Recombinant ribonuclease proteins

FIELD OF THE INVENTION
This invention relates to the production of ribonuclease molecules which are toxic to cells of interest.
BACKGROUND OF THE INVENTION
Ribonucleases such as ribonuclease A ("RNase A") and their cytotoxicity toward tumor cells are well documented from studies performed in the 1960s and 1970s and reviewed in Roth, J., 1963, Cancer Res. 23:657-666. Human serum was also discovered to contain several RNases (Reddi, E., 1975, Biochem. Biophys. Res. Commun. 67:110-118, Blank et al., Human body fluid ribonucleases: detection, interrelationships and significance 1-203-209 (IRL Press, London, 1981)) that are expressed in a tissue specific manner. The proteins involved in the host defense activity of the eosinophil are homologous to RNases and express RNase activity (Gleich et al., 1986, Proc. Natl. Acad. Sci., USA 83:3146-3150; Slifman et al., 1986, J. Immunol, 137:2913-2917). Thus, human serum RNases were believed to also have host defense activities.
Further to these early studies was the discovery that an anti-tumor protein from oocytes of Rana pipiens has homology to RNase A (Ardelt et al., 1991, J. Biol. Chem. 266:245-251). This protein has been termed ONCONASE.RTM., Alfacell Corporation, N.J. See also e.g., Darzynkiewicz et al. (1988) Cell Tissue Kinet. 21, 169-182, Mikulski et al. (1990) Cell Tissue Kinet. 23, 237-246. This protein is also described in U.S. Pat. No. 4,888,172. Phase I and Phase I/II clinical trials of ONCONASE.RTM. as a single therapeutic agent in patients with a variety of solid tumors (Mikulski et al. (1993) Int. J. of Oncology 3, 57-64) or combined with tamoxifen in patients with advanced pancreatic carcinoma have recently been completed (Chun et al. (1995) Proc Amer Soc Clin Oncol 14 No. 157, 210). Conjugation of ONCONASE.RTM. to cell-type-specific ligands increased its potency towards tumor cells (Rybak et al. (1993) Drug Delivery 1, 3-10). Taken together, these results indicate that ONCONASE.RTM. has properties that are advantageous for the generation of a potent selective cell killing agent.
However, since this is not a human-derived protein, it is prone to stimulating undesirable immune responses when used in humans. Thus, it would be desirable to retain the potent cytotoxic properties of this molecule while reducing its immunogenicity in humans. Further, it would be desirable to produce derivations of this molecule recombinantly so that it may be better chemically conjugated or recombinantly joined to other molecules for targeting to specific cells. Until the invention described herein, it has proven difficult to recombinantly express an active cytotoxic molecule related to ONCONASE.RTM.. Though it was thought that the methionine-glutamic acid amino terminal end of the recombinant molecule prohibited the molecule from having significant enzymatic activity, a means to solve this problem has not been forthcoming until the invention herein.
Further, although advances in protein design techniques promise to alleviate some of the immunogenicity associated with the antibody portion of immunotoxins (Bird et al., 1988, Science 242:423; Huston et al., 1988, Proc Natl Acad Sci USA 85:5879; Ward et al., 1989, Nature 341:544), no solution has been forthcoming for the immunogenicity of the toxin portion other than immunosuppression of the patients (Khazaeli et al., 1988, Proceedings of AACR 29:418). Thus, there is a continuing need for methods and compositions that would reduce the immunogenicity of the Rana pipiens-derived toxic moiety.
Non-cytotoxic human members of the RNase A superfamily linked to tumor associated antigens by chemical (Rybak et al.(1991) J. Biol. Chem 266, 1202-21207, Newton et al. (1992) J. Biol. Chem. 267, 19572-19578) or recombinant means (Rybak et al. Proc. Natl. Acad. Sci. U.S.A. 89, 3165, Newton et al. (1994) J Biol Chem. 269, 26739-26745 have been shown to offer a strategy for selectively killing tumor cells with less immunogenicity than current strategies employing plant and bacterial toxins Rybak, S. M. & Youle, R. J. (1991) Immunol. and Allergy Clinics of North America 11:2, 359-380. Human-derived ribonucleases of interest include eosinophil-derived neurotoxin (EDN) and angiogenin.
SUMMARY OF THE INVENTION
We have discovered how to construct RNases which are highly cytotoxic and which are modifications of the native ONCONASE.RTM. (nOnc). When the nOnc was expressed recombinantly it was not found to have significant cytotoxicity. Our modified versions (rOnc), however, are highly cytotoxic and otherwise retain the advantages of the native ONCONASE.RTM. molecules, while in some cases they also have increased cytotoxic properties. The rOnc molecules may be used alone or conveniently used to form chemical conjugates, as well as to form targeted recombinant immunofusions. These rOnc molecules can be used to decrease tumor cell growth. An effective recombinant form of nOnc advantageously permits the recombinant molecule to be fused to other therapeutic or targeting molecules of interest recombinantly. Further, the rOnc molecule can be modified to enhance cytotoxicity as will be seen below. Our nOnc-derived molecules are also desirable because nOnc is a unique ribonuclease in that it can be administered alone directly to patients to decrease and inhibit tumor cell growth without the use of a targeting agent.
The present invention also includes methods of selectively killing cells using a rOnc joined to a ligand to create a selective cytotoxic reagent of the present invention. The method comprises contacting the cells to be killed with a cytotoxic reagent of the present invention having a ligand binding moiety that specifically delivers the reagent to the cells to be killed. This method of the present invention may be used for cell separation in vitro by selectively killing unwanted types of cells, for example, in bone marrow prior to transplantation into a patient undergoing marrow ablation by radiation, or for killing leukemia cells or T-cells that would cause graft-versus-host disease. The toxins can also be used to selectively kill unwanted cells in culture.
Humanized versions of our rOnc molecules are also described which graft portions of mammalian or human-derived RNases such as angiogenin or human eosinophil derived neurotoxin (EDN) to the rOnc-derived molecules. A preferred embodiment of the invention is a molecule where the amino terminal end of EDN is placed onto the amino terminal end of the rOnc molecules. The surprising properties of these hybrid proteins with regard to ribonuclease activity and in vitro anti-tumor effects are described.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE LEGENDS
FIG. 1 shows the deduced amino acid sequence of the Rana clone 9 (SEQ ID NO:2), described below and sequence alignment with the amino acid sequence of nOnc (SEQ ID NO:1). The bold print indicates identical residues between nOnc and Rana clone 9. The dots indicate missing amino acids in the PCR clone.
FIGS. 2A and 2B show the configuration of the DNA constructs exemplified in the examples. The PCR product obtained from Rana pipiens DNA is identified as Rana 9. The N-and C-termini are synthetically filled in and identified as Onc in the constructs encoding [Met-(-1)]rOnc or EDN for the N-terminal EDN/Onc hybrid. Corresponding amino acid residues are indicated below each construct. FIG. 2B shows the sequence alignment of the N-terminal sequences of nOnc (SEQ ID NO:3), rEDN (SEQ ID NO:4), [Met-(-1)]rOnc containing a Gly (G) instead of Asp in position 20 (SEQ ID NO:5), rEDN.sub.(1-21) rOnc with an Asp in amino acid position 26 (SEQ ID NO:6) and rEDN.sub.(1-21) rOncG26 with a Gly in position 26 (SEQ ID NO:7). Bold letters indicate conserved residues, capital letters show the sequence deduced from Rana clone 9.
FIGS. 3A-3D show the inhibition of protein synthesis in human tumor cells by nOnc, rEDN, [Met-(-1)]rOnc or hybrid proteins. Cells (10.sup.4) were plated in individual 96-well microtiter culture plates and treated with varying concentrations of each agent for 48 h. Cell viability was determined as described in the Example Section below. Results from more than one individual experiment were combined to yield the mean data points. Standard errors of the means, when they are greater than the symbol, are shown. Cell lines: ACHN, renal cancer (FIG. 3A); MDA-MB-231 (FIG. 3B) and HS 578T (FIG. 3D), breast cancer; SF-539 (FIG. 3C), CNS, cancer. EDN (open triangles); nOnc (open squares); [Met-(-1)]rOnc, (solid triangles); rEDN.sub.(1-21) rOnc, (open circles); rEDN.sub.(1-21) rOncG26 (solid circles).
FIG. 4 shows a sequence alignment of some members of the RNase A superfamily: Frog lectin is from Rana catesbeiana, ONCONASE.RTM., EDN, ECP (human eosinophil cationic protein), Ang is bovine angiogenin, Seminal is bovine seminal RNase, and RNase A is bovine pancreatic RNase A (SEQ ID NOs:8, 1 and 9-13, respectively). Amino acids conserved in all members are capitalized, and active site residues H12, K41, and H119 (RNase A numbering) are marked with an asterisk.
FIG. 5 shows the inhibition of protein synthesis by MetSerOnc and MetSer- or MetGlu-OncFvs. The cytotoxic effect of the single chain antibody rOnc fusion proteins; E6FB[Met-(-1)]SerrOnc (closed circles), [Met-(-1)]SerrOncAngFBE6 (open squares) and [Met-(-1)]GlurOncFBE6 (closed squares) were compared to the non-targeted recombinant protein, [Met-(-1)]SerrOnc (open circles), by determining inhibition of protein synthesis in SF 539 cells. Cells were plated into 96-well microtiter plates in Dulbecco's minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum. Additions were made in a total volume of 10 .mu.l and the plates were incubated at 37.degree. for 3 days. Phosphate buffered saline containing 0.1 mCi of [.sup.14 C]leucine was added for 2-4 h and the cells were harvested onto glass fiber filters using a PHD cell harvester, washed with water, dried with ethanol and counted. The results were expressed as per cent of [.sup.14 C]leucine incorporation in the mock-treated wells.
FIGS. 6A and 6B show inhibition of protein synthesis in an assay as described for FIGS. 3A-3D using cell line SF539, human glioma cells and rOnc fusion proteins designated MetLysTryrOnc (open circles, FIG. 6A); MetAlaAlaTyrOnc (closed circles, FIG. 6A); and rOnc fusion proteins with signal peptides, MetKDELSerrOnc (open circles, FIG. 6B) and MetNLSSerrOnc (closed circles FIG. 6B).
FIG. 7 shows inhibition of protein synthesis in an assay as described for FIGS. 3A-3D using cell line SF539, human glioma cells and comparing three fusion proteins corresponding to MetSerOnc (SEQ ID NO:39 with a Met-Ser amino terminal end): MetSerOnc (closed circles), MetSerOncC4 (MetSerOnc with a Cys at amino acid position 5 of SEQ ID NO:39, closed squares) and MetSerOncC72 (MetSerOnc with a Cys at amino acid position 73 of SEQ ID NO:39, open circles).

DETAILED DESCRIPTION
This invention provides highly active and cytotoxic ribonuclease molecules which can be used to selectively kill and target cells, particularly tumor cells. In some embodiments the molecules are designed to incorporate sequences from human derived ribonucleases which are also highly active and cytotoxic, but which have the further advantage in that they are less immunogenic in humans. The rOnc molecules of the present invention are those which are recombinant nOnc-derived sequences.
The nOnc molecule has an amino acid sequence set forth in SEQ ID NO:1. Bovine pancreatic RNase A has an amino acid sequence set forth in SEQ ID NO:13. Unless otherwise indicated, the amino acid sequence positions described herein use as a frame of reference the bovine pancreatic RNase A sequence in SEQ ID NO:13 as this is the reference sequence commonly used in the RNase field. It should be understood that such position designations do not indicate the number of amino acids in the claimed molecule per se, but indicate where in the claimed molecule the residue occurs when the claimed molecule sequence is aligned with bovine RNase.
The rOnc molecules described and claimed herein will preferably have cysteine residues at amino acid positions corresponding to amino acid positions 26, 40, 58, 84, 95 and 110; a lysine at position 41 and a histidine at position 119 with reference to the bovine RNase A, SEQ ID NO:13 (such positions correspond to amino acid position numbers 19, 30, 48, 68, 75 and 90 and 87 and 104 of the nOnc sequence respectively set out in SEQ ID NO:1).
The rOnc molecules of this invention are those that have measurable ribonuclease activity, as defined below. The ribonucleases will also have (a) an amino terminal end beginning with a methionine which is followed by any amino acid other than glutamic acid (Glu); (b) a cysteine at amino acid positions 26, 40, 58, 84, 95 and 110; a lysine at position 41 and a histidine at position 119, such positions being determined with reference to those in the amino acid sequence of bovine RNase A (SEQ ID NO:13), and (c) an nOnc-derived amino acid sequence.
Preferably, the rOnc molecules will have an amino terminal end selected from the group consisting of:
Met-Ala;
Met-Ala-Ala-Ser;
Met-Arg;
Met-(J);
Met-Lys-(J);
Met-Arg-(J);
Met-Lys;
Met-Lys-Pro;
Met-Lys-(J)-Pro (SEQ ID NO:14);
Met-Lys-Pro-(J) (SEQ ID NO:15);
Met-Asn;
Met-Gln;
Met-Asn-(J);
Met-Gln-(J);
Met-Asn-(J)-Pro (SEQ ID NO:16);
Met-(J)-Lys;
Met-(J)-Lys-Pro (SEQ ID NO:17); and
Met-(J)-Pro-Lys (SEQ ID NO:18);
where (J) is Ser, Tyr or Thr.
Further, it is preferred that the rOnc molecules be modified so that the aspartic acid of amino acid position 2 of nOnc (position 4 with reference to the sequence of bovine RNase A) is deleted or replaced by Ala or Asn.
In alternative forms of the rOnc molecules, the molecules will employ an amino terminal end encoded by a sequence derived from the amino terminal end of EDN followed by a sequence from rOnc. In such forms, it is preferred that the amino acid sequence is one selected from the group consisting of those sequences substantially identical to those of a formula:
Met(-1)EDN.sub.(1-m) Onc.sub.(n-104)
wherein Met(-1) refers to an amino terminal residue of Met; wherein EDN.sub.(1-m) refers to a contiguous sequence of amino acids of a length beginning at amino acid position 1 of EDN (SEQ ID NO:9) and continuing to and including amino acid position "m" of EDN; wherein Onc.sub.(n-104) refers to a sequence of contiguous amino acids beginning at amino acid position "n" and continuing to and including amino acid position 104 as set out in SEQ ID NO:1; such that:
when m is 21, n is 16 or 17;
when m is 22, n is 17;
when m is 20, n is 16;
when m is 19, n is 15;
when m is 18, n is 14;
when m is 17, n is 12 or 13;
when m is 16, n is 11, 12, 13 or 14;
when m is 15, n is 10;
when m is 14, n is 9;
when m is 13, n is 8; and
when m is 5, n is 1.
In other alternative embodiments, the rOnc molecule will be fused at the carboxyl end to a sequence from angiogenin, such as the sequence exemplified in SEQ ID NO:11 or that at amino acid positions 101 to 107 of SEQ ID NO:20. The nucleic acid sequence for human angiogenin is known and is set out in U.S. patent application Ser. No. 08/125,462.
Preferred rOnc nucleic acid sequences are those that encode preferred rOnc amino acid sequences which are substantially identical to those in SEQ ID NOs:20, 22, 24, 26, 28 and 30 (corresponding nucleic acid sequences are set out in SEQ ID NOs:19, 21, 23, 25, 27 and 29, respectively). Most preferred rOnc amino acid sequences are those that are substantially identical to those set forth in SEQ ID NOs:20, 22, 24 and 26. Their corresponding nucleic acid sequences are also preferred and are set out in SEQ ID NOs:19, 21, 23 and 25, including conservatively modified variants thereof. The most preferred sequence includes SEQ ID NO:22, one which employs an amino terminal end comprising 1 to 21 (typically 21) amino acids of the amino terminal end of EDN grafted on to 16 to 104 amino acids of the nOnc sequence, with amino acid residue 20 in nOnc (Asp) being replaced with Gly. Preferred rOnc sequences further will contain optionally a Cys at a position corresponding to amino acid position 5, or 73 or Ala at amino acid position 88 in place of Cys with reference to SEQ ID NO:39.
Comparisons of the rOnc sequences provided here can be made to described sequences in the pancreatic RNase A superfamily. Many of such members are known and include, but are not limited to, frog lectin from Rana catesbeiana (Titani et al., Biochemistry 26:2189 (1987)); ONCONASE.RTM. (Ardelt, W. et al., J. Biol. Chem. 266:245 (1991)); eosinophil derived neurotoxin (EDN) (Rosenberg et al., supra); human eosinophil cationic protein (ECP) (Rosenberg et al., J. Exp. Med. 170:163 (1989)); angiogenin (Ang) (Fett, J. W. et al., Biochemistry 24:5480 (1985)); bovine seminal RNase (Preuss et al., Nuc. Acids. Res. 18:1057 (1990)); and bovine pancreatic RNase (Beintama et al., Prog. Biophys. Mol. Biol. 51:165 (1988)), references for all such proteins are incorporated by reference herein. Amino acid sequence alignment for such RNases are also set out in FIG. 4 and in Youle et al., Crit. Rev. Ther. Drug. Carrier Systems 10:1-28 (1993) and in U.S. patent application Ser. No. 08/125,462, which is incorporated by reference herein.
DEFINITIONS
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (New York), and Hale and Marham (1991) The Harper Collins Dictionary of Biology, Harper Perennial, NY provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
The terms "measurable ribonuclease activity" or "significant ribonuclease activity" refer to a molecule which has an IC.sub.50 (ng/ml) of less than 40 when added to a rabbit reticulocyte lysate assay wherein protein synthesis is inhibited as measured by the incorporation of [.sup.35 S]methionine into acid precipitable protein. IC.sub.50 is the concentration of protein necessary to inhibit protein synthesis by 50% in the assay. The lysate assay may be done as described in the Promega lysate assay kit which is commercially available from Promega Corporation, Madison, Wis. Ribonuclease activity using high molecular weight RNA and tRNA is determined at 37.degree. C. through the formation of perchloric acid soluble nucleotides following published protocols (Newton, D. L., et al. (1996) Biochemistry 35:545-553). With poly(A,C) UpG and poly U, ribonuclease activity is assayed according to DePrisco et al., and Libonati and Floridi (DePrisco, R., et al. (1984) Biochimica et Biophysica Acta 788:356-363; Libonati, M. et al. (1969) European J. Biochem. 8:81-87). Activity is assayed by measuring the increase with time in absorbance at 260 nm. Incubation mixtures (1 ml of 10 mM imidazole, 0.1 M NaCl, pH 6.5 or pH 7) contain substrate and appropriate amounts of enzyme solution at 25.degree. C. The in vitro translation assay (St. Clair, D. K., et al. (1987) Proc. Natl. Acad. Sci. 84:8330-8334) and the cell viability assays using the (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) (MMT) (Mossman, T. (1983) J. Immunol. Methods 65:55-63) are performed as previously described (Pearson, J. W., et al. (1991) J. Natl. Cancer Inst. 83:1386-1391).
An "nOnc-derived" amino acid sequence is one that contains at least one string of six contiguous amino acids which is identical to a contiguous sequence of six amino acids selected from the group of sequences beginning at amino acid positions 1 (with Glu replacing pyroGlu), 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 41, 42, 43, 44, 45, 46, 47, 50, 52, 54, 56, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 80, 81, 82, 84, 85, 86, 87, 91, 92, 93, 95, or 96 of the nOnc amino acid sequence (SEQ ID NO:1).
"Conservatively modified variations" of a particular nucleic acid sequence refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. Furthermore, one of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are "conservatively modified variations" where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton (1984) Proteins W. H. Freeman and Company.
The terms "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment. The rOncs described herein are isolated and biologically pure since they are recombinantly produced in the absence of unrelated Rana pipiens proteins. They may, however, include heterologous cell components, a ligand binding moiety, a label and the like.
The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof. A nucleic acid encodes another nucleic acid where it is the same as the specified nucleic acid, or complementary to the specified nucleic acid.
An "expression vector" includes a recombinant expression cassette which includes a nucleic acid which encodes a rOnc polypeptide which can be transcribed and translated by a cell. A recombinant expression cassette is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a target cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes a nucleic acid to be transcribed, and a promoter.
The term "recombinant" when used with reference to a protein indicates that a cell expresses a peptide or protein encoded by a nucleic acid whose origin is exogenous to the cell. Recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also express genes found in the native form of the cell wherein the genes are re-introduced into the cell by artificial means, for example under the control of a heterologous promoter.
The term "subsequence" in the context of a particular nucleic acid or polypeptide sequence refers to a region of the nucleic acid or polypeptide equal to or smaller than the particular nucleic acid or polypeptide. "Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York. Generally, highly stringent wash conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and ph. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T.sub.m point for a particular probe. Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The term "identical" in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins or peptides it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., U.S.A.).
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482; by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) Computer Applications in the Biosciences 5: 151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90; Huang, et al. (1992) Computer Applications in the Biosciences 8, 155-65, and Pearson, et al. (1994) Methods in Molecular Biology 24, 307-31. Alignment is also often performed by inspection and manual alignment.
The term "substantial identity" or "substantial similarity" in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80%, or more preferably 85% sequence identity to the reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acid residues. An indication that two polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5.degree. C. to 20.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The term "specifically deliver" as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule. Specific delivery typically results in greater than 2 fold, preferably greater than 5 fold, more preferably greater than 10 fold and most preferably greater than 100 fold increase in amount of delivered molecule (per unit time) to a cell or tissue bearing the target molecule as compared to a cell or tissue lacking the target molecule or marker.
The term "residue" as used herein refers to an amino acid that is incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
A "fusion protein" or when a molecule is "joined" to another refers to a chimeric molecule formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein or the joined molecules may be formed by the chemical coupling of the constituent molecules or it may be expressed as a single polypeptide from a nucleic acid sequence encoding a single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone.
A "ligand" or a "ligand binding moiety", as used herein, refers generally to all molecules capable of specifically delivering a molecule, reacting with or otherwise recognizing or binding to a receptor on a target cell. Specifically, examples of ligands include, but are not limited to, antibodies, lymphokines, cytokines, receptor proteins such as CD4 and CD8, solubilized receptor proteins such as soluble CD4, hormones, growth factors, and the like which specifically bind desired target cells.
Making rOnc-derived Nucleic Acids and Polypeptides
Several specific nucleic acids encoding rOnc-derived polypeptides are described herein. These nucleic acids can be made using standard recombinant or synthetic techniques. Given the nucleic acids of the present invention, one of skill can construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which encode the same polypeptide. Cloning methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3; and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.
The nucleic acid compositions of this invention, whether RNA, cDNA, genomic DNA, or a hybrid of the various combinations, are isolated from biological sources or synthesized in vitro. The nucleic acids of the invention are present in transformed or transfected cells, in transformed or transfected cell lysates, or in a partially purified or substantially pure form.
In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q.beta.-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) are found in Berger, Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual (2nd Ed) Vol. 1-3; and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.
Oligonucleotides for use as probes, e.g., in in vitro rOnc nucleic acid amplification methods, or for use as nucleic acid probes to detect rOnc nucleic acids are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. Purification of oligonucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom. 255:137-149. The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560.
One of skill will recognize many ways of generating desired alterations in a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, Giliman and Smith (1979) Gene 8:81-97; Roberts et al. (1987) Nature 328:731-734 and Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual (2nd Ed) Vol. 1-3; Innis, Ausbel, Berger, Needham VanDevanter and Mullis (all supra).
Polypeptides of the invention can be synthetically prepared in a wide variety of well-known ways. Polypeptides of relatively short size are typically synthesized in solution or on a solid support in accordance with conventional techniques. See, e.g., Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154. Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols. See, e.g., Stewart and Young (1984) Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. Polypeptides are also produced by recombinant expression of a nucleic acid encoding the polypeptide followed by purification using standard techniques.
Making Conservative Modifications of the Nucleic Acids and Polypeptides of the Invention
One of skill will appreciate that many conservative variations of the sequences disclosed yield a substantially identical rOnc. For example, due to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions of a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid. Similarly, conservative amino acid substitutions, in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties (see, the definitions section, supra), are also readily identified as being highly similar to a disclosed amino acid sequence, or to a disclosed nucleic acid sequence which encodes an amino acid. Such conservatively substituted (or modified) variations of each explicitly disclosed sequence are a feature of the present invention.
One of skill will recognize many ways of generating alterations in a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, Giliman and Smith (1979) Gene 8:81-97, Roberts et al. (1987) Nature 328:731-734 and Sambrook, Innis, Ausbel, Berger, Needham VanDevanter and Mullis (all supra).
Most commonly, polypeptide sequences are altered by changing the corresponding nucleic acid sequence and expressing the polypeptide. However, polypeptide sequences are also optionally generated synthetically using commercially available peptide synthesizers to produce any desired polypeptide (see, Merrifield, and Stewart and Young, supra).
One of skill can select a desired nucleic acid or polypeptide of the invention based upon the sequences provided and upon knowledge in the art regarding ribonucleases generally. The physical characteristics and general properties of RNases are known to skilled practitioners. The specific effects of some mutations in RNases are known. Moreover, general knowledge regarding the nature of proteins and nucleic acids allows one of skill to select appropriate sequences with activity similar or equivalent to the nucleic acids and polypeptides disclosed in the sequence listings herein. The definitions section herein describes exemplary conservative amino acid substitutions.
Finally, most modifications to nucleic acids and polypeptides are evaluated by routine screening techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of a polypeptide can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a target nucleic acid, redox or thermal stability of a protein, thermal histeresis, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.
rOnc Fusion Proteins and Other Therapeutic Moieties
The rOnc molecules may also include pharmacological agents or encapsulation systems containing various pharmacological agents. They typically will include a ligand to act as a targeting molecule to direct the rOnc to desired cells. The rOnc may be attached directly to a ligand or an antisense molecule which will assist in delivering the rOnc. See, for example, SEQ ID NOS:40-61. The rOnc can also be engineered to contain a nuclear localization signal ("NLS") such as that described in amino acid positions 1 to 7 in SEQ ID NO:32 (and SEQ ID NO:31) to direct the rOnc within the cell. Alternatively, the Met at position 8 and the corresponding nucleic acids at positions 22-24 of SEQ ID NO:31 has been and can be omitted. The nucleic acid sequence for the NLS is nucleic acids 1-21 of SEQ ID NO:31. A signal peptide is also exemplified at amino acid positions 1-25 of SEQ ID NO:63.
The rOnc molecules are uniquely adapted for gene therapy applications. They can be fused to other therapeutic agents, for example, they could be fused to an anti-B cell lymphoma antibody. For example, as will be explained in more detail below, rOnc molecules recombinantly fused to an anti-transferrin receptor antibody or an anti-colon cancer antibody were active. As mentioned above, nOnc has anti-tumor effects in vivo and preferentially kills rapidly dividing cells stimulated by serum or growth promoting agents such as ras. The molecules are readily internalized in the cell. Their activity can be further facilitated by joining them to a nuclear localization signal and the like to redirect the molecules within the cell. Of particular use in tumor cells would be to target telomerase, an enzyme subject to degradation by RNase.
We have found that Onc synergizes with ras in microinjection studies. This means that Onc and ras have to be together in the cell. Onc does not synergize with ras when it enters the cell via its own routing. A CAAX (SEQ ID NO:33) motif is required to localize ras at the plasma membrane (C=Cys, A=an aliphatic amino acid, X=S,M,C,A, or Q, an example is Cys-Val-Ile-Met (SEQ ID NO:34)). Importantly this type of sequence has been shown to target heterologous proteins to the plasma membrane (Hancock, J., Cadwallader, K., Paterson, H. and C. Marshall (1991) EMBO J. 10:4033). It would be desirable to join the rOnc gene with DNA encoding a CAAX (SEQ ID NO:33) signal as given in the example, or KDEL as described below.
Telomerase is being investigated as a "universal cancer target" (G. B. Morin, JNCI. (1995) 87:859). It is an RNA protein that is located in the nucleus. It has been shown that antisense to telomerase RNA can inhibit the function of the enzyme and block the growth of cancer cells (J. Feng et al., Science (1995) 269:1236). RNase can also destroy the activity of the enzyme. Onc can also destroy the activity of the enzyme when incubated with a cell extract containing telomerase. An NLS/Onc molecule (such as that set out in SEQ ID NO: 32) can be made to route Onc to the nucleus so that it can degrade telomerase. The NLS we used has been shown to redirect proteins to the nucleus for the aim of interfering with the function of a nuclear antigen (S. Biocca, M. S. Neuberger and A. Cattaneo, (1990) 9:101). Our NLS/Onc molecule is effective in killing cells.
An amino terminal sequence to the recombinant molecule may be preferred where it is desirable to translocate the molecule into the cytosol of target cells. Such signal peptide is typically inserted at the amino end of the protein. For example, the first amino acids of the recombinant molecules described herein (after Met) could be KDEL (SEQ ID NO:64) and would accomplish signalling the molecule to the endoplasmic reticulum. Amino acid sequences which include KDEL, repeats of KDEL, or other sequences that function to maintain or recycle proteins into the endoplasmic reticulum, referred to here as "endoplasmic retention sequences" may be employed.
Optionally, the rOnc molecule attached to a ligand may include an encapsulation system, such as a liposome or micelle that contains an additional therapeutic composition such as a drug, a nucleic acid (e.g. an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art. See, for example, U.S. Pat. No. 4,957,735, Connor et al., Pharm. Ther., 28: 341-365 (1985).
One of skill will appreciate that the ligand molecule or other therapeutic component and the rOnc molecule may be joined together in any order. Thus, where the ligand is a polypeptide, the rOnc molecule may be joined to either the amino or carboxy termini of the ligand or may also be joined to an internal region of either molecule as long as the attachment does not interfere with the respective activities of the molecules.
The molecules may be attached by any of a number of means well-known to those of skill in the art. Typically the rOnc will be conjugated, either directly or through a linker (spacer), to the ligand. However, where both the rOnc and the ligand or other therapeutic are polypeptides it is preferable to recombinantly express the chimeric molecule as a single-chain fusion protein.
In one embodiment, the rOnc molecule is chemically conjugated to another molecule (e.g. a cytotoxin, a label, a ligand, or a drug or liposome). Means of chemically conjugating molecules are well-known to those of skill.
The procedure for attaching an agent to an antibody or other polypeptide targeting molecule will vary according to the chemical structure of the agent. Polypeptides typically contain a variety of functional groups; e.g., carboxylic acid (COOH) or free amine (--NH.sub.2) groups, which are available for reaction with a suitable functional group on an rOnc molecule to bind the other molecule thereto.
Alternatively, the ligand and/or rOnc molecule may be derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Ill.
A "linker", as used herein, is a molecule that is used to join two molecules. The linker is capable of forming covalent bonds to both molecules. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where both molecules are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). However, in a preferred embodiment, the linkers will be joined to the alpha carbon amino and carboxyl groups of the terminal amino acids.
A bifunctional linker having one functional group reactive with a group on a particular agent, and another group reactive with an antibody, may be used to form a desired immunoconjugate. Alternatively, derivatization may involve chemical treatment of the ligand, e.g., glycol cleavage of the sugar moiety of a glycoprotein antibody with periodate to generate free aldehyde groups. The free aldehyde groups on the antibody may be reacted with free amine or hydrazine groups on an agent to bind the agent thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation of free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are also known (See U.S. Pat. No. 4,659,839).
Many procedure and linker molecules for attachment of various compounds including radionuclide metal chelates, toxins and drugs to proteins such as antibodies are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987) which are incorporated herein by reference. In particular, production of various immunotoxins is well-known within the art and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann, Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443 which are incorporated herein by reference.
In some circumstances, it is desirable to free the rOnc from the ligand when the chimeric molecule has reached its target site. Therefore, chimeric conjugates comprising linkages which are cleavable in the vicinity of the target site may be used when the effector is to be released at the target site. Cleaving of the linkage to release the agent from the ligand may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. When the target site is a tumor, a linker which is cleavable under conditions present at the tumor site (e.g. when exposed to tumor-associated enzymes or acidic pH) may be used.
A number of different cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example, includes a description of immunoconjugates comprising linkers which are cleaved at the target site in vivo by the proteolytic enzymes of the patient's complement system. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.
Production of rOnc Molecules or Fusion Proteins
Where the molecules of interest are relatively short (i.e., less than about 50 amino acids) they may be synthesized using standard chemical peptide synthesis techniques. Where two molecules of interest are relatively short the chimeric molecule may be synthesized as a single contiguous polypeptide. Alternatively the molecules may be synthesized separately and then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule thereby forming a peptide bond. Alternatively, the molecules may each be condensed with one end of a peptide spacer molecule thereby forming a contiguous fusion protein.
Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984) which are incorporated herein by reference.
In a preferred embodiment, the chimeric fusion proteins of the present invention are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.
DNA encoding the fusion proteins of this invention, as well as the rOnc molecules themselves, may be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22: 1859-1862 (1981); and the solid support method of U.S. Pat. No. 4,458,066, all incorporated by reference herein.
Chemical synthesis produces a single-stranded oligonucleotide. This may be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Alternatively, subsequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence.
In a preferred embodiment, DNA encoding fusion proteins or rOnc molecules of the present invention may be cloned using DNA amplification methods such as polymerase chain reaction (PCR). If two molecules are joined together, one of skill will appreciate that the molecules may be separated by a peptide spacer consisting of one or more amino acids. Generally the spacer will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
The nucleic acid sequences encoding the rOnc molecules or the fusion proteins may be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant protein gene will be operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
The expression vectors or plasmids of the invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, the recombinant rOnc or fusion proteins can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically.
Accordingly, this invention also provides for host cells and expression vectors comprising the nucleic acid sequences described above.
Further, the present invention includes a method of selectively killing cells using a rOnc joined to a ligand to create a selective cytotoxic reagent of the present invention. The method comprises contacting the cells to be killed with a cytotoxic reagent of the present invention having a ligand binding moiety that specifically delivers the reagent to the cells to be killed. This method of the present invention may be used for cell separation in vitro by selectively killing unwanted types of cells, for example, in bone marrow prior to transplantation into a patient undergoing marrow ablation by radiation, for killing leukemia cells or T-cells that would cause graft-versus-host disease.
For methods of use in vivo, preferably the mammalian protein of the reagent used in this method is endogenous to the species in which the reagent is intended for use. Preferably, for use in humans, the cytotoxic reagent is a fusion protein comprising a humanized chimeric antibody and a humanized rOnc. Specific in vivo methods of this invention include a method for the chemotherapeutic alleviation of cancer in mammals comprising administering a cytotoxic amount of a selective cytotoxic reagent according to the present invention. The methods are particularly useful for treating tumors sensitive to the cytotoxic reagents. Tumors of particular interest include pancreatic, colon, breast and kidney tumors.
Pharmaceutical Compositions
The rOnc molecules and fusion proteins employing them of this invention are useful for parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recognized that the subject molecules and fusion proteins and pharmaceutical compositions of this invention, when administered orally, must be protected from digestion. This is typically accomplished either by complexing the protein with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the protein in an appropriately resistant carrier such as a liposome. Means of protecting proteins from digestion are well known in the art.
The pharmaceutical compositions of this invention are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ. The compositions for administration will commonly comprise a solution of the chimeric molecule dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of therapeutic molecule in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
Thus, a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
The compositions containing the present rOnc molecules or the fusion proteins or a cocktail thereof (i.e., with other proteins) can be administered for therapeutic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease, in a cytotoxic amount, an amount sufficient to kill cells of interest. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health.
Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient.
All patents, applications and publications cited herein are incorporated by reference herein. The following examples are provided for illustrative purposes only and are not to be construed as limiting the invention in any way.
EXAMPLES
Example I
Cloning and Expression of rOnc and Onc Conjugates with EDN
A. Materials. Native ONCONASE.RTM. ("nOnc") (SEQ ID NO:1) Ardelt et al. (1991) J. Biol. Chem. 256, 245-251 and recombinant human EDN ("rEDN") (SEQ ID NO:9) Newton et al. (1994) J Biol Chem. 269, 26739-26745 were purified from Rana pipiens oocytes, NASCO, Fort Atkinson, Wis. and Escherichia coli, respectively, as described. Antibodies to the denatured proteins were prepared by Assay Research, Inc., College Park, Md. Reagents for performing PCR, and direct cloning of PCR products, were obtained from Perkin-Elmer Corp., Norwalk, Conn. and from Invitrogen, San Diego, Calif. respectively. Substrates for the ribonuclease assays were purchased from Sigma, St. Louis, Mo. and Boehringer Mannheim, Indianapolis, Ind. The materials and their sources used in the construction and expression of the recombinant proteins as well as the rabbit reticulocyte lysate are described by Newton et al., Biochemistry 35:545 (1996).
B. PCR Cloning of Onconase. Rana pipiens genomic DNA was isolated according to standard procedures using proteinase K Maniatis, T., Fritsch, E. F. & Sambrook, J. Molecular Cloning, a laboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). A series of degenerate primers were designed to correspond to amino acids in various regions of the published nOnc sequence Ardelt et al. (1991) J. Biol. Chem. 256, 245-251. The PCR reaction was performed according to the manufacturer's instructions using 15 .mu.g of genomic DNA in 100 .mu.l. All reagents except the DNA were combined and incubated at 95.degree. C. for 8 min to inactivate any residual proteinase K before the addition of the Taq DNA polymerase. PCR was performed for 40 cycles of denaturation at 94.degree. C. for 1 min, annealing for 2 min at 55.degree. C. and primer extension for 2 min at 72.degree. C. Several pairs of primers yielded products of the expected size. The largest product (252 bp) was obtained using the forward primer encoding amino acid residues 15-23 (AG(GA)GATGT(GT)GATTG(TC)GATAA(CT)ATCATG) (SEQ ID NO:35) and the reverse primer encoding amino acid residues 90-98 (TGTGA(AG)AA(CT)CAGGC(AC)CC(TA)GT(GT)CA(CT)TTT) (SEQ ID NO:36). This fragment was subcloned into pCR.TM.II by TA cloning and a clone carrying an insert of the appropriate size was directly sequenced and found to encode amino acid residues 16-98 of nOnc ("Rana 9") (SEQ ID NO:2). The corresponding nucleic acid sequence is set out in SEQ ID NO:37.
C. Plasmid Construction, Expression, Protein Purification and in Vitro Assays. The N- and C-termini of nOnc were reconstructed using the PCR technique of splicing by overlap extension Horten et al. (1990) BioTechniques 8, 528-532 with amino acid residues 1-15 of nOnc or amino acid residues 1-21 of EDN at the N-terminal and amino acid residues 99-104 of nOnc at the C-terminal. The assembled genes were inserted between the XbaI and BamHI sites of the bacterial expression vector, pET-11d, Novagen, Madison, Wis. All procedures were accomplished essentially as described in Newton et al. (1994) J Biol Chem. 269, 26739-26745. The plasmids were expressed in BL21(DE3) E. coli cells as recommended by the supplier, Novagen, Madison Wis. The fusion proteins were isolated from inclusion bodies, denatured, renatured and dialyzed as described Newton et al. (1994) J Biol Chem. 269, 26739-26745 before being applied to a CM-Sephadex C-50 column, Pharmacia Biotech Inc., Piscataway, N.J. The proteins were eluted with a NaCl gradient (0-0.5M) in 20 mM Tris-HCl, pH 7.5, containing 10% glycerol. Final purification to >95% was achieved by size exclusion chromatography on Sephadex G-100 equilibrated and eluted with 5% formic acid. The proteins were pooled, concentrated by amicon ultrafiltration using a YM3 membrane (or lyophilized), Amicon, Beverly, Mass. and dialyzed against 20 mM Tris-HCl, pH 7.5, containing 10% glycerol before being assayed.
Ribonuclease activity using high molecular weight RNA and tRNA was determined following published protocols, Newton et al. (1994) J Neurosci 14, 538-544 at 37.degree. C. through the formation of perchloric acid soluble nucleotides following published protocols (Newton et al. (1996) Biochem. 35:545-553). With poly (A,C), UpG and poly U, ribonuclease activity was assayed spectrophotometrically according to DePrisco et al., and Libonati and Florida DePrisco et al. (1984) Biochimica et Biophysica Acta 788, 356-363, Libonati, M. & Floridi, A. (1969) European J. Biochem. 8, 81-87. Briefly, activity was assayed by measuring the increase in absorbance at 260 nm. Incubation mixtures (1 ml of 10 mM imidazole, 0.1 M NaCl, pH 6.5 or pH 7) contained substrate and appropriate amounts of enzyme solution at 25.degree. C. The in vitro translation assay, St. Clair et al. (1987) Proc Natl Acad Sci 84, 8330-8334, and the cell viability assays, Pearson et al. (1991) J Natl Cancer Inst 83, 1386-1391, using the (3-[4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide; Thiazolyl blue] (MTT) Mossman, T. (1983) J. Immunol. Methods 65, 55-63 were performed as previously described.
D. Cloning and Expression of [Met-(-1)] rOnc and rOnc chimeras. Eight different oligonucleotide primers were designed to correspond to specific regions in the primary amino acid structure of nOnc, Ardelt et al. (1991) J. Biol. Chem. 256, 245-251 and amplification of Rana pipiens genomic DNA was carried out in a thermal cycler, as described above. A primer pair corresponding to amino acid residues 15 to 23 and 90 to 98 of nOnc, respectively, generated a 252 bp fragment. That PCR product, here denoted Rana clone 9, was cloned into pCR.TM.II and sequence analysis confirmed that the PCR product encoded Onc (104 amino acids, total) from amino acid residue 16 to 98 (FIG. 1).
The entire recombinant Onc ("rOnc") gene (SEQ ID NO:38) was constructed by PCR extension and cloned into an expression vector using methodology previously described Newton et al. (1994) J Biol Chem. 269, 26739-26745. The amino and carboxyl termini of rOnc were completed by inserting the first 15 and last 6 amino acid residues of nOnc, respectively. The configuration of the semi-synthetic rOnc gene is depicted at the top of FIG. 2A. The primers were designed to overlap with the DNA sequence of the Rana clone 9 PCR product. The plasmid was expressed in BL21(DE3) E. coli and the recombinant protein was isolated from inclusion bodies as described in Newton et al. (1994) J Biol Chem. 269, 26739-26745 before being applied to a CM Sephadex C-50 column. Final purification to >95% was achieved by size exclusion chromatography. The rOnc obtained from the bacteria in this expression system contains an extra methionine at the amino terminal [Met-(-1)] (SEQ ID NO:39) in contrast to the authentic pyroglutamyl amino acid residue (<Glu-1) of the native protein (SEQ ID NO:1).
To humanize [Met-(-1)] rOnc while maintaining the alignment of the active site residues (FIG. 2B), the N-terminal of Rana clone 9 was also reconstituted with oligonucleotides that coded for the first 21 amino acid residues of a human eosinophil RNase, EDN (FIG. 2B, rEDN.sub.(1-21) Onc). PCR cloning can result in sequence errors. Indeed, the DNA sequence of the gene encoding EDN.sub.(1-21) Onc contained an A to G substitution resulting in a change from Asp to Gly at position 26 in the chimera (residue 20 in nOnc) and is designated as rEDN.sub.(1-21) rOncG.sub.26 in FIG. 2B. Another plasmid containing encoding rEDN.sub.(1-21) rOnc was sequenced and found to have the correct DNA sequence. Since the mutation resulted in the substitution of a charged amino acid with a small neutral residue the mutant chimera was also expressed and characterized for activity. In addition, [Met-(-1)]rOnc was mutated at position 20 from Asp to Gly (rOncGly.sub.20, FIGS. 2A and 2B).
E. Ribonuclease activity of Onc, EDN, [Met-(-1)]rOnc and hybrid rOnc proteins. Both nOnc (Lin, J. J., et al. (1994) Biochem Biophys Res Commun 204, 156-162) and EDN (Saxena et al. (1992) J. Biol. Chem. 267, 21982-21986) are potent inhibitors of in vitro translation in the rabbit reticulocyte lysate by mechanisms that depend upon their respective nucleolytic activities. As depicted in Table 1, the addition of nOnc or EDN to a rabbit reticulocyte lysate caused the inhibition of protein synthesis as measured by the incorporation of [.sup.35 S]methionine into acid precipitable protein. Whereas both nOnc and EDN inhibited protein synthesis with IC.sub.50s of 0.2 and 1.3 ng/ml, respectively, [Met-(-1)]rOnc, [Met-(-1)]rOncG20, and rEDN.sub.(1-21) rOncG26 were considerably less potent (IC.sub.50s 98, 28 and 28 ng/ml, respectively). The least active RNase in this assay was rEDN.sub.(1-21) rOnc with an IC.sub.50 of 1600 ng/ml. Placental ribonuclease inhibitor (PRI) binds tightly to EDN and inhibits its enzymatic activity, Sorrentino et al. (1992) J. Biol. Chem 267, 14859-14865, yet nOnc activity is very little affected by PRI, Wu, Y. N., et al. (1993) Journal of Biological Chemistry 268, 10686-10693 and Table 1, despite its homology to EDN and other members of the pancreatic RNase superfamily. In this regard, it is interesting that the activity of rEDN.sub.(1-21) rOnc is, like nOnc, barely affected by PRI while the hybrid RNase with the Gly mutation now behaves more like EDN in that its activity is significantly inhibited (21 fold) by PRI.
The ribonuclease activity of these proteins was also assessed in assays using high and low molecular weight substrates. As shown in Table 2, EDN and nOnc have different substrate specificities consistent with previously published results (Ardelt et al. (1991) J. Biol. Chem. 256, 245-251, Sorrentino et al. (1992) J. Biol. Chem 267, 14859-14865, Ardelt et al. (1994) Protein Sci 3, Suppl. 1, 137). Consistent with the results presented in Table 1, [Met-(-1)]rOnc (SEQ ID NO:39) and rEDN.sub.(1-21) rOnc were much less active with all of the substrates (non detectable or very little activity under the assay conditions employed). Surprisingly, the Gly containing hybrid protein, manifested significant ribonuclease activity especially under conditions optimal for EDN enzymatic activity. EDN is more active at a neutral pH (Sorrentino et al. (1992) J. Biol. Chem 267, 14859-14865) and as seen in Table 2 there is a marked increase in EDN degradation of tRNA at pH 7.5 compared to pH 6 (42.3 fold). Also, behaving like EDN, the Gly-containing hybrid increases in activity with a pH shift from 6 to 7.5 (21.7 fold) while nOnc loses activity at pH 7.5 consistent with its pH optimum that ranges from 6-6.5 (Ardelt et al. (1991) J. Biol. Chem. 256, 245-251, Ardelt et al. (1994) Protein Sci 3, Suppl. 1, 137). The enhanced EDN-like activity of the Gly-containing hybrid protein is also evidenced by its behavior with poly(A,C) which is an excellent substrate for EDN. As seen in Table 2, only rEDN.sub.(1-21) rOncG26 expresses almost 50% of the enzymatic activity of EDN with this substrate whereas the activity of the other RNases are negligible. Similar results were observed with poly(U). In contrast, there was no detectable activity of rEDN or rEDN.sub.(1-21) rOncG26 with UpG, an optimal Onconase substrate (Ardelt et al. (1994) Protein Sci 3, Suppl. 1, 137). In summary, both [Met-(-1)]rOnc and rEDN.sub.(1-21) rOnc are less enzymatically active than nOnc or rEDN. Although, rEDN.sub.(1-21) rOncG26 expresses significant EDN-like enzymatic activity when assayed using defined substrates and conditions optimal for EDN, it is not as active as EDN in any assay. This could result from an impaired enzyme substrate interaction or from the use of suboptimal assay conditions for this hybrid enzyme.
TABLE 1______________________________________Activity of [Met-(-1)]rOnc or Hybrid Proteins in Rabbit ReticulocyteLysate compared to rEDN or nOnc in the Presence or Absence of PRI IC.sub.50.sup.a (ng/ml) (-)PRI (+)PRI Fold Difference______________________________________nOnc 0.2 0.24 1.2rEDN 1.3 >40 >30.7[Met-(-1)]rOnc 96 140 1.4[Met-(-1)]rOncG20 28 24 0.9rEDN.sub.(1-21) rOnc 1600 3200 2rEDN.sub.(1-21) rOncG26 28 600 21______________________________________ .sup.a IC.sub.50 is the concentration of protein necessary to inhibit protein synthesis by 50% in the rabbit reticulocyte lysate. Data points result from the average of at least three assays.
TABLE 2__________________________________________________________________________Activity of RNases on Different Substrates RNase Activity (units/mg protein).sup.aSubstrate Assay pH rEDN nOnc [Met-(-1)]rOnc rEND.sub.1-21r Onc rEDN.sub.1-21r OncG.sub.26__________________________________________________________________________Yeast RNA.sup.a 6.0 6000 560 0.01 8 120tRNA.sup.a,c 6.0 1100 390 12 4 340tRNA.sup.a,c 7.5 46000 60 50 130 7400poly (A,C).sup.b 7.0 8000 0.04 5 4.5 3900UpG.sup.b 6.5 0.05 0.18 <0.01 <0.01 <0.01poly U.sup.b 7.0 16.5 0.15 0.20 0.35 4.5__________________________________________________________________________ .sup.a RNase activity was quantitated through the formation of perchloric acid soluble nucleotides. Units are defined as the changes in A.sub.260 per minute calculated from the slopes of the linear part of the assays. Each value is the average of 2-3 assays in separate experiments. .sup.b Spectrophotometric assays were performed according to Deprisco et al. (1984) and Libonati and Floridi (1969) as described in Materials and Methods. Units are defined as the changes in A.sub.260 per minute calculated from the slopes of the linear part of the assays. Each value i the average of two or more determinatinos. .sup.c [Met(-1)]rOncG20 had no detectable activity.
F. Inhibition of protein synthesis in four human tumor cell lines by RNases. The cytotoxic effect of [Met-(-1)]rOnc and the two hybrid RNases were compared to rEDN and nOnc by determining cell viability using the MTT assay. As depicted in FIG. 3, nOnc decreased tumor cell viability in all four human tumor cell lines. At the concentrations shown, rEDN had no effect on the viability of any of the cell lines. In contrast to nOnc, [Met-(-1)]rOnc as well as [Met-(-1)]rOncG20 was consistently less cytotoxic in all four cell lines. Yet, rEDN.sub.(1-21) rOncG26 was more cytotoxic than nOnc in ACHN, human renal carcinoma cells and equally cytotoxic in the MDA-MB-231 human breast carcinoma cell line. Although rEDN.sub.(1-21) rOncG26 was less active than nOnc in the SF-539 and HS 578T human glioma and breast cancer cell lines, respectively, it was still more active than [Met-(-1)]rOnc or rEDN.sub.(1-21) rOnc protein containing Asn at position 26.
G. Structural Analysis of the hybrid RNases. Modeling the hybrid RNase was based on the alignment of the structures for Onc (Mosimann S. C., Ardelt W., James M. N. G., (1994), Refined 1.7 A X-ray crystallographic structure of P-30 protein, an amphibian ribonuclease with anti-tumor activity (J Mol Biol 236, 1141-1153) and EDN (Mosimann S. C., Newton D. L., Youle R. J., James M., X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83A resolution J Mol Biol). This and subsequent alignments were carried out using ALIGN (Satow Y., Cohen G. H., Padlan E. A., Davies D. R., (1986), J. Mol Biol 190, 593-604).
H. Modeling the structures of the hybrid RNases. The coordinates for Onc and EDN were superimposed on the basis of C.sup..alpha. trace alignment. Residues in conserved zones, particularly in the active site, showed very little displacement when comparing both structures (global r.m.s.d. of 1.44 A for 90 C.sup..alpha. atom pairs). The hybrid protein was modeled by manual rebuilding and geometry regularization using TOM (Cambillau C., Horjales E., (1987), J. Mol Graph 5, 174-177). Subsequently, the models for rEDN.sub.(1-21) rOnc and rEDN.sub.(1-21) rOncG26 were assigned an overall B-factor of 15 A.sup.2 for all non-hydrogen atoms and independently subject to 300 cycles of positional energy minimization with the program XPLOR (Brunger A. (1992) XPLOR: a system for X-ray crystallography and NMR., New Haven: Yale University Press). The minimization yielded virtually identical structures in both cases, the highest distance based on C.sup..alpha. trace alignment being 0.44 for the C.sup..alpha. of the mutated residue 26. The geometry quality of the final models were assessed with PROCHECK (Laskowski R. A., MacArthur M. W., Moss D. S., Thornton J. M., (1993), J Appl Crystallogr 26, 283-291).
The structural basis for the marked differences in activity between the Gly and Asp containing hybrid RNases are not obvious from modeling these proteins especially since residue 26 is distant from the active site. When the highly homologous structure of RNase A complexed with a pentanucleotide (Fontecilla-Camps J. C., deLorens R., leDu M. H., Cuchillo C. M., (1994), J. Biol Chem 269, 21526-21531) was superimposed on the structure of the hybrid protein model, the nucleotide was observed also to be distant from the region of the mutation. However, the arrangement of the polynucleotide chain in the different RNases does not necessarily have to coincide. In the structure of EDN, a second sulfate ion was found in addition to the one in the active site (Mosimann S. C., Newton D. L., Youle R. J., James M., X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83A resolution J Mol Biol). This second sulfate is likely replacing a phosphate from the nucleotide to be cleaved, but no phosphate ion is located in the equivalent position in the RNase A-pentanucleotide complex. Moreover, one of the phosphates in this complex is forming a salt bridge with Lys-66, a residue which has no counterpart in Onc since it is located in a loop with a different topology in both molecules. Thus, whether the difference in enzymatic activity between the Asp and Gly mutants in the chimera is related to a change in the binding affinity for the substrate remains an open question.
Although the structural basis for the difference in the activities of the two EDN-Onc hybrids is not clear, the EDN-like behavior of the rEDN.sub.(1-21) rOncG26 hybrid can likely be attributed to the configuration of the N-terminal region since both the pyroglutamic acid in nOnc and Lys-1 in EDN are located in the area of the active site (Mosimann S. C., Ardelt W., James M. N. G., (1994), Refined 1.7 A X-ray crystallographic structure of P-30 protein, an amphibian ribonuclease with anti-tumor activity J Mol Biol 236, 1141-1153; Mosimann S. C., Newton D. L., Youle R. J., James M., X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83A resolution J Mol Biol). In addition, the introduction of a Gly mutation in [Met-(-1)]rOnc did not significantly affect enzymatic activity. The preference of U over C in the B1 subsite of RNase A has been related to the presence of a particular residue (Asp-83) (DelCardayre S. B., Raines R. T., (1995), A residue to residue hydrogen bond mediates the nucleotide specificity of ribonuclease A J Mol Biol 252, 328-336). The corresponding residue in nOnc is also an aspartic acid (Asp-67), while in EDN this position is occupied by a histidine (His-82). EDN is more active toward poly (A,C), suggesting that it "prefers" C in the B1 subsite, possibly because it contains a histidine residue as opposed to the aspartic acid in nOnc and RNase A. Taken together, this could explain the decreased activity of the Gly containing hybrid relative to rEDN since, according to this hypothesis, the presence of the Asp residue contributed by the rOnc sequence would favor the binding of U over C. With regard to the difference in PRI inhibition, the superposition between the hybrid proteins and RNase A demonstrates that Asp-26 in the EDN-Onc chimeras is in the equivalent position to Asn-27 in RNase A that has been reported to be in contact with PRI (Kobe B., Deisenhofer J., (1995), Nature 374, 183-186). In addition, Asp-24 in both chimeras is very close to this region. Thus, the accumulation of negative charges in this area could prevent binding by the inhibitor. If so, the substitution of Gly for Asp would decrease the negative charge and restore the binding capacity.
Example II
rOnc-Antibody Fusion Proteins
Additional rOnc-antibody and ligand proteins have been produced and are highly active. E6FB[Met-(-1)]SerrOnc is an rOnc molecule having the amino acid sequence set out in SEQ ID NO:64 and includes the Fv sequence from antibody E6, an anti-transferrin receptor antibody. See sequences for E6 at amino acid positions 1-237 in SEQ ID NO:41. "FB" refers to a linker used to link the antibody and the rOnc portion of the molecule and is found at nucleic acid positions 712 through 750 in SEQ ID NO:40. E6FB[Met-(-1)]SerrOnc (SEQ ID NO:64) includes a Ser at amino acid position 252 instead of a Glu. Similar hybrid molecules have been made. The nucleic acid and amino acid sequences for Met-NLS(signal peptide)-Gln-rOncFBE6 are set out on SEQ ID NOS:42 and 43. Another E6/rOnc molecule is designated Met-Ser-rOncA87FBE6 and is found on SEQ ID NOS:44 and 45. "A87" refers to the fact that an Ala occurs at amino acid position 87.
Met-Ser-rOnc-Ang-FBE6 is set out on SEQ ID NOS:46 and 47.
E6FBMet-Ser-rOnc is set out on SEQ ID NOS:48 and 49.
Met-Glu-rOncFBE6 is set out on SEQ ID NOS:50 and 51.
Met-Ser-rOncFBE6 is set out on SEQ ID NOS:50 and 51, with the exception that Ser replaces Glu at amino acid position 2.
MOC31 and MOC162 refer to anti-colon cancer antibodies directed against the 17-1-A pancarcinoma antigen which were obtained from Dr. Hennie Hoogenboom. The Fv region of these antibodies was fused to rOnc. The nucleic acid and amino acid sequences for MetSerrOnc A87 FBMOC31 are set out on SEQ ID NOS:52 and 53. The nucleic acid and amino acid sequences for MOC31FBMetSerrOnc are set out on SEQ ID NOS:54 and 55. The nucleic acid and amino acid sequences for MetSerrOncFBMOC161 are set out on SEQ ID NOS:56 and 57.
The ligand, IL2 (interleukin 2) was recombinantly fused to rOnc as well. See SEQ ID NOS:58 and 59 for IL2FBMetSerrOnc. See SEQ ID NOS:60 and 61 for MetSerrOncFBIL2.
Inhibition of protein synthesis in SF539 cells (which bear the transferrin receptor) was measured, as described above, for [Met-(-1)Ser]rOnc, E6FB[Met-(-1)Ser]rOnc; [Met-(-1)Ser]rOnc-AngFBE6 and [Met-(-1)Glu]rOncFBE6 constructs and compared with nOnc. The results are shown on Table 3. The three E6 constructs, in particular, had a very high level of activity--up to 45 fold difference over the two non-E6 molecules. See also FIG. 5. MetSerOncAng molecule was made corresponding to amino acids 1-107 of SEQ ID NO:47.
TABLE 3______________________________________Activity of modified rOnc and modified rOncFvs on protein synthesisRNase IC.sub.50 (nM) Fold Difference______________________________________nOnc 10 1[Met-(-1)Ser]rOnc 8 NSDE6FB[Met-(-1)Ser]rOnc 0.22 45[Met-(-1)Ser]rOnc-AngFBE6 0.27 37[Met-(-1)Glu]rOncFBE6 0.50 20______________________________________ The concentrations necessary to inhibit protein synthesis by 50% in SF539 human glioma cells. NSD, no significant difference.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 64- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 104 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..104#/label= nOnc OTHER INFORMATION:#"native ONCONASE (Registered#from Rana pipiens"emark)- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 1#/note= "Xaa = pyroglutamic acid"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- Xaa Asp Trp Leu Thr Phe Gln Lys Lys His Il - #e Thr Asn Thr Arg Asp# 15- Val Asp Cys Asp Asn Ile Met Ser Thr Asn Le - #u Phe His Cys Lys Asp# 30- Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pr - #o Val Lys Ala Ile Cys# 45- Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Th - #r Thr Ser Glu Phe Tyr# 60- Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cy - #s Lys Tyr Lys Leu Lys#80- Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Gl - #u Asn Gln Ala Pro Val# 95- His Phe Val Gly Val Gly Ser Cys 100- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 83 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..83#/note= "Rana clone 9 peptide from Rana pipiens g - #enomic DNA"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 15- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 30- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 45- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 60- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro#80- Val His Phe- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..28#/note= "N-terminal sequence of nOnc#in place of pyroglutamic acid in position 1"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- Glu Asp Trp Leu Thr Phe Gln Lys Lys His Il - #e Thr Asn Thr Arg Asp# 15- Val Asp Cys Asp Asn Ile Met Ser Thr Asn Le - #u Phe# 25- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 34 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..34#/note= "N-terminal sequence of:#eosinophil-derivedmbinant#(rEDN)" neurotoxin- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Ph - #e Glu Thr Gln His Ile# 15- Asn Met Thr Ser Gln Gln Cys Thr Asn Ala Me - #t Gln Val Ile Asn Asn# 30- Tyr Gln- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..28#/note= "N-terminal sequence of: [Met-(-1)]- #rOncG20, containing a Gly to Asp subst - #itution at position 20 of [Met-(-1)]- #rOnc, and without the extra#Met from the E. coliinal#expression system"erial- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Glu Asp Trp Leu Thr Phe Gln Lys Lys His Il - #e Thr Asn Thr Arg Asp# 15- Val Asp Cys Gly Asn Ile Met Ser Thr Asn Le - #u Phe# 25- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 34 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..34#/note= "N-terminal sequence of: rEDN1-21rOnc - #, without the extra#Met from the E. coliinal#expression system"erial- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Ph - #e Glu Thr Gln His Ile# 15- Asn Met Thr Ser Gln Asp Val Asp Cys Asp As - #n Ile Met Ser Thr Asn# 30- Leu Phe- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 34 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..34#/note= "N-terminal sequence of: rEDN1-21rOnc - #G26, containing a Gly to Asp substitution - # at position 26 of rEDN1-21rOnc - #, and without the extra#Met from the E. coliinal#expression system"erial- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Ph - #e Glu Thr Gln His Ile# 15- Asn Met Thr Ser Gln Asp Val Asp Cys Gly As - #n Ile Met Ser Thr Asn# 30- Leu Phe- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 111 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..111#/note= "Frog Lectin from RanaN: catesbeiana"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- Glu Asn Trp Ala Thr Phe Gln Gln Lys His Il - #e Ile Asn Thr Pro Ile# 15- Ile Asn Cys Asn Thr Ile Met Asp Asn Asn Il - #e Tyr Ile Val Gly Gly# 30- Gln Cys Lys Arg Val Asn Thr Phe Ile Ile Se - #r Ser Ala Thr Thr Val# 45- Lys Ala Ile Cys Thr Gly Val Ile Asn Met As - #n Val Leu Ser Thr Thr# 60- Arg Phe Gln Leu Asn Thr Cys Thr Arg Thr Se - #r Ile Thr Pro Arg Pro#80- Cys Pro Tyr Ser Ser Arg Thr Glu Thr Asn Ty - #r Ile Cys Val Lys Cys# 95- Glu Asn Gln Tyr Pro Val His Phe Ala Gly Il - #e Gly Arg Cys Pro# 110- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 134 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..134#/note= "Human eosinophil-derived#(EDN)" neurotoxin- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Ph - #e Glu Thr Gln His Ile# 15- Asn Met Thr Ser Gln Gln Cys Thr Asn Ala Me - #t Gln Val Ile Asn Asn# 30- Tyr Gln Arg Arg Cys Lys Asn Gln Asn Thr Ph - #e Leu Leu Thr Thr Phe# 45- Ala Asn Val Val Asn Val Cys Gly Asn Pro As - #n Met Thr Cys Pro Ser# 60- Asn Lys Thr Arg Lys Asn Cys His His Ser Gl - #y Ser Gln Val Pro Leu#80- Ile His Cys Asn Leu Thr Thr Pro Ser Pro Gl - #n Asn Ile Ser Asn Cys# 95- Arg Tyr Ala Gln Thr Pro Ala Asn Met Phe Ty - #r Ile Val Ala Cys Asp# 110- Asn Arg Asp Gln Arg Arg Asp Pro Pro Gln Ty - #r Pro Val Val Pro Val# 125- His Leu Asp Arg Ile Ile 130- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 133 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..133#/note= "Human eosinophil cationic protein ( - #ECP)"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Arg Pro Pro Gln Phe Thr Arg Ala Gln Trp Ph - #e Ala Ile Gln His Ile# 15- Ser Leu Asn Pro Pro Arg Cys Thr Ile Ala Me - #t Arg Ala Ile Asn Asn# 30- Tyr Arg Trp Arg Cys Lys Asn Gln Asn Thr Ph - #e Leu Arg Thr Thr Phe# 45- Ala Asn Val Val Asn Val Cys Gly Asn Gln Se - #r Ile Arg Cys Pro His# 60- Asn Arg Thr Leu Asn Asn Cys His Arg Ser Ar - #g Phe Arg Val Pro Leu#80- Leu His Cys Asp Leu Ile Asn Pro Gly Ala Gl - #n Asn Ile Ser Asn Cys# 95- Arg Tyr Ala Asp Arg Pro Gly Arg Arg Phe Ty - #r Val Val Ala Cys Asp# 110- Asn Arg Asp Pro Arg Asp Ser Pro Arg Tyr Pr - #o Val Val Pro Val His# 125- Leu Asp Thr Thr Ile 130- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 125 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..125#/note= "Bovine angiogenin (Ang)"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- Ala Gln Asp Asp Tyr Arg Tyr Ile His Phe Le - #u Thr Gln His Tyr Asp# 15- Ala Lys Pro Lys Gly Arg Asn Asp Glu Tyr Cy - #s Phe His Met Met Lys# 30- Asn Arg Arg Leu Thr Arg Pro Cys Lys Asp Ar - #g Asn Thr Phe Ile His# 45- Gly Asn Lys Asn Asp Ile Lys Ala Ile Cys Gl - #u Asp Arg Asn Gly Gln# 60- Pro Tyr Arg Gly Asp Leu Arg Ile Ser Lys Se - #r Glu Phe Gln Ile Thr#80- Ile Cys Lys His Lys Gly Gly Ser Ser Arg Pr - #o Pro Cys Arg Tyr Gly# 95- Ala Thr Glu Asp Ser Arg Val Ile Val Val Gl - #y Cys Glu Asn Gly Leu# 110- Pro Val His Phe Asp Glu Ser Phe Ile Thr Pr - #o Arg His# 125- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 124 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..124#/note= "Bovine seminal RNase"N:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:- Lys Glu Ser Ala Ala Ala Lys Phe Glu Arg Gl - #n His Met Asp Ser Gly# 15- Asn Ser Pro Ser Ser Ser Ser Asn Tyr Cys As - #n Leu Met Met Cys Cys# 30- Arg Lys Met Thr Gln Gly Lys Cys Lys Pro Va - #l Asn Thr Phe Val His# 45- Glu Ser Leu Ala Asp Val Lys Ala Val Cys Se - #r Gln Lys Lys Val Thr# 60- Cys Lys Asn Gly Gln Thr Asn Cys Tyr Gln Se - #r Lys Ser Thr Met Arg#80- Ile Thr Asp Cys Arg Glu Thr Gly Ser Ser Ly - #s Tyr Pro Asn Cys Ala# 95- Tyr Lys Thr Thr Gln Val Glu Lys His Ile Il - #e Val Ala Cys Gly Gly# 110- Lys Pro Ser Val Pro Val His Phe Asp Ala Se - #r Val# 120- (2) INFORMATION FOR SEQ ID NO:13:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 124 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..124#/note= "Bovine pancreatic RNase A"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:- Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gl - #n His Met Asp Ser Ser# 15- Thr Ser Ala Ala Ser Ser Ser Asn Tyr Cys As - #n Gln Met Met Lys Ser# 30- Arg Asn Leu Thr Lys Asp Arg Cys Lys Pro Va - #l Asn Thr Phe Val His# 45- Glu Ser Leu Ala Asp Val Gln Ala Val Cys Se - #r Gln Lys Asn Val Ala# 60- Cys Lys Asn Gly Gln Thr Asn Cys Tyr Gln Se - #r Tyr Ser Thr Met Ser#80- Ile Thr Asp Cys Arg Glu Thr Gly Ser Ser Ly - #s Tyr Pro Asn Cys Ala# 95- Tyr Lys Thr Thr Gln Ala Asn Lys His Ile Il - #e Val Ala Cys Glu Gly# 110- Asn Pro Val Val Pro Val His Phe Asp Ala Se - #r Val# 120- (2) INFORMATION FOR SEQ ID NO:14:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 3#/note= "Xaa = Ser, Tyr or Thr":- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:- Met Lys Xaa Pro- (2) INFORMATION FOR SEQ ID NO:15:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 4#/note= "Xaa = Ser, Tyr or Thr":- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:- Met Lys Pro Xaa1- (2) INFORMATION FOR SEQ ID NO:16:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 3#/note= "Xaa = Ser, Tyr or Thr":- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:- Met Asn Xaa Pro1- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "Xaa = Ser, Tyr or Thr":- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:- Met Xaa Lys Pro1- (2) INFORMATION FOR SEQ ID NO:18:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "Xaa = Ser, Tyr or Thr":- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:- Met Xaa Pro Lys1- (2) INFORMATION FOR SEQ ID NO:19:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 321 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..321#/note= "MetSerOnc99Ang117"TION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- ATC TCA GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Ile Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT TGT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95# 321T GTT CAG TCA ATT TTC CGT CGT CC - #GVal His Phe Val Gln Ser Ile Phe Arg Arg Pr - #o# 105- (2) INFORMATION FOR SEQ ID NO:20:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 107 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:- Ile Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gln Ser Ile Phe Arg Arg Pr - #o# 105- (2) INFORMATION FOR SEQ ID NO:21:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 333 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..333#/note= "EDNGlyOnc" INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:- ATG AAA CCG CCG CAG TTC ACT TGG GCT CAG TG - #G TTC GAA ACT CAG CAT 48Met Lys Pro Pro Gln Phe Thr Trp Ala Gln Tr - #p Phe Glu Thr Gln His# 15- ATC AAC ATG ACT TCT CAG GAT GTT GAT TGT GG - #T AAT ATC ATG TCA ACA 96Ile Asn Met Thr Ser Gln Asp Val Asp Cys Gl - #y Asn Ile Met Ser Thr# 30- AAC TTG TTC CAC TGC AAG GAC AAG AAC ACT TT - #T ATC TAT TCA CGT CCT 144Asn Leu Phe His Cys Lys Asp Lys Asn Thr Ph - #e Ile Tyr Ser Arg Pro# 45- GAG CCA GTG AAG GCC ATC TGT AAA GGA ATT AT - #A GCC TCC AAA AAT GTG 192Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Il - #e Ala Ser Lys Asn Val# 60- TTA ACT ACC TCT GAG TTT TAT CTC TCT GAT TG - #C AAT GTA ACA AGC AGG 240Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cy - #s Asn Val Thr Ser Arg# 80- CCT TGC AAG TAT AAA TTA AAG AAA TCA ACT AA - #T AAA TTT TGT GTA ACT 288Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr As - #n Lys Phe Cys Val Thr# 95- TGT GAA AAT CAG GCA CCA GTT CAT TTT GTT GG - #A GTT GGA TCT TGT 33 - #3Cys Glu Asn Gln Ala Pro Val His Phe Val Gl - #y Val Gly Ser Cys# 110- (2) INFORMATION FOR SEQ ID NO:22:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 111 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:- Met Lys Pro Pro Gln Phe Thr Trp Ala Gln Tr - #p Phe Glu Thr Gln His# 15- Ile Asn Met Thr Ser Gln Asp Val Asp Cys Gl - #y Asn Ile Met Ser Thr# 30- Asn Leu Phe His Cys Lys Asp Lys Asn Thr Ph - #e Ile Tyr Ser Arg Pro# 45- Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Il - #e Ala Ser Lys Asn Val# 60- Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cy - #s Asn Val Thr Ser Arg# 80- Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr As - #n Lys Phe Cys Val Thr# 95- Cys Glu Asn Gln Ala Pro Val His Phe Val Gl - #y Val Gly Ser Cys# 110- (2) INFORMATION FOR SEQ ID NO:23:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 315 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..315#/note= "MetTyrrOnc"INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:- ATG TAT GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Tyr Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ACT TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT TGT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95# 315 GA GTT GGA TCT TGTVal His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:24:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 105 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:- Met Tyr Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:25:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 315 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..315#/note= "MetSerrOnc"INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:- ATG TCA GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ACT TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT TGT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95# 315 GA GTT GGA TCT TGTVal His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:26:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 105 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:- Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:27:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 318 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..318#/note= "MetLysTyrrOnc"ORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:- ATG AAA TAT GAT TGG CTT ACA TTT CAG AAA AA - #A CAC ATC ACA AAC ACA 48Met Lys Tyr Asp Trp Leu Thr Phe Gln Lys Ly - #s His Ile Thr Asn Thr# 15- AGG GAT GTT GAT TGT GAT AAT ATC ATG TCA AC - #A AAC TTG TTC CAC TGC 96Arg Asp Val Asp Cys Asp Asn Ile Met Ser Th - #r Asn Leu Phe His Cys# 30- AAG GAC AAG AAC ACT TTT ATC TAT TCA CGT CC - #T GAG CCA GTG AAG GCC 144Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pr - #o Glu Pro Val Lys Ala# 45- ATC TGT AAA GGA ATT ATA GCC TCC AAA AAT GT - #G TTA ACT ACC TCT GAG 192Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Va - #l Leu Thr Thr Ser Glu# 60- TTT TAT CTC TCT GAT TGC AAT GTA ACA AGC AG - #G CCT TGC AAG TAT AAA 240Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Ar - #g Pro Cys Lys Tyr Lys# 80- TTA AAG AAA TCA ACT AAT AAA TTT TGT GTA AC - #T TGT GAA AAT CAG GCA 288Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Th - #r Cys Glu Asn Gln Ala# 95# 318 TT GGA GTT GGA TCT TGTPro Val His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:28:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 106 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:- Met Lys Tyr Asp Trp Leu Thr Phe Gln Lys Ly - #s His Ile Thr Asn Thr# 15- Arg Asp Val Asp Cys Asp Asn Ile Met Ser Th - #r Asn Leu Phe His Cys# 30- Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pr - #o Glu Pro Val Lys Ala# 45- Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Va - #l Leu Thr Thr Ser Glu# 60- Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Ar - #g Pro Cys Lys Tyr Lys# 80- Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Th - #r Cys Glu Asn Gln Ala# 95- Pro Val His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:29:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 321 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..321#/note= "MetAlaAlaTyrrOnc"ATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:- ATG GCT GCT TAT GAT TGG CTT ACA TTT CAG AA - #A AAA CAC ATC ACA AAC 48Met Ala Ala Tyr Asp Trp Leu Thr Phe Gln Ly - #s Lys His Ile Thr Asn# 15- ACA AGG GAT GTT GAT TGT GAT AAT ATC ATG TC - #A ACA AAC TTG TTC CAC 96Thr Arg Asp Val Asp Cys Asp Asn Ile Met Se - #r Thr Asn Leu Phe His# 30- TGC AAG GAC AAG AAC ACT TTT ATC TAT TCA CG - #T CCT GAG CCA GTG AAG 144Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Ar - #g Pro Glu Pro Val Lys# 45- GCC ATC TGT AAA GGA ATT ATA GCC TCC AAA AA - #T GTG TTA ACT ACC TCT 192Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys As - #n Val Leu Thr Thr Ser# 60- GAG TTT TAT CTC TCT GAT TGC AAT GTA ACA AG - #C AGG CCT TGC AAG TAT 240Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Se - #r Arg Pro Cys Lys Tyr# 80- AAA TTA AAG AAA TCA ACT AAT AAA TTT TGT GT - #A ACT TGT GAA AAT CAG 288Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Va - #l Thr Cys Glu Asn Gln# 95# 321T CAT TTT GTT GGA GTT GGA TCT TG - #TAla Pro Val His Phe Val Gly Val Gly Ser Cy - #s# 105- (2) INFORMATION FOR SEQ ID NO:30:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 107 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:- Met Ala Ala Tyr Asp Trp Leu Thr Phe Gln Ly - #s Lys His Ile Thr Asn# 15- Thr Arg Asp Val Asp Cys Asp Asn Ile Met Se - #r Thr Asn Leu Phe His# 30- Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Ar - #g Pro Glu Pro Val Lys# 45- Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys As - #n Val Leu Thr Thr Ser# 60- Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Se - #r Arg Pro Cys Lys Tyr# 80- Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Va - #l Thr Cys Glu Asn Gln# 95- Ala Pro Val His Phe Val Gly Val Gly Ser Cy - #s# 105- (2) INFORMATION FOR SEQ ID NO:31:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 336 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..336#/note= "NLSMetSerrOnc"ORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:- CCC AAG AAG AAG CGG AAG GTG ATG TCA GAT TG - #G CTT ACA TTT CAG AAA 48Pro Lys Lys Lys Arg Lys Val Met Ser Asp Tr - #p Leu Thr Phe Gln Lys# 15- AAA CAC ATC ACA AAC ACA AGG GAT GTT GAT TG - #T GAT AAT ATC ATG TCA 96Lys His Ile Thr Asn Thr Arg Asp Val Asp Cy - #s Asp Asn Ile Met Ser# 30- ACA AAC TTG TTC CAC TGC AAG GAC AAG AAC AC - #T TTT ATC TAT TCA CGT 144Thr Asn Leu Phe His Cys Lys Asp Lys Asn Th - #r Phe Ile Tyr Ser Arg# 45- CCT GAG CCA GTG AAG GCC ATC TGT AAA GGA AT - #T ATA GCC TCC AAA AAT 192Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Il - #e Ile Ala Ser Lys Asn# 60- GTG TTA ACT ACC TCT GAG TTT TAT CTC TCT GA - #T TGC AAT GTA ACA AGC 240Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser As - #p Cys Asn Val Thr Ser# 80- AGG CCT TGC AAG TAT AAA TTA AAG AAA TCA AC - #T AAT AAA TTT TGT GTA 288Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Th - #r Asn Lys Phe Cys Val# 95- ACT TGT GAA AAT CAG GCA CCA GTT CAT TTT GT - #T GGA GTT GGA TCT TGT 336Thr Cys Glu Asn Gln Ala Pro Val His Phe Va - #l Gly Val Gly Ser Cys# 110- (2) INFORMATION FOR SEQ ID NO:32:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 112 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:- Pro Lys Lys Lys Arg Lys Val Met Ser Asp Tr - #p Leu Thr Phe Gln Lys# 15- Lys His Ile Thr Asn Thr Arg Asp Val Asp Cy - #s Asp Asn Ile Met Ser# 30- Thr Asn Leu Phe His Cys Lys Asp Lys Asn Th - #r Phe Ile Tyr Ser Arg# 45- Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Il - #e Ile Ala Ser Lys Asn# 60- Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser As - #p Cys Asn Val Thr Ser# 80- Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Th - #r Asn Lys Phe Cys Val# 95- Thr Cys Glu Asn Gln Ala Pro Val His Phe Va - #l Gly Val Gly Ser Cys# 110- (2) INFORMATION FOR SEQ ID NO:33:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "Xaa = an aliphatic amino acid, Ala, Leu, - # Ile, Val, or Pro"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 3#/note= "Xaa = an aliphatic amino acid, Ala, Leu, - # Ile, Val or Pro"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 4#/note= "Xaa = Ser, Met, Cys, Ala or Gln"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:- Cys Xaa Xaa Xaa1- (2) INFORMATION FOR SEQ ID NO:34:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:- Cys Val Ile Met1- (2) INFORMATION FOR SEQ ID NO:35:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 27 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:# 27 ATAA YATCATG- (2) INFORMATION FOR SEQ ID NO:36:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 27 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:# 27 CWGT KCAYTTT- (2) INFORMATION FOR SEQ ID NO:37:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 249 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: 1..249#/note= "Rana 9"HER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:- GATGTTGATT GTGATAATAT CATGTCAACA AACTTGTTCC ACTGCAAGGA CA - #AGAACACT 60- TTTATCTATT CACGTCCTGA GCCAGTGAAG GCCATCTGTA AAGGAATTAT AG - #CCTCCAAA 120- AATGTGTTAA CTACCTCTGA GTTTTATCTC TCTGATTGCA ATGTAACAAG CA - #GGCCTTGC 180- AAGTATAAAT TAAAGAAATC AACTAATAAA TTTTGTGTAA CTTGTGAAAA TC - #AGGCACCA 240# 249- (2) INFORMATION FOR SEQ ID NO:38:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 315 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..315#/note= "[Met-(-1)]rOnc"RMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:- ATG GAG GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Glu Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT TGT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95# 315 GA GTT GGA TCT TGTVal His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:39:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 105 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:- Met Glu Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys# 105- (2) INFORMATION FOR SEQ ID NO:40:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1065 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1065#/note= "sFvFBMetGluOnc"RMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:- GAC ATC AAG ATG ACC CAG TCT CCA TCT TCC AT - #G TAT GCA TCT CTA GGA 48Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Me - #t Tyr Ala Ser Leu Gly# 15- GAG AGA GTC ACT TTC ACT TGC AAG GCG AGT CA - #G GAC ATT AAT AAC TAT 96Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gl - #n Asp Ile Asn Asn Tyr# 30- TTA TGC TGG TTC CAG CAG AAA CCA GGG AAA TC - #T CCT AAG ACC CTG ATC 144Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Se - #r Pro Lys Thr Leu Ile# 45- TAT CGT GCA AAC AGA CTG GTA GAT GGG GTC CC - #A TCA AGG TTC AGT GGC 192Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pr - #o Ser Arg Phe Ser Gly# 60- AGT GGA TCT GGA CAA GAT TAT TCT CTC ACC AT - #T AGC AGC CTG GAG TAT 240Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Il - #e Ser Ser Leu Glu Tyr# 80- GAA GAT ATG GGA ATT TAT TAT TGT CTA CAG TA - #T GAT GAG TTT CCG TAC 288Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Ty - #r Asp Glu Phe Pro Tyr# 95- ACG TTC GGA GGG GGG ACC AAG CTG GAA ATA AA - #A GGA GGC GGT GGC TCG 336Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ly - #s Gly Gly Gly Gly Ser# 110- GGC GGT GGC GGA TCG GGT GGC GGC GGC TCT GA - #G GTT CAG CTC CAG CAG 384Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gl - #u Val Gln Leu Gln Gln# 125- TCT GGG ACT GTA CTG GCA AGG CCT GGG GCT TC - #A GTG AAG ATG TCC TGC 432Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Se - #r Val Lys Met Ser Cys# 140- AAG GCT TCT GGC TAC ACC TTT TCC AGC TAC TG - #G ATG CAC TGG ATA AAA 480Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Tr - #p Met His Trp Ile Lys145 1 - #50 1 - #55 1 -#60- CAG AGG CCT GGA CAG GGT CTG GAC TGG ATT GT - #C GCT ATT GAT CCT CGA 528Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Va - #l Ala Ile Asp Pro Arg# 175- AAT AGT GAT ACT ATT TAC AAC CCG CAA TTC AA - #A CAC AAG GCC AAA CTG 576Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Ly - #s His Lys Ala Lys Leu# 190- ACT GCA GTC ACC TCC ACC AGC ACT GCC TAC AT - #G GAA CTC AAC AGC CTG 624Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Me - #t Glu Leu Asn Ser Leu# 205- ACA AAT GAG GAC TCT GCG GTC TAT TAC TGT AC - #C CCT CTT TAT TAC TTT 672Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Th - #r Pro Leu Tyr Tyr Phe# 220- GAC TCC TGG GGC CAA GGC ACC ACT CTC ACA GT - #C TCC TCA GCC AAG AAA 720Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Va - #l Ser Ser Ala Lys Lys225 2 - #30 2 - #35 2 -#40- CTG AAC GAC GCT CAG GCG CCG AAG AGT GAT AT - #G GAG GAT TGG CTT ACA 768Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Me - #t Glu Asp Trp Leu Thr# 255- TTT CAG AAA AAA CAC ATC ACA AAC ACA AGG GA - #T GTT GAT TGT GAT AAT 816Phe Gln Lys Lys His Ile Thr Asn Thr Arg As - #p Val Asp Cys Asp Asn# 270- ATC ATG TCA ACA AAC TTG TTC CAC TGC AAG GA - #C AAG AAC ACT TTT ATC 864Ile Met Ser Thr Asn Leu Phe His Cys Lys As - #p Lys Asn Thr Phe Ile# 285- TAT TCA CGT CCT GAG CCA GTG AAG GCC ATC TG - #T AAA GGA ATT ATA GCC 912Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cy - #s Lys Gly Ile Ile Ala# 300- TCC AAA AAT GTG TTA ACT ACC TCT GAG TTT TA - #T CTC TCT GAT TGC AAT 960Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Ty - #r Leu Ser Asp Cys Asn305 3 - #10 3 - #15 3 -#20- GTA ACA AGC AGG CCT TGC AAG TAT AAA TTA AA - #G AAA TCA ACT AAT AAA1008Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Ly - #s Lys Ser Thr Asn Lys# 335- TTT TGT GTA ACT TGT GAA AAT CAG GCA CCA GT - #T CAT TTT GTT GGA GTT1056Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Va - #l His Phe Val Gly Val# 350# 1065Gly Ser Cys 355- (2) INFORMATION FOR SEQ ID NO:41:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 355 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:- Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Me - #t Tyr Ala Ser Leu Gly# 15- Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gl - #n Asp Ile Asn Asn Tyr# 30- Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Se - #r Pro Lys Thr Leu Ile# 45- Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pr - #o Ser Arg Phe Ser Gly# 60- Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Il - #e Ser Ser Leu Glu Tyr# 80- Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Ty - #r Asp Glu Phe Pro Tyr# 95- Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ly - #s Gly Gly Gly Gly Ser# 110- Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gl - #u Val Gln Leu Gln Gln# 125- Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Se - #r Val Lys Met Ser Cys# 140- Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Tr - #p Met His Trp Ile Lys145 1 - #50 1 - #55 1 -#60- Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Va - #l Ala Ile Asp Pro Arg# 175- Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Ly - #s His Lys Ala Lys Leu# 190- Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Me - #t Glu Leu Asn Ser Leu# 205- Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Th - #r Pro Leu Tyr Tyr Phe# 220- Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Va - #l Ser Ser Ala Lys Lys225 2 - #30 2 - #35 2 -#40- Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Me - #t Glu Asp Trp Leu Thr# 255- Phe Gln Lys Lys His Ile Thr Asn Thr Arg As - #p Val Asp Cys Asp Asn# 270- Ile Met Ser Thr Asn Leu Phe His Cys Lys As - #p Lys Asn Thr Phe Ile# 285- Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cy - #s Lys Gly Ile Ile Ala# 300- Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Ty - #r Leu Ser Asp Cys Asn305 3 - #10 3 - #15 3 -#20- Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Ly - #s Lys Ser Thr Asn Lys# 335- Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Va - #l His Phe Val Gly Val# 350- Gly Ser Cys 355- (2) INFORMATION FOR SEQ ID NO:42:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1137 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1137#/note= "SigPepGlnOncFBE6"ATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:- ATG GGT CTG GAA AAA TCT CTG ATC CTG TTC CC - #G CTG TTC TTC CTG CTG 48Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pr - #o Leu Phe Phe Leu Leu# 15- CTG GGT TGG GTT CAG CCG TCT CTG GGT CAG GA - #T TGG CTT ACA TTT CAG 96Leu Gly Trp Val Gln Pro Ser Leu Gly Gln As - #p Trp Leu Thr Phe Gln# 30- AAA AAA CAC ATC ACA AAC ACA AGG GAT GTT GA - #T TGT GAT AAT ATC ATG 144Lys Lys His Ile Thr Asn Thr Arg Asp Val As - #p Cys Asp Asn Ile Met# 45- TCA ACA AAC TTG TTC CAC TGC AAG GAC AAG AA - #C ACT TTT ATC TAT TCA 192Ser Thr Asn Leu Phe His Cys Lys Asp Lys As - #n Thr Phe Ile Tyr Ser# 60- CGT CCT GAG CCA GTG AAG GCC ATC TGT AAA GG - #A ATT ATA GCC TCC AAA 240Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gl - #y Ile Ile Ala Ser Lys# 80- AAT GTG TTA ACT ACC TCT GAG TTT TAT CTC TC - #T GAT TGC AAT GTA ACA 288Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Se - #r Asp Cys Asn Val Thr# 95- AGC AGG CCT TGC AAG TAT AAA TTA AAG AAA TC - #A ACT AAT AAA TTT TGT 336Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Se - #r Thr Asn Lys Phe Cys# 110- GTA ACT TGT GAA AAT CAG GCA CCA GTT CAT TT - #T GTT GGA GTT GGA TCT 384Val Thr Cys Glu Asn Gln Ala Pro Val His Ph - #e Val Gly Val Gly Ser# 125- TGT GCC AAG AAA CTG AAC GAC GCT CAG GCG CC - #G AAG AGT GAT GAC ATC 432Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pr - #o Lys Ser Asp Asp Ile# 140- AAG ATG ACC CAG TCT CCA TCT TCC ATG TAT GC - #A TCT CTA GGA GAG AGA 480Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Al - #a Ser Leu Gly Glu Arg145 1 - #50 1 - #55 1 -#60- GTC ACT TTC ACT TGC AAG GCG AGT CAG GAC AT - #T AAT AAC TAT TTA TGC 528Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Il - #e Asn Asn Tyr Leu Cys# 175- TGG TTC CAG CAG AAA CCA GGG AAA TCT CCT AA - #G ACC CTG ATC TAT CGT 576Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Ly - #s Thr Leu Ile Tyr Arg# 190- GCA AAC AGA CTG GTA GAT GGG GTC CCA TCA AG - #G TTC AGT GGC AGT GGA 624Ala Asn Arg Leu Val Asp Gly Val Pro Ser Ar - #g Phe Ser Gly Ser Gly# 205- TCT GGA CAA GAT TAT TCT CTC ACC ATT AGC AG - #C CTG GAG TAT GAA GAT 672Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Se - #r Leu Glu Tyr Glu Asp# 220- ATG GGA ATT TAT TAT TGT CTA CAG TAT GAT GA - #G TTT CCG TAC ACG TTC 720Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Gl - #u Phe Pro Tyr Thr Phe225 2 - #30 2 - #35 2 -#40- GGA GGG GGG ACC AAG CTG GAA ATA AAA GGA GG - #C GGT GGC TCG GGC GGT 768Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gl - #y Gly Gly Ser Gly Gly# 255- GGC GGA TCG GGT GGC GGC GGC TCT GAG GTT CA - #G CTC CAG CAG TCT GGG 816Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gl - #n Leu Gln Gln Ser Gly# 270- ACT GTA CTG GCA AGG CCT GGG GCT TCA GTG AA - #G ATG TCC TGC AAG GCT 864Thr Val Leu Ala Arg Pro Gly Ala Ser Val Ly - #s Met Ser Cys Lys Ala# 285- TCT GGC TAC ACC TTT TCC AGC TAC TGG ATG CA - #C TGG ATA AAA CAG AGG 912Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met Hi - #s Trp Ile Lys Gln Arg# 300- CCT GGA CAG GGT CTG GAC TGG ATT GTC GCT AT - #T GAT CCT CGA AAT AGT 960Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Il - #e Asp Pro Arg Asn Ser305 3 - #10 3 - #15 3 -#20- GAT ACT ATT TAC AAC CCG CAA TTC AAA CAC AA - #G GCC AAA CTG ACT GCA1008Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Ly - #s Ala Lys Leu Thr Ala# 335- GTC ACC TCC ACC AGC ACT GCC TAC ATG GAA CT - #C AAC AGC CTG ACA AAT1056Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Le - #u Asn Ser Leu Thr Asn# 350- GAG GAC TCT GCG GTC TAT TAC TGT ACC CCT CT - #T TAT TAC TTT GAC TCC1104Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Le - #u Tyr Tyr Phe Asp Ser# 365# 1137A GGC ACC ACT CTC ACA GTC TCC TC - #ATrp Gly Gln Gly Thr Thr Leu Thr Val Ser Se - #r# 375- (2) INFORMATION FOR SEQ ID NO:43:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 379 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:- Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pr - #o Leu Phe Phe Leu Leu# 15- Leu Gly Trp Val Gln Pro Ser Leu Gly Gln As - #p Trp Leu Thr Phe Gln# 30- Lys Lys His Ile Thr Asn Thr Arg Asp Val As - #p Cys Asp Asn Ile Met# 45- Ser Thr Asn Leu Phe His Cys Lys Asp Lys As - #n Thr Phe Ile Tyr Ser# 60- Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gl - #y Ile Ile Ala Ser Lys# 80- Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Se - #r Asp Cys Asn Val Thr# 95- Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Se - #r Thr Asn Lys Phe Cys# 110- Val Thr Cys Glu Asn Gln Ala Pro Val His Ph - #e Val Gly Val Gly Ser# 125- Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pr - #o Lys Ser Asp Asp Ile# 140- Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Al - #a Ser Leu Gly Glu Arg145 1 - #50 1 - #55 1 -#60- Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Il - #e Asn Asn Tyr Leu Cys# 175- Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Ly - #s Thr Leu Ile Tyr Arg# 190- Ala Asn Arg Leu Val Asp Gly Val Pro Ser Ar - #g Phe Ser Gly Ser Gly# 205- Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Se - #r Leu Glu Tyr Glu Asp# 220- Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Gl - #u Phe Pro Tyr Thr Phe225 2 - #30 2 - #35 2 -#40- Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gl - #y Gly Gly Ser Gly Gly# 255- Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gl - #n Leu Gln Gln Ser Gly# 270- Thr Val Leu Ala Arg Pro Gly Ala Ser Val Ly - #s Met Ser Cys Lys Ala# 285- Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met Hi - #s Trp Ile Lys Gln Arg# 300- Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Il - #e Asp Pro Arg Asn Ser305 3 - #10 3 - #15 3 -#20- Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Ly - #s Ala Lys Leu Thr Ala# 335- Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Le - #u Asn Ser Leu Thr Asn# 350- Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Le - #u Tyr Tyr Phe Asp Ser# 365- Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Se - #r# 375- (2) INFORMATION FOR SEQ ID NO:44:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1074 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1074#/note= "MetSerOncA87FBE6"ATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:- ATG TCA GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT GCT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Ala Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- GTT CAT TTT GTT GGA GTT GGA TCT TGT GCC AA - #G AAA CTG AAC GAC GCT 336Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- CAG GCG CCG AAG AGT GAT GAC ATC AAG ATG AC - #C CAG TCT CCA TCT TCC 384Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Th - #r Gln Ser Pro Ser Ser# 125- ATG TAT GCA TCT CTA GGA GAG AGA GTC ACT TT - #C ACT TGC AAG GCG AGT 432Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ph - #e Thr Cys Lys Ala Ser# 140- CAG GAC ATT AAT AAC TAT TTA TGC TGG TTC CA - #G CAG AAA CCA GGG AAA 480Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gl - #n Gln Lys Pro Gly Lys145 1 - #50 1 - #55 1 -#60- TCT CCT AAG ACC CTG ATC TAT CGT GCA AAC AG - #A CTG GTA GAT GGG GTC 528Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Ar - #g Leu Val Asp Gly Val# 175- CCA TCA AGG TTC AGT GGC AGT GGA TCT GGA CA - #A GAT TAT TCT CTC ACC 576Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gl - #n Asp Tyr Ser Leu Thr# 190- ATT AGC AGC CTG GAG TAT GAA GAT ATG GGA AT - #T TAT TAT TGT CTA CAG 624Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Il - #e Tyr Tyr Cys Leu Gln# 205- TAT GAT GAG TTT CCG TAC ACG TTC GGA GGG GG - #G ACC AAG CTG GAA ATA 672Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gl - #y Thr Lys Leu Glu Ile# 220- AAA GGA GGC GGT GGC TCG GGC GGT GGC GGA TC - #G GGT GGC GGC GGC TCT 720Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gly Gly Gly Gly Ser225 2 - #30 2 - #35 2 -#40- GAG GTT CAG CTC CAG CAG TCT GGG ACT GTA CT - #G GCA AGG CCT GGG GCT 768Glu Val Gln Leu Gln Gln Ser Gly Thr Val Le - #u Ala Arg Pro Gly Ala# 255- TCA GTG AAG ATG TCC TGC AAG GCT TCT GGC TA - #C ACC TTT TCC AGC TAC 816Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Ser Ser Tyr# 270- TGG ATG CAC TGG ATA AAA CAG AGG CCT GGA CA - #G GGT CTG GAC TGG ATT 864Trp Met His Trp Ile Lys Gln Arg Pro Gly Gl - #n Gly Leu Asp Trp Ile# 285- GTC GCT ATT GAT CCT CGA AAT AGT GAT ACT AT - #T TAC AAC CCG CAA TTC 912Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Il - #e Tyr Asn Pro Gln Phe# 300- AAA CAC AAG GCC AAA CTG ACT GCA GTC ACC TC - #C ACC AGC ACT GCC TAC 960Lys His Lys Ala Lys Leu Thr Ala Val Thr Se - #r Thr Ser Thr Ala Tyr305 3 - #10 3 - #15 3 -#20- ATG GAA CTC AAC AGC CTG ACA AAT GAG GAC TC - #T GCG GTC TAT TAC TGT1008Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Se - #r Ala Val Tyr Tyr Cys# 335- ACC CCT CTT TAT TAC TTT GAC TCC TGG GGC CA - #A GGC ACC ACT CTC ACA1056Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gl - #n Gly Thr Thr Leu Thr# 350#1074 AC CATVal Ser Ser His His His 355- (2) INFORMATION FOR SEQ ID NO:45:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 358 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:- Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Ala Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Th - #r Gln Ser Pro Ser Ser# 125- Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ph - #e Thr Cys Lys Ala Ser# 140- Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gl - #n Gln Lys Pro Gly Lys145 1 - #50 1 - #55 1 -#60- Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Ar - #g Leu Val Asp Gly Val# 175- Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gl - #n Asp Tyr Ser Leu Thr# 190- Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Il - #e Tyr Tyr Cys Leu Gln# 205- Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gl - #y Thr Lys Leu Glu Ile# 220- Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gly Gly Gly Gly Ser225 2 - #30 2 - #35 2 -#40- Glu Val Gln Leu Gln Gln Ser Gly Thr Val Le - #u Ala Arg Pro Gly Ala# 255- Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Ser Ser Tyr# 270- Trp Met His Trp Ile Lys Gln Arg Pro Gly Gl - #n Gly Leu Asp Trp Ile# 285- Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Il - #e Tyr Asn Pro Gln Phe# 300- Lys His Lys Ala Lys Leu Thr Ala Val Thr Se - #r Thr Ser Thr Ala Tyr305 3 - #10 3 - #15 3 -#20- Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Se - #r Ala Val Tyr Tyr Cys# 335- Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gl - #n Gly Thr Thr Leu Thr# 350- Val Ser Ser His His His 355- (2) INFORMATION FOR SEQ ID NO:46:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1086 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1083#/note= "MetSerOncAngsFv"MATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:- ATG TCA GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT GTT GTT GCT TG - #T GAA AAT GGC TTA CCT 288Lys Lys Ser Thr Asn Lys Phe Val Val Ala Cy - #s Glu Asn Gly Leu Pro# 95- GTC CAC TTG GAT CAG TCA ATT TTC CGT CGT CC - #G GCC AAG AAA CTG AAC 336Val His Leu Asp Gln Ser Ile Phe Arg Arg Pr - #o Ala Lys Lys Leu Asn# 110- GAC GCT CAG GCG CCG AAG AGT GAT GAC ATC AA - #G ATG ACC CAG TCT CCA 384Asp Ala Gln Ala Pro Lys Ser Asp Asp Ile Ly - #s Met Thr Gln Ser Pro# 125- TCT TCC ATG TAT GCA TCT CTA GGA GAG AGA GT - #C ACT TTC ACT TGC AAG 432Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Va - #l Thr Phe Thr Cys Lys# 140- GCG AGT CAG GAC ATT AAT AAC TAT TTA TGC TG - #G TTC CAG CAG AAA CCA 480Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Tr - #p Phe Gln Gln Lys Pro145 1 - #50 1 - #55 1 -#60- GGG AAA TCT CCT AAG ACC CTG ATC TAT CGT GC - #A AAC AGA CTG GTA GAT 528Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Al - #a Asn Arg Leu Val Asp# 175- GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA TC - #T GGA CAA GAT TAT TCT 576Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Se - #r Gly Gln Asp Tyr Ser# 190- CTC ACC ATT AGC AGC CTG GAG TAT GAA GAT AT - #G GGA ATT TAT TAT TGT 624Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Me - #t Gly Ile Tyr Tyr Cys# 205- CTA CAG TAT GAT GAG TTT CCG TAC ACG TTC GG - #A GGG GGG ACC AAG CTG 672Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gl - #y Gly Gly Thr Lys Leu# 220- GAA ATA AAA GGA GGC GGT GGC TCG GGC GGT GG - #C GGA TCG GGT GGC GGC 720Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gl - #y Gly Ser Gly Gly Gly225 2 - #30 2 - #35 2 -#40- GGC TCT GAG GTT CAG CTC CAG CAG TCT GGG AC - #T GTA CTG GCA AGG CCT 768Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Th - #r Val Leu Ala Arg Pro# 255- GGG GCT TCA GTG AAG ATG TCC TGC AAG GCT TC - #T GGC TAC ACC TTT TCC 816Gly Ala Ser Val Lys Met Ser Cys Lys Ala Se - #r Gly Tyr Thr Phe Ser# 270- AGC TAC TGG ATG CAC TGG ATA AAA CAG AGG CC - #T GGA CAG GGT CTG GAC 864Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pr - #o Gly Gln Gly Leu Asp# 285- TGG ATT GTC GCT ATT GAT CCT CGA AAT AGT GA - #T ACT ATT TAC AAC CCG 912Trp Ile Val Ala Ile Asp Pro Arg Asn Ser As - #p Thr Ile Tyr Asn Pro# 300- CAA TTC AAA CAC AAG GCC AAA CTG ACT GCA GT - #C ACC TCC ACC AGC ACT 960Gln Phe Lys His Lys Ala Lys Leu Thr Ala Va - #l Thr Ser Thr Ser Thr305 3 - #10 3 - #15 3 -#20- GCC TAC ATG GAA CTC AAC AGC CTG ACA AAT GA - #G GAC TCT GCG GTC TAT1008Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Gl - #u Asp Ser Ala Val Tyr# 335- TAC TGT ACC CCT CTT TAT TAC TTT GAC TCC TG - #G GGC CAA GGC ACC ACT1056Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Tr - #p Gly Gln Gly Thr Thr# 350# 1086 CA CAT CAC CAT TAGTAGLeu Thr Val Ser Ser His His His# 360- (2) INFORMATION FOR SEQ ID NO:47:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 360 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:- Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Val Val Ala Cy - #s Glu Asn Gly Leu Pro# 95- Val His Leu Asp Gln Ser Ile Phe Arg Arg Pr - #o Ala Lys Lys Leu Asn# 110- Asp Ala Gln Ala Pro Lys Ser Asp Asp Ile Ly - #s Met Thr Gln Ser Pro# 125- Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Va - #l Thr Phe Thr Cys Lys# 140- Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Tr - #p Phe Gln Gln Lys Pro145 1 - #50 1 - #55 1 -#60- Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Al - #a Asn Arg Leu Val Asp# 175- Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Se - #r Gly Gln Asp Tyr Ser# 190- Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Me - #t Gly Ile Tyr Tyr Cys# 205- Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gl - #y Gly Gly Thr Lys Leu# 220- Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gl - #y Gly Ser Gly Gly Gly225 2 - #30 2 - #35 2 -#40- Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Th - #r Val Leu Ala Arg Pro# 255- Gly Ala Ser Val Lys Met Ser Cys Lys Ala Se - #r Gly Tyr Thr Phe Ser# 270- Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pr - #o Gly Gln Gly Leu Asp# 285- Trp Ile Val Ala Ile Asp Pro Arg Asn Ser As - #p Thr Ile Tyr Asn Pro# 300- Gln Phe Lys His Lys Ala Lys Leu Thr Ala Va - #l Thr Ser Thr Ser Thr305 3 - #10 3 - #15 3 -#20- Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Gl - #u Asp Ser Ala Val Tyr# 335- Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Tr - #p Gly Gln Gly Thr Thr# 350- Leu Thr Val Ser Ser His His His# 360- (2) INFORMATION FOR SEQ ID NO:48:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1065 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1065#/note= "sFvOncMetSer"FORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:- GAC ATC AAG ATG ACC CAG TCT CCA TCT TCC AT - #G TAT GCA TCT CTA GGA 48Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Me - #t Tyr Ala Ser Leu Gly# 15- GAG AGA GTC ACT TTC ACT TGC AAG GCG AGT CA - #G GAC ATT AAT AAC TAT 96Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gl - #n Asp Ile Asn Asn Tyr# 30- TTA TGC TGG TTC CAG CAG AAA CCA GGG AAA TC - #T CCT AAG ACC CTG ATC 144Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Se - #r Pro Lys Thr Leu Ile# 45- TAT CGT GCA AAC AGA CTG GTA GAT GGG GTC CC - #A TCA AGG TTC AGT GGC 192Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pr - #o Ser Arg Phe Ser Gly# 60- AGT GGA TCT GGA CAA GAT TAT TCT CTC ACC AT - #T AGC AGC CTG GAG TAT 240Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Il - #e Ser Ser Leu Glu Tyr# 80- GAA GAT ATG GGA ATT TAT TAT TGT CTA CAG TA - #T GAT GAG TTT CCG TAC 288Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Ty - #r Asp Glu Phe Pro Tyr# 95- ACG TTC GGA GGG GGG ACC AAG CTG GAA ATA AA - #A GGA GGC GGT GGC TCG 336Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ly - #s Gly Gly Gly Gly Ser# 110- GGC GGT GGC GGA TCG GGT GGC GGC GGC TCT GA - #G GTT CAG CTC CAG CAG 384Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gl - #u Val Gln Leu Gln Gln# 125- TCT GGG ACT GTA CTG GCA AGG CCT GGG GCT TC - #A GTG AAG ATG TCC TGC 432Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Se - #r Val Lys Met Ser Cys# 140- AAG GCT TCT GGC TAC ACC TTT TCC AGC TAC TG - #G ATG CAC TGG ATA AAA 480Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Tr - #p Met His Trp Ile Lys145 1 - #50 1 - #55 1 -#60- CAG AGG CCT GGA CAG GGT CTG GAC TGG ATT GT - #C GCT ATT GAT CCT CGA 528Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Va - #l Ala Ile Asp Pro Arg# 175- AAT AGT GAT ACT ATT TAC AAC CCG CAA TTC AA - #A CAC AAG GCC AAA CTG 576Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Ly - #s His Lys Ala Lys Leu# 190- ACT GCA GTC ACC TCC ACC AGC ACT GCC TAC AT - #G GAA CTC AAC AGC CTG 624Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Me - #t Glu Leu Asn Ser Leu# 205- ACA AAT GAG GAC TCT GCG GTC TAT TAC TGT AC - #C CCT CTT TAT TAC TTT 672Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Th - #r Pro Leu Tyr Tyr Phe# 220- GAC TCC TGG GGC CAA GGC ACC ACT CTC ACA GT - #C TCC TCA GCC AAG AAA 720Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Va - #l Ser Ser Ala Lys Lys225 2 - #30 2 - #35 2 -#40- CTG AAC GAC GCT CAG GCG CCG AAG AGT GAT AT - #G TCA GAT TGG CTT ACA 768Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Me - #t Ser Asp Trp Leu Thr# 255- TTT CAG AAA AAA CAC ATC ACA AAC ACA AGG GA - #T GTT GAT TGT GAT AAT 816Phe Gln Lys Lys His Ile Thr Asn Thr Arg As - #p Val Asp Cys Asp Asn# 270- ATC ATG TCA ACA AAC TTG TTC CAC TGC AAG GA - #C AAG AAC ACT TTT ATC 864Ile Met Ser Thr Asn Leu Phe His Cys Lys As - #p Lys Asn Thr Phe Ile# 285- TAT TCA CGT CCT GAG CCA GTG AAG GCC ATC TG - #T AAA GGA ATT ATA GCC 912Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cy - #s Lys Gly Ile Ile Ala# 300- TCC AAA AAT GTG TTA ACT ACC TCT GAG TTT TA - #T CTC TCT GAT TGC AAT 960Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Ty - #r Leu Ser Asp Cys Asn305 3 - #10 3 - #15 3 -#20- GTA ACA AGC AGG CCT TGC AAG TAT AAA TTA AA - #G AAA TCA ACT AAT AAA1008Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Ly - #s Lys Ser Thr Asn Lys# 335- TTT TGT GTA ACT TGT GAA AAT CAG GCA CCA GT - #T CAT TTT GTT GGA GTT1056Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Va - #l His Phe Val Gly Val# 350# 1065Gly Ser Cys 355- (2) INFORMATION FOR SEQ ID NO:49:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 355 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:- Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Me - #t Tyr Ala Ser Leu Gly# 15- Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gl - #n Asp Ile Asn Asn Tyr# 30- Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Se - #r Pro Lys Thr Leu Ile# 45- Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pr - #o Ser Arg Phe Ser Gly# 60- Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Il - #e Ser Ser Leu Glu Tyr# 80- Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Ty - #r Asp Glu Phe Pro Tyr# 95- Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ly - #s Gly Gly Gly Gly Ser# 110- Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gl - #u Val Gln Leu Gln Gln# 125- Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Se - #r Val Lys Met Ser Cys# 140- Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Tr - #p Met His Trp Ile Lys145 1 - #50 1 - #55 1 -#60- Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Va - #l Ala Ile Asp Pro Arg# 175- Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Ly - #s His Lys Ala Lys Leu# 190- Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Me - #t Glu Leu Asn Ser Leu# 205- Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Th - #r Pro Leu Tyr Tyr Phe# 220- Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Va - #l Ser Ser Ala Lys Lys225 2 - #30 2 - #35 2 -#40- Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Me - #t Ser Asp Trp Leu Thr# 255- Phe Gln Lys Lys His Ile Thr Asn Thr Arg As - #p Val Asp Cys Asp Asn# 270- Ile Met Ser Thr Asn Leu Phe His Cys Lys As - #p Lys Asn Thr Phe Ile# 285- Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cy - #s Lys Gly Ile Ile Ala# 300- Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Ty - #r Leu Ser Asp Cys Asn305 3 - #10 3 - #15 3 -#20- Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Ly - #s Lys Ser Thr Asn Lys# 335- Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Va - #l His Phe Val Gly Val# 350- Gly Ser Cys 355- (2) INFORMATION FOR SEQ ID NO:50:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1074 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1074#/note= "MetGluOncFBE6"ORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:- ATG GAG GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Glu Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT TGT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- GTT CAT TTT GTT GGA GTT GGA TCT TGT GCC AA - #G AAA CTG AAC GAC GCT 336Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- CAG GCG CCG AAG AGT GAT GAC ATC AAG ATG AC - #C CAG TCT CCA TCT TCC 384Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Th - #r Gln Ser Pro Ser Ser# 125- ATG TAT GCA TCT CTA GGA GAG AGA GTC ACT TT - #C ACT TGC AAG GCG AGT 432Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ph - #e Thr Cys Lys Ala Ser# 140- CAG GAC ATT AAT AAC TAT TTA TGC TGG TTC CA - #G CAG AAA CCA GGG AAA 480Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gl - #n Gln Lys Pro Gly Lys145 1 - #50 1 - #55 1 -#60- TCT CCT AAG ACC CTG ATC TAT CGT GCA AAC AG - #A CTG GTA GAT GGG GTC 528Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Ar - #g Leu Val Asp Gly Val# 175- CCA TCA AGG TTC AGT GGC AGT GGA TCT GGA CA - #A GAT TAT TCT CTC ACC 576Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gl - #n Asp Tyr Ser Leu Thr# 190- ATT AGC AGC CTG GAG TAT GAA GAT ATG GGA AT - #T TAT TAT TGT CTA CAG 624Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Il - #e Tyr Tyr Cys Leu Gln# 205- TAT GAT GAG TTT CCG TAC ACG TTC GGA GGG GG - #G ACC AAG CTG GAA ATA 672Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gl - #y Thr Lys Leu Glu Ile# 220- AAA GGA GGC GGT GGC TCG GGC GGT GGC GGA TC - #G GGT GGC GGC GGC TCT 720Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gly Gly Gly Gly Ser225 2 - #30 2 - #35 2 -#40- GAG GTT CAG CTC CAG CAG TCT GGG ACT GTA CT - #G GCA AGG CCT GGG GCT 768Glu Val Gln Leu Gln Gln Ser Gly Thr Val Le - #u Ala Arg Pro Gly Ala# 255- TCA GTG AAG ATG TCC TGC AAG GCT TCT GGC TA - #C ACC TTT TCC AGC TAC 816Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Ser Ser Tyr# 270- TGG ATG CAC TGG ATA AAA CAG AGG CCT GGA CA - #G GGT CTG GAC TGG ATT 864Trp Met His Trp Ile Lys Gln Arg Pro Gly Gl - #n Gly Leu Asp Trp Ile# 285- GTC GCT ATT GAT CCT CGA AAT AGT GAT ACT AT - #T TAC AAC CCG CAA TTC 912Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Il - #e Tyr Asn Pro Gln Phe# 300- AAA CAC AAG GCC AAA CTG ACT GCA GTC ACC TC - #C ACC AGC ACT GCC TAC 960Lys His Lys Ala Lys Leu Thr Ala Val Thr Se - #r Thr Ser Thr Ala Tyr305 3 - #10 3 - #15 3 -#20- ATG GAA CTC AAC AGC CTG ACA AAT GAG GAC TC - #T GCG GTC TAT TAC TGT1008Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Se - #r Ala Val Tyr Tyr Cys# 335- ACC CCT CTT TAT TAC TTT GAC TCC TGG GGC CA - #A GGC ACC ACT CTC ACA1056Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gl - #n Gly Thr Thr Leu Thr# 350#1074 AC CATVal Ser Ser His His His 355- (2) INFORMATION FOR SEQ ID NO:51:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 358 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:- Met Glu Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Th - #r Gln Ser Pro Ser Ser# 125- Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ph - #e Thr Cys Lys Ala Ser# 140- Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gl - #n Gln Lys Pro Gly Lys145 1 - #50 1 - #55 1 -#60- Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Ar - #g Leu Val Asp Gly Val# 175- Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gl - #n Asp Tyr Ser Leu Thr# 190- Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Il - #e Tyr Tyr Cys Leu Gln# 205- Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gl - #y Thr Lys Leu Glu Ile# 220- Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gly Gly Gly Gly Ser225 2 - #30 2 - #35 2 -#40- Glu Val Gln Leu Gln Gln Ser Gly Thr Val Le - #u Ala Arg Pro Gly Ala# 255- Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Ser Ser Tyr# 270- Trp Met His Trp Ile Lys Gln Arg Pro Gly Gl - #n Gly Leu Asp Trp Ile# 285- Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Il - #e Tyr Asn Pro Gln Phe# 300- Lys His Lys Ala Lys Leu Thr Ala Val Thr Se - #r Thr Ser Thr Ala Tyr305 3 - #10 3 - #15 3 -#20- Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Se - #r Ala Val Tyr Tyr Cys# 335- Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gl - #n Gly Thr Thr Leu Thr# 350- Val Ser Ser His His His 355- (2) INFORMATION FOR SEQ ID NO:52:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1095 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1095#/note= "MetSerOncA87FBMOC31"ON:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:- ATG TCA GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT GCT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Ala Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- GTT CAT TTT GTT GGA GTT GGA TCT TGT GCC AA - #G AAA CTG AAC GAC GCT 336Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- CAG GCG CCG AAG AGT GAT CAG GTG AAG CTG CA - #G CAG TCA GGA CCT GAG 384Gln Ala Pro Lys Ser Asp Gln Val Lys Leu Gl - #n Gln Ser Gly Pro Glu# 125- CTG AAG AAG CCT GGA GAG ACA GTC AAG ATC TC - #C TGC AAG GCT TCT GGG 432Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Se - #r Cys Lys Ala Ser Gly# 140- TAC ACC TTC ACA AAC TAT GGA ATG AAC TGG GT - #G AAG CAG GCT CCA GGA 480Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Va - #l Lys Gln Ala Pro Gly145 1 - #50 1 - #55 1 -#60- AAG GGT TTA AAG TGG ATG GGC TGG ATA AAC AC - #C TAC ACT GGA GAG TCA 528Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Th - #r Tyr Thr Gly Glu Ser# 175- ACA TAT GCT GAT GAC TTC AAG GGA CGG TTT GC - #C TTT TCT CTA GAA ACC 576Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Al - #a Phe Ser Leu Glu Thr# 190- TCT GCC AGC GCT GCC TAT TTG CAG ATC AAC AA - #C CTC AAA AAT GAG GAC 624Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn As - #n Leu Lys Asn Glu Asp# 205- ACG GCT ACA TAT TTC TGT GCA AGA TTC GCT AT - #T AAG GGG GAC TAC TGG 672Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala Il - #e Lys Gly Asp Tyr Trp# 220- GGC CAA GGG ACC ACG GTC ACC GTC TCC TCA GG - #T GGA GGC GGT TCA GGC 720Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gl - #y Gly Gly Gly Ser Gly225 2 - #30 2 - #35 2 -#40- GGA GGT GGC TCT GGC GGT GGC GGA TCG GAC AT - #T GTG CTA ACC CAG TCT 768Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Il - #e Val Leu Thr Gln Ser# 255- CCA TTC TCC AAT CCA GTC ACT CTT GGA ACA TC - #A GCT TCC ATC TCC TGC 816Pro Phe Ser Asn Pro Val Thr Leu Gly Thr Se - #r Ala Ser Ile Ser Cys# 270- AGG TCT ACT AAG AGT CTC CTA CAT AGT AAT GG - #C ATC ACT TAT TTG TAT 864Arg Ser Thr Lys Ser Leu Leu His Ser Asn Gl - #y Ile Thr Tyr Leu Tyr# 285- TGG TAT CTG CAG AAG CCA GGC CAG TCT CCT CA - #G CTC CTG ATT TAT CAG 912Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gl - #n Leu Leu Ile Tyr Gln# 300- ATG TCC AAC CTT GCC TCA GGA GTC CCA GAC AG - #G TTC AGT AGC AGT GGG 960Met Ser Asn Leu Ala Ser Gly Val Pro Asp Ar - #g Phe Ser Ser Ser Gly305 3 - #10 3 - #15 3 -#20- TCA GGA ACT GAT TTC ACA CTG AGA ATC AGC AG - #A GTG GAG GCT GAG GAT1008Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Ar - #g Val Glu Ala Glu Asp# 335- GTG GGT GTT TAT TAC TGT GCT CAA AAT CTA GA - #A ATT CCT CGG ACG TTC1056Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Gl - #u Ile Pro Arg Thr Phe# 350# 1095A GGC ACC AAG CTG GAA ATC AAA CGG GC - #G GCC GCAGly Gly Gly Thr Lys Leu Glu Ile Lys Arg Al - #a Ala Ala# 365- (2) INFORMATION FOR SEQ ID NO:53:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 365 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:- Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Ala Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- Gln Ala Pro Lys Ser Asp Gln Val Lys Leu Gl - #n Gln Ser Gly Pro Glu# 125- Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Se - #r Cys Lys Ala Ser Gly# 140- Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Va - #l Lys Gln Ala Pro Gly145 1 - #50 1 - #55 1 -#60- Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Th - #r Tyr Thr Gly Glu Ser# 175- Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Al - #a Phe Ser Leu Glu Thr# 190- Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn As - #n Leu Lys Asn Glu Asp# 205- Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala Il - #e Lys Gly Asp Tyr Trp# 220- Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gl - #y Gly Gly Gly Ser Gly225 2 - #30 2 - #35 2 -#40- Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Il - #e Val Leu Thr Gln Ser# 255- Pro Phe Ser Asn Pro Val Thr Leu Gly Thr Se - #r Ala Ser Ile Ser Cys# 270- Arg Ser Thr Lys Ser Leu Leu His Ser Asn Gl - #y Ile Thr Tyr Leu Tyr# 285- Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gl - #n Leu Leu Ile Tyr Gln# 300- Met Ser Asn Leu Ala Ser Gly Val Pro Asp Ar - #g Phe Ser Ser Ser Gly305 3 - #10 3 - #15 3 -#20- Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Ar - #g Val Glu Ala Glu Asp# 335- Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Gl - #u Ile Pro Arg Thr Phe# 350- Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Al - #a Ala Ala# 365- (2) INFORMATION FOR SEQ ID NO:54:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1098 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1098#/note= "MOC31FBMetSerOnc"ATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:- GGT CAG GTG AAG CTG CAG CAG TCA GGA CCT GA - #G CTG AAG AAG CCT GGA 48Gly Gln Val Lys Leu Gln Gln Ser Gly Pro Gl - #u Leu Lys Lys Pro Gly# 15- GAG ACA GTC AAG ATC TCC TGC AAG GCT TCT GG - #G TAC ACC TTC ACA AAC 96Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gl - #y Tyr Thr Phe Thr Asn# 30- TAT GGA ATG AAC TGG GTG AAG CAG GCT CCA GG - #A AAG GGT TTA AAG TGG 144Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gl - #y Lys Gly Leu Lys Trp# 45- ATG GGC TGG ATA AAC ACC TAC ACT GGA GAG TC - #A ACA TAT GCT GAT GAC 192Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Se - #r Thr Tyr Ala Asp Asp# 60- TTC AAG GGA CGG TTT GCC TTT TCT CTA GAA AC - #C TCT GCC AGC GCT GCC 240Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Th - #r Ser Ala Ser Ala Ala# 80- TAT TTG CAG ATC AAC AAC CTC AAA AAT GAG GA - #C ACG GCT ACA TAT TTC 288Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu As - #p Thr Ala Thr Tyr Phe# 95- TGT GCA AGA TTC GCT ATT AAG GGG GAC TAC TG - #G GGC CAA GGG ACC ACG 336Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr Tr - #p Gly Gln Gly Thr Thr# 110- GTC ACC GTC TCC TCA GGT GGA GGC GGT TCA GG - #C GGA GGT GGC TCT GGC 384Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gl - #y Gly Gly Gly Ser Gly# 125- GGT GGC GGA TCG GAC ATT GTG CTA ACC CAG TC - #T CCA TTC TCC AAT CCA 432Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Se - #r Pro Phe Ser Asn Pro# 140- GTC ACT CTT GGA ACA TCA GCT TCC ATC TCC TG - #C AGG TCT ACT AAG AGT 480Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cy - #s Arg Ser Thr Lys Ser145 1 - #50 1 - #55 1 -#60- CTC CTA CAT AGT AAT GGC ATC ACT TAT TTG TA - #T TGG TAT CTG CAG AAG 528Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Ty - #r Trp Tyr Leu Gln Lys# 175- CCA GGC CAG TCT CCT CAG CTC CTG ATT TAT CA - #G ATG TCC AAC CTT GCC 576Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gl - #n Met Ser Asn Leu Ala# 190- TCA GGA GTC CCA GAC AGG TTC AGT AGC AGT GG - #G TCA GGA ACT GAT TTC 624Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gl - #y Ser Gly Thr Asp Phe# 205- ACA CTG AGA ATC AGC AGA GTG GAG GCT GAG GA - #T GTG GGT GTT TAT TAC 672Thr Leu Arg Ile Ser Arg Val Glu Ala Glu As - #p Val Gly Val Tyr Tyr# 220- TGT GCT CAA AAT CTA GAA ATT CCT CGG ACG TT - #C GGT GGA GGC ACC AAG 720Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Ph - #e Gly Gly Gly Thr Lys225 2 - #30 2 - #35 2 -#40- CTG GAA ATC AAA CGG GCG GCC GCA GCC AAG AA - #A CTG AAC GAC GCT CAG 768Leu Glu Ile Lys Arg Ala Ala Ala Ala Lys Ly - #s Leu Asn Asp Ala Gln# 255- GCG CCG AAG AGT GAT ATG TCA GAT TGG CTT AC - #A TTT CAG AAA AAA CAC 816Ala Pro Lys Ser Asp Met Ser Asp Trp Leu Th - #r Phe Gln Lys Lys His# 270- ATC ACA AAC ACA AGG GAT GTT GAT TGT GAT AA - #T ATC ATG TCA ACA AAC 864Ile Thr Asn Thr Arg Asp Val Asp Cys Asp As - #n Ile Met Ser Thr Asn# 285- TTG TTC CAC TGC AAG GAC AAG AAC ACT TTT AT - #C TAT TCA CGT CCT GAG 912Leu Phe His Cys Lys Asp Lys Asn Thr Phe Il - #e Tyr Ser Arg Pro Glu# 300- CCA GTG AAG GCC ATC TGT AAA GGA ATT ATA GC - #C TCC AAA AAT GTG TTA 960Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Al - #a Ser Lys Asn Val Leu305 3 - #10 3 - #15 3 -#20- ACT ACC TCT GAG TTT TAT CTC TCT GAT TGC AA - #T GTA ACA AGC AGG CCT1008Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys As - #n Val Thr Ser Arg Pro# 335- TGC AAG TAT AAA TTA AAG AAA TCA ACT AAT AA - #A TTT TGT GTA ACT TGT1056Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Ly - #s Phe Cys Val Thr Cys# 350- GAA AAT CAG GCA CCA GTT CAT TTT GTT GGA GT - #T GGA TCT TGT#1098Glu Asn Gln Ala Pro Val His Phe Val Gly Va - #l Gly Ser Cys# 365- (2) INFORMATION FOR SEQ ID NO:55:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 366 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:- Gly Gln Val Lys Leu Gln Gln Ser Gly Pro Gl - #u Leu Lys Lys Pro Gly# 15- Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gl - #y Tyr Thr Phe Thr Asn# 30- Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gl - #y Lys Gly Leu Lys Trp# 45- Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Se - #r Thr Tyr Ala Asp Asp# 60- Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Th - #r Ser Ala Ser Ala Ala# 80- Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu As - #p Thr Ala Thr Tyr Phe# 95- Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr Tr - #p Gly Gln Gly Thr Thr# 110- Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gl - #y Gly Gly Gly Ser Gly# 125- Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Se - #r Pro Phe Ser Asn Pro# 140- Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cy - #s Arg Ser Thr Lys Ser145 1 - #50 1 - #55 1 -#60- Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Ty - #r Trp Tyr Leu Gln Lys# 175- Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gl - #n Met Ser Asn Leu Ala# 190- Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gl - #y Ser Gly Thr Asp Phe# 205- Thr Leu Arg Ile Ser Arg Val Glu Ala Glu As - #p Val Gly Val Tyr Tyr# 220- Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Ph - #e Gly Gly Gly Thr Lys225 2 - #30 2 - #35 2 -#40- Leu Glu Ile Lys Arg Ala Ala Ala Ala Lys Ly - #s Leu Asn Asp Ala Gln# 255- Ala Pro Lys Ser Asp Met Ser Asp Trp Leu Th - #r Phe Gln Lys Lys His# 270- Ile Thr Asn Thr Arg Asp Val Asp Cys Asp As - #n Ile Met Ser Thr Asn# 285- Leu Phe His Cys Lys Asp Lys Asn Thr Phe Il - #e Tyr Ser Arg Pro Glu# 300- Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Al - #a Ser Lys Asn Val Leu305 3 - #10 3 - #15 3 -#20- Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys As - #n Val Thr Ser Arg Pro# 335- Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Ly - #s Phe Cys Val Thr Cys# 350- Glu Asn Gln Ala Pro Val His Phe Val Gly Va - #l Gly Ser Cys# 365- (2) INFORMATION FOR SEQ ID NO:56:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1065 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1065#/note= "MetSerOncFBMOC161"TION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:- ATG TCA GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT TGT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- GTT CAT TTT GTT GGA GTT GGA TCT TGT GCC AA - #G AAA CTG AAC GAC GCT 336Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- CAG GCG CCG AAG AGT GAT CAG GTC CAA CTG CA - #G CAG TCA GGA ACT GAG 384Gln Ala Pro Lys Ser Asp Gln Val Gln Leu Gl - #n Gln Ser Gly Thr Glu# 125- CTG ATA AGG CCT GGG ACT TCA GTG AAG ATA TC - #C TGT AAG GCT TCT GGA 432Leu Ile Arg Pro Gly Thr Ser Val Lys Ile Se - #r Cys Lys Ala Ser Gly# 140- TAC GCC TTC ACT GAC TAC TGG CTA GGT TGG GT - #A AAA CAC AGG CCT GGA 480Tyr Ala Phe Thr Asp Tyr Trp Leu Gly Trp Va - #l Lys His Arg Pro Gly145 1 - #50 1 - #55 1 -#60- CAT GGA CTT GAG TGG ATT GGA GAT ATT TAC CC - #T GGA AGT GAT AAT ACT 528His Gly Leu Glu Trp Ile Gly Asp Ile Tyr Pr - #o Gly Ser Asp Asn Thr# 175- TAC TAC AAT GAG AAA TTC AAG GGC AAA GCC AC - #A CTG ACT ACA GAC AAA 576Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Th - #r Leu Thr Thr Asp Lys# 190- TCC TCG AGC ACA GCC TAT ATG CAG CTC AGT AG - #C CTG ACA TCT GAG GAC 624Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Se - #r Leu Thr Ser Glu Asp# 205- TCT GCT GTC TAT TTC TGT GCA AGG GGC CTT AA - #A GGA GAC TAC TGG GGC 672Ser Ala Val Tyr Phe Cys Ala Arg Gly Leu Ly - #s Gly Asp Tyr Trp Gly# 220- CAA GGG ACC ACC GTC ACC GTC TCC TCA GGT GG - #A GGC GGT TCA GGC GGA 720Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gl - #y Gly Gly Ser Gly Gly225 2 - #30 2 - #35 2 -#40- GGT GGC TCT GGC GGT GGC GGA TCG GAC ATC CA - #G ATG ACC CAG TCT CCA 768Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gl - #n Met Thr Gln Ser Pro# 255- TCC TCA CTG TCT GCA TCT CTG GGA GGC AAA GT - #C ACC ATC ACT TGC AAG 816Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Va - #l Thr Ile Thr Cys Lys# 270- GCA AGC CAA GAC ATT AAG AAG TCT ATA GCT TG - #G TAC CAA CAC AAG CCT 864Ala Ser Gln Asp Ile Lys Lys Ser Ile Ala Tr - #p Tyr Gln His Lys Pro# 285- GGA AAA GGT CCT AGG CTG CTC ATT CAT TAC AC - #A TCT ACA TTA CAG CCA 912Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Th - #r Ser Thr Leu Gln Pro# 300- GGC ATC CCA TCA AGG TTC AGT GGA AGT GGG TC - #T GGT GAA GAA TAT TCC 960Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Se - #r Gly Glu Glu Tyr Ser305 3 - #10 3 - #15 3 -#20- TTC AGC ATC AGC AAC CTG GAG CCT GAA GAT AT - #T GCA ACT TAT TAT TGT1008Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Il - #e Ala Thr Tyr Tyr Cys# 335- CAA CAG TAT GAT AAT CTT CGG ACG TTC GGT GG - #A GGC ACC AAG CTG GAG1056Gln Gln Tyr Asp Asn Leu Arg Thr Phe Gly Gl - #y Gly Thr Lys Leu Glu# 350# 1065Leu Lys Arg 355- (2) INFORMATION FOR SEQ ID NO:57:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 355 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:- Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- Gln Ala Pro Lys Ser Asp Gln Val Gln Leu Gl - #n Gln Ser Gly Thr Glu# 125- Leu Ile Arg Pro Gly Thr Ser Val Lys Ile Se - #r Cys Lys Ala Ser Gly# 140- Tyr Ala Phe Thr Asp Tyr Trp Leu Gly Trp Va - #l Lys His Arg Pro Gly145 1 - #50 1 - #55 1 -#60- His Gly Leu Glu Trp Ile Gly Asp Ile Tyr Pr - #o Gly Ser Asp Asn Thr# 175- Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Th - #r Leu Thr Thr Asp Lys# 190- Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Se - #r Leu Thr Ser Glu Asp# 205- Ser Ala Val Tyr Phe Cys Ala Arg Gly Leu Ly - #s Gly Asp Tyr Trp Gly# 220- Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gl - #y Gly Gly Ser Gly Gly225 2 - #30 2 - #35 2 -#40- Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gl - #n Met Thr Gln Ser Pro# 255- Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Va - #l Thr Ile Thr Cys Lys# 270- Ala Ser Gln Asp Ile Lys Lys Ser Ile Ala Tr - #p Tyr Gln His Lys Pro# 285- Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Th - #r Ser Thr Leu Gln Pro# 300- Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Se - #r Gly Glu Glu Tyr Ser305 3 - #10 3 - #15 3 -#20- Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Il - #e Ala Thr Tyr Tyr Cys# 335- Gln Gln Tyr Asp Asn Leu Arg Thr Phe Gly Gl - #y Gly Thr Lys Leu Glu# 350- Leu Lys Arg 355- (2) INFORMATION FOR SEQ ID NO:58:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 753 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..753#/note= "IL2FBMetSerOnc"RMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:- GCA CCT ACT TCA ACT TCT ACA AAG AAA ACA CA - #G CTA CAA CTG GAG CAT 48Ala Pro Thr Ser Thr Ser Thr Lys Lys Thr Gl - #n Leu Gln Leu Glu His# 15- TTA CTG CTG GAT TTA CAG ATG ATT TTG AAT GG - #A ATT AAT AAT TAC AAG 96Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gl - #y Ile Asn Asn Tyr Lys# 30- AAT CCC AAA CTC ACC AGG ATG CTC ACA TTT AA - #G TTT TAC ATG CCC AAG 144Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Ly - #s Phe Tyr Met Pro Lys# 45- AAG GCC ACA GAA CTG AAA CAT CTT CAG TGT CT - #A GAA GAA GAA CTC AAA 192Lys Ala Thr Glu Leu Lys His Leu Gln Cys Le - #u Glu Glu Glu Leu Lys# 60- CCT CTG GAG GAA GTG CTA AAT TTA GCT CAA AG - #C AAA AAC TTT CAC TTA 240Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Se - #r Lys Asn Phe His Leu# 80- AGA CCC AGG GAC TTA ATC AGC AAT ATC AAC GT - #A ATA GTT CTG GAA CTA 288Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Va - #l Ile Val Leu Glu Leu# 95- AAG GGA TCT GAA ACA ACA TTC ATG TGT GAA TA - #T GCT GAT GAG ACA GCA 336Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Ty - #r Ala Asp Glu Thr Ala# 110- ACC ATT GTA GAA TTT CTG AAC AGA TGG ATT AC - #C TTT TGT CAA AGC ATC 384Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Th - #r Phe Cys Gln Ser Ile# 125- ATC TCA ACA CTG ACT GCC AAG AAA CTG AAC GA - #C GCT CAG GCG CCG AAG 432Ile Ser Thr Leu Thr Ala Lys Lys Leu Asn As - #p Ala Gln Ala Pro Lys# 140- AGT GAT ATG TCA GAT TGG CTT ACA TTT CAG AA - #A AAA CAC ATC ACA AAC 480Ser Asp Met Ser Asp Trp Leu Thr Phe Gln Ly - #s Lys His Ile Thr Asn145 1 - #50 1 - #55 1 -#60- ACA AGG GAT GTT GAT TGT GAT AAT ATC ATG TC - #A ACA AAC TTG TTC CAC 528Thr Arg Asp Val Asp Cys Asp Asn Ile Met Se - #r Thr Asn Leu Phe His# 175- TGC AAG GAC AAG AAC ACT TTT ATC TAT TCA CG - #T CCT GAG CCA GTG AAG 576Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Ar - #g Pro Glu Pro Val Lys# 190- GCC ATC TGT AAA GGA ATT ATA GCC TCC AAA AA - #T GTG TTA ACT ACC TCT 624Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys As - #n Val Leu Thr Thr Ser# 205- GAG TTT TAT CTC TCT GAT TGC AAT GTA ACA AG - #C AGG CCT TGC AAG TAT 672Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Se - #r Arg Pro Cys Lys Tyr# 220- AAA TTA AAG AAA TCA ACT AAT AAA TTT TGT GT - #A ACT TGT GAA AAT CAG 720Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Va - #l Thr Cys Glu Asn Gln225 2 - #30 2 - #35 2 -#40# 753T CAT TTT GTT GGA GTT GGA TCT TG - #TAla Pro Val His Phe Val Gly Val Gly Ser Cy - #s# 250- (2) INFORMATION FOR SEQ ID NO:59:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 251 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:- Ala Pro Thr Ser Thr Ser Thr Lys Lys Thr Gl - #n Leu Gln Leu Glu His# 15- Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gl - #y Ile Asn Asn Tyr Lys# 30- Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Ly - #s Phe Tyr Met Pro Lys# 45- Lys Ala Thr Glu Leu Lys His Leu Gln Cys Le - #u Glu Glu Glu Leu Lys# 60- Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Se - #r Lys Asn Phe His Leu# 80- Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Va - #l Ile Val Leu Glu Leu# 95- Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Ty - #r Ala Asp Glu Thr Ala# 110- Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Th - #r Phe Cys Gln Ser Ile# 125- Ile Ser Thr Leu Thr Ala Lys Lys Leu Asn As - #p Ala Gln Ala Pro Lys# 140- Ser Asp Met Ser Asp Trp Leu Thr Phe Gln Ly - #s Lys His Ile Thr Asn145 1 - #50 1 - #55 1 -#60- Thr Arg Asp Val Asp Cys Asp Asn Ile Met Se - #r Thr Asn Leu Phe His# 175- Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Ar - #g Pro Glu Pro Val Lys# 190- Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys As - #n Val Leu Thr Thr Ser# 205- Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Se - #r Arg Pro Cys Lys Tyr# 220- Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Va - #l Thr Cys Glu Asn Gln225 2 - #30 2 - #35 2 -#40- Ala Pro Val His Phe Val Gly Val Gly Ser Cy - #s# 250- (2) INFORMATION FOR SEQ ID NO:60:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 768 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..765#/note= "MetSerOncFBIL2"RMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:- ATG TCA GAT TGG CTT ACA TTT CAG AAA AAA CA - #C ATC ACA AAC ACA AGG 48Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- GAT GTT GAT TGT GAT AAT ATC ATG TCA ACA AA - #C TTG TTC CAC TGC AAG 96Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- GAC AAG AAC ACT TTT ATC TAT TCA CGT CCT GA - #G CCA GTG AAG GCC ATC 144Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- TGT AAA GGA ATT ATA GCC TCC AAA AAT GTG TT - #A ACT ACC TCT GAG TTT 192Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- TAT CTC TCT GAT TGC AAT GTA ACA AGC AGG CC - #T TGC AAG TAT AAA TTA 240Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- AAG AAA TCA ACT AAT AAA TTT TGT GTA ACT TG - #T GAA AAT CAG GCA CCA 288Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- GTT CAT TTT GTT GGA GTT GGA TCT TGT GCC AA - #G AAA CTG AAC GAC GCT 336Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- CAG GCG CCG AAG AGT GAT GCA CCT ACT TCA AC - #T TCT ACA AAG AAA ACA 384Gln Ala Pro Lys Ser Asp Ala Pro Thr Ser Th - #r Ser Thr Lys Lys Thr# 125- CAG CTA CAA CTG GAG CAT TTA CTG CTG GAT TT - #A CAG ATG ATT TTG AAT 432Gln Leu Gln Leu Glu His Leu Leu Leu Asp Le - #u Gln Met Ile Leu Asn# 140- GGA ATT AAT AAT TAC AAG AAT CCC AAA CTC AC - #C AGG ATG CTC ACA TTT 480Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Th - #r Arg Met Leu Thr Phe145 1 - #50 1 - #55 1 -#60- AAG TTT TAC ATG CCC AAG AAG GCC ACA GAA CT - #G AAA CAT CTT CAG TGT 528Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Le - #u Lys His Leu Gln Cys# 175- CTA GAA GAA GAA CTC AAA CCT CTG GAG GAA GT - #G CTA AAT TTA GCT CAA 576Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Va - #l Leu Asn Leu Ala Gln# 190- AGC AAA AAC TTT CAC TTA AGA CCC AGG GAC TT - #A ATC AGC AAT ATC AAC 624Ser Lys Asn Phe His Leu Arg Pro Arg Asp Le - #u Ile Ser Asn Ile Asn# 205- GTA ATA GTT CTG GAA CTA AAG GGA TCT GAA AC - #A ACA TTC ATG TGT GAA 672Val Ile Val Leu Glu Leu Lys Gly Ser Glu Th - #r Thr Phe Met Cys Glu# 220- TAT GCT GAT GAG ACA GCA ACC ATT GTA GAA TT - #T CTG AAC AGA TGG ATT 720Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Ph - #e Leu Asn Arg Trp Ile225 2 - #30 2 - #35 2 -#40- ACC TTT TGT CAA AGC ATC ATC TCA ACA CTG AC - #T CAT CAC CAT# 762Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Th - #r His His His# 250# 768- (2) INFORMATION FOR SEQ ID NO:61:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 254 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:- Met Ser Asp Trp Leu Thr Phe Gln Lys Lys Hi - #s Ile Thr Asn Thr Arg# 15- Asp Val Asp Cys Asp Asn Ile Met Ser Thr As - #n Leu Phe His Cys Lys# 30- Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Gl - #u Pro Val Lys Ala Ile# 45- Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Le - #u Thr Thr Ser Glu Phe# 60- Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pr - #o Cys Lys Tyr Lys Leu# 80- Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cy - #s Glu Asn Gln Ala Pro# 95- Val His Phe Val Gly Val Gly Ser Cys Ala Ly - #s Lys Leu Asn Asp Ala# 110- Gln Ala Pro Lys Ser Asp Ala Pro Thr Ser Th - #r Ser Thr Lys Lys Thr# 125- Gln Leu Gln Leu Glu His Leu Leu Leu Asp Le - #u Gln Met Ile Leu Asn# 140- Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Th - #r Arg Met Leu Thr Phe145 1 - #50 1 - #55 1 -#60- Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Le - #u Lys His Leu Gln Cys# 175- Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Va - #l Leu Asn Leu Ala Gln# 190- Ser Lys Asn Phe His Leu Arg Pro Arg Asp Le - #u Ile Ser Asn Ile Asn# 205- Val Ile Val Leu Glu Leu Lys Gly Ser Glu Th - #r Thr Phe Met Cys Glu# 220- Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Ph - #e Leu Asn Arg Trp Ile225 2 - #30 2 - #35 2 -#40- Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Th - #r His His His# 250- (2) INFORMATION FOR SEQ ID NO:62:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 387 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..387#/note= "SigPepOnc" INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:- ATG GGT CTG GAA AAA TCT CTG ATC CTG TTC CC - #G CTG TTC TTC CTG CTG 48Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pr - #o Leu Phe Phe Leu Leu# 15- CTG GGT TGG GTT CAG CCG TCT CTG GGT CAG GA - #T TGG CTT ACA TTT CAG 96Leu Gly Trp Val Gln Pro Ser Leu Gly Gln As - #p Trp Leu Thr Phe Gln# 30- AAA AAA CAC ATC ACA AAC ACA AGG GAT GTT GA - #T TGT GAT AAT ATC ATG 144Lys Lys His Ile Thr Asn Thr Arg Asp Val As - #p Cys Asp Asn Ile Met# 45- TCA ACA AAC TTG TTC CAC TGC AAG GAC AAG AA - #C ACT TTT ATC TAT TCA 192Ser Thr Asn Leu Phe His Cys Lys Asp Lys As - #n Thr Phe Ile Tyr Ser# 60- CGT CCT GAG CCA GTG AAG GCC ATC TGT AAA GG - #A ATT ATA GCC TCC AAA 240Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gl - #y Ile Ile Ala Ser Lys# 80- AAT GTG TTA ACT ACC TCT GAG TTT TAT CTC TC - #T GAT TGC AAT GTA ACA 288Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Se - #r Asp Cys Asn Val Thr# 95- AGC AGG CCT TGC AAG TAT AAA TTA AAG AAA TC - #A ACT AAT AAA TTT TGT 336Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Se - #r Thr Asn Lys Phe Cys# 110- GTA ACT TGT GAA AAT CAG GCA CCA GTT CAT TT - #T GTT GGA GTT GGA TCT 384Val Thr Cys Glu Asn Gln Ala Pro Val His Ph - #e Val Gly Val Gly Ser# 125# 387Cys- (2) INFORMATION FOR SEQ ID NO:63:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 129 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:- Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pr - #o Leu Phe Phe Leu Leu# 15- Leu Gly Trp Val Gln Pro Ser Leu Gly Gln As - #p Trp Leu Thr Phe Gln# 30- Lys Lys His Ile Thr Asn Thr Arg Asp Val As - #p Cys Asp Asn Ile Met# 45- Ser Thr Asn Leu Phe His Cys Lys Asp Lys As - #n Thr Phe Ile Tyr Ser# 60- Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gl - #y Ile Ile Ala Ser Lys# 80- Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Se - #r Asp Cys Asn Val Thr# 95- Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Se - #r Thr Asn Lys Phe Cys# 110- Val Thr Cys Glu Asn Gln Ala Pro Val His Ph - #e Val Gly Val Gly Ser# 125- Cys- (2) INFORMATION FOR SEQ ID NO:64:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 355 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..355#/note= "E6FB[Met-(-1)]SerrOnc":- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:- Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Me - #t Tyr Ala Ser Leu Gly# 15- Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gl - #n Asp Ile Asn Asn Tyr# 30- Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Se - #r Pro Lys Thr Leu Ile# 45- Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pr - #o Ser Arg Phe Ser Gly# 60- Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Il - #e Ser Ser Leu Glu Tyr#80- Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Ty - #r Asp Glu Phe Pro Tyr# 95- Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ly - #s Gly Gly Gly Gly Ser# 110- Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gl - #u Val Gln Leu Gln Gln# 125- Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Se - #r Val Lys Met Ser Cys# 140- Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Tr - #p Met His Trp Ile Lys145 1 - #50 1 - #55 1 -#60- Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Va - #l Ala Ile Asp Pro Arg# 175- Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Ly - #s His Lys Ala Lys Leu# 190- Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Me - #t Glu Leu Asn Ser Leu# 205- Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Th - #r Pro Leu Tyr Tyr Phe# 220- Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Va - #l Ser Ser Ala Lys Lys225 2 - #30 2 - #35 2 -#40- Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Me - #t Ser Asp Trp Leu Thr# 255- Phe Gln Lys Lys His Ile Thr Asn Thr Arg As - #p Val Asp Cys Asp Asn# 270- Ile Met Ser Thr Asn Leu Phe His Cys Lys As - #p Lys Asn Thr Phe Ile# 285- Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cy - #s Lys Gly Ile Ile Ala# 300- Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Ty - #r Leu Ser Asp Cys Asn305 3 - #10 3 - #15 3 -#20- Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Ly - #s Lys Ser Thr Asn Lys# 335- Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Va - #l His Phe Val Gly Val# 350- Gly Ser Cys 355__________________________________________________________________________

Recombinant ribonuclease proteins

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Ardelt et al., J. Biol. Chem., 266(1): 245-251, Jan. 5, 1991.
Newton et al. J. Biol. Chem., 269(43): 26739-26745, Oct. 28, 1994.