Crystal structures of human CD40 in complex with monoclonal antibodies dacetuzumab and bleselumab.

CD40 is a member of the tumor necrosis factor receptor superfamily, and it is widely expressed on immune and non-immune cell types. The interaction between CD40 and the CD40 ligand (CD40L) plays an essential function in signaling, and the CD40/CD40L complex works as an immune checkpoint molecule. CD40 has become a therapeutic target, and a variety of agonistic/antagonistic anti-CD40 monoclonal antibodies (mAbs) have been developed. To better understand the mode of action of anti-CD40 mAbs, we determined the X-ray crystal structures of dacetuzumab (agonist) and bleselumab (antagonist) in complex with the extracellular domain of human CD40, respectively. The structure reveals that dacetuzumab binds to CD40 on the top of cysteine-rich domain 1 (CRD1), which is the domain most distant from the cell surface, and it does not compete with CD40L binding. The binding interface of bleselumab spread between CRD2 and CRD1, overlapping with the binding surface of the ligand. Our results offer important insights for future structural and functional studies of CD40 and provide clues to understanding the mechanism of biological response. These data can be applied to developing new strategies for designing antibodies with more therapeutic efficacy.

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection.

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing.

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

Preclinical development of anti-BCMA immunotoxins targeting multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy that is incurable for the majority of patients. New treatments are urgently needed. Recombinant immunotoxins (RITs) are chimeric proteins that are composed of the Fv or Fab portion of an antibody fused to a bacterial toxin. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein and an ideal target for antibody-based treatments for MM. METHODS: RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in Escherichia coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays. We also measured their binding to human and mouse serum albumins and to BCMA and measured their serum half-life in mice. RESULTS: Using Fvs from different anti-BCMA antibodies, we produced RITs that specifically kill BCMA-expressing MM cells in vitro. To increase the serum half-life in vivo, we generated RITs that are fused with albumin-binding domains (ABDs). All RITs with ABDs have some decreased activity compared to the parent RIT, which is not due to decreased binding to BCMA. CONCLUSIONS: Various new anti-BCMA immunotoxins were produced and evaluated. None of these were better than LMB-75 (anti-BCMA BM306-disulfide-stabilized Fv-LRggs) supporting the further preclinical development of LMB-75.

Clinical significance of MUC13 in pancreatic ductal adenocarcinoma.

BACKGROUND: Poor prognosis of pancreatic cancer (PanCa) is associated with lack of an effective early diagnostic biomarker. This study elucidates significance of MUC13, as a diagnostic/prognostic marker of PanCa. METHODS: MUC13 was assessed in tissues using our in-house generated anti-MUC13 mouse monoclonal antibody and analyzed for clinical correlation by immunohistochemistry, immunoblotting, RT-PCR, computational and submicron scale mass-density fluctuation analyses, ROC and Kaplan Meir curve analyses. RESULTS: MUC13 expression was detected in 100% pancreatic intraepithelial neoplasia (PanIN) lesions (Mean composite score: MCS = 5.8; AUC >0.8, P < 0.0001), 94.6% of pancreatic ductal adenocarcinoma (PDAC) samples (MCS = 9.7, P < 0.0001) as compared to low expression in tumor adjacent tissues (MCS = 4, P < 0.001) along with faint or no expression in normal pancreatic tissues (MCS = 0.8; AUC >0.8; P < 0.0001). Nuclear MUC13 expression positively correlated with nodal metastasis (P < 0.05), invasion of cancer to peripheral tissues (P < 0.5) and poor patient survival (P < 0.05; prognostic AUC = 0.9). Submicron scale mass density and artificial intelligence based algorithm analyses also elucidated association of MUC13 with greater morphological disorder (P < 0.001) and nuclear MUC13 as strong predictor for cancer aggressiveness and poor patient survival. CONCLUSION: This study provides significant information regarding MUC13 expression/subcellular localization in PanCa samples and supporting the use anti-MUC13 MAb for the development of PanCa diagnostic/prognostic test.

CD21(-/low) marginal zone B cells highly express Fc receptor-like 5 protein and are killed by anti-Fc receptor-like 5 immunotoxins in hepatitis C virus-associated mixed cryoglobulinemia vasculitis.

OBJECTIVE: Hepatitis C virus (HCV) is associated with B cell lymphoproliferative disorders, including mixed cryoglobulinemia (MC) vasculitis and B cell non-Hodgkin's lymphoma. The expansion of clonal and autoreactive rheumatoid factor-bearing CD21(-/low) marginal zone (MZ) B cells was demonstrated in patients with HCV-associated MC vasculitis. Fc receptor-like (FCRL) proteins comprise a family of immunoregulatory proteins preferentially expressed on B lineage cells. The goal of this study was to investigate the expression of FCRL proteins 1-5 on B cells from patients with HCV-associated MC vasculitis. METHODS: Expression of FCRL proteins 1-5 was assessed by flow cytometry on B cells from 15 HCV-infected patients with type II MC (7 of whom had B cell non-Hodgkin's lymphoma), 20 HCV-infected patients without MC, and 20 healthy donors. To evaluate FCRL-5 as an immunotherapy target in HCV-associated MC vasculitis, 2 anti-FCRL-5 recombinant immunotoxins were produced using anti-FCRL-5 monoclonal antibodies and Pseudomonas exotoxin. RESULTS: Expression of FCRLs 2, 3, and 5 was markedly increased while expression of FCRL-1 was decreased on clonal CD21(-/low) MZ B cells, as compared with other B cell subsets, from HCV-infected patients and healthy donors. However, there was no difference in the pattern of FCRL expression between HCV-MC patients with lymphoma and those without lymphoma. The anti-FCRL-5 immunotoxins showed specific cytotoxicity against FCRL-5-expressing clonal CD21(-/low) MZ B cells isolated from HCV-infected patients as well as FCRL-5-transfected cell lines. No cytotoxicity against T cells or conventional B cells was observed. CONCLUSION: These findings suggest that FCRL-5-targeting therapies could be a specific treatment for HCV-associated MC vasculitis and other FCRL-5-positive autoimmune B cell disorders.

CD164 and FCRL3 are highly expressed on CD4+CD26- T cells in Sézary syndrome patients.

Sézary syndrome (SS) cells express cell surface molecules also found on normal activated CD4 T cells. In an effort to find a more specific surface marker for malignant SS cells, a microarray analysis of gene expression was performed. Results showed significantly increased levels of mRNA for CD164, a sialomucin found on human CD34+ hematopoietic stem cells, and FCRL3, a molecule present on a subset of human natural T regulatory cells. Both markers were increased in CD4 T cells from SS patients compared with healthy donors (HD). Flow cytometry studies confirmed the increased expression of CD164 and FCRL3 primarily on CD4+CD26- T cells of SS patients. Importantly, a statistically significant correlation was found between an elevated percentage of CD4+CD164+ T cells and an elevated percentage of CD4+CD26- T cells in all tested SS patients but not in patients with mycosis fungoides and atopic dermatitis or HD. FCRL3 expression was significantly increased only in patients with high tumor burden. CD4+CD164+ cells displayed cerebriform morphology and their loss correlated with clinical improvement in treated patients. Our results suggest that CD164 can serve as a marker for diagnosis and for monitoring progression of cutaneous T-cell lymphoma (CTCL)/SS and that FCRL3 expression correlates with a high circulating tumor burden.

Human Fc receptor-like 5 binds intact IgG via mechanisms distinct from those of Fc receptors.

Fc receptor-like (FCRL) 5 regulates B cell Ag receptor signaling and has been reported to bind aggregated IgG. Using surface plasmon resonance, we analyzed the interaction of native IgG samples with FCRL5, revealing a complex binding mechanism, where isotype is just one factor. FCRL5 bound IgG1 and IgG4 with ~1 µM KD, whereas the interaction with IgG3 was a magnitude weaker. However, IgG2 samples displayed a wide range of affinities, indicating that additional factors affect binding. We used a panel of 19 anti-FCRL5 mAbs with defined reactivity to identify domains involved in ligand binding. Six mAbs blocked IgG binding, indicating critical roles of FCRL5 domains 1 and 3, as well as epitopes at the domain 1/2 and domain 2/3 boundaries. We found that only glycosylated IgG containing both Fab arms and the Fc region bound with high affinity. Furthermore, the presence of sialic acid in the IgG carbohydrate altered FCRL5 binding. The interaction of IgG and FCRL5 consisted of two kinetic components, suggesting a complex binding mechanism. We established that the IgG-Fc and IgG-F(ab')2 fragments bind FCRL5 independently but with low affinity, revealing the mechanism behind the two-step binding of whole IgG. This complex binding mechanism is distinct from that of Fc receptors, which bind through the Fc. We propose that FCRL5 is a new type of receptor that recognizes intact IgG, possibly enabling B cells to sense Ig quality. Recognition of undamaged IgG molecules by FCRL5 could allow B cells to engage recently produced Abs.

Fc receptor-like 5 promotes B cell proliferation and drives the development of cells displaying switched isotypes.

The biological roles of B cell membrane proteins in the FCRL family are enigmatic. FCRL proteins, including FCRL5, were shown to modulate early BCR signaling, although the subsequent, functional consequences of receptor engagement are poorly understood. We found that FCRL5 surface protein itself was induced temporarily upon BCR stimulation of human, naive B cells, indicating precise control over timing of FCRL5 engagement. Cross-linking of FCRL5 on cells induced to express FCRL5 enhanced B cell proliferation significantly. This enhancement required costimulation of the BCR and TLR9, two signals required for optimal proliferation of naive B cells, whereas T cell help in the form of anti-CD40 and IL-2 was dispensable. In addition, we found that FCRL5 stimulation generated a high proportion of cells displaying surface IgG and IgA. Optimal development of cells expressing switched isotypes required T cell help, in addition to stimuli found necessary for enhanced proliferation. Surprisingly, cells that developed upon FCRL5 stimulation simultaneously displayed surface IgM, IgG, and IgA. Cells expressing multiple Ig isotypes were described in hairy cell leukemia, a disease in which FCRL5 is overexpressed. Enhanced proliferation and downstream isotype expression upon FCRL5 stimulation could reflect a physiological role for FCRL5 in the expansion and development of antigen-primed B cells. In addition, FCRL5 may promote growth of malignant cells in hairy cell leukemia and other FCRL5-expressing tumors.

Fc receptor-like 3 protein expressed on IL-2 nonresponsive subset of human regulatory T cells.

Fc receptor-like 3 (FCRL3) is a cell surface protein homologous to Fc receptors. The FCRL3 gene is present in humans but not in mice. We found that FCRL3 protein is expressed on 40% of human naturally occurring CD4(+) regulatory T (nTreg) cells (CD4(+)CD25(+)CD127(low)). Sorted nTreg cells with the surface phenotype FCRL3(+) and FCRL3(-) were both hypoproliferative to TCR stimulation and both suppressive on proliferation of conventional T cells (CD4(+)CD25(-)) in vitro. They both expressed forkhead box p3 (Foxp3) protein, the intracellular regulatory T cell marker. However, in contrast to FCRL3(-) nTreg cells, FCRL3(+) nTreg cells were not stimulated to proliferate by the addition of exogenous IL-2. In addition, Foxp3(+) cells induced from conventional T cells by TGF-beta treatment did not exhibit FCRL3 expression. These results suggest that the FCRL3(+) subset of human nTreg cells identified in this study arise in vivo and Foxp3 expression alone is not sufficient to induce FCRL3 expression. FCRL3 may be involved in human-specific mechanisms to control the generation of nTreg cells.

Volume-sensitive Cl(-) channel as a regulator of acquired cisplatin resistance.

The platinum-based drug cisplatin (cis-diamminedichloroplatinum (II)) is widely used in cancer therapy. However, cancer cells can develop resistance after exposure to cisplatin. Recently, many studies have pointed to the involvement of plasma membrane ion channels in a cell's response to cisplatin. Our group has found that pretreatment with cisplatin enhanced the activity of volume-sensitive C-channels in human epidermoid cancer KB cells; cisplatin-resistant KCP-4 cells derived from KB cells, on the other hand, lacked functional expression of these channels. This suggests that the activity of volume-sensitive Cl(-) channels is an important factor in determining the sensitivity of cancer cells to cisplatin. Furthermore, when volume-sensitive Cl(-) channel function was partially restored in cisplatin-resistant KCP-4 cells treated with a histone deacetylase inhibitor, KCP-4 cells exhibited a restoration of sensitivity to cisplatin; this increased sensitivity was inhibited by a volume-sensitive Cl(-) channel blocker. We therefore propose that impaired activity of the volume-sensitive Cl(-) channel is involved in the acquired cisplatin resistance of these cancer cells. In this review, we will outline the relationship between volume-sensitive Cl(-) channels, cisplatin-induced apoptosis, and cisplatin resistance. Activating the volume-sensitive outwardly-rectifying Cl(-) channel may be a new strategy in treating clinical cisplatin resistance.

Expression of POTE protein in human testis detected by novel monoclonal antibodies.

The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

FCRL1 on chronic lymphocytic leukemia, hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins.

FCRL1 (Fc receptor-like 1) is a cell-surface membrane protein belonging to FCRL family and is preferentially expressed on B cells. To evaluate FcRL1 as an immunotherapy target for B-cell malignancies, we prepared anti-FCRL1 mAbs without cross-reactivity to other FCRL family proteins and analyzed FCRL1 protein expression on malignant cells from patients and on B-cell lines. Frequent FCRL1 expression was observed by flow cytometry on 12 B-cell non-Hodgkin lymphoma (B-NHL) cell lines and many patient samples: 12 of 14 chronic lymphocytic leukemia (CLL), 7 of 7 follicular lymphoma (FL), 13 of 17 hairy cell leukemia (HCL), and 2 of 3 mantle cell lymphoma (MCL). Two recombinant immunotoxins, E3(Fv)-PE38 and E9(Fv)-PE38, were constructed. Both immunotoxins bound to FCRL1-positive cells with similar affinities (3.4 and 3.2 nM) and were cytotoxic to cell lines, but E9(Fv)-PE38 was 4- to 20-fold more cytotoxic than E3(Fv)-PE38. The concentrations that inhibited response by 50% (IC(50)s) of E9(Fv)-PE38 on 11 different FCRL1-positive cell lines ranged from 1.0 ng/mL to 90 ng/mL and correlated with the FCRL1 expression levels. Our results suggest that anti-FCRL1 immunotoxin E9(Fv)-PE38 exhibits remarkably specific cytotoxicity and merits further evaluation for the treatment of FCRL1-positive malignancies, including CLL, HCL, FL, MCL, and other B-NHL.

Palmitoylation of POTE family proteins for plasma membrane targeting.

The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

Impaired activity of volume-sensitive Cl- channel is involved in cisplatin resistance of cancer cells.

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.

Evolution and expression of chimeric POTE-actin genes in the human genome.

We previously described a primate-specific gene family, POTE, that is expressed in many cancers but in a limited number of normal organs. The 13 POTE genes are dispersed among eight different chromosomes and evolved by duplications and remodeling of the human genome from an ancestral gene, ANKRD26. Based on sequence similarity, the POTE gene family members can be divided into three groups. By genome database searches, we identified an actin retroposon insertion at the carboxyl terminus of one of the ancestral POTE paralogs. By Northern blot analysis, we identified the expected 7.5-kb POTE-actin chimeric transcript in a breast cancer cell line. The protein encoded by the POTE-actin transcript is predicted to be 120 kDa in size. Using anti-POTE mAbs that recognize the amino-terminal portion of the POTE protein, we detected the 120-kDa POTE-actin fusion protein in breast cancer cell lines known to express the fusion transcript. These data demonstrate that insertion of a retroposon produced an altered functional POTE gene. This example indicates that new functional human genes can evolve by insertion of retroposons.

Sandwich ELISAs for soluble immunoglobulin superfamily receptor translocation-associated 2 (IRTA2)/FcRH5 (CD307) proteins in human sera.

BACKGROUND: The immunoglobulin superfamily receptor translocation-associated 2 (IRTA2) gene encodes both membrane and secreted forms of the B-cell differentiation antigen (also identified as FcRH5). The membrane form is highly expressed on the surface of hairy cell leukemia (HCL) cells from patients. This study aimed to develop immunoassays for soluble IRTA2/FcRH5 proteins in human serum. METHODS: Two sandwich ELISAs for soluble IRTA2/FcRH5 were designed using two pairs of monoclonal antibodies specific to four different epitopes on IRTA2/FcRH5. RESULTS: In both ELISAs, the lower limit of quantitation for soluble IRTA2/FcRH5 in human serum was 30 ng/mL. The analytical recovery of 0.3-2.1 ng/mL of IRTA2/FcRH5-Fc used as the standard, from a 100-fold dilution of IRTA2/FcRH5-free sera, was 94-106% for ELISA #1 and 89-97% for ELISA #2. Between-assay coefficients of variance were 7.7-17.6% for ELISA #1 and 7.7-32.7% for ELISA #2. Both ELISAs detected soluble IRTA2/FcRH5 protein in sera from normal donors (median 169 ng/mL in ELISA #1 and 146 ng/mL in ELISA #2, n=123) without correlations to gender or age. A marked increase in soluble IRTA2/FcRH5 levels was observed in samples from patients with HCL (medians 719 ng/mL in ELISA #1 and 754 ng/mL in ELISA #2, n=44). CONCLUSIONS: These ELISAs may be useful for monitoring soluble IRTA2/FcRH5 in HCL and other B-cell malignancies.

Epstein-Barr virus nuclear antigen 2 induces FcRH5 expression through CBF1.

Fc-receptor homolog 5 (FcRH5) is a recently identified B-cell membrane protein of unknown function. In Burkitt lymphoma cell lines with chromosome 1q21 abnormalities, FcRH5 expression is deregulated, implicating FcRH5 in lymphomagenesis. Epstein-Barr virus infects and immortalizes B cells, and is implicated in the etiology of several tumors of B-cell origin. Overexpression of genes located on 1q21-25 has been proposed as a surrogate for Epstein-Barr virus in Burkitt lymphoma. We now report that Epstein-Barr virus nuclear antigen 2 (EBNA2) markedly induces the expression of the FcRH5 gene, encoded on chromosome 1q21. Induction occurred in the absence of other viral proteins and did not require de novo protein synthesis. EBNA2 lacks a DNA-binding domain and can target responsive genes through the host DNA binding protein CBF1. We show that induction of FcRH5 by EBNA2 is strictly CBF1 dependent, as it was abolished in CBF1-deficient cells. Accordingly, EBNA2 targeted CBF1 binding sites present in the FcRH5 promoter in vivo, as detected by chromatin immunoprecipitation. These results identify FcRH5 as a novel, direct target of EBNA2 that may contribute to the development of Epstein-Barr virus-associated tumors.

Cell membrane-specific epitopes on CD30: Potentially superior targets for immunotherapy.

Because CD30 is highly expressed on Hodgkin's lymphoma and anaplastic large cell lymphoma, it is a promising target for immunotherapy. Soluble CD30, the extracellular domain of CD30 that is shed from the cells, can reduce the effects of CD30-targeting agents by competitive binding. In this study, we identified two epitopes on membrane-associated CD30 that are missing on soluble CD30 probably because of a conformational change upon shedding. These epitopes are potentially superior targets for immunotherapy because targeting them should be free from the competitive effects of soluble CD30. We studied 27 anti-native CD30 mAbs that were assigned to 8 different topographical epitopes. Soluble CD30 was prepared from culture supernatants of L540 cells or Karpas 299 cells. In an ELISA, the mAbs to two epitopes, Ep2 (amino acids 107-153) and Ep7 (amino acids 282-338), showed less than a 2% average cross-reactivity to soluble CD30 compared with a CD30-Fc fusion protein. In addition, these mAbs bound to CD30 on cells in the presence of an excess of soluble CD30. These epitopes (Ep2 and Ep7) are, therefore, more efficiently presented on cell-associated CD30 than on soluble CD30 (membrane-specific epitopes). Also, soluble CD30 in the sera of mice bearing L540 tumors did not form immune complexes with the membrane-specific mAbs analyzed by size-exclusion chromatography. In contrast, mAbs to the other epitopes reacted with both soluble CD30 and membrane CD30. Our results suggest that it may be possible to find membrane-specific epitopes on other immunotherapy target molecules.

The PATE gene is expressed in the accessory tissues of the human male genital tract and encodes a secreted sperm-associated protein.

The PATE gene is expressed in prostate and testis. To determine if PATE is expressed in other accessory tissues of the male genital tract, RT-PCR of the epididymis and seminal vesicle was performed. PATE mRNA was highly expressed in the epididymis and seminal vesicle. In situ hybridization of the testis showed PATE mRNA is strongly expressed in the spermatogonia. The PATE gene encodes a 14-kDa protein with a predicted signal sequence and a cleavage site between residues G21 and S22. To determine if PATE is a secreted protein, 293T cells were transfected with a pcDNA-PATE-myc-His plasmid and protein immunoprecipitated with anti-myc monoclonal antibody. Western blot analysis showed the presence of PATE-myc-His protein was in the medium and the cell lysate. Confocal microscopy demonstrated that PATE-myc-His protein is found in the endoplasmic reticulum. The polyclonal antibody SOL-1 was generated by immunization of rabbits with recombinant PATE protein expressed and purified from Escherichia coli. Western blots were performed on extracts of prostate, testis, seminal vesicle and ejaculated spermatozoa, but PATE protein was only detected in the spermatozoa. Immunostaining of sperm smears revealed that PATE is located in a band-like pattern in the sperm head. Our data indicate that PATE is made by various sexual accessory tissues and secreted into the semen where it becomes associated with sperm, suggesting that PATE is a novel sperm-associated protein with a possible role in mammalian sperm maturation.