L523S, an RNA-binding protein as a potential therapeutic target for lung cancer.

Approaches to vaccine-based immunotherapy of human cancer may ultimately require targets that are both tumour-specific and immunogenic. In order to generate specific antitumour immune responses to lung cancer, we have sought lung cancer-specific proteins that can be targeted for adjuvant vaccine therapy. By using a combination of cDNA subtraction and microarray analysis, we previously reported the identification of an RNA-binding protein within the KOC family, L523S, to be overexpressed in squamous cell cancers of the lung. We show here that L523S exhibits significant potential for vaccine immunotherapy of lung cancer. As an oncofetal protein, L523S is normally expressed in early embryonic tissues, yet it is re-expressed in a high percentage of nonsmall cell lung carcinoma. The specificity of L523S expression in lung cancer was demonstrated by both mRNA and protein measurements using real-time PCR, Western blot, and immunohistochemistry analyses. Furthermore, we show that immunological tolerance of L523S is naturally broken in lung cancer patients, as evidenced by detectable antibody responses to recombinant L523S protein in eight of 17 lung pleural effusions from lung cancer patients. Collectively, our studies suggest that L523S may be an important marker of malignant progression in human lung cancer, and further suggest that treatment approaches based on L523S as an immunogenic target are worthy of pursuit.

WT1-specific serum antibodies in patients with leukemia.

WT1 is an oncogenic protein expressed by the Wilms' tumor gene and overexpressed in the majority of acute myelogenous leukemias (AMLs) and chronic myelogenous leukemias (CMLs). The current study analyzed the sera of patients with AML and CML for the presence of antibodies to full-length and truncated WT1 proteins. Sixteen of 63 patients (25%) with AML had serum antibodies reactive with WT1/full-length protein. Serum antibodies from all 16 were also reactive with WT1/NH2-terminal protein. By marked contrast, only 2 had reactivity to WT1/COOH-terminal protein. Thus, the level of immunological tolerance to the COOH terminus may be higher than to the NH2 terminus. The WT1/COOH-terminal protein contains four zinc finger domains with homology to other self-proteins. By implication, these homologies may be related to the increased immunological tolerance. Results in patients with CML were similar with antibodies reactive to WT1/full-length protein detectable in serum of 15 of 81 patients (19%). Antibodies reactive with WT1/NH2-terminal protein were present in the serum of all 15, whereas antibodies reactive with WT1/COOH-terminal protein were present in only 3. By contrast to results in leukemia patients, antibodies reactive with WT1/full-length protein were detected in only 2 of 96 normal individuals. The greater incidence of antibody in leukemia patients provides strong evidence that immunization to the WT1 protein occurred as a result of patients bearing malignancy that expresses WT1. These data provide further stimulus to test therapeutic vaccines directed against WT1 with increased expectation that the vaccines will be able to elicit and/or boost an immune response to WT1.

Vaccination with Her-2/neu DNA or protein subunits protects against growth of a Her-2/neu-expressing murine tumor.

The present study utilizes an in vivo murine tumor expressing human Her-2/neu to evaluate potential Her-2/neu vaccines consisting of either full length or various subunits of Her-2/neu delivered in either protein or plasmid DNA form. Our results demonstrate that protective immunity against Her-2/neu-expressing tumor challenge can be achieved by vaccination with plasmid DNA encoding either full length or subunits of Her-2/neu. Partial protective immunity was also observed following vaccination with the intracellular domain (ICD), but not extracellular domain (ECD), protein subunit of Her-2/neu. The mechanism of protection elicited by plasmid DNA vaccination appeared to be exclusively CD4 dependent, whereas the protection observed with ICD protein vaccination required both CD4 and CD8 T cells.

Mass spectrometric identification of mtb81, a novel serological marker for tuberculosis.

We have used serological proteome analysis in conjunction with tandem mass spectrometry to identify and sequence a novel protein, Mtb81, which may be useful for the diagnosis of tuberculosis (TB), especially for patients coinfected with human immunodeficiency virus (HIV). Recombinant Mtb81 was tested by an enzyme-linked immunosorbent assay to detect antibodies in 25 of 27 TB patients (92%) seropositive for HIV as well as in 38 of 67 individuals (57%) who were TB positive alone. No reactivity was observed in 11 of 11 individuals (100%) who were HIV seropositive alone. In addition, neither sera from purified protein derivative (PPD)-negative (0 of 29) nor sera from healthy (0 of 45) blood donors tested positive with Mtb81. Only 2 of 57 of PPD-positive individuals tested positive with Mtb81. Sera from individuals with smear-positive TB and seropositive for HIV but who had tested negative for TB in the 38-kDa antigen immunodiagnostic assay were also tested for reactivity against Mtb81, as were sera from individuals with lung cancer and pneumonia. Mtb81 reacted with 26 of 37 HIV(+) TB(+) sera (70%) in this group, compared to 2 of 37 (5%) that reacted with the 38-kDa antigen. Together, these results demonstrate that Mtb81 may be a promising complementary antigen for the serodiagnosis of TB.

A multi-epitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in radioimmunoprecipitation-confirmed and consensus-positive sera.

Peptide epitopes of Trypanosoma cruzi have been identified through expression cloning. A tripeptide (2/D/E) containing three epitopes (TcD, TcE, PEP-2) was used in ELISA to detect antibodies to T. cruzi in 239 of 240 consensus-positive sera and 41 of 42 sera confirmed positive by radioimmunoprecipitation assay. The 1 discrepant consensus-positive serum was used to expression-clone a novel gene that contained a repeat sequence. A peptide corresponding to this sequence, TcLo1.2, was specific for T. cruzi. This antigen detected the discrepant consensus-positive serum and enhanced reactivity of low-positive sera in the tripeptide assay. A branched synthetic peptide, 2/D/E/Lo1.2, or a linear recombinant, r2/D/E/Lo1.2, realized all of the diagnostic features of the four epitopes, including the ability to boost reactivity of low-reactive sera. These studies show that peptides and recombinants containing multiple repeat epitopes are powerful tools for developing assays for T. cruzi antibody detection and have direct application in blood screening.

Amino acid residue at codon 268 determines both activity and nucleotide-sugar donor substrate specificity of human histo-blood group A and B transferases. In vitro mutagenesis study.

Histo-blood group A transferase produces A antigens and transfers GalNAc to the acceptor substrate, H structures of glycolipids and glycoproteins. B transferase transfers galactose in place of GalNAc to the same acceptor substrate to synthesize B antigens. We have previously identified four amino acid substitutions between human A and B transferases. Out of these four, substitutions at the last two positions (codons 266 and 268) were found to be crucial for the different donor nucleotide-sugar specificities between A and B transferases as analyzed by gene transfer of chimeric A-B transferase genes. In the present study, we have in vitro mutagenized codon 268 of these two transferase cDNA expression constructs (glycine and alanine in A and B transferases, respectively) and produced substitution constructs with every possible amino acid residue at this position. We examined the activity and specificity of each construct by gene transfer followed by immunodetection of A and B antigens and in vitro enzymatic assay. Amino acid substitution constructs on the A transferase backbone with alanine, serine, and cysteine expressed enzymes with A and B transferase activities. Weak A activity was detected with histidine and phenylalanine constructs while weak B activity was detected with asparagine and threonine constructs. All the other amino acid substitutions at codon 268 on the A transferase backbone showed neither A nor B activity. The glycine construct on the B transferase backbone expressed both A and B transferase activities. Some substitution constructs on the B transferase backbone maintained B activity while some other substitutions abolished the activity. These results show that the side chain of the amino acid residue at 268 of the human A and B transferases is responsible for determining both activity and nucleotide-sugar donor substrate specificity and strongly suggest its direct involvement in the recognition of and binding to the sugar moiety of the nucleotide-sugars.

Genomic organization of human histo-blood group ABO genes.

We have isolated human genomic DNA clones encompassing 30 kbp of the histo-blood group ABO locus. The locations of the exons have been mapped and the nucleotide sequences of the exon-intron boundaries have been determined. The human ABO genes consist of at least seven exons, and the coding sequence in the seven coding exons spans over 18 kb of the genomic DNA. The exons range in size from 28 to 688 bp, with most of the coding sequence lying in exon 7.

Molecular genetic analysis of the ABO blood group system: 3. A(X) and B(A) alleles.

We have employed a PCR approach to determine the nucleotide sequences of the coding region in the last two coding exons of the histo-blood group ABO genes from one A(X) and one B(A) individual. Compared with A1 alleles, the (A(X)) allele has a single nucleotide substitution (T-->A at nucleotide 646) resulting in an amino acid substitution (phenylalanine-->isoleucine at amino acid 216). Compared with B alleles, the B(A) allele has two nucleotide substitutions (T-->C at nt. 657 and A-->G at nt. 703) resulting in an amino acid substitution (serine-->glycine at aa. 235). The amino acid substitution resulting from this B(A) allele is located at the second of the four amino acid substitutions which discriminate human A and B transferases, and the amino acid residue (glycine) is identical to that of A transferase suggesting the involvement of this amino acid or its surrounding area for the recognition and/or binding of the donor nucleotide sugars.

Molecular genetic analysis of the ABO blood group system: 4. Another type of O allele.

We have encountered an allele which seems to be another type of O allele at the human histo-blood group ABO locus. We have determined the nucleotide sequence of this allele over the coding region in the last two coding exons. This allele does not possess the single-nucleotide deletion found common among all the O alleles previously analyzed. Compared with A1 allele, this allele has three nucleotide substitutions resulting in two amino acid substitutions. The introduction of these amino acid substitutions into the A1 transferase expression construct apparently abolished the enzymatic activity of A1 transferase.

Molecular genetic analysis of the ABO blood group system: 1. Weak subgroups: A3 and B3 alleles.

We have determined the nucleotide sequences of the coding region in the last two coding exons of ABO genes (which occupy 91% of the soluble form of A1 transferase) from 7 individuals with weak subgroup phenotypes. Four of the individuals had an A3 phenotype and 3 individuals had a B3 phenotype. We determined the nucleotide sequences based on PCR followed by subcloning and DNA sequencing of the amplified fragments. Two cases of the A3 allele and 1 case of the B3 allele were found to contain a single-base substitution which resulted in an amino acid substitution. However, no other cases of A3 and B3 alleles were found to contain differences in this region. This finding demonstrates for the first time heterogeneity among these weak subgroups at the nucleotide level.

Molecular genetic analysis of the ABO blood group system: 2. cis-AB alleles.

We have determined the nucleotide sequence of the coding region in the last two coding exons of ABO genes from two cis-AB individuals (genotype cis-AB/O) with no consanguinity. In this region, cis-AB alleles from these 2 individuals were identical to one another while different from the A1 allele by two nucleotide substitutions. Both of these nucleotide substitutions result in amino acid substitutions. The first substitution is identical to the one previously found in the A2 allele. The other substitution is found at the fourth position of the four amino acid substitutions which discriminate A1 and B transferases.

Animal histo-blood group ABO genes.

Sequences homologous to the human histo-blood group ABO genes are present in the genomic DNA of various mammals. We have PCR-amplified, subcloned, and sequenced a portion of these genes from several species of primates and found high conservation of the nucleotide as well as the deduced amino acid sequences during evolution.

Human histo-blood group A2 transferase coded by A2 allele, one of the A subtypes, is characterized by a single base deletion in the coding sequence, which results in an additional domain at the carboxyl terminal.

We have identified a possible mutation which characterizes A2 alleles (a minor subtype of A) at the human histo-blood group ABO locus based on polymerase chain reaction (PCR) of genomic DNA, followed by nucleotide sequencing of the amplified fragments. The A2 subtype has a single base deletion near the carboxyl terminal. As a result of frame-shifting, A2 transferase possesses an extra domain. Introduction of this single base deletion into the A1 transferase cDNA expression construct drastically decreased the A transferase activity in DNA-transfected HeLa cells.

Identification in human genomic DNA of the sequence homologous but not identical to either the histo-blood group ABH genes or alpha 1----3 galactosyltransferase pseudogene.

Based on polymerase chain reaction (PCR) and sequencing of the cloned amplified fragments, we identified a homologous sequence to the histo-blood group ABH genes and alpha 1----3 galactosyltransferase pseudogene. The presence of this sequence in human genomic DNA was confirmed by Southern hybridization.