Identification of the target of monoclonal antibody A6H as dipeptidyl peptidase IV/CD26 by LC MS\MS.

The monoclonal antibody (MAb) A6H, originally developed to fetal renal tissues, was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co-stimulate a subpopulation of T cells. The A6H antigen had not been identified heretofore. Antigen from detergent extracts of renal cell carcinoma cells (7860) was immunoabsorbed with A6H-agarose, and the resin-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen had a molecular weight of approximately 120 kDa as determined by Western blots. The 120-kDa protein band was excised and subjected to in-gel tryptic digestion, and the resulting peptides were separated and analyzed by liquid chromatography tandem mass spectrometry (LC MS\MS). The tandem mass spectra of the eluting peptides were used in combination with the SEQUEST computer program to search a human National Cancer Institute (NCI) protein database for the identity of the protein. The target antigen was shown to be dipeptidyl peptidase IV (DPP IV), which is also known as the cluster differentiation antigen CD26. Flow analysis of the expression of the A6H antigen and of CD26 on 7860 cells and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV. Recognition that the A6H antigen is DPP IV/CD26 afforded the opportunity to compare previous studies on A6H with those on other anti-CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co-stimulators of T-cell activation.

Identification of prostate specific membrane antigen (PSMA) as the target of monoclonal antibody 107-1A4 by proteinchip; array, surface-enhanced laser desorption/ionization (SELDI) technology.

Recently we described the generation of the prostate tissue-specific monoclonal antibody (MAb) 107-1A4, its expression pattern and preliminary targeting of human prostate cancer xenografts. In this report we demonstrate that the target antigen for MAb 107-1A4 is prostate-specific membrane antigen (PSMA) using immunoaffinity absorption followed by SDS-PAGE and mass spectrometric analysis of peptides produced by in-gel tryptic digestion. The identity of the antigen has been confirmed by Western blots using MAbs of known specificity. MAb 107-1A4 is not reactive on Western blots. The conformational epitope for 107-1A4 is on the extracellular domain of PSMA. In competition studies, the binding of MAb 107-1A4 to LNCaP cells is inhibited by itself but not by any other of several other anti-PSMA MAbs, suggesting that the epitope may be unique. These results suggest that 107-1A4 is reactive to a conformational epitope in the external domain of PSMA that is unique among the panel of anti-PSMA MAbs in this study. Furthermore this work demonstrates the ability of mass spectroscopy to elucidate antibody-ligand interaction.

Mechanistic studies of the effects of anti-factor H antibodies on complement-mediated lysis.

We have recently reported that complement factor H, a negative regulator of complement-mediated cytotoxicity, is produced and secreted by most bladder cancers. This observation was exploited in the development of the BTA stat and BTA TRAK diagnostic assays, both of which make use of two factor H-specific monoclonal antibodies in sandwich format. Here we show that both antibodies exert interesting effects on the biochemistry of complement activation in in vitro systems. Antibody X13.2 competes with C3b for association with factor H and strongly inhibits factor H/factor I-mediated cleavage of C3b, thereby evidently inactivating a negative regulator of complement; yet, the antibody strongly inhibits complement-mediated lysis as well. Conversely, antibody X52. 1, which does not compete with C3b and has no effect on solution-phase cleavage of C3b, is capable of enhancing complement-mediated lysis of various cell types, including cancer cells, by over 10-fold. Our observations indicate that it is possible to deconvolute the biochemical roles of factor H in complement by means of appropriate inhibitors, a finding with potentially valuable implications for both basic research and cancer therapy.

Complement factor H or a related protein is a marker for transitional cell cancer of the bladder.

The BTAstat and BTA TRAK tests are new immunoassays that detect and measure an antigen in the urine of individuals diagnosed with bladder cancer. As described in this report, the monoclonal antibodies used in these kits were developed by immunizing mice with partially purified protein preparations derived from the urine of patients with bladder cancer. The antigen that is recognized by the monoclonal antibodies was purified from the urine of bladder cancer patients by immunoaffinity chromatography and identified as being either complement factor H (FH) or a closely related protein (CFHrp) by partial amino acid sequence analysis. Like serum FH, the urine antigen was demonstrated to have a complement factor C3b binding site and to accelerate the degradation of C3b in the presence of complement factor I. The culture supernatants from several human bladder, cervical, and renal cancer cell lines contained antigen as determined by immunoassay, and antigen affinity-purified from HeLaS3 culture media was shown to have FH activity. Moreover, the cell lines were shown to make products of the expected sizes by reverse transcription-PCR using FH-specific primers. In contrast, normal human epithelial keratinocytes, a myeloid leukemia cell line, and the colon cancer line LS174T were negative for production of a FH-like protein (CFHrp). We propose that the expression of proteins with FH-like activities may confer a selective growth advantage to cancer cells in vivo by decreasing complement activity, thus aiding their escape from lysis by immune surveillance. Identification of these proteins as cancer products also suggests avenues of chemotherapy or immunotherapy of some cancers.

PR92 antigen in human prostate fluid: elevated levels in prostate cancer.

In this preliminary study, we report that an enzyme-linked immunofluorescence assay (EFLA) was developed for the determination of PR92 antigen in prostatic fluid, utilizing anti-PR92 monoclonal antibody. Fluid samples from 64 patients were assayed. PR92 antigen was expressed as unit per microgram (U/microgram) of prostatic fluid proteins. One hundred percent of men (7 out of 7) less than 50 years of age demonstrated concentrations less than 25 U/micrograms; 91% of men (10 out of 11) with documented carcinoma, and only 9.5% of men (2 out of 21) with benign prostatic hyperplasia, demonstrated concentrations above 230 U/micrograms. The mean concentration of PR92 antigen in prostatic fluid of a group of patients suspected of having prostate cancer (high-risk group; 227 +/- 42 U/micrograms) was significantly greater than that of those with benign prostatic hyperplasia (87 +/- 23 U/micrograms; P = 0.05). Further evaluation of this potential marker and of other antigens within the prostatic fluid is warranted.

OVX1 as a marker for early stage endometrial carcinoma.

BACKGROUND: Endometrial carcinoma is generally diagnosed only after the onset of postmenopausal bleeding. Although most patients with Stage I disease can be cured, the prognosis worsens significantly when the tumor is no longer confined to the uterine corpus. Serum CA 125 is elevated in only 10-20% cases of Stage I and II endometrial carcinoma. A serum tumor marker that can detect early stage endometrial cancer might aid in management of the disease. METHODS: An OVX1 double-determinant radioimmunoassay was used to detect an epitope on a high-molecular-weight mucinlike glycoprotein found in the sera of 45 patients with endometrial cancer. RESULTS: Apparently healthy persons had serum OVX1 antigen levels of 2.23 plus or minus 2.48 U/ml (mean +/- standard deviation). Elevated levels of OVX1 antigen (> 7.2 U/ml) were found in 5% of 184 healthy persons and in 64% of 45 patients with endometrial cancer. OVX1 antigen was elevated in 64% of 36 patients with Stage I, 50% of 2 patients with Stage II, 60% of 5 patients with Stage III, and each of 2 patients with Stage IV endometrial cancer, but only 8.6% of 58 patients with endometriosis. Elevation of serum OVX1 was found more frequently in patients with deep myometrial invasion and with poorly differentiated tumors (P < 0.01). CONCLUSIONS: The OVX1 antigen deserves further evaluation as a marker for early detection of endometrial cancers and as a prognostic factor for women with apparent early stage disease.

OVX1 radioimmunoassay complements CA-125 for predicting the presence of residual ovarian carcinoma at second-look surgical surveillance procedures.

PURPOSE: At second-look surgical surveillance procedures, normal CA-125 levels can be associated with persistent disease in 50% to 60% of patients. A novel radioimmunoassay (RIA) has been evaluated for the ability to identify patients with persistent disease who have normal levels of CA-125. MATERIALS AND METHODS: The OVX1 double-determinant assay used a murine monoclonal antibody to detect an epitope on a high-molecular weight mucin-like glycoprotein. RESULTS: Apparently healthy individuals had serum OVX1 levels of 2.23 +/- 2.48 U/mL (mean +/- SD). Elevated serum OVX1 levels (> 7.2 U/mL) were found in 5% of 184 normal individuals and in 70% of 93 epithelial ovarian cancer patients with clinically evident disease. Among sera from these ovarian cancer patients, OVX1 was elevated in 68% of 76 samples with CA-125 levels more than 35 U/mL and in 76% of 17 samples with CA-125 levels less than 35 U/mL. In serum samples obtained at the time of positive second-look laparotomy, 59% of 41 patients with CA-125 levels less than 35 U/mL had elevated OVX1 antigen levels, whereas 41% of 22 patients with CA-125 levels more than 35 U/mL had elevated serum OVX1 levels. In patients with negative second-look laparotomies, false-positive results were eliminated by increasing the threshold of OVX1 to 10.5 U/mL. At this level, 32% of 41 patients with positive second-look operations had an elevated OVX1 level, despite a normal CA-125 level. When used in combination, CA-125 (> 35 U/mL) and OVX1 (> 10.5 U/mL) detected persistent disease in 56% of 63 patients with positive surveillance procedures, compared with 35% when CA-125 was used alone (P < .05). CONCLUSION: An elevated OVX1 level can alert oncologists to the possibility that ovarian cancer has persisted, despite the return of CA-125 to a normal range.

Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping.

Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA.

Induction of a rat T cell lymphoma-leukemia by magnesium deficiency--a study of fetal defense against maternal neoplasia.

If Sprague-Dawley rats, 25-30 days old, are fed a diet containing 4-5 mg% of Mg, about 25% of survivors develop a large tumor of the thymus within 6-12 weeks. The tumor is composed of lymphoblasts, which seem to arise from the thymic reticuloendothelial system and, at times, disseminate as an acute T cell lymphoma-leukemia of unknown etiology. If the tumor cells are transmitted intraperitoneally to rats, 14-16 days pregnant, a local invasive and generalized disease is established in the mother but not in the fetuses or their domain. However, if the neoplastic cells are injected into the fetal domain, they colonize the fetal tissues. The colonization by tumor cells is most impressive in the extravascular structure of the placental labyrinth but not in the placental syncytiotrophoblastic zone at the maternal-placental junction. This raises the question as to whether this zone may functionally mediate not only the well-known absolute intrauterine fetal defense against maternal metastatic neoplasia, but also the defense of the fetus against maternal immunologic rejection.

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A.

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.

In vitro effects of protease inhibitors on murine natural killer cell activity.

To test whether proteolytic events are involved in natural killer (NK) cell mediated lysis of tumour cells, twenty-three different protease inhibitors were added to in vitro assays of natural killer cell reactivity. Of all of the materials tested, only tosyl-L-lysine chloromethyl ketone (TLCK), tosyl-L-phenylalanine chloromethyl ketone (TPCK) and benzamidine unequivocally inhibited killing at concentrations approaching those needed to affect appropriate purified proteases. All of the effective inhibitors, and none of the others tested, inhibited binding of effector to target cells. The action of TLCK was focused on both effector and target cells, in that cytolysis was completely inhibited by a 1 hr pretreatment of effectors with 10(-4) M TLCK, and 60% inhibited by a 1 hr treatment of targets only.

Detection and partial characterization of two bovine pregnancy-specific proteins.

Two pregnancy-specific proteins were detected by immunoelectrophoresis using antisera developed to homogenates of bovine extraembryonic membranes. Antisera also reacted to extracts of endometrium from pregnant cows and extraembryonic fluids. However, antisera did not react with a preparation presumed to be bovine placental lactogen, fetuin, extracts of various somatic tissues from pregnant cows or extracts of endometrium from nonpregnant cows. One of the proteins had an estimated molecular weight of 65,000-70,000, an isoelectric point of 4.6-4.8 and yielded a reaction of identity with bovine alpha 1-fetoprotein by immunodiffusion. The second protein yielded a reaction of identity with bovine alpha 1-fetoprotein by immunodiffusion. The second protein had no immunological cross-reactivity with the known proteins or organ extracts which were tested. The molecular weight and isoelectric point was 47,000-53,000 and 4.0-4.4, respectively. These data demonstrate the presence of at least 2 pregnancy-specific proteins in cattle.

Primary structures of two low molecular weight proteinase inhibitors from potatoes.

The amino acid sequences of two low molecular weight proteinase inhibitors from Russet Burbank potatoes have been determined. One of these, a chymotrypsin inhibitor, is a peptide of 52 amino acid residues, while the second inhibitor, which is specific for trypsin, contains 51 amino acid residues. These peptides are highly homologous, differing at only nine positions. At position 38, the chymotrypsin inhibitor possesses leucine and the trypsin inhibitor an arginine. This difference probably represents the P1 sites, which are consistent with the respective specificities of the two inhibitors. The inhibitors are also homologous with potato inhibitor II and with an inhibitor previously isolated from eggplants.

Specificity of the carboxypeptidase inhibitor from potatoes.

Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor. However, several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low affinity (K(i) about 10 micromolar) for a carboxypeptidase G(1) from an acinetobacterium was found in this study.

Amino acid sequence of a carboxypeptidase inhibitor from tomato fruit.

The amino acid sequence of a 37 residue carboxypeptidase inhibitor from tomato fruit has been determined. The amino terminus was shown to be 2-oxopyrrolidine-5-carboxylic acid by digestion of reduced and S-carboxymethylated inhibitor with pyroglutamate aminopeptidase. The remainder of the sequence was assigned by analysis of peptides which had been generated by specific cleavage at the Asp4-Pro5 bond under acid conditions and by treatment with trypsin. The amino acid sequence of this inhibitor is identical with that of an analogous inhibitor from potatoes in 26 positions, and two of the replacements are highly conservative. The identification of the nonconservative replacements has been used to better define regions of the inhibitor which are not believed to contribute significantly to the free energy of association of the enzyme-inhibitor complex.

Purification and characterization of the carboxypeptidase isoinhibitors from potatoes.

Three zones of carboxypeptidase inhibitory activity were observed when heat-stable extracts of potato tubers (cv. Russet Burbank) were chromatographed on carboxymethyl cellulose. The isoinhibitors found in these zones were denoted I, II, and III based upon their order of elution from this column. The predominant form (II) had previously been suggested to be a mixture of two polypeptides (IIa and IIb) differing in that IIa possessed an additional residue of glutamine (Hass et al. 1975 Biochemistry 14: 1334). These closely related isoinhibitors (IIa and IIb) were separated by equilibrium ion exchange chromatography and characterized. Isoinhibitor I was shown to be identical to II except for two replacements, Ser-30 --> Ala and Arg-32 --> Gly. These replacements had no significant effect on apparent K(i) values toward either carboxypeptidase A or B. Isoinhibitor III, which was identical to II except that it lacked the amino terminal pyrrolidone carboxylic acid and following glutamine residue, was also functionally indistinguishable from II in inhibition studies. It was concluded that at least two and possibly as many as five genes code for the various isoinhibitor species which are present in potato tubers.