Generation of anti-idiotypic reagents in the EGFRvIII tumor-associated antigen system

The use of anti-idiotype (anti-id) vaccines for immunotherapy of human cancers is attractive, as immunization with true anti-id reagents (Ab2 beta) has been shown to induce both cellular and humoral immunity, frequently when the original antigen does not, or when a state of anergy to the self-expressed tumor-associated antigen exists. The aim of this study was to investigate the potential of an anti-id vaccine approach to the glioma-associated antigen epidermal growth factor receptor variant III (EGFRvIII) for human clinical trials. By using conventional methodology, seven rat mAbs specific for the binding site of the murine anti-EGFRvIII-specific mAb Y10, as defined by the ability to inhibit the binding of mAb Y10 to EGFRvIII expressed on cells or as purified protein, were generated, and a subset (3/7) was found to be true Ab2 beta, as defined by the ability to induce the formation of antibody directed against EGFRvIII in two species (mouse and rabbit) when used as immunogen. The ability of these three Ab2 beta to elicit a protective anti-tumor response when used as a vaccine in the syngeneic, subcutaneous C57Bl/6-B16mseEGFRvIII tumor model was investigated. Following vaccination with one Ab2 beta mAb (2C7), 6/20 mice failed to develop tumor upon challenge, and 3/20 mice with outgrowing tumors exhibited dramatic regression of incipient tumors. Vaccination with a second mAb (5G8) resulted in one tumor-free survivor and one tumor regressor; vaccination with the third Ab2 beta mAb (7D3) did not confer protection, but did significantly increase the latency period until tumor outgrowth in all vaccinated recipients. The ability of Ab2 beta mAb 2C7 to induce an anti-EGFRvIII response in non-human primates was investigated by using the saponin adjuvant approved for human clinical trial, QS-21. Three of three macaques produced anti-EGFRvIII titers, as detected on EGFRvIII-expressing cells by both ELISA and fluorescence-activated cytometric analysis, following six immunizations with Ab2 beta mAb 2C7 and QS-21. The results obtained confirm that an anti-id response in the EGFRvIII antigen system can be induced in rodents, rabbits, and non-human primates, and it may prove a useful adjunct to immunotherapeutic approaches to EGFRvIII-positive gliomas, breast carcinomas, and non-small-cell lung tumors.     

Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines.

Human cervical carcinoma cell lines that harbor human papillomavirus (HPV) have been reported to retain selectively and express HPV sequences which could encode viral E6 and E7 proteins. The potential importance of HPV E6 to tumors is suggested further by the observation that bovine papillomavirus (BPV) E6 can induce morphologic transformation of mouse cells in vitro. To identify HPV E6 protein, a polypeptide encoded by HPV-16 E6 was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 18-kd protein in two human carcinoma cell lines known to express HPV-16 RNA and in mouse cells morphologically transformed by HPV-16 DNA. The 18-kd E6 protein was distinct from a previously identified HPV-16 E7 protein. The HPV-16 E6 antibodies were found to be type specific in that they did not recognize E6 protein in cells containing HPV-18 sequences and reacted weakly, if at all, to BPV E6 protein. The results demonstrate that human tumors containing HPV-16 DNA can express an E6 protein product. They are consistent with the hypothesis that E6 may contribute to the transformed phenotype in human cervical cancers that express this protein.     

Normal Human Fibroblasts Immortalized by Introduction of Human Papillomavirus Type 16 (HPV-16) E6-E7 Genes

This report demonstrates that normal human fibroblasts can be immortalized by the introduction of HPV-16 E6-E7 genes. We designed zinc-inducible expression plasmids with HPV-16 E6, E7 or both. Each plasmid was introduced into normal human fibroblasts (TIG-3 cells) using lipofection methods. Only trans-fectants with the HPV-16 E6-E7 zinc-inducible expression plasmid, which were cultured in medium supplemented with 100 μM ZnSO₄, overcame crisis and could be cultured over 200 population doubling levels (PDLs). These cell lines showed the reactivation of telomerase after crisis, and morphological alterations were also observed.     

Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidler's method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1 x 10(5)/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1 x 10(5)/mouse)-inoculated mice survived 31.4+/-4.4 (7), 25.7+/-2.8 (7), 23.6+/-1.5 (7) and 25.3+/-2.3 (7) days [mean+/-S.D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.     

Defective 3A trophoblast-endometrial cell adhesion and altered 3A growth and survival by human papillomavirus type 16 oncogenes

Human papillomaviruses (HPVs) are found in trophoblasts of spontaneous abortions and replicate in these cells in culture. We used recombinant adeno-associated viruses (rAAV) to introduce the HPV-16 E6 and E7 oncogenes into 3A trophoblasts. AAV/E7/Neo-infected 3A trophoblasts died rapidly, but AAV/E6/Neo- and AAV/E6-E7/Neo-infected cells grew more rapidly than AAV/Neo-infected 3A cells and parental 3A. After G418 selection, the resulting E6-E7/3A and E6/3A cell lines were found to be highly defective for binding RL95 and HEC endometrial cells compared to Neo/3A and parental 3A. Serum requirements and soft agar colony formation analysis showed that E6-E7/3A had the most malignant phenotype, followed by E6/3A, with parental 3A cells having the lowest. E6/3A and E6-E7/3A were also immortal. Thus, HPV-16 oncogene expression may lead to outright trophoblast death, defective endometrial cell recognition, or a malignant phenotype. Any of these changes might lead to disruption/dysfunction of the trophoblast layer/gestational loss.     

TriVax-HPV: an improved peptide-based therapeutic vaccination strategy against human papillomavirus-induced cancers

Background

Therapeutic vaccines for cancer are an attractive alternative to conventional therapies, since the later result in serious adverse effects and in most cases are not effective against advanced disease. Human papillomavirus (HPV) is responsible for several malignancies such as cervical carcinoma. Vaccines targeting oncogenic viral proteins like HPV16-E6 and HPV16-E7 are ideal candidates to elicit strong immune responses without generating autoimmunity because: (1) these products are not expressed in normal cells and (2) their expression is required to maintain the malignant phenotype. Our group has developed peptide vaccination strategy called TriVax, which is effective in generating vast numbers of antigen-specific T cells in mice capable of persisting for long time periods.

Materials and methods

We have used two HPV-induced mouse cancer models (TC-1 and C3.43) to evaluate the immunogenicity and therapeutic efficacy of TriVax prepared with the immunodominant CD8 T-cell epitope HPV16-E7_49-57, mixed with poly-IC adjuvant and costimulatory anti-CD40 antibodies.

Results

TriVax using HPV16-E7_49-57 induced large and persistent T-cell responses that were therapeutically effective against established HPV16-E7 expressing tumors. In most cases, TriVax was successful in attaining complete rejections of 6–11-day established tumors. In addition, TriVax induced long-term immunological memory, which prevented tumor recurrences. The anti-tumor effects of TriVax were independent of NK and CD4 T cells and, surprisingly, did not rely to a great extent on type-I or type-II interferon.

Conclusions

These findings indicate that the TriVax strategy is an appealing immunotherapeutic approach for the treatment of established viral-induced tumors. We believe that these studies may help to launch more effective and less invasive therapeutic vaccines for HPV-mediated malignancies.     

A "chimeric" C57l-derived Ly49 inhibitory receptor resembling the Ly49D activation receptor.

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D(B6) in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A(B6), including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4(+) LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O(C57L)-bound H-2D(d) and lysis by 4E4(+) C57L LAK cells is inhibited by target H-2D(d). The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A-H-2D(d) complex. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.     

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8⁺ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.     

Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.

The study of human papillomavirus type 16 (HPV-16) replication has been impaired because of the lack of a cell culture system that stably maintains viral replication. Recently, cervical epithelial cell populations that stably maintain HPV-16 replicons at a copy number of approximately 1,000 per cell were derived from an HPV-16-infected patient (W12 cell clone 20863 [W12-E cells]). We used neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques to characterize HPV-16 DNA replication in these cells. When W12-E cells were maintained in an undifferentiated state mimicking the nonproductive stage of the life cycle, HPV-16 DNA was found to replicate primarily by theta structures in a bidirectional manner. The initiation site of HPV-16 DNA replication was mapped to approximately nucleotide 100, and the termination site was mapped to between nucleotides 3398 and 5990. To study the productive stage of HPV-16 DNA replication, W12-E cells were grown under culture conditions that promote differentiation of epithelial cell types. Under these conditions, where virus-like particles were detected, the mode of viral DNA replication changed from theta structure to what is apparently a rolling circle mode. Additionally, CIN 612-9E cells, which were derived from an HPV-31-infected patient and harbor HPV-31 extrachromosomally, exhibited the same switch in the mode of DNA replication upon induction of differentiation. These data argue that a fundamental switch in the mechanism of viral DNA replication occurs during the life cycle of the papillomavirus.     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

Cloning and characterization of a novel activating Ly49 closely related to Ly49A.

The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.     

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

An anti-idiotype vaccine elicits a specific response to N-glycolyl sialic acid residues of glycoconjugates in melanoma patients

We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.     

人乳头瘤病毒16型亚基因DNA体外转化功能的细胞学研究

利用HZIP16和HZIP16K(见材料和方法)质粒,将人乳头瘤病毒16型(HPV-16)的全早期区基因及其开放读码框架(ORF)E6-E7分别转入ψ2细胞,所产生的重组病毒能诱导NIH3T3细胞发生转化。转化细胞具有恶性细胞的生物学和形态学特征,可在0.3％软琼脂中形成集落,可使裸鼠致瘤。Southern blot证明,HPV-16 E6-E7 ORFs序列以整合形式存在于转化细胞和裸鼠肿瘤细胞DNA中,表明HPV-16 DNA具有体外诱导NIH3T3细胞恶性转化的作用,E6-E7 ORFs是诱导细胞转化的关键基因。     

Specific N-methylations of HPV-16 E7 peptides alter binding to the retinoblastoma suppressor protein.

Complex formation between the human papilloma virus type 16 E7 protein (HPV-16 E7) and the retinoblastoma growth suppressor protein (RB) is believed to contribute to the process of cellular transformation that leads to cervical carcinoma. Genetic analysis of the HPV-16 E7 protein has shown that the segment of E7 homologous to the conserved region 2 of adenovirus 5 E1A protein is involved in both RB binding and E7-mediated cell transformation. We have previously shown that a peptide colinear with HPV-16 E7 residues 21-29 was able to block immobilized species of E7 from binding to RB protein. The current study reports the effects of different chemical modifications of this peptide. One type of modification, methylation of the alpha-amino nitrogens contributed by Leu22, Tyr25, and Leu28, resulted in a 45-fold increase in E7/RB binding antagonist activity. This increased antagonist activity is sequence-specific since methylation of the amino groups contributed by Tyr23, Cys24, or Glu26 resulted in a profound loss of binding antagonist activity. Using a newly developed binding assay we determined that the apparent dissociation constant for recombinant HPV-16 E7 protein binding to recombinant human RB protein is 1.3 nM. The peptide Ac[N-MeLeu22,N-Me-Tyr25,N-MeLeu28]-(21-29)-E7 amide was determined to be a competitive inhibitor of HPV-16 E7 binding to RB with a Ki value of 32 nM.     

Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody

We report in this study the generation of a novel rat mAb that recognizes mouse plasmacytoid dendritic cells (pDC). This Ab, named 120G8, stains a small subset of CD11c(low) spleen cell with high specificity. This population produces high amounts of IFN-alpha upon in vitro viral stimulation. Both ex vivo- and in vitro-derived 120G8(+) cells display a phenotype identical with that of the previously described mouse pDC (B220(high)Ly6C(high)Gr1(low)CD11b(-)CD11c(low)). Mice treated with 120G8 mAb are depleted of B220(high)Ly6C(high)CD11c(low) cells and have a much-reduced ability to produce IFN-alpha in response to in vivo CpG stimulation. The mAb 120G8 stains all and only B220(high)Ly6C(high)CD11c(low) pDC in all lymphoid organs. Immunohistochemical studies performed with this mAb indicate that pDC are located in the T cell area of spleen, lymph nodes, and Peyer's patches. Although the Ag recognized by 120G8 is not yet known, we show that its expression is up-regulated by type I IFN on B cells and DC. Using this mAb in immunofluorescence studies demonstrates strain- and organ-specific differences in the frequency of pDC and other DC subsets. 129Sv mice have a much higher frequency of pDC, together with a lower frequency of conventional CD8alpha(+)CD11c(high) DC, compared with C57BL/6 mice, both in spleen and blood. The higher ability of 129Sv mice to produce IFN-alpha in vivo is related to a higher number of pDC, but also to a higher ability of pDC from 129Sv mice to produce IFN-alpha in vitro in response to viral stimulation.