridoma technique using axillary lymph node of breast cancer patients. 4.6/6 is a monoclonal IgG established by the mouse-human hybridoma procedure using peripheral blood lymphocytes of a healthy investigator working with the breast cancer cell line T47D which secretes HuMTV antigens. Two anti-idiotypic (Id) antibodies were produced by immunizing rabbits with B11 or 4.6/6. Following exhaustive adsorption on unrelated human-Ig, fetal calf serum and purification on B11 or 4.6/6 affinity columns, the anti-Id antibodies were shown to react specifically with their respective Id. The binding of these anti-Id antibodies to the respective Id was specifically inhibited by prior incubation of the Id with MMTV and/or HuMTV antigens. Rabbit anti-B11 and rabbit anti-4.6/6 anti-Id antibodies were employed to immunize female C3Heb mice. Following immunizations, humoral and cellular immune responses to MMTV-related antigens could be demonstrated. The sera of the mice contained anti-MMTV antibodies and delayed-type hypersensitivity was specifically expressed when irradiated cells of the Mm5MT line (which carry surface MMTV antigens) were injected. Our results support the notion that anti-Id antibodies harboring the internal image can immunize animals against tumor cells bearing on their surface viral-associated antigens.     

Induction of anti-viral immune response by immunization with monoclonal anti-idiotype antibodies directed to private idiotopes

Syngeneic monoclonal anti-idiotypic antibodies were raised against idiotopes on neutralizing monoclonal antibodies with specificity for feline leukemia virus and canine parvovirus. The anti-idiotypic antibodies were shown to recognize paratope-related private idiotopes. Mice were injected with the monoclonal anti-idiotypic antibodies and the sera of these mice were screened for antiviral reactivities. Antibodies to both feline leukemia and canine parvovirus could be induced as determined by ELISA. These results suggest that anti-idiotypic antibodies which detect private idiotopes and thus do not represent internal images of viral antigens may be considered as candidates for the induction of antiviral immunity.     

宫颈癌抗独特型单克隆抗体的制备和鉴定

目的：制备宫颈癌抗独特型单克隆抗体并鉴定其抗原模拟特性。 方法和结果： 以识别小鼠和人宫颈癌相关抗原分子共同表位的单克隆抗体AU14-1（Ab1）为免疫原在含免疫反应剂的无血清培养液中致敏小鼠脾细胞，并将其与SP2/0融合，经筛选和克隆化，建成一株能分泌抗独特型单克隆抗体（Ab2）的杂交瘤细胞系。Ab2的抗原模拟特性经ELISA、结合和竞争抑制试验以及免疫组化染色，表明为Ab2β，具有宫颈癌细胞膜表面抗原的“内影像”。 结论： 获得一株具有宫颈癌抗原内影像的抗独特型单克隆抗体。     

In vitro stimulation prior to fusion generates antigen-binding human-human hybridomas

Production of useful human monoclonal antibodies has been limited by the inability to reliably generate and isolate antigen-specific B cells by in vivo immunization. An in vitro culture system employing antigen and mitogen to stimulate lymphocytes derived from solid lymphoid organs has been developed. Human tonsilar or splenic lymphocytes were stimulated in vitro with antigen and mitogen in short term culture and then fused with either of two enzyme deficient human B cell lines. This approach appears to expand antigen-specific B cell clones prior to fusion resulting in the production of a significant number of antigen-binding human hybridoma antibodies. The system has been effective in the production of human monoclonal antibodies following stimulation with KLH-ARS, a soluble antigen, and intact group B streptococcus, a particulate antigen. Hybridomas have been produced by fusion with two distinct parental human B cell lines supporting the previously reported observation that human B lymphoblastoid cell lines representing different stages of B cell differentiation may be useful fusion partners. The utility of the in vitro stimulation system in producing human-human hybridomas secreting antibody directed against two distinct classes of antigens establishes this approach as a generally useful method for the production of human monoclonal antibodies.     

Hybridomas producing human monoclonal antibodies against varicella-zoster virus

Hybridomas producing human monoclonal antibodies (mAb) against varicella-zoster virus (VZV) were generated by fusing human splenic lymphocytes with mouse myeloma cells. Before cell fusion, lymphocytes were stimulated in vitro with viral antigens and pokeweed mitogen. This combination synergistically increased the generation of VZV-specific hybridomas. Five established hybridomas have been stably producing mAb for at least 9 months. These mAb, designated V1, V2, V6, V8 and V9, were of the IgG1, lambda isotype. They bound to all 6 tested VZV strains but not to other herpes viruses, with the exception that V1 bound to herpes simplex virus (HSV) as well as VZV. Immunoprecipitation analysis showed that V1, V6 and V9 recognized glycoprotein gpII, whereas V2 and V8 recognized gpI. In addition, V1 reacted with the gB glycoprotein of HSV. All these mAb neutralized viral infectivity. The neutralizations by V2 and V8 were more effective and more complement dependent than those by V1, V6 and V9. Immunofluorescence tests revealed that all these mAb bound to the surface membrane of VZV-infected cells. These results suggest that cell fusion between in vitro stimulated lymphocytes and mouse myeloma cells is a reliable method for the generation of hybridomas capable of stable production of human mAb. The human mAb thus developed may provide a new means of passive immunization of humans against VZV infection.     

分泌人源性单克隆抗体永生化淋巴细胞系的构建

目的;构建分泌人源性单允隆抗体（ＭｃＡｂ）的永生化Ｂ淋巴细胞系。方法：用正丁醇法提取的人卵巢癌细胞系ａｏｌ１０／１７抗原在含免疫活化剂的无血清培基中免疫人淋巴细胞,将制备ａｏ １０／１７细胞ＤＮＡ转染经体外免疫的淋巴细胞。采用ＥＬＩＳＡ筛选和有限稀释法克隆化。     

Demonstration of anti-idiotypic antibodies directed against IgM rheumatoid factor in the serum of rheumatoid arthritis patients.

We have identified the presence of anti-idiotypic activity against IgMRF in the sera of RA patients. Only patients seropositive for IgMRF had significant levels of anti-idiotypic activity, while seronegative patients and normal volunteers did not. When this anti-idiotypic activity was affinity-purified from a single RA patient, two separate binding activities were identified. IgG antibodies were pepsin-digested to F(ab')2 fragments before affinity-purification to remove the Fc portion capable of binding to IgMRF. Anti-idiotypic F(ab')2 fragments of IgG were eluted from an IgMRF-Sepharose 4B column. These F(ab')2 bound preferentially to IgMRF bearing an idiotype recognized by the anti-idiotypic murine monoclonal 17.109. A second anti-idiotypic F(ab')2 was affinity purified using rabbit anti-human Fc antibody bound to Sepharose 4B. These eluted antibodies behaved as the internal image of IgG, binding five out of seven IgMRF's tested. The binding of both anti-idiotypic F(ab')2 was inhibited with human IgG. The presence of both IgMRF and anti-idiotypic antibodies directed against it in the sera of RA patients suggests that anti-idiotypic antibodies alone are not capable of inhibiting the production of rheumatoid factor.     

Peptidomimics of antigen are present in variable region of heavy and light chains of anti-idiotypic antibody and function as surrogate antigen for perpetuation of immunological memory

Peptide antigens composed of relevant B cell and T cell epitopes, capable of inducing protective immune response against the whole pathogen, are potentially safe, alternative vaccine antigens for prevention of wide range of diseases. Here, we show that short peptides derived from internal image sequences of anti-idiotypic antibody (peptidomimics) can function as both B and T cell epitopes and perpetuate antigen specific immunological memory. We have sequenced the variable regions of heavy and light chains of the anti-idiotypic antibody specific to rinderpest virus hemagglutinin protein and predicted T cell epitopes in these sequences by an immuno-informatics approach. We have studied the interaction of these epitopes with MHC class I by in vitro assays and in silico analysis by molecular modeling of the idiopeptide-MHC complexes as well as antigen-derived peptide-MHC complexes. The functional capacity of anti-idiotypic antibody derived peptides to stimulate antigen specific T cells in vitro was tested. The ability of peptidomimics to proliferate the immune splenocytes in vitro was 10 times more when compared with that of a control peptide taken from the constant region of immunoglobulin. Similarly three- to fivefold more amounts of IL-2 and IFN-gamma were secreted by immune splenocytes in response to in vitro re-stimulation with peptidomimics. Further, we have provided evidence for the generation of antibodies against peptidomimics in memory response generated on antigen or anti-idiotypic antibody immunizations. In summary, our experiments suggest that peptidomimics are generated in the body after antigen immunization and may have important roles in vivo in regulating antigen specific immunological memory.     

Cyclophosphamide treatment used to manipulate the immune response for the production of monoclonal antibodies

After immunization with a complex mixture of antigens, a considerable bias toward obtaining monoclonal antibodies to immunodominant determinants exists. By selectively killing antigen-stimulated lymphocytes, the cytotoxic drug cyclophosphamide can be used to manipulate the bias of the normal immune response. Cyclophosphamide has been used to tolerize mice to one set of antigens followed by immunization with a similar but slightly different set of antigens. This approach yields an enhanced frequency of antibodies that distinguish the two sets of antigens. Cyclophosphamide treatment has also allowed us to produce monoclonal antibodies to weakly immunogenic glycosaminoglycans and to obtain a high frequency of apparently anti-idiotypic antibodies.     

Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5 antigen of porcine reproductive and respiratory syndrome virus

Syngeneic monoclonal anti-idiotypic antibodies (Mab2s) were generated against idiotypic antibodies to membrane glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) using the sequential immunization method. Six of 12 Mab2s possessed potential internal image characteristics by recognizing a common idiotype on murine and swine anti-GP5 antibodies. Further serological characterization demonstrated that one of the Mab2 (Mab2-5G2) represents internal image anti-idiotope which mimicked the GP5 antigen that inhibited the interaction between idiotypic anti-GP5 antibodies and GP5 antigen, its reaction with the idiotypic anti-GP5 antibody was inhibited by GP5 antigen and detected the common Id on anti-PRRSV antibodies from pigs that were experimentally infected with PRRSV. In addition, Mab2-5G2 identified a soluble protein on MA-104 and porcine alveolar macrophages. These results indicate that Mab2-5G2 may be a useful candidate as an alternative PRRSV serodiagnostic reagent and a useful probe to study PRRSV-cell interaction.     

Anti-idiotypic antibodies to monoclonal antibodies that neutralize coxsackievirus B4 do not recognize viral receptors

We have made anti-idiotypic antibodies in rabbits against three mouse monoclonal neutralizing antibodies with specificities for independent epitopes on Coxsackievirus B4. Each of these anti-idiotypic antibodies was found to react specifically with the immunizing monoclonal antibody in radioimmunoassays and did not react with the other monoclonal antibodies. In addition, the anti-idiotypic antibodies specifically inhibited the function (i.e., virus neutralization) of the immunizing antibody. These anti-idiotypic antibodies were tested for their ability to recognize receptors for Coxsackievirus B4 as measured by their ability to inhibit the attachment of radiolabeled virus to cellular receptors. The anti-idiotypic antibodies did not block the binding of Coxsackievirus B4 to monkey kidney cells. Moreover, when tested for their ability to bind to other receptor positive cells, none of the anti-idiotypic antibodies bound above control levels. Anti-idiotypic antibodies did induce a small anti-anti-idiotypic antibody response in mice when tested by radioimmunoassay; however, little if any virus neutralizing activity was found in the sera of these mice. Our results contrast to those reported for anti-idiotypic antibodies in several other virus systems and suggest that not all anti-idiotypic antibodies made against neutralizing antibodies are capable of eliciting an antiviral immune response or binding to viral viral receptors on cells.     

Immune response to bovine viral diarrhea virus induced by anti-idiotypic antibodies.

We have previously prepared rabbit anti-idiotypic antibodies (anti-Ids) against the neutralizing monoclonal antibodies (MAbs) specific for the gp53 of bovine viral diarrhea virus (BVDV). The anti-Ids, purified by sequential immunoaffinity chromatography, inhibited the immunizing MAb from binding to the original antigens and blocked BVDV infection of cell cultures. This study evaluated immune responses in mice to the purified anti-Id reagents. BVDV-specific neutralizing antibodies were induced by the anti-Ids. The antisera (Ab3) induced by the anti-Ids immunoprecipitated gp53 from BVDV-infected Madin-Darby bovine kidney (MDBK) cell lysates. However, lymphocyte-proliferative responses were specific only for the respective immunizing antigens. These results suggest that the anti-Ids may bear an internal image of the gp53 to stimulate production of antibody but not to stimulate a virus-specific cellular immune response in mice.     

Polyspecificity of human monoclonal antibodies reactive with Mycobacterium leprae, mitochondria, ssDNA, cytoskeletal proteins, and the acetylcholine receptor

The origin of autoantibodies against ubiquitous autoantigens (e.g., single-stranded (SS) DNA, cytoskeletal proteins, mitochondria) is obscure. Patients with lepromatous leprosy have many such autoantibodies in their serum. In order to study the polyspecificities of human autoantibodies expressed during infection with Mycobacterium leprae we prepared human monoclonal antibodies derived from the fusion of peripheral blood lymphocytes of a patient with lepromatous leprosy to the human lymphoblastoid line GM 4672. Hybridomas were tested for binding to a DNAse-treated sonicate of M. leprae and a panel of autoantigens. Of the primary (uncloned) cultures, 14% bound ssDNA, 35% bound M. leprae, 11% bound both M. leprae and ssDNA, and 16% bound to mitochondria. Several also bound to the acetylcholine receptor of Torpedo marmorata. Monoclonal antibodies derived from separate primary cultures revealed similar cross-reactions between several autoantigens and M. leprae. In addition, one antibody was identified which bound to mitochondria and the acetylcholine receptor, and which was recognized by an anti-idiotypic antibody which bears the "internal image" of the acetylcholine receptor. These results suggest that antigenic mimicry may play a role in eliciting autoantibody expression from the immune repertoire.     

Interaction of antibodies with human glomerular epithelial cells

We tested the hypothesis that the pathogenesis of human idiopathic membranous glomerulonephritis is similar to that of Heymann glomerulonephritis, a model of membranous glomerulonephritis induced in rats by immunization with renal brush border preparations; the characteristic subepithelial deposits result from interaction of antibodies with a brush border antigen (gp330) expressed on the plasma membrane of glomerular visceral epithelial cells (GEC), followed by redistribution and shedding of gp330 immune complexes. The experiments were performed in cultured glomerular visceral epithelial cells, in living monkeys and rats, and in isolated perfused human, monkey, and rat kidneys. Antigens from plasma membranes of human renal brush border vesicles (HBBV) and GEC vesicles (HGECV) and their corresponding polyclonal and monoclonal antibodies reactive with human and monkey GEC were prepared. First, polyclonal antibodies to HGECV bound diffusely to cultured GEC; monoclonal antibody 8G5, recognizing a 60-kDa protein, mainly bound to the coated pits and apical invaginations; both polyclonal HGECV and 8G5 monoclonal antibodies induced antigen redistribution (capping) at 37 degrees C. Second, monkeys were actively or passively immunized, and isolated human and monkey kidneys were perfused with the antibodies. Active immunization with HBBV induced tubular immune deposits, whereas active immunization with HGECV did not provoke renal lesions. After passive immunization HBBV and HGECV antibodies bound diffusely to glomerular cells, and subepithelial deposits were observed during the autologous phase; in contrast, 8G5 induced early (day 3) granular deposits. Third, fine granular deposits developed in glomeruli of human and monkey kidneys perfused for 4 hours at 37 degrees C with 8G5; these deposits were more difficult to detect by electron microscopy than those occurring in kidneys of Lewis rats perfused with sheep antiHBBV. The results show that some antibodies redistribute antigens at the surface of human and monkey GEC in vitro, in vivo, and ex vivo and induce formation of granular deposits in human glomerular capillary walls. Failure to induce more severe lesions in human and monkey kidneys may be ascribed to lack of GEC antigens comparable to rat gp330, insufficient cross linking by monoclonal antibody, lack or insufficient concentration of epitope-specific antibodies, insufficient time of kidney perfusion, or a combination of these factors.     

Induction of anti-progesterone immunity and pregnancy blocking by anti-progesterone anti-idiotypes. Variable efficacy of polyclonal Ab2 antibodies directed against a panel of closely related Ab1 antibodies.

Polyclonal rabbit anti-idiotypes (Ab2) have been raised against three mouse monoclonal antiprogesterone Ab1 antibodies (DB3, 11/32, 11/64) closely related in VH and VL sequences. The anti-idiotypes were characterized for specificity and used to immunize groups of female mice. The latter responded with production of anti-progesterone (Ab3) antibodies, confirming the ability of anti-idiotypes to mimic the immunogenicity of a steroid. The response to one of the anti-idiotypic reagents (anti-DB3-id) was 5-10 times stronger than those to the others, despite close sequence homology between the idiotypes. Moreover, immunization with anti-DB3-id led to a reduction in fertility rate from 90% (control) to 30%, whereas immunization with the other anti-idiotypes was without effect. Sequence and structural comparisons suggest that residues associated with VH CDR3 and VL CDR3 may have a key role in determining the efficiency of anti-idiotypic immunization against progesterone. The variability in outcome of using anti-idiotypic reagents against a defined panel of related antibodies is relevant to the use of anti-idiotypes as surrogate antigens.     

Monoclonal antibodies to amoxicillin express different idiotypes determined by anti-idiotype antibodies production

Penicillins are β-lactam antibiotics able to generate several antigenic determinants that are recognized by the immune system. To study the differences in the antigen binding site of two monoclonal antibodies (Mab) specific to amoxicillin, polyclonal rabbit anti-idiotypic antibodies were produced. One Mab, AO3.2 (IgG2a), specific to a structure formed by the acyl-side chain structure and a part of the nuclear region of amoxicillin. The second one, AO6.2 (IgE), is specific to the side chain of amoxicillin, although it also recognizes the side chain of other penicillins (penicillin G and ampicillin). These antibodies were used to immunize rabbits in order to produce polyclonal anti-idiotipic antibodies, which were purified in several steps by affinity chromatography. The specificity and cross-reactivity studies were made by ELISA and ELISA inhibition. The results suggest that the anti-Id antibodies produced are the internal image of the antigen, since the binding to their specific idiotype is blocked mainly by the original hapten (amoxicillin): in 98% of the cases with anti-id-1 (induced against AO3.2) and in 59% with anti-id-2 (induced against AO6.2). The absence of cross-reactivity of each anti-idiotypic antibody with the different Mabs specific to amoxicillin shows that the idiotypes induced by the same hapten have differences that are reflected by the nonrecognition of these anti-idiotypes. We conclude that such a small molecule as amoxicillin can present several antigenic determinants that induce a panel of antibody specificities especially directed against the side chain.     

Anti-idiotypic antibodies to anti-DR in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) sera were evaluated for anti-idiotypic (anti-id) antibodies to HLA-DR antigens (anti-DR) using an ELISA method with murine monoclonal anti-DR antibody-coated microtiter plates incubated serially with either normal or RA sera and peroxidase-conjugated goat anti-human IgG (Fc specific). Specificity was examined using other monoclonal antibodies including anti-Leu 3a, OKM5, OKT8, anti-cytochrome c, and anti-breast tumor antigen. Significant binding of 11/33 (33%) RA to anti-DR was found compared with 0/44 normals (P less than 0.001). Two groups were identified: RA sera reacting with anti-DR and anti-Leu 3a and sera which did not bind to anti-DR but bound to irrelevant monoclonal antibodies. Anti-DR reactivity was differentiated from anti-Leu 3a by competitive inhibition studies. Binding of whole sera and IgG from RA patients to anti-DR was significantly inhibited by DR+ cell extract. The same extract was not inhibitory after selective removal of DR antigen by adsorption on an anti-DR-Sepharose column. These data suggest that anti-id antibodies are directed against the antigen-binding site of id. We conclude that some RA patients have anti-id antibodies potentially involved in immunoregulation of anti-DR antibodies.     

Discrimination of antibodies against antigens of different MHC loci in human sera by monoclonal antibody-specific immobilization of leukocyte antigens

To detect human antibodies against antigens of different major histocompatibility complex loci, particularly of class II specificity, a newly developed enzyme immunoassay for platelet antibodies was adapted for the use of lymphocytes as target cells. Peripheral blood lymphocytes, phytohemagglutinin-stimulated T cells, or Epstein-Barr virus-transformed B cells were simultaneously incubated with a monomorphic class- or locus-specific monoclonal antibody and the human antibody to be investigated. After solubilization, cell lysates were transferred to an enzyme-linked immunosorbent assay tray coated with a goat anti-mouse Ig antibody. Following immobilization of the monoclonal antibody/antigen complexes, human major histocompatibility complex antibodies were detected by addition of enzyme-labeled goat anti-human Ig. By means of this technique human antibodies against different major histocompatibility complex molecules present in the same sample could be clearly distinguished. Application of the monoclonal antibody-specific immobilization of lymphocyte antigens assay is presented by several examples. Of these, identification of DP-specific antibodies as well as serological DP typing are of particular interest.     

Cross-reactivity of mouse monoclonal antibodies produced by in vitro or in vivo immunization

The effect of different immunization schemes on the resulting antibody specificity was investigated. The cross-reactivity of monoclonal antibodies produced by in vitro vs. in vivo immunization was tested, using a solid phase enzyme immunoassay. Ten different monoclonal antibodies were tested against 15 different antigens. There was no difference in cross-reactivity between the two types of antibodies when tested against antigens coated onto the plastic wells in a buffer solution.When the antigens were dried onto the plastic wells the IgM monoclonal antibodies produced by in vitro immunization exhibited a somewhat different reactivity pattern. However, the assay design was shown to be of more importance than the immunization procedure when determining antibody specificities.     

Bovine viral diarrhea virus-specific neutralizing antibodies induced by anti-idiotypic antibodies.

Two murine neutralizing monoclonal antibodies (MAbs), 4D8 and 6D11, recognizing epitopes on gp53, a surface glycoprotein of bovine viral diarrhea virus (BVDV), were used to generate anti-idiotypic antibodies (anti-ids) in a calf. The polyclonal anti-ids were isolated from serum by affinity chromatography on their respective Ab-1-Sepharose columns, followed by repeated adsorption on isotype-matched antibody-Sepharose columns. The anti-ids reacted specifically with their respective Ab-1, but not with isotype-matched controls. They also inhibited the binding of their Ab-1 to BVDV in a concentration-dependent manner. Mice immunized with the two anti-id preparations developed antibodies to BVDV, which neutralized the virus in vitro.