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HER2/neu is an oncogene amplified and over-expressed in 20-30% of breast adenocarcinomas. Treatment with the humanized monoclonal antibody trastuzumab has shown efficacy in combination with cytotoxic agents, although resistance occurs over time. Novel approaches are needed to further increase antibody efficacy. In this study, we provide evidence in a mouse breast cancer therapeutic tumor model that the combination of active immunization with a modified HER2/neu DNA vaccine and passive infusion of an anti-HER2/neu monoclonal antibody leads to significant regression of established tumors. Our data indicate that combination therapy with a HER2/neu DNA vaccine and trastuzumab may have clinical activity in breast cancer patients., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Prostate-specific membrane antigen is a type II membrane glycoprotein that is expressed in benign and neoplastic prostatic tissue and has been recently shown to be also expressed in the neovasculature of various solid malignant tumors including renal cell carcinoma. Renal cell carcinoma is a heterogeneous group of tumors with distinct morphologic and genetic characteristics and clinical behaviors. We performed immunohistochemical studies on formalin-fixed, paraffin-embedded archival material from 75 nephrectomies, using antibodies 13D6 against prostate-specific membrane antigen and CD31 against endothelial cells. The study included 30 clear cell renal cell carcinomas, and 15 of each of papillary and chromophobe renal cell carcinoma and oncocytoma. The extent and intensity of staining were assessed semiquantitatively. In all cases, immunoreactivity was detected only in the tumor-associated neovasculature and not in tumor cells. Clear cell renal cell carcinoma showed the most diffuse staining pattern, where 24/30 cases or 80% had >50% reactive vessels, followed by chromophobe renal cell carcinoma (9/15; 60%) and oncocytoma (5/15, 33%). No diffuse staining was detected in any of the papillary renal cell carcinomas and only focal staining was detected in 11 cases (11/15; 73%). Staining intensity was the strongest in clear cell renal cell carcinoma (25/30; 83%) followed by chromophobe renal cell carcinoma (9/15; 60%), oncocytoma (8/15, 53%) and papillary renal cell carcinoma (5/15; 33%). In summary, prostate-specific membrane antigen is expressed in tumor-associated neovasculature of the majority of renal cortical tumors and is most diffusely and intensely expressed in clear cell renal cell carcinoma and least in papillary renal cell carcinoma. The differences in the expression of prostate-specific membrane antigen in renal cell carcinoma subtypes provide further evidence of the biological diversity of these tumors, and diagnostic and therapeutic applications of such expression can be expanded to include subtypes of renal cell carcinoma.

Purpose: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge., Experimental Design: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge., Results: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell-mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in naïve hosts., Conclusions: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.

Background: We have previously shown that xenogenic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8., Methods: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma., Results: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anit-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models., Discussion: A local immune responses to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may act more as an adjuvant than an immunologic target.

Background: We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8., Methods: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma., Results: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models., Discussion: A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

Purpose: Plasmid DNAs encoding cytokines enhance immune responses to vaccination in models of infectious diseases and cancer. We compared DNA adjuvants for their ability to enhance immunity against a poorly immunogenic self-antigen expressed by cancer., Experimental Design: DNAs encoding cytokines that affect T cells [interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and the chemokine CCL21] and antigen-presenting cells [granulocyte macrophage colony-stimulating factor (GM-CSF)] were compared in mouse models as adjuvants to enhance CD8+ T-cell responses and tumor immunity. A DNA vaccine against a self-antigen, gp100, expressed by melanoma was used in combination with DNA encoding cytokines and cytokines fused to the Fc domain of mouse IgG1 (Ig)., Results: We found that (a) cytokine DNAs generally increased CD8+ T-cell responses against gp100; (b) ligation to Fc domains further enhanced T-cell responses; (c) adjuvant effects were sensitive to timing of DNA injection; (d) the most efficacious individual adjuvants for improving tumor-free survival were IL-12/Ig, IL-15/Ig, IL-21/Ig, GM-CSF/Ig, and CCL21; and (e) combinations of IL-2/Ig+IL-12/Ig, IL-2/Ig+IL-15/Ig, IL-12/Ig+IL-15/Ig, and IL-12/Ig+IL-21/Ig were most active; and (f) increased adjuvanticity of cytokine/Ig fusion DNAs was not related to higher tissue levels or greater stability., Conclusions: These observations support the potential of cytokine DNA adjuvants for immunization against self-antigens expressed by cancer, the importance of timing, and the enhancement of immune responses by Fc domains through mechanisms unrelated to increased half-life.

Prostate-specific membrane antigen (PSMA) is a prototypical differentiation antigen expressed on normal and neoplastic prostate epithelial cells, and on the neovasculature of many solid tumors. Monoclonal antibodies specific for PSMA are in development as therapeutic agents. Methodologies to actively immunize against PSMA may be limited by immunologic ignorance and/or tolerance that restrict the response to self-antigens. Our studies have previously shown that xenogeneic immunization with DNA vaccines encoding melanosomal differentiation antigens induces immunity in a mouse melanoma model. Here we apply this approach to PSMA to establish proof of principle in a mouse model. Immunization with xenogeneic human PSMA protein or DNA induced antibodies to both human and mouse PSMA in mice. Monoclonal antibodies specific for mouse PSMA were generated to analyze antibody isotypes and specificity for native and denatured PSMA at the clonal level. Most antibodies recognized denatured PSMA, but C57BL/6 mice immunized with xenogeneic PSMA DNA followed by a final boost with xenogeneic PSMA protein yielded autoantibodies that reacted with native folded mouse PSMA. Monoclonal antibodies were used to confirm the expression of PSMA protein in normal mouse kidney. These results establish the basis for clinical trials to test PSMA DNA vaccines in patients with solid tumors that either express PSMA directly or that depend on normal endothelial cells expressing PSMA for their continued growth.

Xenogeneic DNA vaccination can elicit tumor immunity through T cell and antibody-dependent effector mechanisms. Blockade of CTLA-4 engagement with B7 expressed on APCs has been shown to enhance T cell-dependent immunity. We investigated whether CTLA-4 blockade could increase T-cell responses and tumor immunity elicited by DNA vaccines. CTLA-4 blockade enhanced B16 tumor rejection in mice immunized against the melanoma differentiation antigens tyrosinase-related protein 2 and gp100, and this effect was stronger when anti-CTLA-4 was administered with booster vaccinations. CTLA-4 blockade also increased the T-cell responses to prostate-specific membrane antigen (PSMA) when given with the second or third vaccination. Based on these pre-clinical studies, we suggest that anti-CTLA-4 should be tested with xenogeneic DNA vaccines against cancer and that special attention should be given to sequence and schedule of administration.

Immunotherapy is currently being investigated as a treatment for patients with asymptomatic, recurrent prostate cancer manifested only by a rising prostate-specific antigen (PSA) level. Several different approaches to active immunization against antigens found on cancer cells have been explored. Immunization with DNA overcomes many of the obstacles noted in previous studies. Injection of plasmid DNA encoding a xenogeneic differentiation antigen (prostate-specific membrane antigen [PSMA]) is a potent means to induce antibody and T-cell responses to these otherwise poorly immunogenic self proteins. Use of the xenogeneic DNA (ie, human PSMA DNA injected into mouse) has been shown to be an absolute requirement to overcome immunologic tolerance. We are currently conducting a phase I trial of human and mouse PSMA DNA vaccines in patients with recurrent prostate cancer, based on preclinical experiments described below.

Cancer poses a difficult problem for immunotherapy because it arises from the host's own tissues. Many of the target antigens are tissue-specific molecules shared by cancer cells and normal cells. Thus, these are weak antigens that do not typically elicit immunity. In addition, tumors have several features that make their recognition and destruction by the immune system difficult. Despite these obstacles, several strategies for developing effective tumor immunity have been developed. Crucial to these approaches is the discovery and understanding of the molecular identity of antigens and the mechanisms involved in tumor immunity. In this review, strategies to overcome immune ignorance and tolerance are discussed.

The transcription factor TFII-I has been shown to bind independently to two distinct promoter elements, a pyrimidine-rich initiator (Inr) and a recognition site (E-box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII-I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E-box elements. The primary structure of TFII-I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix-loop/span-helix homology. These unique structural features suggest that TFII-I may have the capacity for multiple protein-protein and, potentially, multiple protein-DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII-I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E-box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions.

The use of chemiluminescence and the ELISA spot assay for identifying rare immunoglobulin switch variants is described. The technique utilizes nitrocellulose membranes and allows rapid screening of a large number of cells. The number of spots can be recorded either manually or automatically by using a commercially available colony counting program. This modification of the ELISA spot assay makes it less labor intensive and time consuming and can be adapted for the search for rare cells secreting small amounts of Ig or other macromolecules.

The Xenopus laevis TFIIIA promoter contains a motif that has been implicated in promoter activation in late-stage oocytes and contains the sequence (-269) CACGTG (-264). A cDNA encoding a protein (B1) that binds to this element has been cloned from X. laevis and Xenopus borealis ovarian cDNA libraries. We show that this protein is a member of the helix-loop-helix family of regulatory proteins and contains 80% sequence identity with the human adenovirus major late transcription factor (MLTF or USF). A survey of B1 protein expression during oogenesis and embryogenesis revealed both oocyte-specific and somatic cell-specific B1 protein-DNA complexes. Immunological data, RNA blot analysis, and proteolytic clipping band shift assays indicated that these complexes most likely represent altered forms of a single B1 polypeptide. Implications for TFIIIA gene regulation during development are discussed.

We isolated full-length cDNAs encoding the 43-kD form of human upstream stimulatory factor (USF), a cellular factor required for efficient transcription of the adenovirus major late (AdML) promoter in vitro. Sequence analysis showed USF to be a member of the c-myc-related family of DNA-binding proteins. Using proteins translated in vitro, we identified a DNA-binding domain near the carboxyl terminus, which includes both a helix-loop-helix motif and a leucine repeat. We show that USF interacts with its target DNA as a dimer. The leucine repeat is required for efficient DNA binding of the intact protein and for interactions between full-length and truncated USF proteins. Interestingly, it is not required for DNA binding of the isolated helix-loop-helix domain. The structure of different cDNA clones indicates that USF RNA is differentially spliced, and alternative exon usage may regulate the levels of functional USF protein.

We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The S1 nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect.

We report here a 54 base pair deletion in the CH3 exon of the alpha gene in the mouse myeloma W3129. This deletion results in a loss of 18 amino acids and a change from a glycine to a serine at position 464. The extent of the deletion was determined by sequencing a portion of CH3 cloned from a variant of W3129, and S1 nuclease protection showed the deletion pre-exists in the parental cell line. The deletion removes the donor splice site normally used in joining CH3 to the alpha membrane (MB) exon when forming MB-specific mRNA. Examination of cytoplasmic RNA by blot hybridization and S1 nuclease protection using MB-specific probes showed a complete lack of membrane mRNA in W3129 and its derivatives. An RNA transcript of unknown origin and function which includes sequences from the CH3-MB intron was seen in W3129 and in J558, an IgA, lambda myeloma with specificity for alpha (1----3) linked dextrans. We discuss the possible influence of the mutation on the W3129 protein. In contrast to the other myelomas studied in this laboratory, light chain loss variants are readily isolated from W3129 and are stable in their production of heavy chain [Dackowski and Morrison (1981) Proc. natn. Acad. Sci. U.S.A. 78, 7091-7095].

The function of sequences 3' of Ig genes in controlling their expression has been investigated by analyzing mutants of Ig-producing cells and by gene transfection experiments. A mutant of an IgA producing myeloma was isolated whose steady-state level of heavy chain mRNA and protein was decreased. Analysis of the mutant showed it had deleted at least 4 kb of DNA immediately 3' of the alpha gene and introduced at least 5 kb of non-Ig sequence in its place. Examination of nuclear RNA showed no accumulation of aberrant transcripts or altered processing patterns. Instead, the transcription rate of heavy chain in the mutant was approximately 1/7 of that in its parent. This mutant suggests that sequences 3' of Ig genes facilitate their transcription; alternatively, the non-Ig sequences may act to depress transcription. A complete gamma 2b heavy chain gene containing both the secreted and membrane exons was transfected into lymphoid cells. The ratio of membrane/secreted Ig mRNA produced by the transfectants was found to reflect the phenotype of the recipient cell; myelomas made mostly secreted mRNA while lymphomas made only a slight excess of secreted mRNA. When a heavy chain was used with a deletion of sequences within the IVS between the secreted and membrane exons which left the AATAA poly A addition signal intact, but removed the site of cleavage and polyadenylation, the processing ratio was altered so that predominantly membrane Ig was produced in transfected myeloma cells. The alteration in processing could be a result of the deletion of the normal site for poly A addition; alternatively it could be a result of the deletion of another sequence which is recognized by processing enzymes. A 13 bp sequence (GTCCTGGTTCTTT), was found to be highly conserved both in position and sequence in human and mouse gamma chain genes. When a gene with a deletion which left the poly A addition site and the conserved sequence intact was used for transfection, the processing pattern was found to be identical to that of a wild type heavy chain gene.

The human gene-specific upstream stimulatory transcription factor (USF) is required, both in vivo and in vitro, for maximal expression of the major late promoter (MLP) of adenovirus. We report here the complete purification and identification of USF from HeLa cell nuclei. The protein was followed throughout its purification using a quantitative filter binding assay. With a combination of classical purification techniques and fast-flow protein liquid chromatography, USF can be purified to homogeneity starting either with a standard HeLa cell nuclear extract or with a higher salt extract from (lysed) HeLa cell nuclei (nuclear pellet extract). Approximately 20,000-fold purification from the nuclear pellet extract and 80,000-fold from the nuclear extract are necessary to obtain homogeneous preparations of the transcription factor. A maximum of 20,000 molecules of USF appear to be present in HeLa cells. Two major forms of the USF protein can be distinguished both by their slightly different mobilities in sodium dodecyl sulfate gel electrophoresis (apparent molecular weights 44,000 and 43,000, respectively) and by different electrophoretic mobilities of the corresponding protein-DNA complexes. Both forms of USF are heat-stable and interact with the MLP as monomers. Antibodies elicited against purified HeLa USF interact with the transcription factor bound to the MLP upstream element.