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DNA vaccination can elicit the production of anti-tumor antibodies, thus obviating the need to continuously administer passive antibody. This vaccination strategy is particularly important where antibodies have proven to be effective anti-tumor agents. To amplify antibody responses against weak tumor antigens, we previously designed DNA-fusion vaccines incorporating tumor sequences linked to microbial genes. By using a safe idiotypic (Id) antigen from a B cell tumor fused to a fragment C (FrC) sequence from tetanus toxin, we induced both anti-Id and anti-FrC antibodies. It was important to determine whether the antigen itself, either injected or released from residual tumor cells, would boost the antibody response. Id protein not only failed to boost the response, but permanently and rapidly inhibited it by ablating Id-specific memory B cells. In contrast, an Id protein-FrC conjugate boosted both Id-specific and FrC-specific responses. Strikingly, the depletion of CD4+ T cells converted the Id protein-FrC conjugate vaccine into an inhibitor. These findings support the hypothesis that the activation of memory B cells by a DNA vaccine encoding a protein antigen, in the presence of the protein itself, depends completely on T cell help. Furthermore, by using knockout mice, we have shown that inhibition of the Id-specific memory B cells by the Id protein is largely independent of the FcgammaRIIB and, hence, independent of immune complexes. The principles revealed by using a DNA vaccine have implications for all cancer vaccines designed to induce and maintain antibody responses against weak autologous tumor antigens.

The association of a specific autoantibody response with distinct disease phenotypes is observed in both autoimmune diseases and cancer. Although the underlying mechanisms remain unclear, it is likely that unique properties of disease-specific autoantigens expressed in the relevant target cells play a role. It has recently been observed that the majority of autoantigens targeted across the spectrum of systemic autoimmune diseases (but not nonautoantigens) are selectively cleaved by the cytotoxic lymphocyte granule protease granzyme B (GB), generating unique fragments not observed during other forms of cell death. Although susceptibility of a molecule to cleavage by GB strongly predicts autoantigen status, the significance of this association is unclear. We used hepatocellular carcinoma and the hepatocellular carcinoma autoantigen, nucleophosmin/B23, as a model system to define the unique features of disease-specific autoantigens in the relevant disease microenvironment. These studies revealed a striking, selective susceptibility of B23 to cleavage by GB in extracts of neoplastic liver. The increased sensitivity of tumor B23 to proteolysis by GB was accompanied by slightly increased mobility on SDS/PAGE, altered subcellular localization, enrichment of an SDS-stable oligomeric form of B23, and recognition by a conformation-specific antibody detecting a B23 epitope ending at the GB cleavage site. In vitro studies demonstrated that this unique B23 conformation and resultant increased susceptibility to cleavage by GB arise when B23 translation is initiated at methionine-7. We propose that unique features of autoantigens in the disease-relevant microenvironment may regulate susceptibility to cleavage by GB and their selection by the specific autoimmune response.

Cancer patients and tumor-bearing mice possess serum antibodies that recognize antigens expressed by cancer cells at the time of diagnosis. After diagnosis, cancers progress to more aggressive stages, most often by acquiring new genetic changes that can give rise to new proteins, some of which are antigenic. However, at these relatively later stages of tumor growth, it remains unclear whether, when, and how a host can generate de novo antibody responses against these newly appearing tumor antigens. To this end, we used a tamoxifen-regulated Cre-loxP system, MerCreMer, to induce genetic recombination in cancer cells of well-established tumors, resulting in increased enhanced green fluorescence protein (EGFP) expression. These late tumor-bearing mice generated specific IgG antibodies against EGFP within 3 wk after antigen induction. Mice generated these antibody responses in the presence of preexisting anti-tumor antibody responses. Preexisting CD4(+) T cell responses to already expressed tumor antigens likely enhanced antibody responses to the induced EGFP antigen. By analogy, new antibody responses in cancer patients may identify genetic changes occurring in a growing tumor and indicate imminent tumor progression.

Autologous serum antibodies to molecules that are aberrantly expressed in tumors represent potential biomarkers for early diagnosis of cancer. In this study, we identified the homeobox gene HOXA7 as encoding an antigen in epithelial tumors of the ovary. These tumors are thought to arise from the simple epithelium lining the ovarian surface, but they often resemble the specialized epithelia derived from the müllerian ducts. Expression of HOXA7 was detected in ovarian tumors exhibiting müllerian-like features and correlated with the generation of anti-HOXA7 antibodies by patients. In contrast, it was observed that healthy women lack anti-HOXA7 antibodies (P < 0.0001) and that HOXA7 expression is absent from normal ovarian surface epithelium. Interestingly, HOXA7 expression was detected in the müllerian-like epithelium lining inclusion cysts in normal ovaries and in the müllerian duct-derived epithelium of normal fallopian tubes. Furthermore, ectopic expression of HOXA7 enhanced the epithelial phenotype of immortalized ovarian surface epithelial cells, as indicated by the appearance of cobblestone morphology, induction of E-cadherin expression, and down-regulation of vimentin. These findings associate aberrant HOXA7 expression with the müllerian-like differentiation of epithelial ovarian tumors and suggest diagnostic utility of serum antibodies to HOXA7.

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewis(y) (Le(y)), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Le(y) as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Le(y) conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Le(y)-serine epitopes and the Pam(3)Cys immunostimulating moiety was found to be superior to a similar construct containing only one Le(y)-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Le(y)-serine Pam(3)Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.

Cancer-testis antigen NY-ESO-1 is one of the most immunogenic tumor antigens defined to date. Spontaneous humoral and CD8+ T-cell responses to NY-ESO-1 are detected in 40-50% of patients with advanced NY-ESO-1-expressing tumors. A clinical trial was initiated to study the immunological effects of intradermal vaccination with 3 HLA-A2-binding NY-ESO-1 peptides in 12 patients with metastatic NY-ESO-1-expressing cancers. Seven patients were NY-ESO-1 serum antibody negative, and five patients were NY-ESO-1 serum antibody positive at the outset of the study. Primary peptide-specific CD8+ T-cell reactions and delayed-type hypersensitivity responses were generated in four of seven NY-ESO-1 antibody-negative patients. Induction of a specific CD8+ T-cell response to NY-ESO-1 in immunized antibody-negative patients was associated with disease stabilization and objective regression of single metastases. NY-ESO-1 antibody-positive patients did not develop significant changes in baseline NY-ESO-1-specific T-cell reactivity. However, stabilization of disease and regression of individual metastases were observed in three of five immunized patients. These results demonstrate that primary NY-ESO-1-specific CD8+ T-cell responses can be induced by intradermal immunization with NY-ESO-1 peptides, and that immunization with NY-ESO-1 may have the potential to alter the natural course of NY-ESO-1-expressing tumors.

To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66) derived from the parental murine mAb T84. 66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA.

The p53 protein is an attractive target for immunotherapy, because mutations in the p53 gene are the most common genetic alterations found in human tumors. These mutations result in high levels of p53 protein in the tumor cell, whereas the expression level of wild-type p53 in nonmalignant tissue is usually much lower. Several canarypox virus recombinants expressing human or murine p53 in wild-type or mutant form were constructed. Immunization with these viruses protected BALB/c mice from a challenge with an isogenic and highly tumorigenic mouse fibroblast tumor cell line expressing high levels of mutant p53. The tumor protection was equally effective regardless of whether wild-type or mutant p53 was used for the immunization, indicating that the immunologic response was not dependent on any particular p53 mutation and that immunization with this live virus vaccine works effectively against mutant p53 protein expressed in a tumor cell. In tumors escaping immunologic rejection, the expression of the p53 protein was commonly down-regulated.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen-expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti-globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy.

The mouse monoclonal antibody MEM136 (mAb1) is directed against an epitope on human melanoma-associated proteoglycan antigen (MPG). This epitope is also present on various normal human and subhuman tissues. A monoclonal murine anti-idiotope (anti-Id) antibody (mAb2), designated I-Mel-2, was generated against MEM136 and used as a surrogate antigen for the MPG molecule. I-Mel-2 was tested in cynomolgus monkeys (Macaca fascicularis) for its ability to induce anti-MPG humoral responses. All monkeys immunized with Ab2 developed specific anti-anti-idiotype (Ab3) responses that were capable of inhibiting binding of Ab2 to Ab1. Furthermore, I-Mel-2 immune monkey serum contained anti-MPG antibodies (Ab1') that bound to MPG-positive but not to MPG-negative melanoma cell lines. Monkeys immunized with Colo38 melanoma cells (membrane-bound MPG antigen) did not contain anti-MPG antibodies that inhibited the binding of two distinct anti-MPG mAb 125I-labeled MEM136 or 125I-labeled 225.28 to Colo38 cells. The induction of anti-MPG responses in monkeys did not cause any apparent side effects in animals, despite the fact that the MPG antigen is expressed by many normal tissues. The affinity-purified, I-Mel-2 idiotype-specific, Ab3 immunoprecipitated MPG antigen from melanoma cells. Furthermore, the I-Mel-2-induced Ab3 inhibited melanoma cell invasion in an in vitro assay, implying that these antibodies have biological significance.

Lymphocytes from lymph nodes obtained at mastectomy in breast cancer patients have been fused with murine nonproducer myeloma cells to obtain human-mouse hybridoma cultures that synthesize human monoclonal antibodies. To date, 52 hybridoma cultures synthesizing either human IgG or human IgM have been obtained from lymph nodes of 13 patients. Ig production was stable in many of these cloned cultures through 60-200 days of observation. Levels of human Ig synthesis ranged from 0.1 to 20 microgram/ml of supernatant fluid. The immunological reactivities of the human Igs were assayed on tissue sections by using the immunoperoxidase technique. Several of the human monoclonal antibodies demonstrated preferential binding to human mammary tumor cells. One human IgM monoclonal antibody was used to discriminate between mammary carcinoma cells (from 55 of 59 patients) and normal mammary epithelial cells, stroma, or lymphocytes of the same breast. Decreased binding to some of the benign breast tumors tested and to selected non-breast adenocarcinomas was also observed. This same human monoclonal antibody, however, reacted significantly with metastatic mammary carcinoma cells in lymph nodes of breast cancer patients, with no binding to normal lymphocytes or to stroma of the same node. These studies demonstrate that stable clones of human-mouse hybridomas can be generated by using lymph nodes of mastectomy patients and that clones can be selected which synthesize human monoclonal antibodies reactive with human mammary carcinoma cells.

A monoclonal antibody (M2A, IgG2a) was produced against a cultured human ovarian epithelial adenocarcinoma cell line, HEY. Monoclonal antibody M2A reacted with a glycoprotein of molecular weight 40,000 on the surface of HEY cells. The affinity constant of the monoclonal antibody M2A for HEY cells was 10(9) M-1, and the number of binding sites on HEY cells was 2 X 10(4) per cell. The monoclonal antibody produced positive immunoperoxidase staining of fetal (but not adult) testis and of seminomas and dysgerminomas but did not stain various normal adult tissues or other gonadal or extragonadal tumors. Monoclonal antibody M2A may be useful for confirming a histological diagnosis of seminoma and dysgerminoma.

We fused spleen lymphocytes obtained from mice immunized against a human melanoma cell line and melanoma-mouse hybrid cells with the P3 X 63 Ag8 mouse myeloma in order to produce hybrids secreting antibodies against a human melanoma. Antibodies secreted by individual hybrids were tested for their reaction with a panel of human melanoma, colorectal carcinoma, and normal cells in an indirect radioimmunoassay, and they displayed different specificities and crossreactivities. Some reacted only with melanomas, whereas others crossreacted with normal human or human colorectal carcinoma cells. By analysis of competitive binding of mixtures of monoclonal antibodies, it was possible to delineate different epitopes on melanomas. Hybrids growing in nude mice and producing antimelanoma antibody suppressed growth of melanoma tumors.

We have constructed a recombinant vaccinia virus, v-p97NY, which expresses the human melanoma-associated glycoprotein p97. Immunization with v-p97NY could induce humoral and cell-mediated immunity to p97, including delayed-type hypersensitivity, in mice and in two of two monkeys (Macaca fascicularis). The fact that an immune response was induced also in monkeys is important because normal cells from monkeys, but not from mice, express a low level of cross-reactive p97. Mice immunized with v-p97NY rejected transplants of syngeneic mouse melanoma expressing p97. A rejection response could be detected also when immunization was started 2 days after tumor transplantation, irrespective of whether the transplanted cells grew subcutaneously or as lung metastases. Evidence was obtained that melanoma cells lacking p97 may be killed as "bystanders" at the site of an immune response to melanoma cells expressing p97.

Plasmids have been constructed that direct the synthesis in Escherichia coli of heavy chains and/or light chains of an anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was constructed for expression of a truncated form of heavy chain (Fd' fragment) in E. coli. Functional CEA-binding activity was obtained by in vitro reconstitution in E. coli extracts of heavy chain or Fd' fragment mixed with extracts containing light chain.

We have produced somatic cell hybrids between mouse myeloma cells deficient in hypoxanthine phosphoribosyltransferase IMP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.8) and spleen cells derived from mice primed with either syngeneic or allogeneic cells transformed by simian virus 40. Such hybrids produced antibodies specific for simian virus 40 tumor (T) antigen. Only four of twelve independent hybrid cell cultures produced antibodies against simian virus 40 T antigen that crossreacted with the T antigen induced by BK virus, a human papovavirus isolated from patients who had undergone immunosuppressive therapy.