Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope.

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

Involvement of the prostate and testis expression (PATE)-like proteins in sperm-oocyte interaction.

BACKGROUND: The prostate and testis expression (PATE)-like family of proteins are expressed mainly in the male genital tract. They are localized in the sperm head and are homologous to SP-10, the acrosomal vesicle protein also named ACRV1. Our aim was to characterize the expression and functional role of three PATE-like proteins in the testis and ejaculated sperm. METHODS: The expression and localization of PATE-like proteins in human testis biopsies (n= 95) and sperm cells were assessed by RT-PCR, immunohistochemistry and immunofluorescence staining (at least 600 sperm cells per specimen). The function of the PATE protein was tested by the hemizona assay and hamster egg penetration test (HEPT). RESULTS: PATE and PATE-M genes and proteins were present almost exclusively in germ cells in the testis: immunoflourescence showed that the percentage of germ cells positive for PATE, PATE-M and PATE-B was 85, 50 and 2%, respectively. PATE and PATE-M proteins were localized in the equatorial segment of the sperm head, while PATE-B protein was localized in the post-acrosomal region. A polyclonal antibody (Ab, at 1:50 and 1:200 dilutions) against the PATE protein did not inhibit sperm-zona binding in the hemizona assay (hemizona index of 89.6 ± 10 and 87 ± 36%, respectively). However, there was inhibition of sperm-oolemma fusion and penetration in the HEPT (penetration index: without Ab 7 ± 3.9; Ab dilution of 1:100, 4 ± 3.5; Ab dilution of 1:20, 0.6 ± 1.2, P < 0.001). CONCLUSIONS: Our data suggest that PATE protein is involved in sperm-oolemma fusion and penetration but not sperm-zona binding.

CAPC negatively regulates NF-κB activation and suppresses tumor growth and metastasis.

CAPC, also known as LRRC26, is expressed in normal prostate and salivary gland. We developed a mAb to CAPC and used it to characterize the protein and study its function. CAPC protein was detected in normal prostate and salivary gland, in several human breast cancer cell lines and in the prostate cancer cell line LNCaP. Knockdown of CAPC by siRNA in LNCaP cells enhanced anchorage-independent growth in soft agar. Conversely, overexpression of CAPC in MDA-231 breast cancer cells and A431 epidermoid cancer cells inhibited growth in soft agar and tumorigenesis in nude mice, and suppressed the metastasis of MDA-231 cells to the lung. Overexpression of CAPC downregulated NF-κB activity and its target genes, including GM-CSF (CSF2), CXCL1, IL8 and LTB1. It also suppressed genes encoding the serine protease mesotrypsin (PRSS3) and cystatin SN (CST1). CAPC expressing tumors showed a decrease in the number of proliferating cells and a large increase in ECM. The role of CAPC in the suppression of tumor growth and metastasis may be through its alteration of the tumor microenvironment.

Partial inactivation of Ankrd26 causes diabetes with enhanced insulin responsiveness of adipose tissue in mice.

AIMS/HYPOTHESIS: ANKRD26 is a newly described gene located at 10p12 in humans, a locus that has been identified with some forms of hereditary obesity. Previous studies have shown that partial inactivation of Ankrd26 in mice causes hyperphagia, obesity and gigantism. Hypothesising that Ankrd26 mutant (MT) mice could develop diabetes, we sought to establish whether the observed phenotype could be (1) solely related to the development of obesity or (2) caused by a direct action of ankyrin repeat domain 26 (ANKRD26) in peripheral tissues. METHODS: To test the hypothesis, we did a full metabolic characterisation of Ankrd26 MT mice that had free access to chow or were placed under two different energy-restricted dietary regimens. RESULTS: Highly obese Ankrd26 MT mice developed an unusual form of diabetes in which white adipose tissue is insulin-sensitive, while other tissues are insulin-resistant. When obese MT mice were placed on a food-restricted diet, their weight and glucose homeostasis returned to normal. In addition, when young MT mice were placed on a pair-feeding diet with normal mice, they maintained normal body weight, but showed better glucose tolerance than normal mice, an increased responsiveness of white adipose tissue to insulin and enhanced phosphorylation of the insulin receptor. CONCLUSIONS/INTERPRETATION: These findings show that the ANKRD26 protein has at least two functions in mice. One is to control the response of white adipose tissue to insulin; the other is to control appetite, which when Ankrd26 is mutated, leads to hyperphagia and diabetes in an obesity-dependent manner.

Elevation of soluble CD307 (IRTA2/FcRH5) protein in the blood and expression on malignant cells of patients with multiple myeloma, chronic lymphocytic leukemia, and mantle cell lymphoma.

CD307 is a differentiation antigen expressed in B-lineage cells. One soluble and two membrane-bound forms have been predicted and an enzyme-linked immunosorbent assay (ELISA) for soluble CD307 established. Our goal was to determine if CD307 is expressed on the surface of cells from patients with multiple myeloma (MM), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and other B-cell malignancies and if soluble CD307 levels are elevated in the blood of patients with these B-cell malignancies. Cells and blood were collected from patients. Expression of CD307 was measured by flow cytometry and blood levels of soluble CD307 by ELISA. High soluble CD307 levels were detected in 21/43 (49%) of patients with MM, 36/46 (78%) with CLL and 9/24 (38%) with MCL. Soluble CD307 levels correlated with plasma cell percentages in bone marrow aspirates in MM and total white blood cells in CLL. CD307 on the cell membrane was detected by flow cytometry in 8/8 MM, 23/29 CLL and 4/5 MCL samples. Because CD307 is present on malignant cells from patients with MM, CLL and MCL, CD307 may be a useful therapeutic target for the treatment of these diseases.

IL-4 receptors on human medulloblastoma tumours serve as a sensitive target for a circular permuted IL-4-Pseudomonas exotoxin fusion protein.

Cytotoxins directed to interleukin-4 receptors have shown to mediate relatively selective cytotoxicity against a variety of human cancer cells in vitro and in vivo. In an ongoing Phase I clinical trial, a recombinant protein comprised of circularly permuted IL-4 fused to a mutated form of Pseudomonas exotoxin (the fusion protein termed IL-4(38-37)-PE38KDEL or cpIL4-PE) has shown antitumour activity against malignant glioma. Human medulloblastomas are neuroectodermal tumours that occur in children and have a poor prognosis. The goal of this study was to determine whether human medulloblastoma derived cell lines express interleukin-4 receptor and whether interleukin-4 receptor expression is accompanied by sensitivity to cpIL4-PE. Medulloblastoma cell lines express interleukin-4 receptor at the protein and mRNA levels as determined by binding, indirect immunofluorescence and RT--PCR studies. These cells expressed IL-4Ralpha (also known as IL-4Rbeta) and IL-13Ralpha1 (also known as IL-13Ralpha') chains, however common gamma(c), a component of the interleukin-4 receptor system in immune cells was not detected. Consistent with the expression of IL-4R, cpIL4-PE was found to be highly and specifically cytotoxic to four of five medulloblastoma cell lines. Susceptibility of medulloblastoma cell lines to cpIL4-PE seemed to correlate closely to the functional IL-4 binding sites in general as demonstrated by 125I-IL-4 binding, but did not seem to correlate with mRNA or cell surface immunoreactive receptor protein expression. The sensitivity of medulloblastoma cells to cpIL4-PE could be eliminated by concurrent incubation with IL-4 or IL-13, but not with IL-2. None of these cell lines showed any change in proliferation upon treatment with exogenous IL-4. These studies establish the interleukin-4 receptor as a medulloblastoma-associated target for possible tumour-directed cancer therapy. Further studies are warranted to investigate interleukin-4 receptor expression in primary medulloblastoma tumours and sensitivity to cpIL-4PE in vitro and in vivo.

T-cell receptor gamma chain alternate reading frame protein (TARP) expression in prostate cancer cells leads to an increased growth rate and induction of caveolins and amphiregulin.

Previously, we showed that prostate and prostate cancer cells express a truncated T-cell receptor gamma chain mRNA that uses an alternative reading frame to produce a novel nuclear T-cell receptor gamma chain alternate reading frame protein (TARP). TARP is expressed in the androgen-sensitive LNCaP prostate cancer cell line but not in the androgen-independent PC3 prostate cancer cell line, indicating that TARP may play a role in prostate cancer progression. To elucidate the function of TARP, we generated a stable PC3 cell line that expresses TARP in a constitutive manner. Expression of TARP in PC3 cells resulted in a more rapid growth rate with a 5-h decrease in doubling time. cDNA microarray analysis of 6538 genes revealed that caveolin 1, caveolin 2, amphiregulin, and melanoma growth stimulatory activity alpha were significantly up-regulated, whereas IL-1beta was significantly down-regulated in PC3 cells expressing TARP. We also demonstrated that TARP expression is up-regulated by testosterone in LNCaP cells that express a functional androgen receptor. These results suggest that TARP has a role in regulating growth and gene expression in prostate cancer cells.

Increased sensitivity to cocaine by cholinergic cell ablation in nucleus accumbens.

Chronic exposure to cocaine causes long-lasting behavioral changes associated with cocaine reinforcement and addiction. An important neural substrate for cocaine addiction is the nucleus accumbens (NAc), which receives dopaminergic input from the ventral tegmental area. Although the neural circuit of the NAc is controlled by several other neurotransmitters, their involvement in cocaine addiction remains elusive. In this investigation, we ablated cholinergic interneurons from the adult NAc with immunotoxin-mediated cell targeting and examined the role of acetylcholine transmitter in adaptive behavioral changes associated with cocaine reinforcement and addiction. Acute exposure to cocaine induced abnormal rotation in unilaterally cholinergic cell-eliminated mice. This abnormal turning was enhanced by repeated exposure of cocaine. In bilaterally cholinergic cell-eliminated mice, chronic cocaine administration induced a prominent and progressive increase in locomotor activity. Moreover, these mice showed robust conditioned place preference with a lower dose of cocaine, compared with wild-type littermates. This investigation demonstrates that acetylcholine in the NAc plays a key role in both acute and chronic actions of cocaine.

MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer.

BACKGROUND: With the completion of the human draft genome sequence, efforts are now devoted to identifying new genes. We have developed a computer-based strategy that utilizes the EST database to identify new genes that could be targets for the immunotherapy of cancer or could be involved in the multistep process of cancer. MATERIALS AND METHODS: Utilizing our computer-based screening strategy, we identified a cluster of expressed sequence tags (ESTs) that are highly expressed in breast cancer. Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses demonstrated the tissue specificity of the computer-generated cluster and comparison with the human genome sequence assisted in isolating a full-length cDNA clone. RESULTS: We identified a new gene that is highly expressed in breast cancer. This gene is expressed at moderate levels in normal breast and testis and at very low levels in liver, brain, and placenta. The gene has two major transcripts of 4.5 kb and 4.1 kb. The 4.5-kb transcript is very abundant in breast cancer, and has an open reading frame of 1382 amino acids. The predicted protein sequence of the 4.5-kb transcript reveals that it has high homology with MRP5, a member of multidrug resistant-associated protein family (MRP). There are seven reported members in the MRP family; we designate this gene as MRP8 (ABCC11). The 4.5-kb MRP8 transcript consists of 31 exons and is located in a genomic region of over 80.4 kb on chromosome 16q12.1. The smaller 4.1-kb transcript of MRP8 is found in testis and may initiate within intron 6 of the gene. CONCLUSION: The selective expression of MRP8 (ABCC11), a new member of ATP-binding cassette transporter superfamily could be a molecular target for the treatment of breast cancer.

Efficient ablation by immunotoxin-mediated cell targeting of the cell types that express human interleukin-2 receptor depending on the internal ribosome entry site.

BACKGROUND: Immunotoxin-mediated cell targeting (IMCT) is a technique for conditional genetic ablation of specific cell types. IMCT provides a useful approach for generating animal models for human neurodegenerative disorders. The strategy of IMCT depends on the cytotoxic activity of antiTac-based recombinant immunotoxins that selectively target cells expressing the human interleukin-2 receptor alpha-subunit (IL-2Ralpha). Transgenic mice were generated that express the IL-2Ralpha under the control of an appropriate tissue-specific gene promoter, and they were treated with the recombinant immunotoxins resulting in the ablation of the target cell types. To restrict the expression of IL-2Ralpha transgene in the cell types of interest, it is useful to knock-in the IL-2Ralpha expression cassette into the specific marker gene locus with gene targeting. Moreover, the knock-in of the IL-2Ralpha cassette located downstream of an internal ribosome entry site (IRES) into the 3'-untranslated region of the marker gene enables IL-2Ralpha expression in the restricted cell types while preserving the intact marker gene expression. However, there is a possibility that IRES-dependent expression of the receptor may be less efficient than cap-dependent expression. METHODS AND RESULTS: The efficiency of IRES-dependent IL-2Ralpha expression and immunotoxin responsiveness of the cells expressing the receptor were examined. The IL-2Ralpha gene fused to green fluorescence protein (GFP) (IL-2R/ GFP) was used as the target receptor. Embryonic stem cell clones were isolated that carry two types of bicistronic vectors in which the IL-2R/GFP fusion gene or the chloramphenicol acetyltransferase gene was connected upstream or downstream of IRES. The expression level of IL-2R/GFP protein in the cell clones was evaluated by GFP fluorescence detection and Western blot analysis. The IRES-dependent expression produced the same level of receptor protein as cap-dependent expression. The immunotoxin responsiveness of the cloned cells was evaluated by measuring the colony-forming efficiency in medium containing various amounts of a recombinant immunotoxin. The colony-forming efficiency of the cells expressing IL-2R/ GFP through IRES-dependent expression was reduced together with increasing immunotoxin concentration in a similar dose-dependent manner to the cells expressing the receptor through cap-dependent expression. CONCLUSIONS: The present results indicate that it is possible to effectively use the IRES-dependent expression system for IMCT. The system permits expression of the target receptor in selective cell types by introducing the IRES-driven expression cassette into the 3'-untranslated region of the marker gene locus.

Cse1l is essential for early embryonic growth and development.

The CSE1L gene, the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation as well as in apoptosis. CSE1 and CSE1L are essential genes in Saccharomyces cerevisiae and mammalian cells, as shown by conditional yeast mutants and mammalian cell culture experiments with antisense-mediated depletion of CSE1L. To analyze whether CSE1L is also essential in vivo and whether its absence can be compensated for by other genes or mechanisms, we have cloned the murine CSE1L gene (Cse1l) and analyzed its tissue- and development-specific expression: Cse1l was detected at embryonic day 7.0 (E7.0), E11.0, E15.0, and E17.0, and in adults, high expression was observed in proliferating tissues. Subsequently, we inactivated the Cse1l gene in embryonic stem cells to generate heterozygous and homozygous knockout mice. Mice heterozygous for Cse1l appear normal and are fertile. However, no homozygous pups were born after interbreeding of heterozygous mice. In 30 heterozygote interbreeding experiments, 50 Cse1l wild-type mice and 100 heterozygotes were born but no animal with both Cse1l alleles deleted was born. Embryo analyses showed that homozygous mutant embryos were already disorganized and degenerated by E5.5. This implicates with high significance (P < 0.0001, Pearson chi-square test) an embryonically lethal phenotype of homozygous murine CSE1 deficiency and suggests that Cse1l plays a critical role in early embryonic development.

GDEP, a new gene differentially expressed in normal prostate and prostate cancer.

BACKGROUND: The database of human expressed sequence tags (dbEST) is a potential source for the identification of tissue specific genes. The database contains sequences that originate from cDNA libraries from different tissues cell types and tumors. METHODS: Computer based analysis identified a cluster of sequence homologous ESTs, containing ESTs derived only from human prostate cDNA libraries. The tissue specificity was examined by multiple tissue RNA dot blots and RT-PCR. The new RNA transcript was characterized using northern blot analysis, RACE-PCR, and a ribonuclease protection assay. RESULTS: We have identified a gene differentially expressed in prostate using EST database analysis and experimental studies. We name the gene GDEP for gene differentially expressed in prostate. The major GDEP transcript is about 520 bp long. GDEP RNA was detected in nine prostate tissue samples, four normal and five cancer. Expression in prostate epithelial cells was established by in situ hybridization. Weak expression was detected in the prostate cancer cell line LNCaP. In vitro transcription/translation indicate that the RNA encodes a small 34 amino acid protein. The major transcript consists of two exons with one large intron (> 15 kb). The GDEP gene was mapped to chromosome 4q21.1 by radiation hybrid mapping. CONCLUSIONS: Our data proves that tissue specific genes can be identified by EST database mining. The prostate specificity of GDEP expression indicates that GDEP may be useful in the diagnosis or treatment of prostate cancer. Published 2001 Wiley-Liss, Inc.

Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia.

BACKGROUND: Hairy-cell leukemia that is resistant to treatment with purine analogues, including cladribine, has a poor prognosis. We tested the safety and efficacy of an immunotoxin directed against a surface antigen that is strongly expressed by leukemic hairy cells. METHODS: RFB4(dsFv)-PE38 (BL22), a recombinant immunotoxin containing an anti-CD22 variable domain (Fv) fused to truncated pseudomonas exotoxin, was administered in a dose-escalation trial by intravenous infusion every other day for a total of three doses. RESULTS: Of 16 patients who were resistant to cladribine, 11 had a complete remission and 2 had a partial remission with BL22. The three patients who did not have a response received low doses of BL22 or had preexisting toxin-neutralizing antibodies. Of the 11 patients in complete remission, 2 had minimal residual disease in the bone marrow or blood. During a median follow-up of 16 months (range, 10 to 23), 3 of the 11 patients who had a complete response relapsed and were retreated; all of these patients had a second complete remission. In 2 of the 16 patients, a serious but completely reversible hemolytic-uremic syndrome developed during the second cycle of treatment with BL22. Common toxic effects included transient hypoalbuminemia and elevated aminotransferase levels. CONCLUSIONS: BL22 can induce complete remissions in patients with hairy-cell leukemia that is resistant to treatment with purine analogues.

Synthesis and evaluation of a macrocyclic bifunctional chelating agent for use with bismuth radionuclides.

The detailed synthesis of the bifunctional chelating agent 2-(p-isothiocyanatobenzyl)-1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (BF_PEPA) is reported. This ligand was conjugated to monoclonal antibody B3 and the resultant immunoconjugate radiolabeled with (205,206)Bi. The in vivo stability of the radiolabeled immunoconjugate, and targeting characteristics were determined by biodistribution studies in A431 xenograft tumor-bearing mice sacrificed at 0.5, 1, 2, 4, and 24 hr. Results indicate that BF_PEPA appears to not be a suitable bifunctional chelating agent for sequestering isotopes of Bi(III) for radioimmunotherapy applications.

Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.

Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.

A key role of starburst amacrine cells in originating retinal directional selectivity and optokinetic eye movement.

The directional selectivity of retinal ganglion cell responses represents a primitive pattern recognition that operates within a retinal neural circuit. The cellular origin and mechanism of directional selectivity were investigated by selectively eliminating retinal starburst amacrine cells, using immunotoxin-mediated cell targeting techniques. Starburst cell ablation in the adult retina abolished not only directional selectivity of ganglion cell responses but also an optokinetic eye reflex derived by stimulus movement. Starburst cells therefore serve as the key element that discriminates the direction of stimulus movement through integrative synaptic transmission and play a pivotal role in information processing that stabilizes image motion.

An episomally maintained MDR1 gene for gene therapy.

Potential applications of the MDR1 multidrug transporter in gene therapy include protecting sensitive bone marrow cells against cytotoxic drugs during cancer chemotherapy and serving as a dominant selectable marker when coexpressed with a corrective passenger gene. To address safety concerns associated with integrating viral systems, such as retroviruses, we tested the feasibility of maintaining a nonvirally delivered MDR1 gene (pEpiHaMA) episomally. An MDR1 vector containing the Epstein-Barr virus (EBV) origin of replication (OriP) and its nuclear retention protein (EBNA-1) was transfected into human (KB-3-1) cells. MDR1 was expressed at a higher level in cells carrying the episomal vector, pEpiHaMA, compared with the vector lacking sequences needed for episomal maintenance (pHaMA). Furthermore, more drug-resistant KB-3-1 colonies were obtained on selection after transfection with pEpiHaMA. These observations correlated with longer maintenance of episomes in cells transfected with pEpiHaMA. In addition, episomes could still be recovered for more than 1 month from tumor explants in nude mice that were injected with pEpiHaMA-liposome complexes after drug selection, suggesting that these constructs can be maintained extrachromosomally in vivo.

PRAC: A novel small nuclear protein that is specifically expressed in human prostate and colon.

BACKGROUND: The database of human Expressed Sequence Tags (dbEST) provides a potential source for identification of tissue-specific genes. This database contains sequences that originate from cDNA libraries from particular tumors, organs or cell types. In this report, we have used the EST database to identify PRAC, a novel gene specifically expressed in human Prostate, prostate cancer, Rectum And distal Colon. METHODS: Using a computer based analysis, a cluster of sequence homologous ESTs was identified which contained ESTs derived only from human prostate cDNA libraries. The tissue specificity was examined by multiple tissue RNA dot blots and RT-PCR. The PRAC transcript and protein was identified using Northern blot analysis, RACE-PCR, primer extension, and western blot. RESULTS: PRAC encode a 382 nucleotide RNA found in prostate, rectum, distal colon, and in three prostate cancer cell lines; LNCaP, PC-3 and DU145. This transcript encodes a 6 kDa nuclear protein. The PRAC gene is located on chromosome 17 at position 17q21, about 4 kbp downstream from the homeodomain Hoxb-13 gene. CONCLUSIONS: Our data proves that the EST database can be a useful tool for discovery of prostate-specific genes. The nuclear localization, identification of potential phosphorylation sites, and possible cotranscription with the Hoxb-13 gene suggest that PRAC may have a regulatory role in the nucleus.

Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M.

We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g <--> e' (i, i' +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (T(m) = 28 degrees C) or nanomolar (T(m) = 40 degrees C) range, respectively, but heterodimerize with a T(m) >90 degrees C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 microM and 6 degrees C but that heterodimerize with a T(m) = 74 degrees C or K(d(37)) = 1.1 x 10(-11) M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with T(m) = 73 degrees C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells.