Highly efficient and consistent gene transfer into dendritic cells utilizing a combination of ultraviolet-irradiated adenovirus and poly(L-lysine) conjugates.

Dendritic cells (DCs) are capable of presenting tumor-associated antigens and subsequently play an essential role in T-cell activation. The aim of this study was to develop an efficient method for gene transfer into DCs. These genetically transduced DCs can then be used as potent inducers of specific cell-mediated immune response. When compared with physical methods for gene transfer (lipofection and calcium phosphate precipitation), adenovirus (AdV) vectors proved to be highly efficient for gene transfer into DCs. To overcome concomitant AdV gene expression and potential immunogenicity, AdVs were irradiated with UV. The UV dose was optimized to block AdV transcription without altering AdV receptor binding and endocytosis capacity. We subsequently used a polycationic amino acid compound, poly(L-lysine), to conjugate the irradiated AdVs to transgenes. The resulting complexes were found to mediate a highly efficient transfer of transgenes into DCs, without concomitant expression of AdV gene products. Low titers of irradiated AdVs were sufficient for a consistent gene transfer into DCs. This is the first study to demonstrate efficient, consistent, and practical gene transfer using an UV approach to irradiated AdV-poly(L-lysine) conjugates and should be useful for the development of DC-based tumor vaccine therapies.

Identification of a positive regulatory element responsible for tissue-specific expression of prostate-specific antigen.

The prostate-specific antigen (PSA) promoter (PSA-P) has been identified, characterized, and determined to be tissue specific. Compared with high expression of the genomic PSA gene in prostate cells, expression of the transgene driven by the putative PSA promoter is low. This suggests that the identified promoter may be incomplete or may function optimally with additional regulatory elements. To identify the presence of additional regulatory elements, we screened sequences upstream of the PSA promoter and identified a DNA fragment of 822 bp, which enhances PSA gene expression. Combining the newly identified PSA gene regulatory sequence (PSAR) with our previously identified PSA promoter (PCPSA-P) exhibited enhanced expressional activity in the PSA-producing LNCaP cell line. With the addition of 10 to 100 nM dihydrotestosterone, a more than 1000-fold increase in expression was observed as compared to androgen-negative controls. Furthermore, although the combined regulatory element (PSAR)-PSA promoter (PCPSA-P) sequence resulted in high transgene expression in LNCaP cell lines, the combined regulatory element-promoter sequence resulted in minimal expression in the non-PSA-producing prostate cell line PC-3, renal tumor cell line R11, and cervical adenocarcinoma cell line HeLa. The newly identified 822 bp alone could also function as a promoter. Compared with the combined promoter, however, the 822-bp fragment alone demonstrated lower activity and lower responsiveness to androgen stimulation. Our results suggest that coupling the PSA promoter with an upstream regulatory element results in a marked increase in PSA expression, suggesting that the complete PSA promoter contains two functional domains: a proximal promoter and a distal promoter, which can also function as an enhancer. The enhanced gene expression of the new construct, combined with its tissue specificity and androgen responsiveness, in turn provides a foundation for the development of tissue-specific vectors for prostate cancer gene therapy.

Prostate stem cell antigen is a promising candidate for immunotherapy of advanced prostate cancer.

Immunotherapy of prostate cancer (CaP) may be a promising novel treatment option for the management of advanced CaP. However, the lack of suitable tumor antigens remains a major obstacle for the rational design of vaccines. To characterize potential CaP antigens, we determined the mRNA expression of the prostate-specific genes C1, C2, C5, PAGE-1, and prostate stem cell antigen (PSCA) in hormone-refractory CaP, benign prostatic hyperplasia, CaP cell lines, and CaP specimens. Among these gene products, only expression of PSCA appears to be retained in the majority of advanced CaP samples, as shown by reverse transcription-PCR analyses. Peptide fragments of PSCA presented in the context of major histocompatibility molecules could serve as recognition targets for CD8 T cells, provided these lymphocytes were not clonally deleted or peripherally tolerized. Our goal was to determine whether the human T-cell repertoire could recognize PSCA-derived peptide epitopes in the context of a common class I allele, HLA-A0201. Of nine peptides that, according to HLA-A0201 binding motifs, were candidate ligands of A0201 class I molecules, three peptides were able to stabilize HLA-A0201 molecules on the cell surface. One of the latter peptides, encompassing amino acid residues 14-22, was capable of generating a PSCA-specific T-cell response in a human lymphocyte culture from a patient with metastatic CaP. PSCA-specific CTLs recognized peptide-pulsed targets as well as three prostate carcinoma lines in cytotoxicity assays, indicating that this peptide could be endogenously processed. In conclusion, our findings establish PSCA as a potential target for antigen-specific, T cell-based immunotherapy of prostate carcinoma.

Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1.

A 55 residue peptide corresponding to the nucleocapsid protein of HIV-1 (NCp7) containing two zinc binding domains as well as three truncated peptides were synthesized by Fmoc-based solid phase synthesis using the fragment condensation approach. Circular dichroism (CD) data support a conformational model in trifluoroethanol/buffer solution consisting of two helical segments at the chain ends with two Zn-modules in the center of the molecule. CD titration experiments show that the synthetic protein binds two equivalents of Zn2+ stoichiometrically, and the Zn2+ induced conformational changes are completely reversible by addition of EDTA. NCp7 and its S-acetamidomethylated analog (NCp7-Acm), devoid of the zinc co-ordination centers, exhibit preferential binding to RNA with a Kd = approximately 10(-9) M irrespective of the cysteine modification as determined by filter binding assays. The binding affinity of the NCp7 protein to single-stranded DNA is lower than to RNA. Binding to double-stranded DNA is lower than to ssDNA. The NCp7-Acm protein exhibits reduced single-stranded DNA binding affinity compared to the unmodified protein. Nucleic acid binding analyses with the fragments of NCp7 protein suggest that two basic amino acid stretches are involved in RNA binding of the NCp7.

Single mutations in a gene for a tail fiber component of an Escherichia coli phage can cause an extension from a protein to a carbohydrate as a receptor.

The T-even type Escherichia coli phage Ox2 recognizes the outer membrane protein OmpA as a receptor. This recognition is accomplished by the 266 residue protein 38, which is located at the free ends of the virion's long tail fibers. Host-range mutants had been isolated in three consecutive steps: Ox2----Ox2h5----Ox2h10----Ox2h12, with Ox2h12 recognizing the outer membrane protein OmpC efficiently and having lost some affinity for OmpA. Protein 38 consists, in comparison with these proteins of other phages, of two constant and one contiguous array of four hypervariable regions; the alterations leading to Ox2h12 were all found within the latter area. Starting with Ox2h12, further host-range mutants could be isolated on strains resistant to the respective phage: Ox2h12----h12h1----h12h1.1----h12h1.11----h12 h1.111. It was found that Ox2h12h1.1 (and a derivative of Ox2h10, h10h4) probably uses, instead of OmpA or OmpC, yet another outer membrane protein, designated OmpX. Ox2h12h1.11 was obtained on a strain lacking OmpA, -C and -X. This phage could not grow on a mutant of E. coli B, possessing a lipopolysaccharide (LPS) with a defective core oligosaccharide; Ox2h12h1.111 was obtained from this strain. It turned out that the latter two mutants used LPS as a receptor, most likely via its glucose residues. Selection for resistance to them in E. coli B (ompA+, ompC-, ompX-) yielded exclusively LPS mutants, and in another strain, possessing OmpA, C and X, the majority of resistant mutants were of this type. Isolated LPS inactivated the mutant phages very well and was inactive towards Ox2h12. By recombining the genes of mutant phages into the genome of parental phages it could be shown that the phenotypes were associated with gene 38. All mutant alterations (mostly single amino acid substitutions) were found within the hypervariable regions of protein 38. In particular, a substitution leading to Ox2h12h1.11 (Arg170----Ser) had occurred at the same site that led to Ox2h10 (His170----Arg), which binds to OmpC in addition to OmpA. It is concluded that not only can protein 38 gain the ability to switch from a protein to a carbohydrate as a receptor but can do so using the same domain of the polypeptide.

Amino acid requirements of the nucleocapsid protein of HIV-1 for increasing catalytic activity of a Ki-ras ribozyme in vitro.

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several functions such as specific recognition, dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37 degrees C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme.

The polypurine tract, PPT, of HIV as target for antisense and triple-helix-forming oligonucleotides.

Replication of retroviral RNA into double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by the reverse transcriptase (RT). The PPT is highly conserved among the known HIV-1 retroviral isolates. It occurs twice, once within the coding region of the integrase and the other one adjacent to the 3' LTR. The data presented show that two antisense oligonucleotides, a 20-mer and a 40-mer, complementary to the PPT induce complete blocks of DNA synthesis whereas an antisense oligonucleotide outside the PPT is only slightly inhibitory. Previously polypurine sequences have been used by several groups for triplex-formation. During replication the HIV-polypurine tract, PPT, is present in a RNA-DNA hybrid. Therefore triple-helix formation consisting of RNA-DNA and a third DNA strand covering the PPT region was tested here by protection against RNase H cleavage in vitro. Incubation with a pyrimidine oligonucleotide in parallel orientation to the PPT-RNA shows some protection. GT-pyrimidine-purine mixed oligonucleotides (25-mer) led to protection against RNase H up to 50% independent of their orientation. The data suggest that triple-helix formation may have taken place with the PPT in vitro. Therefore, this highly conserved structure may prove useful in nucleic acid based anti-viral therapy with antisense or triple-helix approaches. Furthermore, the influence of HIV-1 nucleocapsid (NC) protein, NCp15, on reverse transcription is reported. The data show a two- to three-fold stimulatory effect of the NCp15 on RNA directed DNA synthesis.

Current Status of Dendritic Cell-Based Tumor Vaccination.

For several decades, approaches utilizing nonspecific immune stimulants have provided evidence that the immune system, when properly activated, may eradicate cancer cells. However, it was only after the identification of the first human tumor-associated antigen, less than a decade ago, that development of specific vaccination procedures for cancer patients became feasible. Recent insights into the pivotal role of dendritic cells (DCs) for initiation and regulation of immune responses have allowed the design of DC-based tumor vaccination trials. In addition, the development of methods to raise large numbers of DCs from peripheral blood monocytes has paved the way for their clinical application. Tumor-specific vaccination utilizing antigen-loaded autologous DCs, has become practical and applicable to patients and may lead to vigorous antitumor responses. This review outlines recent progress, obstacles still to be overcome, and the future potential of DC-based vaccination. Copyright 2000 S. Karger GmbH, Freiburg

Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

The nucleocapsid (NC) protein of human immunodeficiency virus HIV-1 (NCp7) is responsible for packaging the viral RNA by recognizing a packaging site (PSI) on the viral RNA genome. NCp7 is a molecule of 55 amino acids containing two zinc fingers, with only the first one being highly conserved among retroviruses. The first zinc finger is flanked by two basic amino acid clusters. Here we demonstrate that chemically synthesized NCp7 specifically binds to viral RNA containing the PSI using competitive filter binding assays. Deletion of the PSI from the RNA abrogates this effect. The 35 N-terminal amino acids of NCp7, comprising the first zinc finger, are sufficient for specific RNA binding. Chemically synthesized mutants of the first zinc finger demonstrate that the amino acid residues C-C-C/H-C/H are required for specific RNA binding and zinc coordination. Amino acid residues F16 and T24, but not K20, E21 and G22, located within this zinc finger, are essential for specific RNA binding as well. The second zinc finger cannot replace the first one. Furthermore, mutations in the basic amino acid residues flanking the first zinc finger demonstrate that R3, 7, 10, 29 and 32 but not K11, 14, 33 and 34 are also essential for specific binding. Specific binding to viral RNA is also observed with recombinant NCp15 and Pr55Gag. The results demonstrate for the first time specific interaction of a retroviral NC protein with its PSI RNA in vitro.

Mutations of basic amino acids of NCp7 of human immunodeficiency virus type 1 affect RNA binding in vitro.

The nucleocapsid (NC) protein of human immunodeficiency virus type 1 is required for packaging of viral RNA and for virion assembly. It contains two clusters of basic amino acids, consisting of five and four amino acid residues, flanking the first of its two zinc fingers. These amino acid residues have been mutagenized to neutral ones individually, as well as in various combinations, by site-directed mutagenesis. Wild-type NCp7 and the mutant proteins were expressed as recombinant proteins in Escherichia coli, with six histidines as tags at their amino termini in order to allow efficient purification. The purified proteins were analyzed for RNA binding in vitro with human immunodeficiency virus type 1 5' leader RNA transcribed in vitro. Assays comprised Northwestern blots at various salt concentrations and filter binding tests which allowed determination of the dissociation constants of the various mutants. The results indicated that mutations of the amino acid R-7 and of R-32 and K-33 were more critical for RNA binding than other mutations. Mutation of the other amino acid residues reduced the binding affinity in proportion to the number of mutations. Mutation of seven of the nine basic amino acid residues reduced the binding of RNA by 50- to 90-fold.

Adenovirus-mediated interleukin-2 production by tumors induces growth of cytotoxic tumor-infiltrating lymphocytes against human renal cell carcinoma.

Combination therapy with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes (TILs) demonstrates significant clinical activity in patients with metastatic renal cell carcinoma (RCC). To investigate whether local delivery of IL-2 via gene transfer is capable of improving the potency and efficacy of in vitro propagated TILs as compared with standard growth conditions [400 BRMP U (BU)/ml], a replication-deficient adenovirus expressing the human IL-2 gene under control of the cytomegalovirus (CMV) promoter (Ad-IL-2) has been constructed in our laboratory. RCC-TIL cultures were initiated by directly infecting RCC tumor suspension with Ad-IL-2 at a multiplicity of infection of 10:1. Subsequently the TIL cultures were restimulated with nonirradiated autologous RCC infected with Ad-IL-2 (RCC-Ad-IL-2) every 10 days (TIL/tumor = 50:1). Cell growth, phenotype, cytotoxicity, and cytokine messenger RNA (mRNA) expression were analyzed and compared with TIL growth stimulated with exogenous IL-2 (400 BU/ml). All five TILs tested responded to RCC-Ad-IL-2 activation, and a completed clearance of tumor cells was observed in cultures within 7-10 days. Lysis of nonirradiated RCC-Ad-IL-2 cells by TILs also was observed in cultures 3-5 days after restimulation. The IL-2 concentration in cell culture supernatants was maintained between 10 BU and 35 BU/ml (2 and 7 ng/ml), respectively. When compared with exogenous IL-2, RCC-Ad-IL-2 induced less growth expansion of TILs whereas a reduced CD56+ (23 +/- 14% vs. 44 +/- 13%; p < 0.05) but increased CD3+CD4+ cell population (32 +/- 11% vs. 15 +/- 6%; p < 0.05) with enhanced T cell-receptor use (59 +/- 10% vs. 42 +/- 7%; p < 0.005) was determined. An augmented human leukocyte antigen (HLA)-restricted and tumor-specific cytotoxicity was detected in RCC-Ad-IL-2-expanded TILs (day 35, 15.3 +/- 4.2 LU vs. 4.6 +/- 1.8 LU; p < 0.005). These properties were mediated by the CD8+ and CD4+ T-cell populations, as demonstrated by antibody-blocking assays. A unique cytokine profile also was detected in RCC-Ad-IL-2-induced TILs, which demonstrated an upregulation of both GM-CSF and IL-6 mRNA as compared with TILs expanded in the presence of exogenous IL-2. These data suggest that RCC-Ad-IL-2 is a potent immune stimulant that can be used in vitro as an immunogen to propagate cytotoxic RCC-TILs for adoptive immunotherapy or potentially in vivo by direct injection as a live tumor vaccine.