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  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6489—Metalloendopeptidases (3.4.24)
  • C12N9/6494—Neprilysin (3.4.24.11), i.e. enkephalinase or neutral-endopeptidase 24.11
• • A—HUMAN NECESSITIES
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  • C12Y304/24011—Neprilysin (3.4.24.11), i.e. enkephalinase or neutral endopeptidase 24.11
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  • A61K38/00—Medicinal preparations containing peptides

Definitions

• CALLA common acute lymphoblastic leukemia antigen
• CALLA The common acute lymphoblastic leukemia antigen (CALLA) has been shown to be a 100KD cell surface glycoprotein. It was initially identified on human lymphoblas tic leukemia cells. Ritz, J. et al ., Nature (London), 283:583-585 (1980). Later work showed that CALLA is also expressed by other lymphoid malignancies, such as lymphoblastic, Burkitts, and nodular poorly differentiated lymphocytic lymphomas, and by early lymphoid progenitors from fetal liver and fetal, pediatric and adult bone marrow and fetal and pediatric thymus. Greaves, M.F.
• the present invention relates to cDNA encoding human CALLA, or a fragment thereof.
• the cDNA encodes CALLA capable of binding to an anti CALLA antibody (e.g., the monoclonal antibody J5).
• the primary structure and the function of the encoded human CALLA have been determined.
• the cDNA has been shown to have near identity with the rat and the rabbit zinc metalloendopeptidase, neutral endopeptidase 24.11 (enkephalinase), and to encode functional neutral endopeptidase activity.
• the cDNA or DNA encoding CALLA or a fragment thereof can be inserted into a vector, which can be used to transform cultured cells for the production of CALLA or CALLA fragments.
• CALLA fragments encoded by the DNA are soluble.
• the CALLA fragments of the invention preferably contain none of the hydrophobic transmembrane portion of CALLA or only a portion (generally, six or fewer amino acids) of the transmembrane portion which does not prevent solubilization.
• CALLA fragments of the present invention need not have perfect homology with the corresponding region of naturally-occurring CALLA, and in some instances (e.g., antibody production), less than perfect or total homology is preferred. In general there will be at least about 50% homology between the amino acid sequence of the naturally-occurring CALLA and the amino acid sequence of CALLA or a CALLA fragment of the present invention.
• pure CALLA is now available for use, for example, in the diagnosis and treatment of medical conditions characterized by the presence of cells which express CALLA on their surfaces.
• Figure 1 is a graph of the HPLC profile of a CALLA tryptic digest.
• Figure 2 shows the restriction maps of CALLA cDNA clones.
• Figure 3 is the nucleotide sequence of human
• CALLA cDNA and the predicted amino acid sequence of the encoded human CALLA.
• Figure 4 is a hydropathicity plot of the translated CALLA cDNA sequence.
• Figure 5 is a restriction map of the CALLA cDNA clones used in the pIGTE/N CALLA construct.
• Figure 6 is a diagramatic representation of the pIGTE/N CALLA construct,
• Figure 7 presents a graphic comparison of the relative cell surface CALLA expression on the Nalm-6, J55, A2-3 and A2 - 2 cell lines.
• the present invention relates to DNA encoding all or a portion of human common acute lymphoblastic leukemia antigen (CALLA); to the encoded polypeptide, or a fragment thereof; to methods of making and using the encoded polypeptide or a fragment thereof; and to antibodies raised against and capable of binding CALLA.
• CALLA common acute lymphoblastic leukemia antigen
• cDNA encoding human CALLA which binds to an anti-CALLA antibody has been isolated and sequenced and the encoded polypeptide has been characterized, in terms of its primary structure and its function.
• the nucleotide sequence of cDNA encoding human CALLA and the predicted amino acid sequence of the encoded CALLA are presented in
• Neutral endopeptidase 24.11 enkephalinase is a cell membrane-associated enzyme that cleaves peptide bends on the amino side of hydrophobic amino acids.
• the enzyme was identified in brain as an enkephalinase because it cleaved the Gly3-Phe4 bond of enkephalins. Malfroy, B. e t al . , Nature, 276:523-526 (1978). The enzyme was subsequently found in many other tissues, including kidney, in which it was present at high levels. Llorens, C. & J.C. Schwartz, Eur. J. Pharmacol, 69:113-116 (1981).
• Neutral endopeptidase 24.11 has been shown to react with a variety of physiologically active peptides including chemotactic peptide, substance P and neurotensin, oxytocin, bradykinin, angiotensin I and II and a variety of opioid peptides.
• enkephalinase is a zinc binding metalloendopeptidase
• the chromosomal location of the CALLA encoding gene is 3q21-27, a region rich in metal binding proteins, including transferrin, lactotransferrin, melanotransferrin, the transferrin receptor and ceruloplasmin.
• CALLA protein has been isolated from the Nalm-6 cell line through use of an anti-CALLA monoclonal antibody (J5) and standard techniques.
• the Nalm-6 cell line was originally derived from a patient with chronic myelogenous leukemia in lymphoid blast crisis and is known to express high levels of surface CALLA. Hurwitz e t al . , Cancer, 23:174 (1970); Goldmacher e t al ., J. Immunol ., 136:320
• Nalm-6 cells of a component of 97-100KD in size Digestion with endoglycos idase F resul ted in a decrease of approximately 10KD in the molecular mass of the immunoprecipitated component. This indi cate s that at least 10% of the molecular mass of CALLA results from N-linked carbohydrates.
• the isolated protein was subsequently purified to homogeneity, using techniques which are recognized and described in the Examples. NH 2 -terminal sequence information was obtained from the intact CALLA protein and from derived tryptic peptides and V8 protease fragments, as shown in Table 1.
• CALLA cDNAs were isolated from a Nalm-6 cell line ⁇ gt10 library, using redundant oligonucleotide probes. Shipp, M.A. e t al . , Proc. Natl. Acad. Sci . , USA, 85:4819-4823 (1988).
• the CALLA cDNA sequence predicts a 749 amino acid integral membrane protein which has a single 24 amino acid hydrophobic segment which could function as both a transmembrane region and a signal peptide.
• the extracellular protein segment is made up of the COOH-terminal 700 amino acids, which include six potential N - l inke d glyc o sylat ion sites.
• CALLA- transfected cell lines were subsequently produced and a sensitive enzymatic assay was used, In conjunction with a specific neutral metallopeptidase inhibitor, to demonstrate that CALLA is a functional form of this membrane-bound enzyme.
• CALLA derived from a lymphoblastic leukemia cell line has functional neutral endopeptidase 24.11 activity
• a construct containing the CALLA open reading frame from the leukemic cell line, Nalm-6, under the control of an Immunoglobulin promoter and enhancer was engineered and transfected into the murine myeloma cell line, J558, which lacks cell surface CALLA expression.
• CALLA+ J558 cells were identified by phenotyping with the J5 anti-CALLA monoclonal antibody, sorted and cloned by limiting dilution. Two J5+ subclones, A2-3 and A2-2, which had high and low levels of CALLA expression, respectively, were chosen for further analysis.
• Figure 2 represents a comparison of relative CALLA fluorescence of these two CALLA+ s t ab l e transfectants, the parental CALLA multiple myeloma line, J558, and the CALLA+ acute lymphoblastic leukemia line from which the CALLA cDNA was isolated, Nalm-6.
• J558 lacks detectable cell surface CALLA expression (mean channel fluorescence 0).
• A2-3 and A2-2 express cell surface CALLA.
• A2 - 3 expresses a substantially higher number of CALLA sites per cell than does A2 - 2 (mean channel fluorescence 116.8 and 7.6, respectively).
• Table 2 illustrates the results of the enzymatic assay performed using whole cell suspensions of the individual cell populations.
• Cell suspensions of Nalm-6, A2-3, A2-2 and J558 exhibited neutral endopeptidase activity of 4789, 1906, 446 and 0.2 nanomoles of product per hr per 10 6 cells, respectively. The value of 0.2 nanomoles per hr per 10 6 cells is within the experimental error of no activity.
• A2-2 cell suspensions on neutral endopeptidase activity was also assessed. It resulted in a marke d reduction in activity to 53, 52 and 18 nmols/h/10 6 cells, respectively, (Table 2). Hudgin, R.L. et al., Life Sci. , 29:2593-2601 (1981). As indicated in Table 3, cell lysates from Nalm-6, A2-3 , A2-2, and J558 contained neutral endopeptidase specific activities of 9.96, 2.35, 1.18 and 0.08 nmols/min/mg protein of neutral endopeptidase activity, respectively.
• CALLA is a functional form of active neutral endopeptidase 24.11 (enkephalinase).
• CALLA protein is found on both early normal lymphoid progenitors and their malignant counterparts and that CALLA cDNA from an acute lymphoblastoid leukemia encodes neutral endopeptidase 24.11 activity indicates that the enzyme functions at a critical stage in lymphoid differentiation. This is of particular interest in light of previous studies demonstrating that the cell surface bound enzyme has the potential to mediate a wide range of biological activities in a variety of tissues.
• neutral endopeptidase 24.11 (enkephalinase) has been shown to inactivate endogenous opioid pentapeptides on neurons in brain, chemotactic peptide (f-Met-Leu-Phe) on polymorphonuclear granulocytes and a variety of regulatory peptides on the surface of proximal tubule epithelial cells of the kidney.
• chemotactic peptide f-Met-Leu-Phe
• CALLA/neutral endopeptidase 24.11 In light of the structural identity of the enzyme in various tissues, the biologic activity of CALLA/neutral endopeptidase 24.11 is likely to be dictated by the availability of specific substrates in individual organs, rather than by the presence of different functional forms of the enzyme. The substrate for the cell surface CALLA/neutral endopeptidase 24.11 of early lymphoid progenitors is not yet known.
• CALLA/neutral endopeptidase 24.11 may also react with a small regulatory peptide at the cell surface of lymphoid precursors. Such an action could lead to the inactivation of a physiologically active peptide or convert an inactive form of the peptide to an active one.
• the CALLA substrate for lymphoid precursors may be a previously defined peptide.
• Neutral endopeptidase 24.11 has not previously been Identified on lymphoid cells. It has, however, been detected in nodal tissue. Bowes, M.A. and A.J. Kenny, Biochem. J, 236:801-810 (1986). In porcine lymph nodes, the enzyme is found on a subpopulation of adherent cells with the morphological characteristics of fibroblasts. Bowes, M.A. and A.J. Kenny, Biochem. J, 236:801-810 (1986). These cells are most prevalent in medullary areas and are also found In the center of follicles and encircling them. Of interest, these neutral endopeptidase 24.11+ cells are observed to have clusters of lymphoid cells firmly attached to their cell surface. Bowes, M.A. and A.J. Kenny, Biochem. J,
• Chroni c treatment with morphine induces a selective and specific increase in brain enkephalinase activity, likewise indicating that the enzyme may regulate the local c onc ent r a t i o n of opioid neurotransmitters and that the concentration of such neurotransmitters may also affect enzyme levels.
• CALLA encodes functional lympho i d neutral endopeptidase 24.11, and, thus, it is likely that its peptide ligand will also modulate cell surface CALLA. This may result in efficient internalization of ligand. It is not yet known whether such a putative p ep t ide affects migration, growth or other functional aspects of immature normal or malignant B cells. However, the unequivocal identification of CALLA as functional neutral endopeptidase 24.11 (enkephalinase) and the availability of specific endopeptidase inhibitors should make it possible to assess the role of CALLA in lymphoid development. Uses of DNA Encoding Human CALLA, CALLA, Antibodies Reavtive with CALLA
• DNA encoding human CALLA, CALLA or fragments thereof and antibodies reactive with CALLA have both diagnostic and therapeutic applications.
• the cDNA encoding human CALLA can be used to produce CALLA, by means of techniques known to those skilled in the art (such as those detailed in the Examples). For example, DNA having all or a portion of the nuc l e o t i de sequence of
• Figure 3 can be introduced into a suitable host cell (generally as a component of a vector), in which it is subsequently produced and from which it can be isolated.
• the DNA can be introduced into the cells by known techniques (e.g., co-cultivation, electroporation).
• the DNA introduced in this manner can be isolated, as described herein, or can be DNA encoding functional CALLA (e.g. DNA, referred to as equivalent DNA, which has a sequence sufficiently similar to that represented in Figure 3 to encode functional CALLA).
• cDNA as used herein, is intended to include DNA, produced or obtained by any means, which has the sequence represented in Figure 3 or equivalent DNA.
• CALLA-encoding DNA can be used to produce all or a fragment of CALLA is as follows:
• Human cDNA sequence encoding the CALLA or a desired CALLA fragment (e.g., the sequence enco ding only the portion of CALLA which is exposed beyond the transmembrane region) can be inserted into a suitable expression vector, using conventional techniques.
• a desired cDNA can be inserted into the expression vector described in Ringold U.S. Patent No. 4,656,134, hereby incorporated by reference.
• the resulting plasmld can then be used to transform mammalian host cells, and the human CALLA fragments Isolated and purified from those cells and/or their medium according to conventional methods.
• the polypeptides can be produced in a bacterial host (e.g., E. coli).
• the cDNA encoding the desired fragment can, for example, be inserted into the expression vector described in DeBoer et: al., Proc. Natl. Acad. Sci.,
• the resulting plasmid can then be used to transform E. coli cells, and the CALLA fragment isolated and purified according to conventional methods.
• nucleotide sequence of Figure 3 can also be used as probes to detect, identify and/or quantity DNA encoding CALLA in a sample (e.g., cells) of interest. These probes will generally be labelled (e.g., radioactively, enzymatically, etc.) and can be used in standard techniques.
• CALLA polypeptides of the present invention can be used, for example, for therapeutic purposes.
• Soluble CALLA fragments e.g., all or a portion of the portion of the CALLA region which extends beyond the transmembrane region of a cell.
• Soluble CALLA fragments will be admixed with a pharmaceutically acceptable carrier substance, e.g., saline, and administered by a medically acceptable administration route (e.g., intravenously, intramuscularly, or orally) to patients suffering from medical conditions characterized by the presence of CALLA-bearing cells which are associated with their disease, in an amount sufficient to inhibit proliferation of those CALLA-bearing cells.
• the soluble CALLA fragments will function by competitively inhibiting the natural binding events of surface-bound CALLA, which events are apparently necessary for the proliferation and/or mobility of such cells (e.g., acute lymphoblastic leukemic cells).
• the amount of soluble CALLA fragment administered will be determined on an individual basis, but will generally be from approximately 10 ⁇ g/kg bodyweight to approximately 50 ⁇ g/kg per day. As a result of the present invention, it is possible to make diagnostically useful antibodies which were previously unavailable. Because the amino acid sequence of CALLA has now been provided, any synthetic peptide corresponding to any given region of the molecule can routinely be made, and that peptide used to raise highly specific antibodies.
• Two antibodies each specific to a different region of CALLA, can be used in immunoassays, such as a sandwich assay, which require two different antibodies, which bind to two different sites on the target molecule; such assays are described, for example, in David et al., U.S. Patent No. 4,376,110, the teachings of which are hereby incorporated by reference.
• antibodies reactive with denatured CALLA are antibodies reactive with denatured CALLA (i.e., CALLA which has lost i ts native three-dimensional conformation).
• denatured CALLA i.e., CALLA which has lost i ts native three-dimensional conformation.
• Such antibodies can be made by immunizing animals with CALLA fragments or CALLA analogs containing amino acid substitutions which prevent normal folding of the protein.
• These antibodies will be particularly useful for analysis of pathologic specimens which have been fixed in formalin, a process which causes denaturation of any CALLA present on, for example, cancer cells in a lymph node.
• Antibodies made to native CALLA, which retains its three-dimensional structure are unreactive with denatured CALLA, and thus cannot be used to detect CALLA-bearing cells in formalin-fixed specimens.
• the antibodies reactive with denatured CALLA will be labelled (e.g., with fluorochromes) and can be used in immuno
• CALLA CALLA
• Enkephalins are endogenous opioid-llke pentapeptides present in areas of the central nervous system associated with perception of pain, as well as with other functions. As explained previously, neutral endopeptidase 24.11
• enkaphalinase has been shown to inactivate endogenous opioid pentapeptides on neurons in the brain. That is, neutral endopeptidase 24.11 (enkephalinase) is known to cleave the Gly 3 -Phe 3 amide bond of the op io id pentapeptides, enkephalins, both in vitro and in vivo.
• a parenterally active enkephalinase inhib ito r has been shown to display analgesic properties in human. Malfroy, B. et al., FEBS Letters, 229:206-210 (1988).
• CALLA has functional neutral endopeptidase activity, as well as striking similarity at the amino acid level to human enkephalinase and to rat enkephalinase and rabbit neutral endopeptidase molecules.
• CALLA/neutral endopeptidase 24.11 it should be possible to design a substance capable of interfering with this ability of CALLA/neutral endopeptidase 24.11 to cleave opioid pentapeptides, producing a prolonged and enhanced concentration of endogenous opioids, which, in turn, will produce an analgesic effect.
• an inhibitor of CALLA which will inhibit/reduce leukemic growth or behavior of cells.
• CALLA exhibits enzymatic function which might influence (enhance) leukemic growth or behavior.
• an inhibitor of CALLA which binds to it and reduces or eliminates its enzymatic activity, can be designed and used to inhibit such growth.
• An alternative approach is based on the knowledge presented herein and the fact that there appears to be a natural ligand of CALLA which is involved in leukemic cell growth; this putative natural ligand might bind to CALLA and, after cleavage, become active.
• an "artificial ligand” which inhibits binding of the natural CALLA ligand (e.g., by binding to CALLA and, thus, preventing CALLA natural ligand binding or by binding to the natural ligand, and, again, preventing CALLA natural ligand binding).
• the present invention will now be illustrated by the following examples, which are not intended to be limiting in any manner.
• the Nalm-6 line was utilized as a cellular source in conjunction with the anti-CALLA monoclonal antibody J5.
• Ritz et al. Nature 258:454 (1975).
• the Nalm-6 cell line was originally derived from a patient with chronic myelogenous leukemia in lymphoid blast crisis and is known to express high levels of surface CALLA. Hurwitz et al., Cancer, 23:174 (1979); Goldmacher et al., J. Immunol ., 136:320 (1986).
• J5 antibody was found to immunoprecipitate a structure of 97-100KD from 125 I surface labelled cells.
• Nalm-6 cells (3 ⁇ 10 7 ) were radioiodinated using lactoperoxidase and lysed in RIPA buffer containing 1% Triton X-100, 0.15 M NaCl, ImM PMSF,
• CALLA was eluted with SDS sample buffer containing 5% 2 -mercap toethanol. Endo-F glycosidase treatment was carried out, and aliquots of samples were analyzed with and without enzyme treatment on 10% SDS-PAGE. Luescher and Bron, J. Immunol ., 134:1084 (1985).
• the anti-J5 column was washed sequentially with 50 ml aliquots of 10 mM Tris, pH 8/0.15 M NaCl with 1) 0.05% SDS/0.5% TX-100/1% DOC; 2) 0.5% TX-100, 1% DOC; and 3) 0.5%-Tx-100.
• Bound material was eluted with 0.1 M glycine, pH 3.0/0.5% Triton X-100 and collected in 1 ml fractions in tubes containing 60 ⁇ l.of 1 M Tris, pH 8. Subsequently, 100 ⁇ l 10% SDS was added to each tube at 22°C.
• CALLA Fifty picomoles of purified CALLA were subjected to amino terminal sequencing, and only a 5 picomole signal (XXSESQ) was obtained, suggesting that CALLA was in large part N-terminally blocked to Edman degradation. For this reason, CALLA was digested with trypsin and with V8 protease. Four hundred picomoles of electroeluted CALLA in 50 mM ammonium bicarbonate/0.1% SDS was mixed with V8 protease (Boehringer Mannheim) to give a protein/enzyme ratio of 5:1. After incubation at 37°C for.
• One nanomole of electroeluted CALLA was made 0.1M Tris-HCL pH 8/20 mM dithiothreitol/2% SDS and adjusted 60 minutes later to 50 mM in iodacetic acid.
• the reduced s - carb oxymethylated preparation was then precipitated by the addition of 9 volumes of ethanol at -20°C for 16 hours, dissolved in 0.1M Tris-HCL pH8/2mM CaCl 2 , mixed with TPCK trypsin (Cooper) to give a protein/enzyme ratio of 50:1, and incubated for 16 hours at 37°C.
• CALLA protein, V8 fragments, and tryptic peptides were then analyzed for N-terminal sequence on a gas phase protein sequenator (Applied Biosystems, model 470A) equipped with an in-line 120A PTH analyzer using program 03PTH.
• V8 peptide III contains a sequence Identical to that obtained from the intact CALLA protein, indicating that V8 peptide III is derived from the partially blocked NH 2 -terminus.
• Reverse phase HPLC separation of the product of the tryptic digest yielded at least 80 different peaks ( Figure 1); 8 of these were selected for further HPLC purification with an alternative gradient, yielding eleven tryptic peptides with the sequences shown in Table 1.
• Tryptic peptide VIII and V8 peptide I contain an overlapping amino acid sequence LNYKEDEYFENIIQN . (The lysine residue in TPVIII was likely not cleaved by enzymes because tryptic digestion of lysine residues followed by C-terminal acidic amino acids is ineffective. Cunningham et al., Biochem., 12:4811 (1973)).
• Each double positive clone contained an identical EcoRI fragment approximately 1.6Kb in size and certain clones contained additional EcoRI fragments of smaller size.
• the clone containing the longest cDNA insert (approximately 3.5Kb) was subcloned into the m13 vector mp18 and is referred to as clone 1.2 (12.1).
• Clones 1.1 and 2.1 begin 100BP 5' to the initiation methionine ATG .
• Figure 2 the open reading frame from clones 1.1, 1.2 and 2.1, 2.2 is indicated.
• the 3' untranslated sequence from clones 1.2, 2.2, and 2.3 is 1472 BP in length, ending in a polyA sequence.
• Clone 3.1 (1-8) is identical to the other CALLA cDNAs at its 5' end as shown, but contains an additional 1775 bp of 3' untranslated sequence ending in a polyA tail.
• the nucleotide sequence shown is a composite of clones 1.1, 1.2, and 3.1 ( Figure 3).
• Position 1 (11bp 5' to the ATG) represents the position at which clones 1.1 and 2.1 become identical.
• the translated CALLA cDNA sequence predicts the indicated 750 amino acid protein with six potential N-linked glycosylation sites (Asn-Xaa- Ser/Thr*).
• the eleven CALLA tryptic peptides and three CALLA V8 fragments identified by protein micro - sequencing are underlined in the translated CALLA cDNA sequence.
• the single 24 amino acid hydrophobic segment (amino acids 27-50) is underlined.
• Clone 1.2 ( Figure 2, BP 199 to 3734, Figure 3) has an open reading frame and a translated amino acid sequence which contains all eleven of the tryptic peptides but only two of the three V8 peptides determined by microsequencing of the CALLA protein (Table 1, Figures 2 and 3).
• the translated sequence of clone 1.2 lacks the NH 2 -terminal residues identified from both the intact CALLA protein and V8 peptide III (Table 1, Figure 3).
• the independently derived CALLA cDNA sequences from 1.1/1.2 and 2.1/2.2/2.3 are identical from BP 1 to 3734, Including 11 nucleotides of 5' untranslated sequence.
• Each clone contains an additional unique approximately 100 bp of 5' sequence, possible representing alternative 5' splicing.
• An additional overlapping clone (3.1) was identified which corresponds to the previously characterized clones from its 5' end (bp 2321) through bp 3733; thereafter, clone 3.1 contains an additional 1775 bp of sequence which ends in a polyA tail ( Figures 2 and 3).
• the complete nucleotide sequence derived from CALLA clones 1.1, 1.2 and 3.1 contains an open reading frame of 2250 bases (positions 12-2261) beginning with an ATG methionlne codon which is preceded by a TAG termination codon at bp 6-8 in-frame.
• a canonical polyadenylation signal (AATAAA) is found 25 bp upstream of the polyadenylation site in clone 3.1. Additional canonical polyadenylation signals are found in the 3' untranslated region at bp 3088-3093, 3371-3376, 3792-3797, and 4406-4411.
• the translated CALLA cDNA sequence includes 170 of the 182 amino acid residues identified by microsequencing the intact CALLA protein, the derived tryptic peptides, and the V8 protease fragments (Table 1, Figure 3), providing conclusive evidence that the cDNA represents authentic CALLA.
• the CALLA cDNA predicts a 750 amino acid protein with a polypeptide core MW of 85.5KD and six potential N-linked glycosylation sites
• the translated CALLA cDNA sequence has a single hydrophobic 24 amino acid segment at positions 27-50 with the characteristics of a transmembrane region. This hydrophobic segment is preceded by five basic residues, suggesting that CALLA is oriented such that the NH 2 terminus constitutes the cytoplasmic tail.
• the translated CALLA sequence does not contain an initial hydrophobic NH 2 terminal segment that could function as a signal peptide.
• comparison of the translated cDNA sequence with that derived from the Intact protein and N-terminal V8 fragment (V8III) reveals that the only NH 2 - terminal proteolytic processing which occurs during CALLA synthesis results In removal of the initiation methionine (Table 1, Figure 3).
• Hydrophobicity values were calculated by the PRSTRC algorithm assigned over windows of five amino acids (Ralph eta l . , CABIOS 3:211 (1987)). In Figure 4, positions above the baseline are hydrophobic. Amino acid positions are indicated below the plot and the transmembrane region is shaded. In contrast to previous studies suggesting that
• CALLA is a non-integral membrane protein
• these results indicate that CALLA is a type II integral membrane protein with a short (25 amino acid) NH 2 terminal cytoplasmic tail, a single 24 amino acid hydrophobic region that functions as both an uncleaved internal signal sequence and a transmembrane segment, and a large (700 amino acids) carboxy terminal extracellular domain.
• the fact that all six putative N-linked glycosylation sites are located in the carboxy terminal segment is consistent with this interpretation, as are the 125 I surface labelling studies, given that there are no tyrosine residues in the NH 2 -terminal segment. Searches of the GenBank data base (release
• the region immediately preceding and including the transmembrane segment of CALLA has partial identity (14 amino acids out of 31 amino acids with no gaps) with that of another type II membrane protein, pro-sucrase isomaltase, raising the possibility that the dual function transmembrane segments of certain type II proteins may have common features. Hunziker et al., Cell, 46:227 (1986).
• RNA samples were prepared and analyzed by
• RNAs from a panel of CALLA+ and CALLA- lymphoid cell lines and primary tumors were probed in
• RNA samples were analyzed: Nalm-6; Raji, a CALLA + Burkitts lymphoma cell line; Molt -4, a CALLA+ T cell acute lymphoblas tic leukemia (ALL) cell line; HSB, Jurkat and J77, CALLA- T cell leukemia cell lines; unstimulated peripheral blood mononuclear cells (PBMC); mitogen triggered PBMC ; LAZ 221, a CALLA+ ALL cell line, and LAZ 388, a CALLA- EBV transformed lymphoblas toid line from the same donor as LAZ 221.
• PBMC peripheral blood mononuclear cells
• a 10 ⁇ g sample of poly A selected Nalm-6 RNA was also analyzed.
• Cell lines defined as CALLA positive (by immunofluorescence using the J5 antibody) Including Nalm-6, the ALL cell lines Laz 221 and Molt-4, and the Burkitts lymphoma cell line Raji contain two major CALLA mRNAs: a 3.7Kb and a 5.7Kb CALLA transcript.
• CALLA negative sources including three T cell tumor lines, Jurkat J77. (a second Jurkat clone), and HSB, and EBV transformed lymphoblas toid line, Laz 388, and resting and mitogen-stimulsted peripheral blood lymphocytes, lack these transcripts.
• the 3.7 Kb transcript probably results from utilization of the polyadenylation signal at bp 3792, and the minor mRNAs of 4.5, 3.1 and 2.7 Kb from utilization of polyadenylation signals located at 4406-4411, 3371-3376, and 3088-1093, respectively.
• Anti-CALLA monoclonal antibody reactivity has also been observed on normal granulocytes, a subpopulation of bone marrow stromal cells, certain elements in kidney, fetal intestine, and breast, and certain non-lymphoid tumor cells.
• HB-100 a CALLA+ fibroblast strain
• G361 a CALLA-melanoma line
• CLL-227 a CALLA+ colon carcinoma line
• Nalm-6 for comparison
• granulocyte a CALLA+ fibroblast strain
• the 3.7Kb transcript is the major CALLA mRNA in HB-100 and CCL-227 cells.
• the 3.7 and 5.7Kb CALLA transcripts are both abundant in G-361 cells whereas the 5.7 Kb m RNA is the major CALLA transcript in granulocytes.
• DNA was prepared and analyzed by Southern blotting according to standard protocols. Maniatis e t al ., Molecular Cloning, Cold Spring Harbor, NY,
• BamHI and Hindlll digested genomic DNAs from a panel of CALLA+ and CALLA- sources were Southern blotted and hybridized with the approximately 1.6Kb EcoRI CALLA cDNA fragment.
• Ten micrograms of high molecular weight DNA from PBL; Laz 388; Laz 221; and Nalm-6 was digested with Hindlll or BamHI, and subjected to Southern blot analysis using the 1.6Kb CALLA cDNA probe .
• CALLA The molecular cloning of CALLA and its identification as a type II transmembrane glycoprotein does not make it possible to infer, in a direct way, its role in lymphoid function.
• Proteins in this class have diverse functions ranging from receptors to membrane bound enzymes and include the transferrin receptor, the asialoglycoprotein receptor, influenza viral neuraminidase, gamma glutamyl transpeptidase, prosucrase-isomaltase complex and the invariant chain of HLA .
• CALLA is an integral membrane protein
• Antibody induced modulation of CALLA was noted to be similar to that seen with cell surface receptors such as surface Ig and T3-Ti; it also resembled the specific down regulation or loss of cell surface receptors induced by many peptide hormones.
• CALLA has a relatively short cytoplasmic tail makes it unlikely to serve a direct signal transduction function.
• CALLA expression appears on uncommitted TdT+ lymphoid progenitors and generally declines as the cells display evidence of B cell or T cell commitment, suggesting that the antigen may function in the earliest stages of lymphoid differentiation.
• the fact that CALLA is also expressed by a subpopulation of bone marrow stromal cells raises the possibility that CALLA may participate in the microenvironment necessary for early lymphoid maturation.
• DNA from clone 1.1 was digested with EcoRI and Aval, yielding a 0.435Kb fragment ( Figure 5).
• Aliquots of DNA from clone 2 were digested with Aval and Clal, yielding a 0.499Kb fragment, or with Clal and ApaI, yielding a 1.546Kb fragment, respectively ( Figure 5).
• the plasmid vector pBluescript SK(+) (Stratagene) was digested with EcoRI and Apal.
• the 0.435Kb EcoRI-Aval, 0.499Kb Ava-Clal, 1.546Kb Clal-Apal CALLA cDNA fragments and the EcoRI-Apal cut plasmid vector were gel-purified, l i gate d and used to transform DH5 ⁇ + Eschericia coli (Bethesda Research Labs).
• Recombinants were identified on the basis of blue/white color selection on LB plates containing 100 ⁇ g/ml ampicillin, 80 ⁇ g/ml X-gal and 20 mM lPTG.
• the reconstructed CALLA cDNA containing the intact open reading frame was excised from the plasmid vec to r us in»g Drl - Apal and b lunted on its 3 ' end wi th T4 DNA polymerase.
• the resulting 5' and 3' blunt-ended CALLA cDNA fragment was ligated into EcoR5 digested pBluescript SK(+) which contains a SalI site in the polylinker.
• the fragment was excised with Smal and Apal which cleaved at polylinker sites 3' of the CALLA fragment arid 5' of the CALLA fragment and SalI site, respectively.
• the Apal end was blunted with T4 DNA polymerase and the modified CALLA open reading frame insert was ligated into EcoR5 cut pBluescript SK(+). Following transformation, recombinants containing a resulting 3' SalI end from the polylinker were identified with diagnostic SalI digestions.
• the pIGTE/N plasmid ( Figure 6) contains the human immunoglobulin promoter, both human and murine immunoglobulin enhancers, an SV40 intron and polyadenylation signal, and the gene for
• CMV-neomycIn resistance Following transformation, recombinants were analyzed for orientation using a panel of diagnostic restriction endonucleases.
• a plasmid, designated pIGTE/N CALLA s which contains the CALLA open reading frame in the sense orientation was obtained.
• the pIGTE/N CALLA construct was transfected into the CALLA- murine myeloma cell line, J558, by electroporation as previously described. Patten, H. et al., Proc. Natl. Acad. Sci. USA 81:7161-7165 (1984).
• cells were diluted in 10 ml of RPMI supplemented with 10% fetal calf serum/2% glutamine/100 U/ml penicillin and 100 ⁇ g/ml streptomycin and cultured for 48 hours at 37°C in 5% CO 2 . Thereafter, cells were maintained in RPMI supplemented with 10% fetal calf serum/2% glutamine/100 U/ml penicillin and 100 ⁇ g/ml streptomycin and 900 ⁇ g/ml of G418 for 14 day s and phenotyped for CALLA expression using the anti-CALLA monoclonal antibody, J5. Ritz, J. et al.. Nature, 283:583-585 (1980).
• J5+ cells were selected by fluo r e s c enc e activated cell sorting (Epics V), using standard techniques, and cloned by limiting dilution at 0.5 cells/well in G418-containing media.
• Epics V fluo r e s c enc e activated cell sorting
• CALLA+J558 cells were identified by phenotyping with the J5 anti-CALLA monoclonal antibody, sorted and cloned by limiting dilution. Two J5+ subclones, As-3 and A2-2, which had high and low levels of CALLA expression, respectively, were chosen for further analysis.
• Figure 2 presents a comparison of relative CALLA fluorescence of these two CALLA+ stable transfectants, the parental CALLA multiple myeloma line, J558, and the CALLA+ acute lymphoblastic leukemia line from which the CALLA cDNA was isolated, Nalm-6.
• J558 lacks detectable cell surface CALLA expres s i on (mean channel Fluorescence 0).
• the two J5+ sub clone s express cell surface CALLA.
• A2-3 expresses a substantially higher number of CALLA sites per cell than does A2-2 (mean channel fluorescence 116.8 and 7.6, respectively).
• EXAMPLE 8 Enzymatic Assay for Neutral Endopeptidase 24.11 Activity As described previously, search of the CALLA cDNA sequence against GenBank (release 56, 6/88) revealed striking homologies with the rat and rabbit neutral endopeptidase 24.11, commonly referred to as "enkephalinase". The DASHER program (D.V. Faulkner, Molecular
• Neutral endopeptidase 24 . 11 activity was meas ure d f luorome tr ical ly in a coupled assay using glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide (Enzyme Systems Products) as substrate. Orlowski, M. & S. Wilk, Biochem., 20:4942-4950 (1981). Cleavage of this substrate by neutral endopeptidase 24.11 yields Phe-4-methoxy-2-naphthylamide which, in the presence of aminopeptidase activity, is converted to the fluorescent product 4-methoxy-2-naphthylamine. Reaction mixtures contained 0.1 mM substrate, 100mM MES
• Cell extracts were prepared by resuspending washed cells in 200-400 ⁇ l of 20 mM MES, pH 6.5, containing 1% octyl glycoside
• Table 2 illustrates the results of the enzymatic assay performed using whole cell suspensions of the individual cell populations.
• Cell suspensions of Nalm-6, A2-3, A2-2 and J558 exhibited neutral endopeptidase activity of 4789, 1906, 446 and 0.2 nanomoles of product per hr per 10 6 cells, respectively.
• TABLE 2. DETERMINATION OF NEUTRAL ENDOPEPTIDASE ACTIVITY IN WHOLE CELL SUSPENSIONS
• ND not determined
• cell lysates from Nalm-6, A2-3, A2-2, and J558 contained neutral endopeptidase specific activities of 9.96, 2.35, 1.18 and 0.08 nmols/min/mg protein of neutral endopeptidase activity, respectively.
• the neutral endopeptidase activity associated with the Nalm-6, A2-3 and A2-2 cell lysates was dramatically reduced to 0.20, 0.08 and 0.29 nmols/min/mg protein, respectively (Table 3).

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