WO1994028133A9 - FACTEURS DE DIFFERENCIATION RECOMBINES DE $i(NEU) - Google Patents

FACTEURS DE DIFFERENCIATION RECOMBINES DE $i(NEU)

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WO1994028133A9
WO1994028133A9 PCT/US1994/005769 US9405769W WO9428133A9 WO 1994028133 A9 WO1994028133 A9 WO 1994028133A9 US 9405769 W US9405769 W US 9405769W WO 9428133 A9 WO9428133 A9 WO 9428133A9
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ser
ndf
glu
thr
lys
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PCT/US1994/005769
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  • This invention relates to novel polypeptides (herein referred to as neu differentiation factors), produced by recombinant DNA methods, which interact with and stimulate the neu receptor and modulate cellular function.
  • the invention includes analogs and
  • oncogenic transformation can be induced by constitutive production of growth regulatory factors or by altered forms of their cognate receptors (Yarden, Y., and Ullrich, A., Ann . Rev. Biochem. 57:443-448, 1988).
  • the oncogenic receptors include a family of
  • transmembrane glycoproteins that share a common
  • the neu proto-oncogene also known as HER-2 or c-erbB-2
  • This pl85 neu glycoprotein is known to be present in many epithelial and neural tissues
  • p185 neu or neu gene amplification occurs in approximately twenty percent of breast, stomach, bladder and ovarian adenocarcinomas (Lofts, F.J., and Gullick, W.J., Cancer Treat . Res .
  • Increased p185 neu expression also occurs in certain non-malignant neoplasias, such as adenomatous polyps (D'Emilia, J.K. et al., Oncogene 4:1233-1239, 1989; Cohen, J.A. et al., Oncogene 4:1233- 1239 1989), Barrett's esophagous (Jankowski, J.G. et al., Gut 33:1033-1038, 1992), and polycystic kidneys (Herrera, G., Kidney Int . 40:509-513, 1991).
  • Phosphorylation of the p185 neu receptor on tyrosine residues can be stimulated by proteins purified from several sources (Peles, E. et al . , Cell 69: 205-216, 1992 ; Lupu, R . et al . , Science 249:1552-1555, 1990; Holmes, W.E. et al., Science
  • neu The 44 kDa rat factor, named neu
  • NDF NDF differentiation factor
  • NDF transmembrane glycoprotein precursor
  • the recombinant version of this rat NDF was found to interact with pl85 neu and stimulate tyrosine phosphorylation of the receptor in human tumor cells of breast, colon and neuronal origin (Peles, E. et al., EMBO J. 12:461-971, 1993).
  • In situ hybridization with a NDF probe
  • polypeptides add to the expanding knowledge of NDF-related molecules, which now also includes glial growth factors (Marchionni, M.A. et al., Nature 362:312-318, 1993), as well as chicken acetylcholine receptor
  • non-naturally occurring polypeptides encoded by human nucleotide sequences which function as stimulators and inducers of neu (or Her-2, or c-erbB-2) receptor activities.
  • These polypeptides include
  • recombinant DNA-derived neu receptor stimulating factors expressed from cDNA clones isolated from human tissues and cell lines.
  • Such polypeptides possess an ability to stimulate human pl85 neu tyrosine phosphorylation.
  • These recombinant polypeptides can be termed neu receptor stimulating factors, but are preferably referred to herein as neu differentiation factors (or "NDFs"), based on their ability to induce a differentiated phenotype in certain cell lines.
  • NDFs neu differentiation factors
  • polypeptides are encoded by nucleotide sequences which are also described in detail herein.
  • the isolated DNA sequences provided by the present invention are useful in securing expression of the polypeptides in procaryotic and/or eucaryotic host cells.
  • the present invention specifically provides DNA sequences encoding all or part of the unprocessed amino acid sequences (proNDFs) as well as DNA sequences encoding all or part of the processed (mature) forms of the NDFs and novel recombinant analogs thereof.
  • Such DNA sequences include:
  • cDNA complementary DNA
  • genomic DNA sequences encoding variant forms of human NDF, manufactured DNA sequences (e.g., as by solid phase chemical synthesis) encoding human NDF, fragments of human NDF, and analogs of human NDF.
  • the DNA sequences may incorporate codons facilitating transcription and translation of messenger RNA in host cells.
  • vectors containing such DNA sequences and host cells transformed or transfected with such vectors. Additionally, the invention includes methods of producing biologically active human NDF polypeptides by recombinant DNA techniques, and methods of treating disorders using recombinant NDFs.
  • compositions containing recombinant human NDF polypeptides and antibodies generated with such polypeptides are also encompassed by this invention.
  • Figure 1 depicts a high pressure liquid chromatogram of the trypsin digest of "naturally-occurring" neu receptor stimulating factor purified from media conditioned by ras-transformed rat fibroblasts according to the methods of Peles, E. et al., Cell, 1992, above. The elution profile is shown, and the amino acid sequences obtained from the fractions corresponding to each peak are indicated in conventional single letter designation.
  • the amino acid in parentheses represents an asparagine-linked glycosylation site, while the dotted line represents a longer sequence in which the amino acids were not identified.
  • the inset shows
  • FIG. 2 shows the structure of the mammalian
  • COS-7 cell expression vector pJT-2/NDF COS-7 cell expression vector pJT-2/NDF.
  • the rat NDF cDNA insert is the clone 44 cDNA sequence of Figure 5 [SEQ ID NO: 21].
  • Figure 3 demonstrates stimulation of human neu receptor tyrosine phosphorylation by recombinant rat NDF.
  • Human MDA-MB-453 breast carcinoma cells were incubated with concentrated conditioned media from COS-7 cells transfected with the indicated partially purified cDNA clones (left panel) or with fully purified cDNA clones (right panel). Positive controls comprised
  • COS-7 cells untransfected COS-7 cells (lane designated "COS"), or by cells transfected with pJT-2 plasmids containing
  • Figure 4 shows the nucleotide sequence of rat NDF cDNA [SEQ ID NO: 19] and a deduced amino acid sequence [SEQ ID NO: 20]. Depicted is a combined nucleotide sequence from four rat cDNA clones. The beginning of the clone 44 DNA sequence in particular is indicated by an arrow. Nucleotide numbers and amino acid numbers are given in the left and right columns, respectively. Potential sites of N-linked glycoprotein are marked by asterisks, and cysteine residues found in the presumed extracellular domain are encircled. The overlining indicates peptide sequences that were
  • transmembrane region whereas the dashed underlining indicates the polyadenylation sites.
  • the portions of the protein sequence that contain an immunoglobulin (Ig) homology unit and an epidermal growth factor (EGF) -like motif are indicated on the right hand side.
  • Figure 5 shows the nucleotide sequence of rat NDF cDNA clone 44 [SEQ ID NO: 21], separately, and a deduced amino acid sequence [SEQ ID NO: 22]. Nucleotide numbers are given in the right hand column.
  • Figure 6 depicts the hydropathy profile of the precursor of the rat neu receptor stimulating factor encoded by rat cDNA clone 44.
  • Figure 7 shows the alignment of the amino acid sequence [SEQ ID NO: 23] of the EGF-like domain and the flanking carboxyl terminal sequence of rat NDF from clone 44 with representative members of the EGF family. Alignment and numbering begin at the most amino terminal cysteine residue of the EGF motifs. Amino acid residues are indicated by the single letter code. Dashes
  • NDF rat transforming growth factor ⁇ [SEQ ID NO: 24] (TGF ⁇ ; Marquardt, H. et al., Science
  • MEF myxoma virus growth factor
  • human heparin-binding EGF [SEQ ID NO: 27] (HB-EGF; Higashiyama, S. et al.,
  • Figure 8 shows the alignment of the immunoglobulin (Ig)-like domain of rat NDF [SEQ ID NO: 1]
  • Figure 9 is a schematic presentation of the presumed secondary structure and membrane orientation of the precursor of the neu receptor simulating factor from rat cDNA clone 44. Positions corresponding to the immunoglobulin (Ig)-domain and epidermal growth factor (EGF) motif are shown by thick lines and their cysteine residues are indicated by circles. The disulfide linkage of the Ig domain was directly demonstrated by amino acid sequence analysis (Example ID, below), and the secondary structure of the EGF-domain is based on the homology with the EGF family. Also shown are the three cysteine residues found in the transmembrane domain. Arrows mark the processing sites of the immunoglobulin (Ig)-domain and epidermal growth factor (EGF) motif are shown by thick lines and their cysteine residues are indicated by circles. The disulfide linkage of the Ig domain was directly demonstrated by amino acid sequence analysis (Example ID, below), and the secondary structure of the EGF-domain is based on the homology with the EGF family. Also shown are
  • N-glycosylation site and the short vertical lines represent presumed sites of O-glycosylation.
  • Figure 10 depicts a receptor competition assay using naturally-occurring neu receptor stimulating factor purified from ras-transformed rat fibroblast conditioned media (Peles, E. et al., Cell, 1992, above) which has been radiolabelled (" 125 I-NDF”).
  • the radio-labelled NDF was incubated with human MDA-MB-453 breast carcinoma cells in the absence ("NONE") or presence of conditioned media from COS-7 cells expressing either recombinant neu receptor stimulating factor from rat cDNA clone 44 ("C-NDF") or recombinant TGF ⁇ ("C-TGF ⁇ ").
  • C-NDF recombinant neu receptor stimulating factor from rat cDNA clone 44
  • C-TGF ⁇ recombinant TGF ⁇
  • Figure 11 depicts another receptor competition assay. 125 I-iabeled naturally-occurring NDF was
  • C-TGF ⁇ ⁇ -TGF ⁇
  • Figure 12 shows autoradiograms obtained from Northern blots of mRNA isolated from cultured cells (panels A and C) or freshly isolated tissue of an adult rat (panel B), using rat NDF cDNA clone 44 as a probe. The autoradiograms were obtained after a three-hour
  • Figure 13 shows the nucleotide sequence of rat
  • NDF cDNA clone 4 [SEQ ID NO: 34] and a deduced partial rat (proNDF-ß3) amino acid sequence [SEQ ID NO: 35].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included. The amino acid
  • Figure 14 shows the nucleotide sequence of rat NDF cDNA clone 19 [SEQ ID NO: 36] and a deduced rat (proNDF- ⁇ 2b) amino acid sequence [SEQ ID NO: 37].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 15 shows the nucleotide sequence of rat NDF cDNA clone 20 [SEQ ID NO: 38] and a deduced rat (proNDF- ⁇ 2b) amino acid sequence [SEQ ID NO: 39].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 16 shows the nucleotide sequence of rat NDF cDNA clone 22 [SEQ ID NO: 40] and a deduced rat (proNDF-ß2a) amino acid sequence [SEQ ID NO: 41].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 17 shows the nucleotide sequence of rat
  • NDF cDNA clone 38 [SEQ ID NO: 42] and a deduced rat (proNDF- ⁇ 2a) amino acid sequence [SEQ ID NO: 43].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 18 shows the nucleotide sequence of rat NDF cDNA clone 40 [SEQ ID NO: 44] and a deduced partial rat (proNDF-ß2) amino acid sequence [SEQ ID NO: 45].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 19 shows the nucleotide sequence of rat NDF cDNA clone 41 [SEQ ID NO: 46] and a deduced rat (proNDF-ß2a) amino acid sequence [SEQ ID NO: 47].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 20 shows the nucleotide sequence of rat NDF cDNA clone 42A [SEQ ID NO: 48] and a deduced rat (proNDF-ß4a) amino acid sequence [SEQ ID NO: 49].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 21 shows the nucleotide sequence of rat NDF cDNA clone 42B [SEQ ID NO: 50] and a deduced rat (proNDF- ⁇ 2a) amino acid sequence [SEQ ID NO: 51].
  • Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
  • Figure 22 shows rat proNDF (precursor) structures predicted from the rat cDNA sequences of Figures 4, 5, and 13-21. Boxed areas indicate protein coding regions. "Ig”, “EGF” and “TM” indicate the immunoglobulin-like, EGF-like and transmembrane domains, respectively. The number of amino acid residues in each predicted precursor sequence is given in the right hand column. Dashed lines represent divergent 3'
  • Figure 23 shows the amino acid sequences encoded by different rat proNDF cDNAs.
  • the complete proNDF- ⁇ 2a amino acid sequence from Figure 17 is shown [SEQ ID NO: 43].
  • Divergent sequences in proNDF variants are aligned with the proNDF- ⁇ 2a sequence.
  • ProNDF-ß2a [SEQ ID NO: 126], ß3 [SEQ ID NO: 127] and ß4a [SEQ ID NO: 52] structures were deduced from Rat-1-EJ cDNAs (Table 2, below, and Figures 16, 13, and 20,
  • the NDF-ß1 sequence [SEQ ID NO: 52] was obtained from cDNA amplified from rat brain and spinal cord (See Example 9 and Figure 28, below). Asterisks, carboxyl-terminal amino acids; dashes, gaps introduced to facilitate sequence alignment; overline, putative transmembrane domain; dots, cysteine residues in predicted extracellular domain (the two N-terminal cysteine residues are part of the immunoglobulin-like domain; the six other marked cysteine residues reside in the EGF-like growth factor domain).
  • Figure 24 shows autoradiograms of anti-phosphotyrosine Western blots, using the neu receptor tyrosine phosphorylation assay procedure of Example 3, below. The results with recombinant rat NDF proteins expressed in COS-7 cells from the indicated cDNA clones are shown.
  • COS-7(-) is medium from untransfected COS-7 cells.
  • Figure 25 shows the transient expression of recombinant proNDFs from rat cDNA clones.
  • COS-7 cells were transfected with expression plasmids containing cDNAs encoding the indicated NDF isoforms and were labeled with 35 S-methionine/cysteine.
  • Conditioned media and cell lysates were immunoprecipitated with an
  • Figure 26 shows endoglycosidase treatment of recombinant rat proNDFs expressed in COS-7 cells.
  • COS-7 cells were transfected with proNDF-ß3 and proNDF- ⁇ 2c cDNA expression plasmids and labeled for seventeen hours with 35 S-methionine/cysteine.
  • Radiolabeled COS-7 cell media and lysates were immunoprecipitated with affinity-purified rabbit antibody raised against recombinant rat NDF- ⁇ 2 14-241 .
  • the immunoprecipitates were treated with endoglycosidases as indicated and analyzed by
  • Figure 27 shows the reverse transcription-polymerase chain reaction (RT-PCR) analysis of NDF mRNAs expressed in various rat tissues. Reverse transcription was used to prepare first-strand cDNA from mRNA samples. NDF cDNAs were amplified in PCR reactions with primers specific for the NDF EGF-like domain. Lanes are as follows: 1, Rat-1-EJ; 2, heart; 3, skin; 4, ovary; 5, lung; 6, stomach; 7, spleen; 8, liver; 9, muscle; 10, kidney; 11, brain; 12, spinal cord; 13, cDNA clone 20 (proNDF- ⁇ 2b) 14, cDNA clone 40 (proNDF-ß2); 15, cDNA clone 42A (proNDF-ß4). Positions of DNA size markers (in base pairs) are indicated.
  • Figure 28 shows the nucleotide sequence [SEQ ID NO: 55] of the PCR products obtained from rat spinal cord and brain, and a deduced partial rat NDF-ß1 amino acid sequence [SEQ ID NO: 56]. Nucleotide numbers are given in the left hand column, and amino acid numbers are given in the right hand column. The amino acid numbering corresponds to the amino acid numbering in Figure 23.
  • Figure 29 shows a Northern blot analysis of several human cell lines screened for the presence of NDF-related mRNA with rat NDF clone 44 cDNA probe.
  • Figure 30 shows the nucleotide sequence of human NDF cDNA clone P1 [SEQ ID NO: 3] and a deduced partial amino acid sequence for human proNDF- ⁇ 1a [SEQ ID NO: 4]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
  • Figure 31 shows the composite nucleotide sequence of human proNDF- ⁇ 2b cDNA [SEQ ID NO: 5] and a deduced amino acid sequence [SEQ ID NO: 6]. This sequence was derived from the sequences of clone 43 [SEQ ID NO: 7] and clone 17, shown in Figures 32 and 33, respectively. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Linker sequences added to the 3' end of the cDNA to facilitate cloning are included.
  • Figure 32 shows the nucleotide sequence of human NDF cDNA clone 43 and a deduced human proNDF- ⁇ 2b amino acid sequence [SEQ ID NO: 8]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Linker sequences added to the 3' end of the cDNA to facilitate cloning are included.
  • Figure 33 shows the nucleotide sequence of human NDF cDNA clone 17 [SEQ ID NO: 9] and a deduced partial amino acid sequence of human proNDF- ⁇ 2b [SEQ ID NO: 10]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38. Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included.
  • Figure 34 shows the nucleotide sequence of human NDF cDNA clone 19 [SEQ ID NO: 11] and a deduced partial amino acid sequence of human proNDF- ⁇ 3 [SEQ ID NO: 12]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
  • Figure 35 shows the nucleotide sequence of human NDF cDNA clone P13 [SEQ ID NO: 13] and a deduced partial amino acid sequence for human NDF-ß1a [SEQ ID NO: 14]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
  • Figure 36 shows the nucleotide sequence of human NDF cDNA clone 294-8 [SEQ ID NO: 15] and a deduced partial amino acid sequence for human proNDF-ß2 [SEQ ID NO: 16]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
  • Figure 37 shows the nucleotide sequence of human NDF cDNA clone 33 [SEQ ID NO: 17] and a deduced partial amino acid sequence for human proNDF-ß3 [SEQ ID NO: 18]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
  • Figure 38 is a composite of the amino acid sequences encoded by different human proNDF cDNAs
  • Figure 39 is a schematic diagram of a mammalian cell vector for expression of a chimeric NDF gene comprised of rat and human sequences.
  • Figure 40 shows the induction of tyrosine phosphorylation in MDA-MB-453 cells (using the assay procedure of Example 3) with conditioned medium from COS-7 cells which had been transfected with expression plasmids described in Example 11, below, containing various (human, rat, and human-rat chimera) NDF cDNA clones: Lane 1, control, 100 ⁇ l 10x concentrated conditioned medium from untransfected COS-7 cells; lanes 2 and 3, hNDF- ⁇ 2b; lanes 4 and 5, h-rNDF- ⁇ 2c; lanes 6 and 7, h-rNDF-ß1c; lanes 8 and 9, h-rNDF-ß2c; lanes 10 and 11, h-rNDF- ⁇ 1c; lanes 12 and 13, rNDF- ⁇ 2c; lane 14, positive control of human met-NDF- ⁇ 2 14-241 purified from E. coli . Even-numbered lanes represent 100 ⁇ l 10x concentrated conditioned medium from untransfected COS-7
  • conditioned media, and odd-numbered lanes represent 100 ⁇ l 10x concentrated media.
  • Figure 41 shows SDS-PAGE analysis of a recombinant rat NDF- ⁇ 2 purified from media conditioned by CHO cells with the pDSR ⁇ 2/rNDF- ⁇ 2c expression plasmid described in Example 11. The sizes of molecular weight markers in the left hand lane are indicated in
  • Figure 42 shows the stimulation of MDA-MB-453 cell neu receptor tyrosine phosphorylation by various amounts of recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells.
  • the assay shows the stimulation of MDA-MB-453 cell neu receptor tyrosine phosphorylation by various amounts of recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells. The assay
  • Example 3 Procedure of Example 3 was used.
  • PBSA phosphate buffered saline, or PBS, with 0.1% bovine serum albumin, or BSA
  • BSA bovine serum albumin
  • Figure 43 shows the stimulation of MDA-MB-453 neu receptor tyrosine phosphorylation by recombinant human and rat NDF isoforms purified from E. coli .
  • PBSA was used as a negative control.
  • Figure 44 shows histograms depicting gold-to-red fluorescence ratios for BT-474 cells treated for seven days in culture with recombinant human met-NDF- ⁇ 2 14-241 purified from E. coli and stained with Nile Red.
  • Treatment regimens included the following concentrations of the recombinant NDF: A, 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml. The results show that recombinant human NDF induces accumulation of neutral lipids.
  • Figure 45 shows histograms depicting gold-to-red fluorescence ratios for BT-474 cells treated for seven days in culture with recombinant rat NDF- ⁇ 2 produced by CHO cells and stained with Nile Red.
  • Treatment regimens included the following concentrations of the recombinant NDF: A, 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml.
  • A 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml.
  • B 100 ng/ml
  • C 20 ng/ml
  • D 4 ng/ml
  • Figure 46 shows the growth stimulatory effects of recombinant human met-NDF- ⁇ 2 14-241 on BT-474 cell proliferation in vitro . Cells were treated with
  • Figure 47 shows the growth inhibitory effects of recombinant human met-NDF- ⁇ 2 14-241 on MDA-MB-468 cell proliferation in vitro . Cells were treated with
  • Figure 48 illustrates the effect in vitro of rat met-NDF- ⁇ 2 14 _ 241 on the adhesion on colon epithelial cells. Intestinal crypts were isolated from mouse colons and plated on collagen type IV coated plates.
  • Figure 49 shows indirect immunofluorescence analysis of LIM 1215 colon carcinoma cells cultured in the absence (panel A), or presence (panel B) of 50 ng/ml recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells. Treated cells were sectioned, then stained with monoclonal anti-CEA antibodies
  • Figure 50 shows indirect immunofluorescent analysis of LIM 1863 colon carcinoma cells cultured in the absence (panel A), or presence (panel B) of 50 ng/ml recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells. Treated cells were sectioned, then stained with rabbit anti-TIMP-2 antibodies followed with phycoerythrin-labeled goat anti-rabbit IgG
  • Figure 51 shows measurements of new epithelium in rabbit ear wounds treated with different doses of recombinant human met-NDF- ⁇ 2 14-241 ,
  • Figure 52 shows measurements of the area of new epithelium covering rabbit ear wounds treated with different doses of recombinant human met-NDF- ⁇ 2 14-241 ,
  • Figure 53 shows measurements of the number and percentage of proliferating (BrdU-positive) basal and suprabasal keratinocytes in rabbit ear wounds treated with different doses of recombinant human met-NDF- ⁇ 2 14-241 .
  • DNA sequences of this invention are valuable as products useful in effecting the large scale synthesis of human NDFs by a variety of recombinant techniques.
  • DNA sequences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of the expression of recombinant NDFs and their related products.
  • DNA sequences of the invention are also suitable materials for use as probes in isolating additional human cDNAs and genomic DNA encoding NDFs and other genes encoding related proteins.
  • the DNA sequences of the invention are also suitable materials for use as probes in isolating additional human cDNAs and genomic DNA encoding NDFs and other genes encoding related proteins.
  • sequences may also be useful in various alternative methods of protein synthesis (e.g., in insect host cells) or in genetic therapy in humans and other
  • DNA sequences of the invention are expected to be useful in developing transgenic mammalian species which may serve as eucaryotic "hosts" for production of NDFs and NDF products in quantity. See, generally, Palmiter et al., Science 222, 809-814 (1983).
  • Diagnostic applications of NDF DNA sequences of this invention are also possible, such as for the detection of alterations of genes and of mRNA structure and expression levels.
  • the recombinant human NDF polypeptides of this invention are characterized by being the products of procaryotic or eucaryotic host expression (e.g., by bacterial, yeast, higher plant, insect or mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning or by gene synthesis,
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • procaryote e.g., E. coli
  • non-human mammalian such as COS or CHO, and avian
  • COS non-human mammalian
  • avian e.g., non-human mammalian, such as COS or CHO, and avian
  • recombinant NDFs of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or may be non-glycosylated.
  • the recombinant NDFs of the invention may also include an additional methionine amino acid residue at the amino terminus.
  • the present invention also embraces products such as polypeptide analogs of human NDFs encoded by naturally-occurring mRNAs.
  • Such analogs include
  • fragments of NDF or of NDF precursors fragments of NDF or of NDF precursors (proNDFs).
  • NDF NDF polypeptide products
  • modifications of cDNA and genomic nucleotide sequences can be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogs and derivatives of the described NDFs.
  • Such products share one or more of the biological properties of NDF polypeptide products encoded by naturally-occurring mRNAs.
  • inventions include those which are foreshortened, for example, by deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longer-lasting effects); or which have been altered to delete one or more potential sites for O-glycosylation and/or N-glycosylation or which have one or more
  • cysteine residues deleted or replaced by other residues e.g., alanine or serine residues, and are potentially more easily isolated in active form from microbial systems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target proteins or to receptors on target cells.
  • polypeptide fragments duplicating only a part of the continuous amino acid sequence are also included.
  • any one or more of the products of the invention may have therapeutic utility or utility in other contexts, such as in antagonism of naturally-occurring NDFs.
  • Competitive antagonists may be quite useful in, for example, cases of overproduction of NDF.
  • the present invention also includes that class of polypeptides encoded by portions of the DNA
  • compositions useful in treating are also encompassed by the invention.
  • solubilizers useful in recombinant NDF therapy.
  • a "therapeutically effective amount” as used herein refers to that amount which provides therapeutic effect for a given condition and administration regimen.
  • Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g.,
  • Tween 80, Polysorbate 80 Tween 80, Polysorbate 80
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., sodium metabisulfite
  • Thimerosol, benzyl alcohol) and bulking substances e.g., lactose, mannitol
  • covalent attachment of polymers to the polypeptide to prolong in vivo half-life and to enhance potency for instance, water-soluble polymers as polyethylene glycol, polypropylene glycol and copolymers of polyethylene glycol and polypropylene glycol, see, e.g., Davis et al., U.S. Patent No.
  • particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions will influence the
  • the recombinant NDF polypeptides of this invention are expected to be useful, alone or in
  • the neu receptor is expressed by human epithelial cells of the gastrointestinal, respiratory, urinary and reproductive tracts (Press, M. et al.,
  • recombinant NDFs can be used to promote reepithelialization and restoration of mature, functionally differentiated phenotypes in the new epithelia.
  • recombinant NDFs can be used alone or in combination with cytotoxic agents to inhibit abnormal cellular proliferation.
  • Recombinant NDF polypeptides are expected to be particularly useful the treatment of dermal wounds, cancer, gastrointestinal disorders, kidney diseases, Barrett's esophagus, and diseased or damaged lung.
  • Recombinant neu differentiation factors are useful in the repair of diseased and damaged skin through their ability to stimulate tyrosine
  • Recombinant NDFs can be used to accelerate healing of dermal wounds, such as acute and chronic dermal wounds, excisional wounds, second and third degree burns (partial and full thickness) and
  • Epidermolysis bullosa is a defect in adherence of the epidermis to the underlying dermis, resulting in frequent open, painful blisters, which can cause severe morbidity. Accelerated reepithelialization of these lesions would result in less risk of infection, diminished pain, and less wound care.
  • Recombinant neu differentiation factors are useful in cancer therapy for inhibiting proliferation of certain cancer cells.
  • recombinant NDFs can restore a more mature and functionally differentiated phenotype in certain cancer cells, thereby restoring normal cellular
  • recombinant NDFs can inhibit tumor cell invasion and metastasis by induction of intercellular and extracellular matrix adhesion
  • Cancer cells that are susceptible to NDF treatment are those that express the neu receptor.
  • Tumors in which neu receptor expression or neu gene amplification have been detected include cancers of the breast, ovary, endometrium, cervix, salivary gland, esophagus, lung, stomach, pancreas, colon, kidney, prostate, and bladder, as well as squamous cell
  • carcinomas of the head and neck carcinoid tumors of the gut, glioblastomas, astrocytomas, papillary thyroid carcinomas, sebaceous and sweat gland carcinomas, and hepatocellular carcinomas.
  • NDF proteins such as lung and colon cancers
  • NDF proteins will be particularly susceptible to recombinant NDF therapy, since normal NDF levels can be restored with recombinant NDF polypeptides.
  • tumors with mutated NDF genes will be responsive to NDF
  • Additional inhibition of tumor growth can be achieved through enhancement of chemotherapy by recombinant NDFs.
  • Antibodies and ligands that interact with the neu receptor and with the related EGF receptor enhance tumor cell sensitivity to the chemotherapeutic agent cisplatin (Aboud-Pirak, E. et al. J. Natl . Cancer Inst . 80:1605-1611 1988; Christen, R.D. et al., J. Clin . Invest 86:1632-1640, 1990; Hancock, M. C. et al. Cancer Res . 51:4575-4580 1991; Nishikawa, K. et al., Cancer Res . 52:4758-4765, 1992). Phorbol esters, which
  • NDF mRNA and the neu receptor messenger in normal gastrointestinal tissues predicts that recombinant NDFs will have activities on normal, diseased and injured stomach and intestine.
  • recombinant NDF is a potent inducer of adherence for normal colonic epithelial cells. It is expected that recombinant NDFs can be useful in
  • recombinant NDFs can offer a significant therapeutic improvement in the treatment of gastric ulcers.
  • Duodenal ulcers like gastric ulcers, are treatable, but recombinant NDF therapy to more fully and more rapidly regenerate the mucosal lining of the duodenum would be an important advance.
  • Inflammatory bowel diseases such as Crohn's disease (affecting primarily the small intestine) and ulcerative colitis (affecting primarily the large bowel) are chronic diseases of unknown etiology which result in the destruction of the mucosal surface, inflammation, scar and adhesion formation during repair, and
  • Recombinant NDF therapy to stimulate resurfacing of the mucosal surface can be of benefit in controlling progression of disease.
  • Gut toxicity is a major limiting factor in radiation and chemotherapy treatment regimes.
  • Pretreatment with recombinant NDFs may have a
  • cytoprotective affect on the small intestinal mucosa allowing increased dosages of such therapies while reducing potential fatal side effects of gut toxicity.
  • Barrett's esophagus (a precursor of adenocarcinoma) exhibits esophageal columnar metaplasia with frequent overexpression of the neu receptor
  • Smoke inhalation is a significant cause of morbidity and mortality in the week following a burn injury due to necrosis of the bronchiolar epithelium and the alveoli.
  • Recombinant NDFs are expected to stimulate proliferation and differentiation of the bronchiolar epithelial cells, which express the neu receptor, thereby enhancing repair and regeneration of the lung epithelium damaged by smoke inhalation.
  • kidney diseases with abnormal cell growth and differentiation e.g. autosomal-dominant polycystic kidney disease, acquired dialysis-associated cystic disease, and non-cystic end stage kidneys
  • Recombinant NDF therapy may restore the normal differentiated phenotype to the diseased tissue and also inhibit abnormal
  • Recombinant NDFs through stimulation of the neu receptor present in epithelial cells lining kidney tubules, will also be useful in restoring normal kidney epithelium following ischemic acute tubular necrosis. Liver
  • Neu receptor expression in the liver can be detected in certain hepatitis B virus and hepatitis C virus infectious lesions of the liver (Brunt, E.M., and Swanson, P.E., Am. J. Clin . Pathol . 97:53-61, 1992).
  • Recombinant NDFs can be used to treat such lesions, either alone or in combination with cytotoxic agents.
  • Peripheral nerves can be used to treat such lesions, either alone or in combination with cytotoxic agents.
  • Neu receptor expression is found in Schwann cells associated with transected peripheral nerves undergoing Wallerian degeneration (Cohen, J.A. et al., J. Neurosci . Res . , 1992, above). Recombinant NDFs can be used to regulate Schwann cell proliferation and maturation in cases of peripheral nerve injury or degeneration.
  • NDF DNAs Altered expression of naturally-occurring NDF in diseased tissues (e.g., malignant or premalignant tissues) can be found with the NDF DNAs, NDF mRNA detection methods, anti-NDF antibodies, and NDF
  • Perturbations in the expression of naturally-occurring NDFs are expected to alter normal cell growth and differentiation with pathological consequences such as neoplasia.
  • Other methods can also be used to detect aberrant NDF expression in human tissues. These methods, known to those skilled in the art of analyzing protein and mRNA expression, include immunohistochemical assays, enzyme-linked immunoabsorbent assays, and polymerase chain reaction assays. The identification of premalignant and malignant cells with abnormal NDF expression is particularly useful for the diagnosis of cancer.
  • the NDFs of this invention may also be combined with substances such as radiolabeled molecules, toxins, cytokines, and other compounds useful in tumor treatment, in order to increase localization of these substances on human tumors expressing high levels of the neu receptor.
  • substances such as radiolabeled molecules, toxins, cytokines, and other compounds useful in tumor treatment, in order to increase localization of these substances on human tumors expressing high levels of the neu receptor.
  • polypeptides of the invention will be formulated and dosed according to the specific disorder to be treated, the condition of the individual patient, the site of delivery of the factor, the method of administration, and other circumstances known to the skilled practitioner.
  • polypeptide derivative is an amount that is effective to alter cellular proliferation and differentiation, or to prevent, lessen the worsening of, alleviate or cure the condition for which the polypeptide is administered.
  • activity of the present polypeptide may be enhanced or supplemented with use of one or more additional biologically active or cytotoxic agents which are known to be useful in treating the same condition for which the polypeptide of the invention is being administered, e.g., IL-2 or chemotherapeutic agents for cancer
  • platelet-derived growth factors for wound healing, and so forth.
  • epidermal growth factor for wound healing
  • Biological materials employed in these Examples were obtained as follows.
  • the monoclonal antibody (Ab-3) to the carboxyl terminus of the neu receptor was obtained from Oncogene Science (Uniondale, NY).
  • a monoclonal antibody to phosphotyrosine, PY20, was obtained from Amersham (Arlington Heights, IL).
  • a mouse monoclonal antibody to human ß-casein was obtained through Dr. R.C. Coombes from the Ludwig Institute in London (Earl, H.M., and Mcllhinney, R.A.J., Mol . Immunol . 22: 981-991,
  • Rat1-EJ cell line (ATCC CRL 10984) was generated by transfection of the human EJ ras oncogene into Rat1 fibroblasts as described by Peles, E. et al. in Cell, 1992, above, and by Land, H. et al., in Nature 304:596-602, 1983.
  • the following cell lines were obtained from the American Type Culture Collection
  • MDA-MB-231 (ATCC HTB 26), MDA-MB-453 (ATCC HTB 131), Hs 294T (ATCC HTB 140), SK-BR-3 (ATCC HTB 30), HT-1080 (ATCC CCL 121), BALB/c 3T3 (ATCC CRL 6587) and COS-7 (ATCC CRL 1651).
  • COS-7 cells were cultured in Dulbecco ' s modified Eagle medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah).
  • MDA-MB-453 cells were grown in RPMI Medium 1640 with 15% fetal bovine serum.
  • ras-transformed rat fibroblasts has been described by Peles, E. et al., in Cell, 1992, above). In order to obtain more amino acid sequence information for the design of independent
  • oligonucleotide probes 300 picomoles of the purified rat protein were subjected to partial proteolysis with trypsin, as follows: ten micrograms of the protein were reconstituted in 200 ⁇ l of 0.1 M ammonium bicarbonate buffer (pH 7.8); digestion was conducted with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Serva) at 37°C for eighteen hours, using an enzyme-to-substrate ratio of 1:10.
  • the resulting peptide mixture was separated by reversed phase HPLC and monitored at 215 nm using a Vydac C4 micro column (2.1 mm i.d. ⁇ 15 cm, 300 A) and a Hewlett-Packard 1090 liquid chromatographic system equipped with a diode-array detector and a workstation ( Figure 1).
  • the column was equilibrated with 0.1% trifluoroacetic acid (mobile phase A) and elution was effected with a linear gradient from 0-55% mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) over seventy minutes.
  • the flow rate was 0.2 ml/min and the column temperature was controlled at 25°C.
  • amino acid sequences of the peptides corresponding to the three major eluted fractions were determined by automated Edman degradation. Amino acid sequence analyses of the peptides were performed with a Model 477 protein sequencer (Applied Biosystems, Inc., Foster City, CA) equipped with an on-line
  • PTH phenylthiohydantoinyl amino acid analyzer
  • Model 900 data analysis system Haunkapiller, M.W.
  • cDNA was synthesized with the "Superscript" kit (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD) . Columnfractionated double-strand cDNA was ligated into a SalI- and Notl-digested pJT-2 plasmid vector, to yield plasmids containing cDNA inserts such as depicted in
  • the pJT-2 plasmid vector was derived from the V19.8 vector (ATCC 68124). In particular, the HindIII and SacII cloning sites of V19.8 were changed into SalI and NotI sites by using synthetic oligonucleotide linkers to yield the PJT-2 vector. Plasmids containing cDNA inserts were transformed into DH10B E. coli cells by electroporation (Dower, W.J. et al., Nucleic Acids Res . 16:6127-6145, 1988). B. Preparation of cDNA Probes and Isolation of Clones
  • N-terminal region of NDF as described in Example 1C specifically, residues 5-24 (RGSRGKPGPAEGDPSPALPP) [SEQ ID NO: 71], and residues 7-12 of the T40.4 tryptic peptide (GEYMCK) [SEQ ID NO: 72].
  • RGSRGKPGPAEGDPSPALPP residues 5-24
  • GEYMCK tryptic peptide
  • the synthetic oligonucleotides were end-labeled with ⁇ - 32 P-ATP with T4 polynucleotide kinase and used to screen replicate sets of nitrocellulose filters.
  • the hybridization solution contained 6 x SSC, 50 mM sodium-phosphate (pH 6.8), 0.1% sodium-pyrophosphate, 2 x Denhardt's solution, 50 ⁇ g/ml salmon sperm DNA and 20% formamide (for probe 1) or no formamide (for probe 2). Hybridization was carried out for fourteen hours at 42°C (for probe 1) or 37°C (for probe 2).
  • the filters were washed at either 50°C with 0.5 x SSC/ 0.2% SDS/ 2 mM EDTA (for probe 1) or at 37°C with 2 x SSC/ 0.2% SDS/ 2 mM EDTA (for probe 2). Autoradiography of the filters gave ten clones that hybridized with both probes. These clones were purified by re-plating and probe
  • the clones were transiently expressed in COS-7 cells.
  • the cDNA clones were inserted into the pJT-2 eukaryotic expression vector under the control of the SV40 promoter and 3'-flanked with SV40 termination and poly-adenylation signals.
  • electroporation as follows. 6 ⁇ 10 6 cells in 0.8 ml Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum were transferred to a 0.4 cm cuvette and mixed with 20 ⁇ g of plasmid DNA in 10 ⁇ L of TE solution (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Electroporation was performed at room temperature, 1600 volts and 25 ⁇ F, using a BioRad Gene Pulser apparatus with the pulse controller unit set at 200 ohms. The cells were then diluted into 20 ml of DMEM/10% fetal bovine serum and transferred into a T75 flask (Falcon). After fourteen hours of incubation at 37°C, the medium was replaced with DMEM/1% fetal bovine serum, and the incubation was continued for an additional forty eight hours.
  • DMEM Dulbecco's modified Eagle medium
  • Falcon T75 flask
  • the resulting conditioned media were then evaluated for their ability to stimulate tyrosine phosphorylation of the neu receptor in MDA-MB-453 human breast tumor cells as follows.
  • the conditioned media were filtered through a 0.2-micron sterile filter unit
  • clone 44 cDNA was completely purified by re-plating and by filter
  • clone 44 was selected for further analysis by DNA sequencing.
  • Rat clone 44 cDNA was primarily sequenced using a 373A automated DNA sequencer and "Taq DyeDeoxyTM Terminator" cycle sequencing kits from Applied
  • a 594 base-long untranslated stretch Downstream of the 3' end of this reading frame is a 594 base-long untranslated stretch.
  • the latter includes a poly (A) tail preceded by a polyadenylation signal (AAATAAA). Because no stop codon and recognizable signal peptide sequence were found at the amino terminus of the longest open reading frame, the nucleotide sequences of three other independent positive cDNA clones were analyzed in a similar manner. All three clones included an
  • the combined nucleotide sequence of the rat cDNA clones [SEQ ID NO: 19] is presented in Figure 4, and the clone 44 cDNA sequence is given in Figure 5.
  • the combined sequence spans 2,186 base pairs, including a poly (A) tail, and contains an open reading frame of four hundred and twenty-two residues [SEQ ID NO: 20] if the amino terminal methionine is considered to be the initiator codon.
  • Hydropathy analysis ( Figure 6) revealed no prominent hydrophobic sequence at the amino terminus of the protein that could function as a signal peptide for protein secretion (von Hijne, G.,
  • extracellular domain contains all of the peptide
  • the NDF precursor is expected to accumulate on the surface of cells expressing high levels of NDF mRNAs.
  • Membrane-bound precursor proteins have been found for other members of the EGF growth factor family.
  • Brachmann, R., et al. demonstrated the presence of transforming growth factor ⁇ on the surface of cells expressing transforming growth factor ⁇ mRNA.
  • Molecules that specifically bind proNDF could selectively direct therapeutic molecules to cells overexpressing NDF.
  • monoclonal antibodies raised against recombinant NDF and conjugated to therapeutic molecules could selectively localize the therapeutic molecule on the surface of cells expressing high levels of membrane-bound proNDF.
  • Such cells would include tumor cells with an activated ras gene.
  • Antibody conjugates could be constructed using
  • chemotherapeutic compounds include cytokines, toxins, and others.
  • lymphokines or radionuclides are lymphokines or radionuclides.
  • ⁇ means less than
  • radiolabeled naturally-occurring NDF 125 I-NDF was 3 ⁇ 10 6 cpm/ng.
  • the radiolabeled NDF (10 picomolar) was incubated for sixty minutes at 4°C with monolayers of MDA-MB-453 cells that were grown in a 24-well dish (Costar). This incubation was also performed in the presence of conditioned media from transfected COS-7 cells. Unbound 125 I-NDF was removed by three washes with phosphate-buffered saline (PBS), and the cells were solubilized in 0.1 N NaOH solution containing 0.1% SDS. Radioactivity was determined with a ⁇ -counter.
  • PBS phosphate-buffered saline
  • conditioned medium containing recombinant NDF expressed from the pJT-2/NDF expression vector (Fig. 2) in COS-7 cells reduced the total 125 I-NDF binding by approximately 50%.
  • COS-7 conditioned medium from cells transfected with a pJT-2 expression vector containing a rat TGF ⁇ cDNA (Blasband, A. et al., Mol . Cell Biol . 10: 2111-2121, 1990) was employed.
  • the TGF ⁇ conditioned medium did not inhibit 125 I-NDF binding.
  • the partial inhibitory effect of the recombinant rat NDF conditioned medium may be attributed either to its relatively low concentration in the binding assay, or to high non-specific ligand
  • a covalent crosslinking assay was employed. This was performed by allowing MDA-MB-453 cells to bind 125 I-NDF in the
  • sulfosuccinimidyl suberate (BS 3 , pierce) was added at 1 mM final concentration after one hour of binding at 4°C, followed by a brief wash with PBS. Following forty-five minutes of incubation at 22°C, the monolayers were incubated for ten minutes with quenching buffer (100 mM glycine in PBS, pH 7.4). The cells were then washed twice with ice-cold PBS, lysed in lysis buffer, and the neu protein was immunoprecipitated with quenching buffer (100 mM glycine in PBS, pH 7.4). The cells were then washed twice with ice-cold PBS, lysed in lysis buffer, and the neu protein was immunoprecipitated with quenching buffer (100 mM glycine in PBS, pH 7.4). The cells were then washed twice with ice-cold PBS, lysed in lysis buffer, and the neu protein was immunoprecipitated with quenching
  • the 1.9 kb-long cDNA insert of rat clone 44 was labeled with ⁇ - 32 p-dCTP by the random priming method (Feinberg, A.P., and Vogelstein, B., Anal . Biochem 132: 6-13 , 1983).
  • the conditions of hybridization were as follows: 6 x SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 2 x
  • Denhardt's solution 50 ⁇ g/ml salmon sperm DNA and 50% formamide. Hybridization was carried out for fourteen hours at 42°C, followed by washing for thirty minutes at 60°C with 0.2 x SSC, 0.1% SDS and 2 mM EDTA. The filters were exposed to Kodak XAR x-ray film with an intensifier screen at -70°C for the indicated periods of time.
  • Figure 12 shows that three bands were visualized in the Northern blots with poly (A) selected RNA of Rat-1-EJ fibroblasts. Their molecular sizes corresponded to 6.8, 2.6, and 1.7 kilobases.
  • panel B demonstrates tissue-specific regulation of NDF mRNA expression levels in adult rat tissues.
  • the highest NDF mRNA expression was observed in the spinal cord.
  • NDF mRNAs were also detected in brain tissue. In addition to the mRNAs described above, these tissues express a variant mRNA that is 3.4 kb in size.
  • Other positive tissues include lung, ovary and stomach. Relatively low amounts of the middle-size transcript were displayed by the skin, kidney and heart. The liver, spleen and muscle did not contain detectable NDF mRNA.
  • the Northern blot also shows tissue-specific differences in the relative proportion of the different NDF mRNA species. Each variant mRNA could encode variant NDF proteins with different biological properties.
  • Naturally-occurring NDF may regulate cell proliferation and differentiation through tissue-specific variations in NDF mRNA structure and expression.
  • FIG. 12 panel C, shows that human tumor cells can overexpress NDF mRNA.
  • An initial survey of human tumor cell lines found that HT-1080 fibrosarcoma cells, Hs 294T melanoma cells and MDA-MB-231 mammary adenocarcinoma cells express elevated NDF mRNA levels.
  • the nucleotide sequences for both strands of the NDF coding region were determined for all ten cDNAs using synthetic oligonucleotide primers with fluorescence-based dideoxy-DNA sequencing as described in Example 4.
  • the cDNAs ranged in size from 1.1 kb to 3.2 kb and, presumably, are derived from the 6.8, 2.6 and 1.7 kb NDF mRNAs found in Rat-1-EJ cells (See
  • Example 6 DNA sequence analyses revealed that these ten cDNAs encode six distinct NDF precursor proteins (proNDFs) (Table 2, and Figures 5 and 13-21). The open reading frames in the ten cDNAs ranged from 0.7 kb
  • clone 4 to 2.0 kb (clone 42A).
  • Two of the cDNAs were partial cDNA clones.
  • the clone 4 sequence begins at proNDF codon 11.
  • Clone 40 is truncated at codon 36 of the intracellular domain.
  • Clone 44 has a complete open reading frame, but has a truncated 5'-untranslated sequence.
  • the ten cDNAs encode six homologous NDF precursor proteins comprised of 241 to 662 amino acid residues, which are designated proNDF- ⁇ 2a [SEQ ID NO: 43], proNDF- ⁇ 2b, proNDF- ⁇ 2c, proNDF- ⁇ 2a, proNDF- ⁇ 3 and proNDF ⁇ -4a ( Figures 22, 23 and Table 2).
  • the six predicted proNDFs are identical in the first 213 amino acid residues. This identical region includes: a basic N-terminus which is proteolytically processed at amino acid 14 in naturally occurring rat NDF; an
  • ProNDF cDNAs do not encode N-terminal hydrophobic signal peptide sequences. However, with the exception of proNDF- ⁇ 3, a 23-amino acid hydrophobic region is present in the proNDFs. Carboxy-terminal to this putative transmembrane domain is a cytoplasmic domain that is invariant in the first 157 amino acid residues.
  • variable region of the proNDF ectodomain begins in the EGF-like domain at amino acid position 213.
  • the ⁇ / ⁇ variation alters the
  • the ⁇ / ⁇ sequence variation may alter receptor activation in different cellular contexts.
  • the NDF isoforms may differentially bind receptor heterodimers formed between the NDF receptor and related receptor molecules, such as the 170 kDa EGF receptor (Wada, T. et al., Cell 61:1339-1347, 1990; Spivak-Kroizman, T. et al., J.
  • proNDF- ⁇ proteins share a common sequence at positions 213 to 230 that is distinct from the corresponding NDF- ⁇ sequence. Variation between members of the proNDF- ⁇ subfamily occurs carboxy-terminal to the EGF-like domain, where the ⁇ 1 and ⁇ 4a proteins contain additional amino acid residues. ProNDF- ⁇ 3 cDNA encodes a stop codon in this region, resulting in a smaller protein that lacks the transmembrane and cytoplasmic domains.
  • the different proNDF sequences may alter proteolytic release of 44 kDa isoforms from the larger NDF precursor molecules.
  • the proNDF proteins differ significantly in the number of charged residues located between the EGF-like domain and the hydrophobic
  • the 27-amino acid sequence that is unique to the ⁇ 4a isoform includes nine basic residues and five acidic residues. This sequence introduces several dibasic residues that are potential proteolytic cleavage sites.
  • Cytoplasmic domains with 374, 196 and 157 amino acid residues distinguish proNDFs ⁇ 2a, ⁇ 2b and ⁇ 2c, respectively ( Figures 5, 14, 15, 17, 21, 22, and
  • the proNDF- ⁇ 2 a, b and c isoforms are identical in the first four hundred and twenty-two amino acid
  • the proNDF- ⁇ 2a cytoplasmic domain contains two hundred and seventeen additional amino acid
  • ProNDF- ⁇ 2b cDNA encodes a different 39-amino acid sequence
  • ProNDF- ⁇ 2c has the shortest cytoplasmic domain, with the cDNA having a stop codon at codon position 423 followed by a different 3'-untranslated sequence.
  • NDF proteins in transfected COS-7 was also analyzed by immunoprecipitation of 35 S-labeled proteins.
  • antibodies were raised by immunizing rabbits with recombinant rat met-NDF- ⁇ 2 14 -241 purified from E. coli as described in Example 14 (Section I), below. Rabbits were immunized with an initial injection of 200 ⁇ g of recombinant rat met-NDF- ⁇ 2 14-241 . The antigen was emulsified with complete
  • the resulting purified anti-NDF antibody was used to immunoprecipitate recombinant 35 S-labeled NDF proteins from COS-7 cell cultures expressing the various recombinant precursor proteins.
  • COS-7 cells transfected with proNDF cDNA expression plasmids were grown in 60-mm dishes in DMEM plus 10% FBS at 37°C for forty eight hours. Cells were washed and placed in 1.5 ml DMEM minus methionine and cysteine with 1% dialyzed FBS.
  • the cell lysates were clarified by centrifugation and diluted 1:4 in immunoprecipitation dilution buffer (1.25% Triton X-100; 190 mM NaCl; 60 mM Tris-HCl, pH 7.4; 6 mM EDTA; 10 units/liter trasylol).
  • the radiolabeled COS-7 cell conditioned media and diluted cell lysates were pretreated at 4°C for two hours with 20 ⁇ l of rabbit normal serum and 20 ⁇ l of protein A-Sepharose CL-4B (Sigma) diluted 1:1 in PBS. The protein A-Sepharose was then removed by centrifugation.
  • the pretreated samples were gently agitated at 4°C overnight with affinity-purified anti-NDF antibody (20 ⁇ g/ml) and 20 ⁇ l protein A-Sepharose.
  • the protein A-Sepharose beads were pelleted and washed four times in a washing solution (0.1% Triton X-100; 0.02% SDS; 150 mM NaCl; 50 mM Tris-HCl, pH 7.5; 5 mM EDTA; 10 units/liter trasylol).
  • the washed beads were heated with 60 ⁇ l of SDS gel electrophoresis loading buffer at 100°C for three minutes.
  • the beads were removed by centrifugation and the supernatants analyzed by electrophoresis in 10% SDS-PAGE gels (Novex). Prestained protein molecular weight markers were from Novex. NDF proteins were undetectable in cell lysates and in conditioned media from COS-7 cells transfected with irrelevant cDNAs, and also when samples were immunoprecipitated in the
  • the immunoprecipitated proteins correspond in size to the processed form of NDF purified from media conditioned by Rat-1-EJ cells.
  • the 40 to 45 kDa recombinant NDF proteins were found in both cell lysates and conditioned media. Larger NDF precursor proteins were detected as high molecular weight proteins immunoprecipitable from the cell lysates, but not from conditioned media.
  • the NDF ⁇ 2a, ⁇ 2a, and ⁇ 4a precursor proteins are significantly larger than the NDF ⁇ 2b and ⁇ 2c precursor proteins.
  • immunoprecipitated proteins are presumably the full-length, glycosylated NDF precursor proteins.
  • NDF- ⁇ 2c precursor proteins were found at higher levels than the other precursor isoforms, suggesting somewhat less efficient processing for this variant.
  • proNDF- ⁇ 3 cDNA directed the synthesis of 30- to 35-kDa proteins. These proteins were found in the COS-7 cell lysate, but not in the conditioned medium ( Figure 25).
  • Prestained molecular weight markers were from Novex. Enzymatic deglycoslyation of the 40-44 kDa and 60-75 kDa NDF- ⁇ 2c recombinant proteins reduced their size
  • glycoslyations are more abundant than N-linked
  • NDF- ⁇ 3 is apparently modified prior to proteolytic processing of the proNDF.
  • NDF- ⁇ 3 was resistant to glycanase treatment. This result, and the exclusively intracellular localization of NDF- ⁇ 3, indicates that NDF- ⁇ 3 protein does not enter the secretory pathway. This is consistent with the absence of a significant hydrophobic domain in NDF- ⁇ 3.
  • Amino terminal signal peptide sequences are notably absent from all of the predicted NDF precursor proteins.
  • the different isoforms of proNDF (with the exception of proNDF- ⁇ 3) are all glycoslyated and processed to 40 to 45 kDa biologically active NDF glycoproteins. These proteins are found in the
  • ProNDF- ⁇ 3 which accumulates as an unglycosylated intracellular protein, has essentially the entire proNDF extracellular domain.
  • rat clone 40 cDNA encodes a truncated proNDF- ⁇ 2
  • the intracellular, nonsecreted NDF- ⁇ 3 protein may be
  • NDF- ⁇ 3 may function as a sequestered growth factor that is released when tissues are injured.
  • IL-1 ⁇ A similar role has been proposed for IL-1 ⁇ , which also lacks a signal peptide sequence (Mitzutani, H. et al., J. Clin . Invest . 87:1066-1071, 1991).
  • NDF- ⁇ 3 could have intracellular functions.
  • NDF cDNA sequences encoding the variable portion of the EGF-like domain (e.g. proNDF- ⁇ 2a codon positions 200-241) were amplified using the synthetic oligonueleotide primers 5'-GCGTCTAGATGAAGGACCTGTCAAACCC-3' (sense) [SEQ ID NO: 75] and 5'-GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'-GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'-GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'-GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'-GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'
  • RNA samples (1 ⁇ g) from normal adult female Sprague-Dawley rat (Charles River Laboratory, Wilmington, MA) tissues and from the Rat-1 and Rat-1-EJ cell lines were reverse transcribed to generate first strand cDNA with cDNA
  • PCR reactions employed Perkin Elmer Cetus GeneAmpTM PCR kits. The reactions were in a final volume of 50 ⁇ l and contained 10% of the reverse transcription reaction products or approximately 100 ng of cloned cDNAs as positive controls. Twenty-five cycles in a Perkin Elmer-Cetus GeneAmpTM 9600
  • thermocycler amplified the PCR products. Each cycle included twenty seconds at 94°C, twenty seconds at 55°C, and twenty seconds at 72°C. Ten ⁇ l aliquots of each reaction mixture were analyzed by electrophoresis in 2% agarose gels, followed by ethidium bromide staining.
  • NDF- ⁇ 1 is homologous to human heregulin ⁇ 1 (Holmes et al. Science, 1992, above).
  • Tissue-specific NDF variants were not detected by PCR and DNA sequencing with the exception of
  • a NDF- ⁇ 1 related cDNA (designated ARIA-1) was cloned from an embryonic chick brain cDNA library (Falls et al., Cell, 1993, above). Recombinant ARIA-1
  • NDF and ARIA in situ hybridizations with NDF and ARIA probes detect expression in spinal cord motor neurons, intestinal and dorsal root ganglia, and neuroepithelium lining lateral ventricles of the brain (See, also, Orr-Urtreger, et al., Proc. Natl . Acad. Sci . USA, 1993, above).
  • the conserved 374 amino acid cytoplasmic domain found in proNDFs ⁇ 2a, ⁇ 2a and ⁇ 4a is more than 85% identical to the proHRG (Holmes et al., Science, 1992, above) and proARIA-1 (Falls, D.L. et al., Cell, 1993, above) cytoplasmic domains. All of these cytoplasmic domains have a carboxy-terminal valine residue that may be important for proteolytic processing.
  • ProTGF- ⁇ has a carboxy-terminal valine that is critical for regulated release of TGF- ⁇ (Bosenberg, M.W. et al., Cell 71:1157-1165, 1992). Leucine or isoleucine can substitute for the terminal TGF- ⁇ valine in directing cleavage.
  • ProNDF- ⁇ 2b has a 196 amino acid residue cytoplasmic domain with a carboxy-terminal leucine and is processed efficiently to mature NDF- ⁇ 2.
  • the 157 amino acid proNDF- ⁇ 2c cytoplasmic domain ends with two hydrophilic residues. Accumulation of proNDF- ⁇ 2c within transfected COS-7 cells suggests that this precursor isoform is less efficiently processed. However, processing to mature NDF- ⁇ 2 does occur, demonstrating that NDF release from COS-7 cells does not require a terminal hydrophobic residue. In nontransfected cells, alternative carboxy-termini may be more critical for mature NDF release.
  • RNAs were transferred onto nitrocellulose filters and probed with 32 p-labeled rat NDF clone 44 cDNA (See
  • Example 6 a human kidney adenocarcinoma cell line, A-704 (American Type Culture Collection, ATCC HTB 45) exhibited the highest level of NDF mRNA
  • Double-strand cDNA was synthesized from poly (A) + RNA extracted from A-704 cells. Column-fractionated cDNA was ligated to SalI and NotI digested plasmid vector pJT-2 and transformed into E. coli strain DH10B (GIBCO BRL) or E. coli strain MC1061 (BioRad) by electroporation, using a cDNA kit and procedure
  • Hybridization was at medium stringency (30% formamide, 6x SSC, 2x Denhardt's solution, 100 ⁇ g/ml salmon sperm DNA, 1 mM EDTA, 0.2% SDS, 0.1% sodium pyrophosphate, 50 mM NaH 2 PO 4 , pH 6.8) at 42°C overnight. Filters were washed in a solution of 0.5x SSC, 2 mM EDTA, and 0.2% SDS at 23°C for thirty minutes; then at 42°C for ninety minutes. The filters were exposed overnight to X-ray film at -75°C.
  • NDF- ⁇ 2b human NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF
  • a cDNA library was synthesized with poly (A) + RNA extracted from A-704 cells. Double-strand cDNA was ligated to BstXI linkers and ligated into the BstXI site on plasmid vector pCDNA II (Invitrogen, San Diego, CA). The ligation mixture was used to transform E. coli strain DH10B.
  • Denhardt's solution 100 ⁇ g/ml salmon sperm DNA, 1 mM EDTA, 0.2% SDS, 0.1% sodium pyrophosphate, 50 mM NaH 2 PO 4 , pH 6.8) at 42°C overnight.
  • the filters were washed in a solution made of 0.1x SSC, 2 mM EDTA, 0.2% SDS, at 23°C for 30 min; then at 70°C for 60 min.
  • the filters were then exposed to X-ray films at -75°C overnight.
  • NDF cDNA sequences ( ⁇ 1 kb)
  • This region contains: the spacer domain; the variable EGF-like domain ( ⁇ and ⁇ forms); the variable sequences between the EGF-like domain and the transmembrane domain; the conserved transmembrane domain; and a portion of the cytoplasmic tail.
  • the sense primer was 5'-AGG AAA TGA CAG TGC CTC T-3' [SEQ ID NO: 78].
  • the antisense primer was 5'-TCT CTG GCA TGC CTG AGG-3' [SEQ ID NO: 79]. PCR was carried out for 40 cycles. The reaction conditions were: 94°C, 1 minute; 50°C,
  • the human proNDF cDNA clones are summarized in Table 4, and the composite amino acid sequence for the different human proNDF isoforms is shown in Figure 38.
  • Rat cDNA clone 44 (proNDF- ⁇ 2c, Figure 5) was subjected to PCR using two primers.
  • the 37-base oligonucleotide sense primer [SEQ ID NO: 80] had the sequence: 5'-CGG TCT AGA AGC TTC CAC CAT GTC TGA GCG CAA AGA A-3'. It included 5' XbaI and HindIII restriction enzyme cloning sites followed by the Kozak consensus sequence CCACC, as well as the initial 18 bases of the rat NDF coding sequences.
  • the 30-base oligonucleotide antisense primer [SEQ ID NO: 81] had the sequence: 5'-GCC GTC GAC CTA TTA CCT TTC GCT ATG AGG-3'. It included a Sail site, two tandem translation stop codons, and 15 bases complementary to the sequences encoding the carboxyl end of rat proNDF- ⁇ 2c.
  • the PCR product was digested with XbaI and Sail to generate a -1.4 kb DNA fragment containing sequences encoding the entire rat proNDF- ⁇ 2c. This fragment was then subcloned into the expression vector pDSR ⁇ 2 (published European patent application A20398753) that had been cut with XbaI and SalI. The final plasmid was designated as pDSR ⁇ 2/rNDF- ⁇ 2c.
  • Rat clone 42A cDNA ( Figure 20) encodes proNDF-ß4a, with a unique XbaI site at amino acid positions 244-245.
  • Example 11 included the same 37-base sense primer described in Example 11 [SEQ ID NO: 80] for expression of rat NDF- ⁇ 2c.
  • This primer contained XbaI and HindIII sites, the Kozak consensus sequence CCACC, and the 18 bases
  • the 27-base antisense primer had the following sequence: 5'-GCT CTA GAG GCT TCT CTG TTT CTT GCC-3' [SEQ ID NO: 82] and contained an XbaI site and 19 bases of the upstream sequences adjacent to the XbaI site mentioned above.
  • the rat proNDF-ß4a (clone 42A) cDNA was subjected to PCR using this pair of primers.
  • the PCR product was
  • the second pair of PCR primers included a 33-base sense primer with the following sequences: 5'-GCT CTA GAA AGA AAA TTG GAT CAT AGC CTT GTG-3' [SEQ ID NO: 83], containing an XbaI site and 25 bases of the adjacent sequences downstream from the XbaI site in NDF.
  • the 33-base antisense primer had the sequence 5'-GCC GTC GAC CTA TTA TAC AGC AAT AGG GTC TTG-3' [SEQ ID NO: 84], and contained a Sail site, two tandem translation stop codons and 18 bases complementary to sequences encoding the proNDF-ß4a carboxyl terminus.
  • the rat proNDF-ß4a cDNA was subjected to PCR using this second pair of primers.
  • the PCR product was digested with XbaI and Sail to generate an approximately 1.3 kb DNA fragment which encoded amino acids 245-662.
  • the approximately 730 bp HindIII-XbaI fragment and this approximately 1.3 kb Xba-Sall fragment were ligated with the expression vector pDSR ⁇ 2 which was digested with HindIII and SalI.
  • the resulting plasmid was designated pDSR ⁇ 2/rNDF-ß4a.
  • the initial plasmid made to express recombinant human NDF- ⁇ 2b was constructed following the strategy described above for expresson of rat NDF- ⁇ 2c.
  • Human NDF- ⁇ 2b clone 43 cDNA ( Figure 32) was used as the template DNA for PCR.
  • the two primers used had the following sequences: sense primer (37-mer): 5'-CGG TCT AGA AGC TTC CAC CAT GTC CGA GCG CAA AGA A-3' [SEQ ID NO: 85]; antisense primer (30-mer): 5'-GCC GTC GAC CTA TTA GAG AAT GAA GCC CAA-3' [SEQ ID NO: 86].
  • transfected cell clones expressing recombinant rat proNDF- ⁇ 2c consistently secreted higher levels of NDF protein than the transfected cell clones expressing recombinant human proNDF- ⁇ 2b.
  • some of the human proNDF- ⁇ 2b expressing cell clones had NDF mRNA levels comparable to that of rat NDF- ⁇ 2c clones, but produced much lower levels of NDF protein. Since the cytoplasmic portion of the rat proNDF- ⁇ 2c amino acid sequence somehow augments recombinant NDF production (i.e., affects proNDF stability or enhances the
  • NDF- ⁇ 2b DNA was engineered in this way, many clones were isolated which produced increased amounts of
  • the chimeric human-rat proNDF- ⁇ 2c expression vector was constructed by the following method.
  • Human NDF- ⁇ 2b (clone 43) cDNA ( Figure 32) was subjected to PCR using two PCR primers.
  • the sense primer (39-mer) had the sequence: 5'-GCC GAA GAC GGT CAT GAA GCT TCT GCC GCT GTT TCT TGG-'3 [SEQ ID NO: 87], and included an unique internal XhoI site;
  • the antisense primer (33-mer) had the sequence: 5'-CCT TTC AAA CCC CTC GAG ATA CTT GTG CAA GTG-3' [SEQ ID NO: 88] and included several base changes to create a HindIII site.
  • the resulting PCR product of approximately two hundred and ten base pairs, encoding proNDF- ⁇ 2b amino acids 206-274 and including the
  • transmembrane domain (amino acids 243-265), was digested with XhoI and HindIII and subcloned into plasmid
  • the resulting plasmid was a chimeric molecule encoding the human proNDF- ⁇ 2b extracellular domain, the conserved transmembrane domain (TM), and the rat proNDF- ⁇ 2c cytoplasmic domain.
  • This plasmid was designated as pDSR ⁇ 2/h-rNDF- ⁇ 2c. e. Chimeric human-rat NDF- ⁇ 1c, ß1c and ß2c
  • NDF- ⁇ 2 from a chimeric rat-human DNA construct was also used for expressing recombinant human NDF- ⁇ 1, -ß1 and -ß2 isoforms. Briefly, cDNAs individually encoding human proNDF- ⁇ 1, -ß1 and -ß2 were subjected to PCR using the same primers described above for generating the chimeric human-rat NDF- ⁇ 2c DNA.
  • the -210 bp XhoI-HindIII PCR fragments generated from human NDF cDNA clone P1 as the template represents sequences from human NDF- ⁇ 1; the human NDF cDNA clone P-13 template gave the human NDF-ß1 sequence; and the human NDF cDNA clone 294- 8 template yielded the human NDF-ß2 sequence.
  • the XhoI-HindIII fragments were first subcloned into pGEM7Zf(-).
  • COS-7 cells ATCC CRL 1651
  • pDSR ⁇ 2/hNDF- ⁇ 2b ATCC CRL 1651
  • pDSR ⁇ 2/h-rNDF- ⁇ 2c ATCC CRL 1651
  • pDSR ⁇ 2/h-rNDF-ß1c pDSR ⁇ 2/h-rNDF-ß2c
  • Transfected COS-7 cells were plated at 2 ⁇ 106 cells per 60 mm plate in DMEM with 10% FBS.
  • Conditioned medium (CM) from twenty-four to seventy-two hours post-transfection were collected from the plates and tested in the neu receptor tyrosine phosphorylation assay using MDA-MB-453 carcinoma cells as described in Example 3. As shown in Figure 40, all of the CM samples tested at two concentrations (100 ⁇ l unconcentrated CM, or 100 ⁇ l of 10-fold concentrated CM) were active in the assay.
  • Conditioned medium from untransfected COS-7 cells was negative (Fig. 40, lane 1). EXAMPLE 12
  • the CHO D- cell line requires proline for growth and is routinely grown in high glucose DMEM, supplemented with non-essential amino acids (NEAA), 16 ⁇ M thymidine,
  • Plasmid DNAs were introduced into the CHO D- cells by calcium phosphate precipitation. Approximately 0.8 ⁇ 10 6 cells in 60 mm plates were exposed to 2 to 3 ⁇ g of plasmid DNA together with sufficient mouse spleen DNA to total 10 ⁇ g. The medium was changed the next day. After incubation for another twenty-four hours, the cells were split into eight 100-mm plates containing selective medium (high glucose DMEM with NEAA and 5% dialyzed FBS but without nucleosides). After ten to fourteen days, clones were picked and serum-free
  • Example 3 or analyzed by SDS/polyacrylamide gel
  • recombinant clones were grown in a spinner bottle in a 1:1 mixture of high glucose DMEM and F12 with NEAA, 5% FBS and 2 mM glutamine. Cells were then transferred to 850 cm 2 roller bottles containing the same medium with 2 ⁇ 10 7 cells per bottle. After three to four days at 37°C, the cell monolayers were washed with PBS and re-fed with 150 to 200 ml fresh medium per bottle (as above but lacking FBS). Conditioned medium was
  • Second and third harvests of conditioned media were also produced from the cell monolayer. Conditioned media were harvested and centrifuged at 7,000 x g for twenty minutes at 4°C or filtered using 0.45 ⁇ m cellulose acetate filters and stored frozen at -80°C until purification.
  • Recombinant rat NDF- ⁇ 2 expressed by CHO cells transfected with pDSR ⁇ 2/rNDF- ⁇ 2c was purified by the following procedure. Pooled serum-free conditioned media from harvests of roller bottles containing the transfected CHO cells expressing
  • recombinant rat NDF- ⁇ 2c were cleared by filtration through 0.2 ⁇ filters and concentrated with a Pellicon diafiltration system with 10-kDa molecular size cut-off membrane. Concentrated material was directly loaded onto a column of heparin-Sepharose, pre-equilibrated with 20 mM sodium phosphate buffer pH 7.2 ⁇ 0.1)
  • the dialyzed, clarified sample was then loaded onto a DEAE Sepharose 6B fast flow column which had been pre-equilibrated with dialysis buffer described above.
  • the column was extensively washed until no absorbance at 280 nm could be detected.
  • the column was then developed with a 200 ml gradient of 0.02 M to 0.5M NaCl in 20 mM sodium phosphate buffer, pH 7.2 ⁇ 0.1. Fractions were collected and assayed for NDF content. Ammonium sulfate was added to the pooled NDF fraction obtained from DEAE Sepharose chromatography to achieve a concentration of 2 M.
  • the material was loaded on a Phenyl-Sepharose 4B column (Pharmacia) pre-equilibrated with 20 mM sodium phosphate, pH 7.2 ⁇ 0.1 containing 2 M ammonium sulfate. After loading, the column was washed with starting buffer and developed with a gradient of ammonium sulfate (from 2 M to no salt) in 20 mM sodium phosphate, pH 7.1 ⁇ 0.1. The main peak of activity was pooled and extensively dialyzed against PBS buffer and concentrated by centrifugation using a Centriprep 10 cartridge. IV. Characterization of the purified recombinant rat NDF- ⁇ 2 from CHO cells
  • the purified recombinant rat NDF- ⁇ 2 isoform migrates at a molecular weight of about forty to forty-four kilodaltons, identical to that of the
  • Expression plasmids, pCFM1656 and pCFM3106, used in the following examples can be derived from
  • plasmid pCFM836 (a detailed description of pCFM836 is contained in U.S. Patent No. 4,710,473, incorporated
  • Plasmid pCFM1656 can be derived from pCFM836 plasmid by destroying the two endogenous NdeI restriction sites by end filling with T4 polymerase enzyme followed by blunt end ligation, by replacing the DNA sequence between the unique A atII and ClaI
  • Plasmid pCFM3106 can be derived from pCFM1656 by making a series of site-directed base changes by PCR
  • the base pair changes are as follows: plasmid bp # bp in pCFM1656 bp changed to in
  • Table 5 shows the PCR primer sequences used to amplify NDF coding sequences for insertion into pCFM1656 and pCFM3106.
  • NDF- ⁇ 2 14-241 a DNA fragment encoding NDF- ⁇ 2 14-241 was first constructed. This fragment contained the native cDNA sequence corresponding to amino acid positions 14-241 of human NDF- ⁇ 2.
  • the oligonucleotide primers 409-27 [SEQ ID NO: 97] and 409-28 [SEQ ID NO: 98] were used to amplify the NDF coding sequence from human NDF- ⁇ 2 cDNA plasmid clone 43 ( Figure 32).
  • the 5' forward (sense) primer 409-27 provided an XbaI site, a ribosome-binding sequence, and a methionine start codon followed by the NDF- ⁇ 2 gene sequence starting from codon 14.
  • the 3' backward (antisense) primer 409-28 contained a BamHI site and the complementary sequence of a TAA stop codon and the NDF- ⁇ 2 3' coding sequence ending at codon 241.
  • the PCR fragment amplified from cDNA clone 43 was ligated into the XbaI and BamHI sites of plasmid
  • E. coli strain FM5 ATCC #53911 which is a derivative of strain K-12 with a temperature sensitive ⁇ repressor gene, CI857 (Sussman et al., C. R. Acad. Sci. 254:
  • the transformant was designated Strain 1664.
  • the met-huNDF- ⁇ 2 expression level of Strain 1664 was subsequently improved by optimizing the first 12 codons of the protein coding sequence.
  • a fragment with NdeI and SacII cohesive ends was generated by annealing two chemically synthesized oligonucleotides (436-23 and 436-24) [SEQ ID NOS: 100 and 101,
  • NDF cDNAs isolated from cDNA libraries that encode different human NDF isoforms differ in the sequences 3' to an unique XhoI site in the EGF-like coding region, which makes the XhoI site a convenient cloning site for the construction of different
  • Oligonucleotide 502-10 [SEQ ID NO: 106] containing sequences 5' to the XhoI site was used as the 5' primer, and oligonucleotide 502-11 [SEQ ID NO: 107], which provided the BamHI site and TAA stop codon sequence, was the 3' primer.
  • the resulting PCR fragment amplified from human NDF- ⁇ 1 cDNA clone P1 ( Figure 30) was used to replace the XhoI-BamHI fragment in plasmid pCFM1656-met-huNDF- ⁇ 2 14-241 , and thus generated the NDF- ⁇ 1 expression plasmid pCFM1656-met-huNDF- ⁇ 1 14-249 .
  • Strain 1854 was the designation assigned to the FM5 strain containing this plasmid. d , Recombinant human met-NDF- ⁇ 1 14-246
  • the primers 502-10 and 502-11 [SEQ ID NOS: 106 and 107, respectively] were also used for PCR synthesis of a NDF- ⁇ 1 gene.
  • the XhoI-BamHI fragment in pCFM1656-met-huNDF- ⁇ 2 14-241 was replaced by the PCR fragment amplified from pCFM1656-met-huNDF- ⁇ 1 177 _ 246 (described below in Section e).
  • the FM5 strain harboring the resulting construct pCFM1656-met-huNDF- ⁇ 1 14-246 was designated
  • NDF- ⁇ 1 177 _ 246 A DNA fragment containing the coding sequence for NDF- ⁇ 1 177 _ 246 and necessary cloning and expression elements (cloning sites, ribosome-binding sequence, methionine start codon, and TAA stop codon) was PCR synthesized from the EGF-like region of huNDF- ⁇ 1 cDNA clone 294-1 with primers 494-26 [SEQ ID NO: 105] and 487-15 [SEQ ID NO: 104], and cloned into the XbaI and BamHI sites of pCFM1656.
  • Clone 294-1 is a partial cDNA clone for NDF- ⁇ 1 which encodes amino acids 119 to 410 ( Figure 38). It is isolated by PCR as described in Example 10 (Section IV). Strain 1769 was assigned to this FM5 strain, bearing pCFM1656-met-huNDF- ⁇ 1 177-246 . f. Recombinant human NDF- ⁇ 2 177-241
  • the ⁇ 2 isoform counterpart of pCFM1656-met-huNDF- ⁇ 2 177 _ 241 was constructed similarly with PCR primers 437-29 [SEQ ID NO: 102] and 443-1 [SEQ ID NO: 103] from human cDNA clone 43.
  • the expression level of this resulting strain 1697 with pCFM1656-met-huNDF- ⁇ 2 177- 241 was unsatisfactory. Therefore, secretion was considered as an alternative to produce NDF- ⁇ 2 177-241 .
  • An ompA secretion signal peptide (A) was fused to the cDNA sequence encoding human NDF- ⁇ 2 amino acids 177-241.
  • Oligonucleotide 502-24 [SEQ ID NO: 108] containing an XbaI cloning site, a ribosome-binding sequence, a methionine start codon, degenerate ompA codons, and part of the NDF 5' coding sequences beginning at codon 177 was used as the sense primer together with antisense primer 502-25 [SEQ ID NO: 109] containing sequence around the XhoI site. Using these two primers,
  • fragments containing the ompA signal peptide codons and the NDF- ⁇ 2 coding sequence were PCR synthesized from pCFM1656-met-huNDF- ⁇ 2 177-241 .
  • the fragments were used to substitute the XbaI-XhoI fragment of pCFM1656-met-huNDF- ⁇ 2 177-241 .
  • the FM5 secretion strain harboring pCFM1656-Al-huNDF- ⁇ 2 177 _ 241 was designated Strain 1776.
  • the expression level of secretion strain 1776 was greatly improved. However, the ratio of the processed huNDF- ⁇ 2 177-241 vs. non-processed preprotein was
  • the nucleotide sequence of human NDF- ⁇ 3 differs from human NDF- ⁇ 2 only in the region downstream from the unique XhoI site in the EGF-like domain.
  • the ⁇ 3 segment was PCR amplified with primers 580-31 and 583-16 [SEQ ID NOS: 113 and 114, respectively] from cDNA clone 19.
  • the resulting fragment was then used to replace the XhoI-BamHI fragment of pCFM1656-met-huNDF- ⁇ 2 14-241 in which the N-terminal codons of NDF have been optimized.
  • the new construct, pCFM1656-met-huNDF- ⁇ 3 14- 247 was transformed into FM5, and thus generated Strain 1910.
  • cDNA fragment encoding rat NDF- ⁇ 2 14-241 was PCR amplified from rat cDNA plasmid clone 44 with primers 260-19 [SEQ ID NO: 96] and 425-26 [SEQ ID NO: 99] and cloned into the XbaI and XhoI sites in pCFM1656.
  • the primer design and cloning strategy are similar to the ones used for the cloning of the met-huNDF- ⁇ 2 14-241 sequence described.
  • the FM5 strain containing the resulting expression plasmid pCFM1656-met-ratNDF- ⁇ 2 14-241 was designated as Strain 1685. II . Protein expression a. Fermentor
  • Biolafitte fermentor which contained eight liters of batch medium (80 g of yeast extract, 42 g of ammonium sulfate, 28 g of dibasic potassium phosphate, 32 g of monobasic potassium phosphate, 5 g of sodium chloride, 40 g of glucose, 32 ml of 1 M magnesium sulfate, 5 ml of antifoam Dow P2000, 16 ml of trace metal solution and 16 ml of vitamin solution.
  • the culture was allowed to grow to an optical density of 4-5 at 600 nm before starting phase I feeding.
  • the feed one medium contains 100 g of Bacto tryptone, 100 g of yeast extract, 900 g of glucose, 70 ml of 1 M magnesium sulfate, 20 ml of trace metal solution and 20 ml of vitamin solution in a total volume of two liters.
  • the phase one feed rate was initiated at 13 ml/hr and then adjusted every two hours according to the cell mass determined by optical density.
  • the medium fed into the fermentor allowed the E. coli cells to grow at an exponential rate under a glucose limited condition so that the amount of toxic by-products accumulating in the cell culture was minimal.
  • the temperature during the entire growth phase was set at 30°C to ensure the complete suppression of the transcription from the lambda P L promoter and the tight control of plasmid amplification.
  • the culture pH was kept at 7 with phosphoric acid and ammonium
  • the desired dissolved oxygen level was maintained by adjusting the agitation, air- and oxygen-input rates, and back pressure in the fermentor. As the optical density of the culture reached 30, the
  • the first feed medium was replaced with a second medium (800 g of Bacto tryptone, 400 g of yeast extract, and 440 g of glucose in a total volume of four liters) at a constant feed rate of 250-300 ml/hr to provide enough glucose as energy and amino acids as precursors for the synthesis of recombinant NDF.
  • a second medium 800 g of Bacto tryptone, 400 g of yeast extract, and 440 g of glucose in a total volume of four liters
  • E. coli Strain 1776 was inoculated into a 250 ml flask which contained 100 ml Luria broth plus
  • Recombinant rat met-NDF- ⁇ 2 14-241 was purified from E. coli Strain 1685 by subjecting a clarified cell lysate to anion exchange, cation exchange, hydrophobic interaction, and hydroxyapatite column chromatography.
  • E. coli cell paste was disrupted in 5 mM EDTA, pH 8.5, by two passages through a Niro-Soavi Homogenizer at 14,000 psi.
  • the cell lysate was adjusted to pH 8.5 and centrifuged for one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a JS-4.2 rotor.
  • S-Sepharose fractions containing a prominent recombinant NDF protein of about 34 kDa were identified by SDS-PAGE analysis using a 10% gel (Novex, San Diego) followed by Coomassie blue staining. These fractions were pooled, diluted with an equal volume of water, and adjusted to 1.2 M ammonium sulfate, pH 6.5. The sample was then loaded onto a column containing TSKGEL Butyl-toyopearl 650M (TosoHaas, Montgomeryville, PA). This column was previously equilibrated with 1.2 M ammonium sulfate in PBS, pH 6.5. After washing with
  • the column was eluted with a gradient of 1.2 to 0 M ammonium sulfate in 1x PBS, pH 6.5. Individual fractions were collected. Column fractions containing the recombinant NDF protein were identified by SDS-PAGE. The first NDF peak eluted contained less aggregates and more monomer. Fractions containing this first peak were pooled and dialyzed overnight at 4 C against two changes of 10 mM sodium phosphate, pH 8.5. The dialyzed sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 10 mM sodium phosphate, pH 8.5. The column was washed with 10 mM sodium phosphate, pH 8.5, then eluted with a gradient of 0 to 1 M NaCl in 10 mM sodium
  • a hydroxyapatite (IBF Biotechnics, Columbia, MD) column was used as a fourth chromatography step,.
  • the column was equilibrated with 10 mM sodium phosphate, pH 6.8. After loading the dialyzed S-Sepharose
  • Protein concentrations for recombinant rat met-NDF- ⁇ 2 14-241 were determined by ultraviolet absorption at 280 nm with a Beckman DU-650 spectrophotometer and an extinction coefficient of 0.54 mg -1 cm -1 ml.
  • Reversed-phase HPLC analysis using a HP1090 HPLC with a Vydac C 4 column (Hewlett-Packard Co., Palo Alto, CA) showed that the purified and refolded NDF preparation had a single peak with a retention time of 47.5 minutes in the absence and presence of 6 M guanidineHCl.
  • the addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, observed by the shift in retention time to 50.2 minutes.
  • Recombinant human NDF- ⁇ 2 1-241 was purified from E. coli Strain 1784 by subjecting a clarified cell lysate to anion exchange, cation exchange, and
  • E. coli cell paste was disrupted in 5 mM EDTA, pH 7.5 by two passages through a Niro-Soavi Homogenizer at 14,000 psi.
  • the cell lysate was centrifuged one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a JS-4 .2 rotor.
  • the clarified supernatant was adjusted to pH 8.5 and passed through Q-Sepharose Fast Flow
  • Tris-Cl, pH 8.5 Tris-Cl, pH 8.5.
  • the flow-through fraction from the Q-Sepharose column was loaded onto a S-Sepharose Fast Flow column. Column loading, equilibration and elution conditions were as described for recombinant human met-NDF- ⁇ 2 14-241 .
  • S-Sepharose fractions containing a prominent monomeric recombinant NDF protein were identified by SDS-PAGE analysis, pooled, and loaded onto a column containing TSKGEL Butyl-toyopearl 650M equilibrated with 1.2 M ammonium sulfate, pH 6.5. The column loading and elution conditions were the same as described for rat met-NDF- ⁇ 2 14-241 . Column fractions containing the monomeric recombinant NDF protein were identified by SDS-PAGE, pooled, and dialyzed against three changes of 1x PBS, pH 8.5 at 4°C overnight.
  • the dialyzed sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 8.5.
  • the column was washed with 1x PBS, pH 8.5, and then eluted with a gradient of 0 to 1 M NaCl in 1x PBS, pH 8.5.
  • Column fractions primarily
  • the protein concentration of recombinant human NDF- ⁇ 2 1-241 was determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coefficient of 0.48 mg- 1 cm -1 ml. Reverse phase HPLC analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 45.8 minutes. Addition of
  • Recombinant human met-NDF- ⁇ 2 14-241 was purified from E. coli Strain 1664 by subjecting a clarified cell lysate to anion exchange, cation exchange, hydrophobic interaction, and hydroxyapatite column chromatography.
  • E. coli cell paste was disrupted in of 5 mM EDTA, pH 8.5 by two passages through a Niro-Soavi
  • the cell lysate was adjusted to pH 8.5 and centrifuged one hour at 4 , 100 rpm in a J6-B centrifuge (Beckman), using a JS-4.2 rotor. The clarified supernatant was then passed through
  • S-Sepharose fractions containing a prominent recombinant NDF protein of about 35 kDa were identified by SDS-PAGE analysis using a 10% gel (Novex, San Diego) followed by coomassie blue staining. These fractions were pooled, diluted with an equal volume of H 2 O, and adjusted to 1.4 M ammonium sulfate, pH 6.5. The sample was then loaded onto a column containing TSKGEL Butyl-toyopearl 650M, equilibrated with 1.4 M ammonium
  • a hydroxyapatite (IBF Biotechnics, Columbia, MD) column was used as a fourth chromatography step.
  • the column was equilibrated with 10 mM sodium phosphate, pH 6.8. After loading the dialyzed butyl-toyo fraction, the column was washed with 10 mM sodium phosphate
  • the sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 8.5.
  • the column was washed with 1x PBS, pH 8.5, and then eluted with a gradient of 0 to 1 M NaCl in 1x PBS, pH 8.5.
  • Protein concentrations for recombinant human met-NDF- ⁇ 14-241 were determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coeffiecient of 0.54 mg -1 cm -1 ml.
  • Vydac C4 or Synchropak RP-4 columns were used with a HP1090 HPLC (Hewlett-Packard, Palo Alto, CA). The columns were equilibrated with 97% buffer A (0.1% trifluoroacetic acid in HPLC water) and 3% buffer B (90% acetonitrile, 0.1% trifluoroacetic acid in HPLC water).
  • Recombinant NDF protein (10-50 ⁇ g) was injected in a total volume of 250 ⁇ l.
  • a 40 ⁇ l sample was first treated with 200 ⁇ l of 0.1 M Tris-6 M guanidineHCl, pH 8 and 10 ⁇ l of 0.2 M dithiothreitol for thirty minutes at 23°C.
  • the final concentrations of guanidineHCl and dithiothreitol were 4.8 M and 8 mM, respectively.
  • the columns were eluted with a linear gradient of 3-50% buffer B for the first sixty minutes. The gradient was increased to 95% buffer B for the next ten minutes. Elution was continued for another ten to fifteen minutes with 95% buffer B.
  • the analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 46.4 minutes in the presence and absence of 6 M
  • guanidineHCl The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, observed by the shift in retention time to 49.6 minutes.
  • the N-terminal amino acid sequence analysis for the first ten residues (MKKKERGSGK) [SEQ ID NO: 122] matched the sequence predicted from DNA and included the N-terminal methionine used to initiate translation in E. coli . VII. Purification of recombinant human NDF- ⁇ 2 177-241
  • Recombinant human NDF- ⁇ 2177-241 was purified from E. coli Strain 1776 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange, hydrophobic and anion exchange column chromatography. E. coli cell paste was disrupted in 5 mM EDTA, pH 6.5, by two passages through a Microfluidizer
  • the cell lysate was centrifuged for one hour at 4,100 rpm in a J6-B centrifuge (Beckman) .
  • the pellet was resuspended in 5 mM EDTA, pH 6.5, and centrifuged again for one hour.
  • the pellet was solubilized by mixing with eight volumes of 8 M urea, 20 mM Tris-HCl, pH 8.6, for thirty minutes at room temperature. Reduced glutathione was added to 10 mM and the sample was stirred at room temperature, pH 8.8, for an additional thirty minutes.
  • the denatured, reduced inclusion body protein was diluted 1:20 into a buffer containing 20 mM Tris-HCl, 1 mM EDTA, 0.2 M L-arginine, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.8). The solution was left for sixty minutes at room temperature, then at 4oC for sixteen hours without further stirring.
  • the renatured sample was diluted with two volumes of water and adjusted to pH 4.6 by adding citric acid to 5 mM followed by 5 M HCl. After one hour of centifugation at 4,100, rpm in a J6-B centrifuge, the supernatant was loaded onto a S-Sepharose Fast Flow column. The column was previously equilibrated with 20 mM citric acid, pH 4.6. After loading, the column was washed first with equilibration buffer, then with 20 mM sodium phosphate, pH 6.8. Bound protein was stepwise eluted with 0.5 M NaCl in 1x PBS, pH 7.0.
  • S-Sepharose fractions containing a prominent recombinant NDF protein of about 8 kDa were identified by SDS-PAGE analysis using a 4-20% gradient gel (Novex, San Diego) followed by coomassie blue staining. These fractions were pooled, diluted to 1 mg/ml with 10 mM citric acid, pH 5.0, and adjusted to 1.5 M ammonium sulfate. The sample was then loaded onto a column containing TSKGEL Butyl-toyopearl 650M equilibrated with 1.5 M ammonium sulfate, 10 mM citric acid, pH 5. The column was washed with the equilibration buffer and eluted with a gradient of 1.5 to 0 M ammonium sulfate in 10 mM citric acid, pH 5. Column fractions containing the monomeric
  • recombinant NDF protein were identified by SDS-PAGE and pooled. After concentration in a stirred cell with a YM3 ultrafiltration membrane (Amicon, Danvers, MA), the sample was dialyzed against three changes of 1x PBS overnight at 4oC.
  • the dialyzed sample was diluted with two volumes of water, adjusted to pH 6.9, and passed through a Q-Sepharose Fast Flow column previously equilibrated with 20 mM sodium phosphate, pH 6.9.
  • the flow-through fraction was adjusted to pH 4.5 with 3 M citric acid, then loaded onto a S-Sepharose Fast Flow column
  • Protein concentrations for recombinant human NDF- ⁇ 2 177 _ 241 were determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coefficient of 0.389 mg -1 cm -1 ml. Reverse phase HPLC analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 34.5 minutes. The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, which was observed by a shift in retention time to 43.5 minutes. The N-terminal amino acid sequence analysis for the first fifteen residues (SHLVKCAEKEKTFCV) [SEQ ID NO: 123] matches the sequence predicted from DNA. IX. Purification of recombinant human met-NDF- ⁇ 1 14-249
  • Recombinant human met-NDF- ⁇ 1 14-249 was purified from E. coli Strain 1854 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange and hydrophobic column chromatography.
  • E. coli cell paste was disrupted in 1 mM EDTA, 10 mM Tris-Cl, pH 7.0 by two passages through a Niro-Soavi Homogenizer at 14,000 psi. The cell lysate was centrifuged for one hour at 4,100 rpm in a J6-B
  • Reduced glutathione was added to 10 mM and the sample was stirred at room temperature, pH 8.6, for an additional thirty minutes.
  • the denatured, reduced inclusion body protein was dropped into 50 volumes of 20 mM Tris-HCl, 1 mM EDTA, 0.2 M L-arginine, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.8).
  • the stirring was continued for ten minutes at room temperature. The solution was then left at 4 C overnight.
  • the renatured sample was diluted with one volume of water and adjusted to pH 7.0. After one hour of centifugation at 4,100 rpm in a J6-B centrifuge, the supernatant was loaded onto a S-Sepharose Fast Flow column, previously equilibrated with 1x PBS, pH 7.0.
  • the column was first washed with 1x PBS, pH 7.0, then with 1x PBS, pH 7.0 containing an
  • the dialyzed sample was loaded onto a S- Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 7.0. Bound protein was eluted by a gradient of 0.15-0.80 M NaCl in 1x PBS, pH 7.0.
  • Protein concentrations for recombinant human met-NDF- ⁇ 1 14-249 were determined by ultraviolet absorption at 280 nm with an extinction efficient of 0.486 mg -1 cm -1 ml. Reverse phase HPLC analysis showed that the
  • NDF preparation had a single peak with a retention time of 47.2 minutes.
  • the addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, which was observed by the shift in retention time to 50.4 minutes.
  • the N-terminal amino acid sequence analysis at the first twenty residues matched the sequence predicted from DNA and included the N-terminal methionine residue added to initiate translation in E. coli .
  • the sequence analysis also showed that amino-terminal processing occurred at amino acid residue 15 with the ratio of 4.8%.
  • E. coli cell paste was disrupted in ten volumes of 10mM tris-HCl 5mM EDTA, pH 8.0. The cell lysate was centrifuged, and the pellet was solubilized in five volumes of 8M Urea, pH 8.6, for thirty minutes at room temprature. Dithiothreitol was added to 5mM to complete the reduction. The sample was stirred at room temperature, pH 8.6, for an additional thirty minutes. The solublized, reduced inclusion body protein was added dropwise to fifty volumes of 20 mM Tris-Cl, 1 mM EDTA, 0.2 M L-arginine, 2 M urea, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.6). The stirring was continued for 10 minutes at room temperature. The solution was then left at 4°C overnight.
  • the protein concentration of recombinant human met-NDF- ⁇ 1 14-246 was determined by absorption at
  • Recombinant human met-NDF- ⁇ 1 177-246 was purified from E. coli Strain 1769 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange and reverse phase column chromatography.
  • E. coli cell paste was disrupted in ten volumes of 5mM EDTA, 10mM tris-HCl, pH 8.0. The cell lysate was centrifuged, the pellet was resuspended in 5 mM EDTA, 10 mM Tris-Cl, pH 8.0 and centrifuged again, then solubilized by mixing with five volumes of 8 M urea, pH 8.5, at room temperature for thirty minutes. Reduced glutathione was added to 10mM, and the sample was stirred at room temperature, pH 8.7, for an
  • the solublized, reduced inclusion body protein was added dropwise to fifty volumes of thirty mM Tris-HCl, 1 mM EDTA, 0.2 M
  • CM-Sepharose fractions containing a prominent ⁇ 7 kD recombinant NDF protein were identified by SDS-PAGE analysis. Pooled fractions containing the product were diluted with two volumes of water. The pH was
  • Vydac C4 column from The Separations Group, Hesperia, CA.
  • the C 4 column was previously packed in 80% ethanol, pH 4.0, and equilibrated with 20mM citric acid, pH 4.0.
  • the dialyzed sample was loaded onto a S-Sepharose Fast Flow (Pharmacia) column previously
  • This final product was designated recombinant human met-NDF- ⁇ 1 177-246 .
  • the protein concentration of recombinant human met-NDF- ⁇ 1 177-246 was determined by absorption at 280 nm with an extinction coefficient of 0.66 mg -1 cm -1 ml.
  • Recombinant human met-NDF- ⁇ 3 14-247 was purified from E. coli Strain 1910 by renaturing recombinant NDF protein from a cell lysate pellet traction, followed by cation exchange, hydrophobic extraction and anion exchange column chromatography.
  • E. coli cell paste was disrupted in ten volumes of 5mM tris-HCl, ImM EDTA, pH 8.0.
  • the cell lysate was centrifuged, and the pellet was solubilized in ten volumes of 8M urea, 2mM EDTA, 5mM Dithioerythritol (2, 3-Dihydroxybutane-1.4-dithiol), pH 9 for ninety minutes at room temperature.
  • the solubilized, reduced inclusion body protein was added slowly into twenty five volumes of 20 mM tris-HCl, 0.4 M L-arginine, ImM EDTA, 0.5mM reduced glutathione, pH 8.7. The stirring was continued for five minutes at room temperature. The sample was then left at room
  • the renatured sample was adjusted to pH 4.7 by adding citric acid to 10 mM followed by 5M HCl. After centrifugation, the supernatant was loaded onto a s-sepharose fast flow column. The column was previously equilibrated with 20mM citric acid pH 4.7. After loading, the column was washed first with equilibration buffer, then with 20mM sodium phosphate, 20mM Glycine, pH 8.5; finally with 0.2M NaCl in 20mM sodium phosphate, 20mM glycine, pH 8.5. Bound protein was stepwise eluted with 0.7 M NaCl in 20mM sodium phosphate and 20mM
  • recombinant NDF protein were identified by SDS-PAGE and pooled.
  • the pooled sample was dialyzed against three changes of 1 x PBS pH 7.5.
  • the dialyzed sample was passed through a
  • the protein concentration of recombinant human met-NDF- ⁇ 3 14-247 was determined by absorption at 280 nm with an extinction coefficient of 0.446 mg -1 cm -1 ml. SDS-PAGE analysis using a 10% NOVEX gel followed by
  • NDF preparation had a single peak with a retention time of 47.9 minutes.
  • the addition of both guanidineHCl and dithiothreitol caused the NDF protein to become reduced and unfolded, observed by a shift in retention time to 51.0 minutes.
  • the N-terminal amino sequence analysis of the first nineteen residues matched the sequence predicted from DNA and included the
  • BT-474 cells (ATCC HTB 20) were obtained from the
  • BT-474 cells were seeded in 6-well flat-bottom dishes in 2 mL of "complete" media at a density of 1 ⁇ 10 5 cells per well. After incubation for twenty-four hours at 37°C in a humidified air/5% CO 2 chamber, media were removed and replaced with samples containing either rHuNDF- ⁇ 2 or rRtNDF- ⁇ 2. Plates were re-incubated using the
  • rHuNDF- ⁇ 2 14-241 370 ⁇ g/mL; rRtNDF- ⁇ 2, 492 ⁇ g/mL
  • Diluent consisted of a 1:1 mixture of DMEM (GIBCO-BRL):F-12
  • the FACStar PLUS (Becton Dickinson Immunocytometry Systems, San Jose, CA) was equipped with a 5 W argon laser operated at 488 nm. Gold fluorescence was collected using a narrow band pass filter at 575 ⁇ 13 nm (Becton Dickinson) while red fluorescence was collected beyond 610 nm with a long pass filter (Omega Optical, Brattleboro, VT). Data were acquired and analyzed using the software package Lysis II Version 1.0 (Beckton
  • Nile Red fluorescence can vary:
  • BT-474 cells were treated with recombinant NDF- ⁇ 2 samples for seven days prior to staining with Nile Red and analysis by flow cytometry. Histograms depicting the gold-to-red fluorescence ratio measurements are presented in Figures 44 and 45.
  • Recombinant NDF- ⁇ 2 induced changes in the distribution of cells from the low ratio peak (i.e., "nondifferentiated" cells with few neutral lipid vesicles) to the high ratio peak (i.e.,
  • E. coli-produced rHuNDF- ⁇ 2 and CHO-produced rRtNDF- ⁇ 2 stimulate accumulation of neutral lipids in BT-474 cells in a dose-dependent manner. This is reflected by an increase in the percentage of cells in the high-ratio peak. This effect was most pronounced at the highest concentration tested (100 ng/mL). Thus, treatment with either preparation of NDF- ⁇ 2 can induce a
  • E. coli/human refers to recombinant human met-NDF- ⁇ 2 14-241 purified from E. coll
  • CHO/rat refers to recombinant rat NDF- ⁇ 2 purified from CHO cell supernatenta.
  • the in vitro growth of cancer cells can be modified by numerous growth stimulatory and inhibitory factors (Aaronson, S.A., Science 254:1146-1153, 1991). Some of these, such as EGF (Imai, Y. et al., Cancer Res . 42:4394-4398, 1982), bFGF (Karey, K.P., and Sirbasku, D.A., Cancer Res . 48:4083-4092, 1988), IGF-I (Karey, K.P., and Sirbasku, D.A., Cancer Res . , above) and TGF-ß1 (Knabbe, C. et al., Cell 48 : 417-428, 1987), can regulate the proliferation of rodent and mammalian breast
  • rHuNDF- ⁇ 2 14-241 recombinant human met-NDF- ⁇ 2 14-241 (rHuNDF- ⁇ 2) stimulates, retards or has no affect on the in vitro growth of two human mammary cell lines: BT-474 cells, a ductal carcinoma line that overexpresses the neu/Her-2 receptor, and MDA-MB-468 cells, an
  • rHuNDF- ⁇ 2 was purified from E. coli and re-folded in an active configuration (Example 14).
  • BT-474 ATCC HTB 20
  • MDA-MB-468 ATCC HTB 132 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were
  • MDA-MB-468 were cultured in the same media without L-glutamine and insulin as supplements.
  • Cells were seeded in 96-well flat-bottom microtiter dishes in 100 ⁇ L of cell-specific "complete" media at low density [i.e., 3,000 (BT-474) or 2,000 (MDA-MB-468) cells per well]. After incubation for twenty-four hours at 37°C in a humidified air/5% CO 2 chamber, 100 ⁇ L aliquots with samples of rHuNDF- ⁇ 2 were added which was followed by incubation of the plates using the
  • rHuNDF- ⁇ 2 dilutions were prepared from a stock solution (rHuNDF- ⁇ 2 14-241 , 370 ⁇ g/mL) in diluent (1:1 mix of DMEM (GIBCO):F-12 (GIBCO) with 100 ⁇ M non-essential amino acids (GIBCO) and 2 mM L-glutamine (GIBCO)). All dilutions were tested in quadruplicate. Just prior to analysis, sample media were replaced with 50 ⁇ L per well phenol red-free RPMI 1640 (GIBCO) containing 1 mg/mL MTT (Sigma). Plates were then re-incubated for an additional three hours. The formazan precipitate was solubilized by adding
  • E. coli-expressed rat NDF- ⁇ 2 14 _ 241 was used in a crypt colony formation assay. Results show that this isoform stimulated the attachment of colon crypts on plates coated with rat collagen type IV.
  • Mouse colon crypts were prepared as described by Whitehead et al. (In Vitro Cellular & Developmental Biology, Vol. 23, Number 6, Pages 436-442, 1987). Mice were sacrificed with a lethal dose of CO 2 , and large intestines were isolated. The large intestine was cut longitudinally, rinsed with buffer A(1x PBS containing 0.3 mg/ml of L-Glutamine, 100 units/ml of penicillin, 100 units/ml of streptomycin), and sliced into 0.5 cm pieces. These pieces were washed again with buffer A in a 50 ml conical tube several times. Clean tissue was washed with extraction buffer (0.5mM DTT, 2mM EDTA in buffer A) and incubated with 10 ml of extraction buffer for one hour. The extraction buffer was then
  • Crypts were harvested by shaking the tissue in 5 ml of Solution A. Crypts were transferred to a 15-ml tube.
  • the crypts were distributed in collagen type IV coated 6-well plates (Collaborative Biomedical, Cambridge, MA) at a density of five hundred crypts per well.
  • the medium (RPMI 1640, 0.3 mg/ml L-Glutamine, 100 units/ml penicillin, 100 units/ml streptomycin) contained either 1% or 10% fetal bovine serum (FBS). After twenty-four hours of incubation at 37°C, colonies of attached cells were stained with Gram Crystal Violet and counted. Formation of colonies was FBS dependent with 3-5 fold more colonies in medium containing 10% FBS than in media containing 1% FBS. The increase of colony formation was not due to the general increase of protein in the medium, since the same effect was not observed when BSA was used to substitute FBS.
  • IL-1, IL-2, IL-3, IL-6, IL-8, PDGF, SCF, EGF, TGF- ⁇ , bFGF, aFGF, KGF, G-CSF, and NDF were comparison tested at 10 ng/ml in medium containing 1% FBS. Among these factors, only NDF gave results similar to the stimulation obtained with 10% FBS in this assay. The assay was then repeated with various
  • Colorectal carcinoma is among the most common malignancies in Western societies.
  • the carcinogenic target is the colonic epithelium, which is a highly proliferative organ that undergoes constant growth renewal and differentiation. Tumors are thought to arise from undifferentiated progenitor cells which harbor mutations that disrupt normal growth control mechanisms. Tumors begin as a small benign growth, or adenoma, which later acquire more aggressive growth characteristics and eventually metastasize.
  • Current therapies include surgical resection and stringent chemotherapy in a combined modality prior to metastasis. Unfortunately, tumors that are not cured by surgery respond poorly to follow-up chemotherapy.
  • the HER-2 /neu proto-oncogene encodes a type I transmembrane protein with intrinsic tyrosine kinase activity, which is generally believed to be a growth factor receptor that transmits mitogenic signals.
  • HER-2 /neu is expressed in a variety of tissues including those enriched in epithelial surfaces such as breast, lung and the intestine.
  • the HER-2 gene has previously been shown to be amplified in breast cancer, suggesting its role in tumorigenesis of epithelial cell types
  • ⁇ DF Neu-Differentiation Factor
  • CEA carcinoembryonic antigen
  • the LIM 1215 colorectal carcinoma cell line was provided by Robert Whitehead (Ludwig Institute for Cancer Research, Melbourne) and maintained as adherent monolayer cultures in RPMI 1640 growth media containing 5% FBS, 1 ⁇ g/ml insulin, 10 ⁇ g/ml hydrocortisone and
  • rat NDF CHO-derived NDF- ⁇ 2,
  • the cell cultures were washed with PBS, removed from the dish by scraping with a rubber policeman, then pelleted by centrifugation.
  • the cell pellet was surrounded by O.C.T. embedding compound (#4583, Miles, Inc.) and flash-frozen in liquid nitrogen.
  • the embedded frozen pellets were sectioned using a cryostat fitted with a glass knife, and the frozen sections placed on glass slides. The sections were fixed in absolute acetone at -20°C for six minutes, then air dried.
  • the fixed sections were rehydrated in PBS containing 0.3 ⁇ g/ml BSA (antibody diluent) then incubated with 1 ⁇ g/ml mouse monoclonal antibody to human CEA (MAB 425, Chemicon International, Inc.) in antibody diluent for one hour at 23°C.
  • the slides were washed three times with antibody diluent, then incubated with 1 ⁇ g/ml FITC conjugated sheep anti-mouse IgG antibody (Amersham, Inc.) for approximately thirty minutes at room temperature.
  • the slides were washed, and the area containing the stained section was covered with PBS solution containing 4% n-propyl galate (Sigma Chemical Co., # P-3130) and 90% glycerol to minimize fluorochrome bleaching.
  • the slides were analyzed under a fluorescent microscope at UV 320 nm and photographed using Kodak Gold ASA 100 color film (Figure 49).
  • CEA is detected at significant levels only at the apical portion of the colonic crypt (Benchimol, S. et al. Cell, 1989, above). Intestinal epithelial cells are known to differentiate as they migrate from the basal to apical crypt compartments (Cheng, H., and
  • NDF is capable of regulating cell growth and differentiation control in HER-2 expressing colon carcinoma in vitro .
  • NDF may therefore modulate CEA expression in normal and transformed colonic epithelial cells in vivo. This may prove to be useful in treatment of colon carcinoma by regulating carcinoma cell-cell and cell-matrix interactions by decreasing the mortality and morbidity associated with metastatic colon carcinoma.
  • Tumor metastasis is a complex, multi-step process that begins when individual tumor cells leave the primary site of transformation by degrading their resident extracellular matrix.
  • Matrix degradation is mediated by several classes of proteolytic enzymes, including members of the Zn + -dependent metallo-proteinases, such as type IV collagenase and stromelysin (transin) (Liotta, L.A., et al., Cancer Research
  • the type IV collagenase and stromelysin genes are known to be induced following initiation of the growth response, and to be
  • TIMP-2 tissue inhibitors of metalloproteinase
  • LIM 1863 the metastatic ileoceacal carcinoma cell line LIM 1863.
  • LIM 1863 cells were treated with recombinant rat NDF to determine its effects on TIMP-2 protein expression and found that NDF treatment dramatically increased TIMP-2 expression and secretion. Since NDF is believed to play a role in differentiation of epithelial cell types, NDF-induced expression of TIMP-2 may have a role in inducing
  • TIMP-2 can inhibit metalloproteinases involved in tumor metastasis (DeClerck, Y.A. et al. Cancer Research 52:701-703,
  • NDF treatment may alter the metastatic properties of LIM 1863 cell in vitro, and of other human colon cancers in vivo.
  • the LIM 1863 ileoceacal carcinoma cell line (Whitehead, R.H. et al. Cancer Research 47: 2104-2113 , 1987) was provided by Robert Whitehead (Ludwig Institute for Cancer Research, Melbourne) and maintained in suspension cultures in RPMI 1640 growth media containing 5% FBS, 1 ⁇ g/ml insulin, 10 ⁇ g/ml hydrocortisone and 10 ⁇ M ⁇ -thioglycerol. Confluent cultures are
  • the embedded frozen pellets were sectioned using a cryostat fitted with a glass knife, and the frozen sections were placed on glass slides. The sections were fixed in absolute acetone at -20°C for six minutes, then air dried. The fixed sections were rehydrated in PBS containing 0.3 mg/ml BSA (antibody diluent), then incubated with a 1:300 dilution of rabbit polyclonal anti-TIMP-2 in antibody diluent for one hour at room temperature.
  • the TIMP-2 antiserum provided by Helen Hockmen (Amgen, Inc.), was raised against
  • the LIM 1863 cell line spontaneously forms organoids in suspension consisting of a polarized layer of epithelial cells surrounding a central lumen
  • TIMP-2 expression in cells expressing HER-2 receptors implies that NDF treatment would result in the downstream modulation of metalloproteinase activity in various compartments of the body. This may be useful in regulating tissue remodeling and repair in injured organs. More
  • TIMP-2 HER-2 receptor positive colon cancer cells by NDF treatment may inhibit their ability to metastasize and subsequently invade neighboring tissues. This effect may be difficult to mimic by the addition of even large amounts of recombinant TIMP-2 protein and may decrease the mortality and morbidity associated with metastatic colon carcinoma.
  • the area of epithelium produced in treated and untreated wounds was calculated from cross-sectional bisection for each dose group.
  • a one-way ANOVA and Dunnett's t-test was run for each dose against the control group.
  • Paraffin-embedded 3 ⁇ m sections of tissue were stained using anti-BrdU (Dako Corp., Carpinteria, CA), Avidin-Biotin Complex (Vector Laboratories, Inc., Burlingame, CA), and diaminobenzidine substrate (DAB; Sigma Chemical Co., St. Louis, MO). Sections were digested with 0.1% protease solution, followed by treatment with 2N HCl. Endogenous peroxidase was quenched by exposure to 3% hydrogen peroxide solution. Slides were blocked with a 10% solution of normal horse serum in phosphate buffered saline (PBS), then incubated with anti-BrdU diluted 1:400 in 1% bovine serum albumin.
  • PBS phosphate buffered saline
  • Sections were counterstained with hematoxylin.

Abstract

L'invention concerne des polypeptides non naturels stimulant la phosphorylation du récepteur de neu, lesquels peuvent être préparés par des techniques de génie génétique à partir de séquences nucléotidiques obtenues sur des cellules et des tissus humains. Ces polypetides sont utiles dans le traitement de diverses pathologies humaines impliquant des cellules exprimant le récepteur de neu et répondant à une modulation par les polypeptides. L'invention concerne également des molécules d'ADN codant les polypetides, ou des analogues de ces derniers, des procédés de production par génie génétique des polypeptides ou analogues à partir des molécules d'ADN, des matières biologiques utiles dans ces procédés tels que des vecteurs d'expression et des cellules hôtes transformées ou transfectées, ainsi que des compositions pharmaceutiques contenant lesdits polypeptides.
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US5670342A (en) * 1995-04-06 1997-09-23 Amgen Inc. NDF peptides
US5912326A (en) * 1995-09-08 1999-06-15 President And Fellows Of Harvard College Cerebellum-derived growth factors
US5814308A (en) * 1996-03-26 1998-09-29 Zhang; Ke Methods for the treatment of gastrointestinal tract disorders
IL127892A0 (en) 1996-07-12 1999-10-28 Genentech Inc Gamma-heregulin
WO1998035036A1 (fr) 1997-02-10 1998-08-13 Genentech, Inc. Variants d'hereguline
US6136558A (en) * 1997-02-10 2000-10-24 Genentech, Inc. Heregulin variants
WO1998056804A1 (fr) * 1997-06-13 1998-12-17 Human Genome Sciences, Inc. 86 proteines secretees humaines
AU4469997A (en) * 1997-09-17 1999-04-05 Urs Eppenberger Heregulin-gamma
US20010023241A1 (en) 1998-02-04 2001-09-20 Sliwkowski Mark X. Use of heregulin as a growth factor
US6080845A (en) * 1998-08-05 2000-06-27 Amgen Inc. Monoclonal antibody against utricular epithelium
US6017886A (en) * 1998-08-05 2000-01-25 Amgen Inc. Use of NDF peptide as growth factor for sensory epithelium
JP2004527203A (ja) * 2000-02-28 2004-09-09 デコード ジェネティクス イーエッチエフ． ヒト精神***病遺伝子
WO2001064877A2 (fr) * 2000-02-28 2001-09-07 Decode Genetics Ehf Gene humain de la schizophrenie
WO2007101130A2 (fr) 2006-02-23 2007-09-07 Novocell, Inc. Compositions et procédés utiles pour la culture de cellules différenciables
JP5390624B2 (ja) 2008-11-04 2014-01-15 バイアサイト インク 幹細胞集合体懸濁液組成物、その分化方法
BR112012029611A2 (pt) * 2010-05-21 2017-07-25 Merrimack Pharmaceuticals Inc proteína de fusão biespecífica, composição farmacêutica, método de tratamento de dano ao tecido em um indivíduo, método de promoção da regeração ou sobrevivência do tecido em um indivíduo e molécula de ácido nucleico
KR20130113962A (ko) * 2010-05-28 2013-10-16 마인드-엔알쥐 에스에이 뉴레굴린 이소형, 뉴레굴린 폴리펩타이드 및 이의 용도
AU2012318541B2 (en) 2011-10-06 2018-04-12 Aveo Pharmaceuticals, Inc. Predicting tumor response to anti-ERBB3 antibodies
AU2013248265B2 (en) 2012-11-08 2018-11-01 Viacyte, Inc. Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof
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