CA2245804C - Recombinant ribonuclease proteins - Google Patents

Abstract

The invention relates to ribonucleases derived from a native ribonuclease found in the oocytes of Rana pipiens. Various humanized and recombinant forms of these molecules are described as well as uses for them.

Description

'WO 97/31116 PCT/US97/02588 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, B:iochem. Biophys. Res. Common. 67:110-118, Blank et al., Human body fluid ribonucleases: detection, interrelationships and s~.gnificance 1-203-209 {IRL Press, London, 1981)) that are ea~pressed 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 a1. , 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 Rang pipiens has homology to RNase A (Ardelt et al., 1991, J. Biol. Chem.
256:245-251). This protein has been termed ONCONASE~, Alfacell Corporation, N.J. See also e.g., Darzynkiewicz et a1. (1988) Cell Tissue Ki.rlet. 21, 169-182, Mikulski et a1.
.. 35 (1990) Ce.I1 Tissue Kinet. 23, 237-246. This protein is also described in U.S. Patent No. 4,888,172. Phase I and Phase » I/IT clinical trials of ONCONASE~ as a single therapeutic agent in patients with a variety of solid tumors (Mikulski et a1. (1993) Int. J. of Oncology 3, 57-64) or combined with ta~moxifen in patients with advanced pancreatic carcinoma have

2 recently been completed (Chun et a1. (1995) Proc Amer Soc Clin Onco1 14 No. 157, 210). Conjugation of ONCONASE~ to cell-type-specific ligands increased its potency towards tumor cells (Rybak et a1.(1993} Drug Delivery 1, 3-10). Taken together, these results indicate that ONCONASE° 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 recambinantly express an active cytotoxic molecule related to ONCONASE°. 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 Nat1 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 a1.(1991} J. Biol. Chem 266, 1202-22207, Newton et a1. (1992) J. Biol. Chem. 267, 19572-19578) or recombinant means (Rybak et a1. Proc. Natl. Acad. Sci. IT.S.A. 89, 3165,

3 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 AZIergy Clinics of North Amexica 11:2, 359-380.
l3uman-derived ribonucleases of interest include eosinophil-derived neurotoxin (EDN) and angiogenin.
SUMMARY OF THE INVENTION
IO We have discovered how to construct RNases which are highly cytotoxic and which are modifications of the native ONCONASE~ (nOnc). When the nOnc was expressed recombinantly 3_t was not found to have significant cytotoxicity. Our modified versions {rOnc), however, are highly cytotoxic and L5 atherwise retain the advantages of the native ONCONASE°
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 20 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 cytotaxicity as will 25 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.
30 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 35 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

4 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 alsoo 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.
Various embodiments of this invention provide a ribonuclease molecule comprising: (a) an amino terminal end beginning with a methionine which is followed by any amino acid other than glutamic acid; (b) when aligned for maximum correspondence with SEQ ID N0:13, a cysteine at amino acicL
positions 26, 40, 58, 84, 95 and 110; a lysine at position 41 and a histidine at position 119, and (c) an nOnc-derived amino acid sequence; wherein said ribonuclease molecule has measurable ribonuclease activity.
A ribonuclease of this invention may be one which has an amino terminal end selected from the group consisting of: Met-Ala; Met-Ala-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 N0:14); Met-Lys-Pro-(J) (SEQ ID N0:15);
Met-Asn; Met-Gln; Met-Asn-(J); Met-Gln-(J); Met-Asn-(J)-Pro (SEQ ID N0:16); Met-(J)-Lys; Met-(J)-Lys-Pro (SEQ ID NU:17);
and Met-(J)-Pro-Lys (SEQ ID N0:18); where (J) is Ser, Tyr or Thr. In other embodiments, a ribonuclease of this invention may have an amino terminal end selected from the group consisting of Met-Ser; Met-Tyr or Met-Thr.

6G4 682 0274 CA 02245804 2006-O1-17 04:09:40 p.m. 01-11-2006 4 /5 4a various embodiments of this invention provide a ribonuclease of this invention the amino acid sequence comprises a sequence having the foriaula: Met(-1) eosinophil derived neurotoxin~l_a~ Oac~a-loss, wherein Mat(-1) refers to an S amino terminal residue of Met; wherein eosinophil derived neurotoxin ~1_,~~ refers to a coatifluous sequence of amino acids of a length beginning at amino acid position 1 of eosinophil derived neurotoxin (SEQ ID N0:9) and continuing to and including amino acid position "m" of eosiaophil derived neurotoxin; wherein oac~a_~oa~ refers to a seQueace of contiguous amino acids beginning at amino acid position "n"
an8 continuing to and including amino acid position 104 as set out in S$Q ID N0:1; and wherein "m" is the amino acid position of eosissophil derived neurotoxin selected from the group consisting of 5, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 2a; such that:
when m is 21, n is 16 or 17;
when m is an, n is 17i when m is 20, n is 16s when m i 19 a i's15 f s , when m is 18, n is 14J

when m is 17, n is 1a or 13;

when m is 16, n is 11, 12, 13 or 14;

when m is 15, a is 10;

when m is 14, n is 9;

when m is 13, n is 8; and when m is 5, n is 1.
various embodiments of this invention provide a pharmaceutical composition coanprisiag a cytotoxic amount of a ribonuclease of this invention and a pharmaceutically acceptable carrier.
various embodiments of this invention provide nucleic acids encoding a ribonuclease of this invention as 4b well as vectors and host cells comprising such nucleic acids of this invention.
Various embodiments of this invention provide a method of selectively killing cells in vitro comprising contacting cells to be killed with a ribonuclease of this invention joined to a ligand binding moiety.
Various embodiments of this invention provide the use of a ribonuclease of this invention joined to a ligand binding moiety for selectively killing cells.
Various embodiments of this invention provide the use of a ribonuclease of this invention joined to a ligand.
binding moiety for preparation of a medicament for selectively killing cells.
IS BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE LEGENDS
Figure 1 shows the deduced amino acid sequence of the Rana clone 9 (SEQ ID N0:2), 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.
Figures 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. Figure 2B
shows the sequence alignment of the N-terminal sequences of nOnc (SEQ ID N0:3), rEDN (SEQ ID N0:4), [Met-(-1)]rOnc 4c containing a Gly (G) instead of Asp in position 20 (SEQ ID
N0:5), rEDN~l_2l~rOnc with an Asp in amino acid position 26 (SEQ ID N0:6) and rEDN~l_2lyrOncG26 with a Gly in position 26 (SEQ ID N0:7). Bold letters indicate conserved residues, capital letters show the sequence deduced from Rana clone 9.

Figures 3A-3D show the inhibition of protein ~~ynthesis in human tumor cells by nOnc, rEDN, [Met-(-1)]rOnc or hybrid proteins. Cells (104) were plated in individual 96-well microtiter culture plates and treated with varying

5 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:
A.CHN, 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~l_21)r~nc, (open circles);
rEDN~l_21) rOncG26 (solid circles) .
Figure 4 shows a sequence alignment of some members of the RNase A superfamily: Frog lectin is from Rana catesbeiana, ONCONASE~, EDN, ECP (human eosinophil Cationic protein), Ang is bovine angiogenin, Seminal is bovine seminal R7Uase, 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.
Figure 5 shows the inhibition of protein synthesis by MetSerOnc and MetSer- or MetGlu-OncFvs. The cytotoxic ei°fect of the single chain antibody rOnc fusion proteins;
EEiFB [Met- (-1) ] SerrOnc (closed circles) , [Met- (-1) ] SerrOnc-AngFBE6 (open squares) and [Met-(-1)]GlurOncFHE6 (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 ~ 35 bovine serum. Additions were made in a total volume of 10 u1 and the plates were incubated at 37° for 3 days. Phosphate buffered saline containing 0.1 mCi of [~4C]leucine was added far 2-4 h and the cells were harvested onto glass fiber

6 PCT/US97/02588 filters using a PHIL cell harvester, washed with water, dried with ethanol and counted. The results were expressed as per cent of_[14C]Ieucine incorporation in the mock-treated wells. -Figures 6A and 6B show inhibition of protein synthesis in an assay as described for Figures 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).
lE'igure 7 shows inhibition of protein synthesis in an assay as described for Figures 3A-3D using cell line SF539, human glioma cells and comparing three fusion proteins corresponding to MetSerOnc (SEQ ID N0:39 with a Met-Ser amino terminal end): MetSerOnc (closed circles), MetSerOncC4 (MetSerOnc with a Cys at amino acid position 5 of SEQ ID
N0:39, closed squares) and MetSerOncC72 (MetSerOnc with a Cys at amino acid position 73 of SEQ ID N0:39, 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 N0:13. 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 N0:13 as this is the reference sequence 'WO 97/31116 PCTlUS97/02588

7 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 1I9 with reference to the bovine RNase A, SEQ ID N0:13 (such positions correspond to amino acid position numbers 19, 30, 48, 68, 75 and 90 and 87 and 104 of the nOnc sequence rEaspectively set out in SEQ ID NO:1).
The rOnc molecules of this invention are those that halve 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 N0:13), 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 N0:14) ;
Met-Lys-Pro-(J) (SEQ ID N0:15);
Met-Asn;
Met-Gln;
Met-Asn- (J) ;

8 Met-Gln- (J) ;
Met-Asn- (J) -Pro (SEQ ID N0:16) ;
Met- (J) -Lys;
Met-{J)-Lys-Pro (SEQ ID N0:17); and Met- (J) -Pro-Lys (SEQ ID N0:18) ;
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 ~1-m) Onc ~n_ Z fl4 ) wherein Met(-1) refers to an amino terminal residue of Met; wherein EDNt1-m) refers to a contiguous sequence of amino acids of a length beginning at amino acid position 1 of EDN (SEQ ID N0:9) and continuing to and including amino acid position ~~m~~ of EDN; wherein Onc ~n_lfl4) refers to a sequence of contiguous amino acids beginning at amino acid position ~~n~~
and continuing to and 3.ncluding 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.

9 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 N0:1.1 or that at amino acid positions 101 to 107 of SEQ ID N0:20.
The nucleic acid sequence for human angiogenin is known and is set out in U.S. Patent 5840840.
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:l9, 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:l9, 21, 23 and 25, including conservatively modified variants thereof. The most preferred sequence includes SEQ ID N0:22, one which employs as 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 Cya with reference to SEQ ID N0:39.
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, lbut are not limited to, frog lectin from Rana catesbeiana (Titani et al., Biochemistry 26:2189 (1987)); ONCONASE~ (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 (Heintama et al., Prog. Biophys. Mol. Biol. 51:165 (1988)).

Amino acid sequence alignment for such RNases are also set out in Fig. 4 and in Youle et a3., Crit. Rev. Ther.
Drug. Carrier Systems 10:1-28 (1993) and in U.S. Patent 5840840.
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 Wi:ley and Sons (New York), and Hale and Marham (1991) The Harper Collies 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 ternis 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-IUH 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 ICSO (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 [35S]methionine into acid precipitable protein. IC5o 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, WI. Ribonuclease activity using high molecular weight RNA and tRNA is determined at 37°C
through the formation of perchloric acid soluble nucleotides following published protocols (Newton, D.L., et aI. (1996) WO 97!31116 PCT/LTS97/02588 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 aZ. (i984) .Biochimica et Biophysics Acta 788:356-363; Libonati, M. et a1.
(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°C. The in vitro translation assay (St.
Clair, D.K., et aZ. (1987) Proc. Nat!. 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 a1. (1991) J. Nat!. 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, 9,1, 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 se=quences, 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
al.! 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 sSTnthetically, 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 p7.asmid, 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 Tij ssen ( 1993 ) Laboratory Techniques .i.n 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° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and ph. The Tm 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 Tm 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 prcperties (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 'WO 97/31116 PCT/LTS97/02588 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, California, USA) .
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.
~J~ol. 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, California, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA); 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 a1. (1988) Nucleic Acids Research 16, 10881-90; Huang, et a1. (1992) Computer Applications in the B~osciences 8, 155-65, and Pearson, et a1. (1994) Methods in Molecular Biology 24, 307-31. Alignment is also often performed by inspection and manual alignment.
The term "substantial identity" or "substantial sa.milarity" 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° C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm 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 delivered 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 t:wo or more polypeptides through a peptide bond formed between t:he 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.
~akincr rOnc-derived Nucleic Acids and Polype~ti~des 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 thtese 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 T
to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et a1. (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, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH
Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO
BRL Life Technologies, Inc. (Gaithersberg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, CA, and Applied Biosystems (Foster City, CA), 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.
1n 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 lipase chain reaction (LCR), Q~i-replicase amplification and other RNA polymerase mediated techniques (e. g., NASBA) are found in Berger, Sambrook et a1. (1989) Molecular Cloning - A Laboratory Manual (2nd Ed) Vol. I-3; and Ausubel,_ as well as Mullis et al., (1987) U.S._ Patent No. 4, 683,202; PCR Protocols A Guide to r Methods and Applications (Innis et a1. eds) Academic Press :Lnc. San Diego, CA (1.990) (Innis); Arnheim & Levinson (October :L, 1990) C&E1V 36-47; The Journal Of NIFI Research (1991) 3, 81-94; (Kwoh et aZ. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et a1. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et a1. (1989) J. Clin. Chew 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) ~3iotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560;
Barringer et a1. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology I3: 563-564. Improved methods of cloning i''n 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 a1. (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 Ps=arson and Regnier (1983) J. Chrom. 255:137-149. The seaquence 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 a1. (1987) Nature 328:731-734 and Sambrook et al. (1989) Molecular Cloni.rag - A Laboratory Manual (2nd Ed) Vol. 1-3; Innis, -Ausbel, Berger, Needham VanDevanter and Mullis (a11 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.
'n n rv tive Mo 'fi tions of th Nu i A i an Polypgptides 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 r 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) Gerse 8:81-97, Roberts et a1. (1987) Nature 328:731-734 and Sambrook, Innis, Ausbel, Berger, Needham Zi'anDevanter and Mullis (a11 supra) .
Most commonly, polypeptide sequences are altered by changing the corresponding nucleic acid seguence 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 know~.edge 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.
r~Onc Fusion Proteins and Other Thera~~eutic MoaPties 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 N0:32 (and SEQ ID N0:31) 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 N0:31 has been and can be omitted. The nucleic acid sequence for the NLS is nucleic acids 1-22 of SEQ ID N0:31. A signal peptide is also exemplified at amino acid positions 1-25 of SEQ ID N0:63.
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 N0:33) ~iVVO 97131116 PCT/US97/02588 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., Faterson, 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 N0:33) 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 te=lomerase_ 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 th.e 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
N0:64) 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 em~al oyed .
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. -Patent 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. Poiypeptides typically contain a variety of functional groups; e.g., carboxylic acid (COON) or free amine (-NH2) 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 Illinois.
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.
Patent No. 4,671,958). Procedures for generation of free sulfhydzyl 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. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;
4,680,338; 4,569,789; and 4,589,071; and Horlinghaus et al.
Cancer Res. 47: 4071-4075 (1987), 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 ~:n Clinical Medicine, Academic Press, pp. 168-190 11982), Waldmann, Science, 252: 1657 (1991), U.S. Patent Nos.
4,545,985 and 4,894,443.
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 then the effector is to be released at the target site. Cleaving of the linkage to release the agent j°rom 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 inmnunoconjugates comprising linkers which a;re 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 o:ne 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 aI. J.
Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chew. Co., Rockford, Ill. (1984), 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. Patent No. 4,458,066.

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 enhaneer 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 rnammalian cells. Cells transformed by the plasmids can be ~3elected 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 t:he art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like {see, generally, R. Scopes, Protein Purification, S~pringer-Verlag, N.Y. (1982), Deutscher, Methods in ~;nzymoZ ogy Vo1. 182 : Gui de to Pro tein Purifi ca ti on . , Academi c 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 b:i.nding moiety that specifically delivers the reagent to the ce=lls 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 a.n 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 vi vo 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.
~'harma~eutical 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 'WO 97131116 PCT/US97/02588 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 Remingtorl's .Pharmaceutical Science, 15th ed. , Mack Publishing Company, Euston, Pennsylvania (19$0).
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.
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 aad Bxpressioa of rOac sad Onc conjugates ari th EDN .
A. Materials. Native ONCONASE~ ("nOnc") (SEQ ID NO:1) Ardelt et aI. (1991) J. Biol. Chew. 256, 245-251 and recombinant human EDN ("rEDN") (SEQ ID N0:9) Newton et al. (1994): J Biol Chem. 269, 26739-26745 were purified from Rang pipiens oocytes, NASCO, Fort Atkinson, WI and Escherichia coli, respectively, as described. Antibodies to the denatured proteins were prepared by Assay Research, Inc., College Park, Nm. Reagents for performing PCR, and direct cloning of PCR
products, were obtained from Perkin-Elmer Corp., Norwalk, CT
and from Invitrogen, San Diego, CA respectively. Substrates for the ribonucleaee assays were purchased from Sigma, St.
Louis, MO and Hoehringer Mannheim, Indianapolis, IN. 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).
8. PCR Cloaiag of Onconase. Rana pipiens genomic DNA was isolated according to standard procedures using proteir_ase R
Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular Cloning, a laboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 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. Chew. 256, 245-251. The PCR reaction was perfozmed according to the manufacturer's instructions using 15 ~Cg of genomic DNA in 100 ~cl. All reagents except the DNA were combined and incubated at 95°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°C for 1 min, annealing for 2 min at 55°C and primer extension for 2 min at 72°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 N0:35) and the reverse primer encoding amino acid residues 9 0 - 9 8 ( TGTGA ( AG ) AA ( CT ) CAGGC ( AC ) CC ( TA ) GT ( GT ) CA ( CT ) TTT ) ( S EQ ID
N0:36). This fragment was subcloned into pCR~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 N0:2). The corresponding nucleic acid sequence is set out in SEQ ID N0:37.
C. Plasmid Construction, E.xpressioa, 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 aI. (1990) BioTechniques 8, 528-532 with amino acid residues 1-15 of nOnc or amino acid residues 1-2 1 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-lld, Novagen, Madison, WI. All procedures were accomplished essentially as described in Newton et a1. (1994) J Bio1 Chem. 269, 26739-26745. The plasmids were expressed in BL21(DE3) E. coli cells as recommended by the supplier, Novagen, Madison WI. The fusion proteins were isolated from inclusion bodies, denatured, renatured and dialyzed as described Newton et aI. (1994) J Biol Chem. 269,26739-26745 before being applied to a CM-Sephadex~ C-50 column, Pharmacia Biotech Inc., Piscataway, NJ. The proteins were eluted with a NaCl gradient (0-0.5M) in 20 mM Tris-HC1, 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, MA and dialyzed against 20 mM Tris-HC1, 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 a1. (1994) J Neurosci 14, 538-544 at 37°C through the formation of perchloric acid soluble nucleotides following published protocols (Newton et a1. (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 a1. (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 ar pH 7) contained substrate and appropriate amounts of enzyme solution at 25°C. The in vitro translation assay, St. Clair et a1. (1987) Proc Nat1 Acad Sci 84, 8330-8334, and the cell viability assays, Pearson et aZ.
(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)7 rOn.c and rOna chimeras. Eight different oligonucleotide primers were designed to correspond to specific regions in the primary amino acid structure of nOnc, Ardelt et a1. (1991) J. BioZ.
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 by fragment. That PCR product, here denoted Rana clone 9, was cloned into pCR~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
N0:38) was constructed by PCR extension and cloned into an expression vector using methodology previously described Newton et a3. (1994) J Bio1 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 a.t the top of Fig. 2A. The primers were designed to overlap with the DNA sequence of the Rang clone 9 PCR product. The' plasmid was expressed in HL21(DE3) E. coli and the recombinant protein was isolated from inclusion bodies as described in Newton et a1. (1994) J Bio1 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 th~.s expression system contains an extra methionine at the amino terminal (Met-(-1)] (SEQ ID N0:39) 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. 2H), 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. 2H, rEDN~l_2i)Onc). PCR cloning can result in sequence errors.
Indeed, the DNA sequence of the gene encoding EDN~l-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~l_21)rOncG26 in Fig. 2H. Another plasmid containing encoding rEDNtl_2i)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 (rOncGly2o, Figs.
2A and H).
E. Riboauclease activity of Oac, EDN, IMet-t-1)]rOnc sad hybrid rOac proteias. Hoth nOnc (Lin, J.J., et al. (1994) Biochem Biophys Res Common 204, 156-162) and EDN (Saxena et a1. (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 [3SS3methionine into acid precipitable protein. Whereas both nOnc and EDN inhibited protein synthesis with IC5os of 0.2 and 1.3 ng/ml, respectively, CMe t - ( -1 ) ] rOnc , [Me t - ( -1 ) ] rOncG2 0 , and rEDN ~ 1 _ 21 ) rOncG2 6 we re considerably less potent (ICsos 98, 28 and 28 ng/ml, respectively). The least active RNase in this assay was rEDN~l_21)rOnc with an ICSO 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 a/. (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~l_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 a1. (1991) J. Biol. Chem. 256, 245-251, Sorrentino et a/. (1992) J. Biol. Chem 267, 14859-14865, Ardelt et a1. (1994) Protein Sci 3, Suppl. 1, 137).
Consistent with the results presented in Table 1, [Met- (-1) J rOnc (SEQ ID N0:39 ) and rEDN~l_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 a1. (1991) J.
Biol. Chem. 256, 245-251, Ardelt et a.I. (1994) Protein Sci 3, ~SuppL. 1, 137). The enhanced EDN-like activity of the Gly-containing hybrid protein is also evidenced by its 7behavior with poly(A,C) which is an excellent substrate for EDN. As seen in Table 2, only rEDN~l_2i)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~l_21)rOncG26 with UpG, an optimal Onconase substrate (Ardelt et aI. (1994) Protein Sci 3, Szappl. 1, 137) . In summary, both [Met- (-1) 3 rOnc and rEDN~l_21) rOnc are less e:nzymatically active than nOnc or rEDN. .Although, rEDN~l_2z)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.

WO 97!31116 PCTlLTS97/02588 Table 1 Activity of [Met-(-1)]rOnc or Hybrid Proteins en the Rabbit Reticulocyte Lysate compared to rEDN or nOnc in the Presence or Absence of PRI
ICS' (ng/ml) (-)I'~ O+-)1'~ Fold Difference nOnc 0.2 0.24 1.2 rEDN 1.3 > 40 > 30.7 [Met-{-1)]rOnc96 140 1.4 [Met-(-1)]rOncG2028 24 0.9 rEDN~I_z,~rOnc1600 3200 2 rEDN~I zorOnoG2628 600 21 ~I ~ ~s a bit protein synt esis concentration y 50 m a o protein necessary to rabbit reticulocyte lysate. Data points result from the average of at Ieast three assays.

'WO 97131116 3 9 PCT/US97/02588 Table 2 Activity of RNases on Different Substrates RNase Activity(units/mg _ protein)' _5ubstrate Assay rEDN Onc [Met-(-I)]rOncrEND,_Z~~OncrEDN1_Z~~OncG~
pH n Yeast RNA' 6.0 6000 560 0.01 8 120 1:RNA'' 6.0 1100 390 12 4 340 IRNA' 7.5 46000 60 50 130 7400 poly (A,C)b 7.0 8000 0.04 5 4.5 3gpp UpGb 6.5 0.05 O. <0.01 <0.01 <0.01 poly Ub 7.0 16.5 0.15 0.20 0.35 4.5 ase activity was quanttta ough the ormanon o perc one acid so ub a nucleon es.
mts arc defined as the changes in A~ per minute calculated from the slopes of the linear part of the assays. Each value is the average of 2-3 assays in separate Cxperiments.
6Spectrophotometric assays were performed accordixtg to Deprisco et al. (1984) and Libonati and Floridi (I969) as described in Materials and Methods. Units are defined as the changes in A~ per minute calculated from the slopes of the linear part of the assays. Each value is the average of two or more determinations_ °[rvlet-(-I)]rOncG20 had no detectable activity.

WO 97/31116 PCT/fJS97/02588 F, Inhibition of protein synthesis is four human tumor cell liaes 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~z-2~~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~l_2z~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~2_2i~ 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 Mo1 Bio1 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 Mo3 BioZ). This and subsequent alignments were carried out using ALIGN (Satow Y., Cohen G.H., Padlan E.A., Davies D.R., (1986), J. MoZ Bio1 190, 593-604).
H. Modeliag the structures of the hybrid RNases. The coordinates for Onc and EDN were superimposed on the basis of Ca 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' atom pairs). The hybrid protein was modeled by manual rebuilding and geometry regularization using TOM (Cambillau C., Horjales E., (1987), J. Mol Graph 5, I74-177).

Wo 97/31116 41 PCT/ITS97/02588 Subsequently, the models for rEDN~l_2i) rOnc and rEDNti_ 2:L)rOncG26 were assigned an overall B-factor of 15 A2 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 Ca trace alignment being 0.44 for the Ca 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 App1 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. Bio1 Chean 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 act=ive 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 Mo1 Bio1). 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 a.n 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~l_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 Bio1 236, 1141-1153; Masimann 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 Bio1). 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 Mo1 Bio1 252, 328-336). The corresponding residue in nOnc a.s 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 Bi 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 a.n 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.

'WO 97/31116 43 PCTlIJS97/02588 E:Kample TI. rOne-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 nucleic acid sequence set out in SEQ ID N0:40 and the amino acid sequence set out in SEQ ID
NO:41 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 N0:41. "F8" 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. This molecule includes a Ser at amino acid position 252 instead of a Glu. E6FB[Met-(-I)]GlurOnc refers to the sequence in SEQ ID N0:40. 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 anl~ibodies 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 MOC3IFBMetSerrOnc are set out on SEQ ID NOS:54 and 55. The nucleic acid and amino acid sequences fox 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 a.n 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 E& constructs, in particular, had a very high level of activity -- up to 45 fold difference over the two non-E6 molecules. See also Figure 5. MetSerOncAng molecule was made corresponding to amino acids 1-107 of SEQ ID N0:47.
Table 3 Activity of modified rOnc and modified rOncFvs on protein synthesis fCso (~ Fold Difference nOnc IO 1 [Met-(-1)Ser]rOnc 8 NSD
E6FB[Met-(-i)Ser]rOnc 0.22 45 [Met-{-I)Ser]rOnc-AngFBE6 0.27 37 [Met-(-I)Glu]rOncFBE6 0.50 20 ~'he concentrations necessary to inhi it protein synthesis by S m SF human glioma cells. NSD, no significant difference.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: The Government of the United Sates of America, represented by The Secretary of the Department of Health and Human Services (ii) TITLE OF INVENTION: Recombinant Ribonuclease Proteins (iii) NUMBER OF SEQUENCES: 63 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fetherstonhaugh & Co.
(B) STREET: Box 11560, Vancouver Centre, 2200 - 650 West Georgia Street (C) CITY: Vancouver (D) PROVINCE: B.C.
(E) COUNTRY: Canada (F) POSTAL CODE: V6B 4N8 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,245,804 (B) FILING DATE: 19-FEB-1997 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/011,800 (B) FILING DATE: 21-FEB-1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Fetherstonhaugh & Co.
(C) REFERENCE/DOCKET NUMBER: 40330-1363 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (604) 682-7295 (B) TELEFAX: (604) 682-0274 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 104 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..104 (D) OTHER INFORMATION: /label= nOnc /note= "native ONCONASE (Registered Trademark) from Rana pipiens"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "Xaa = pyroglutamic acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Xaa Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 1..83 (D) OTHER INFORMATION: /note= "Rana clone 9 peptide from Rana pipiens genomic DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 1..28 (D) OTHER INFORMATION: /note= "N-terminal sequence of nOnc with Glu in place of pyroglutamic acid in position 1"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Glu Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 1..34 (D) OTHER INFORMATION: /note= "N-terminal sequence of recombinant eosinophil-derived neurotoxin (rEDN)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Phe Glu Thr Gln His Ile Asn Met Thr Ser Gln Gln Cys Thr Asn Ala Met Gln Val Ile Asn Asn Tyr Gln (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 1..28 (D) OTHER INFORMATION: /note= "N-terminal sequence of [Met-(-1)]rOncG20, containing a Gly to Asp substitution at position 20 of [Met-(-1)]rOnc, and without the extra N-terminal Met from the E. coli bacterial expression system"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Glu Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Gly Asn Ile Met Ser Thr Asn Leu Phe (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 1..34 (D) OTHER INFORMATION: /note= "N-terminal sequence of rEDNl-2lrOnc, without the extra N-terminal Met from the E. coli bacterial expression system"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Phe Glu Thr Gln His Ile Asn Met Thr Ser Gln Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 1..34 (D) OTHER INFORMATION: /note= "N-terminal sequence of rEDNl-2lrOncG26, containing a Gly to Asp substitution at position 26 of rEDNl-2lrOnc, and without the extra N-terminal Met from the E. coli bacterial expression system"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Phe Glu Thr Gln His Ile Asn Met Thr Ser Gln Asp Val Asp Cys Gly Asn Ile Met Ser Thr Asn Leu Phe (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 111 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..111 (D) OTHER INFORMATION: /note= "Frog Lectin from Rana catesbeiana"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Glu Asn Trp Ala Thr Phe Gln Gln Lys His Ile Ile Asn Thr Pro Ile Ile Asn Cys Asn Thr Ile Met Asp Asn Asn Ile Tyr Ile Val Gly Gly Gln Cys Lys Arg Val Asn Thr Phe Ile Ile Ser Ser Ala Thr Thr Val Lys Ala Ile Cys Thr Gly Val Ile Asn Met Asn Val Leu Ser Thr Thr Arg Phe Gln Leu Asn Thr Cys Thr Arg Thr Ser Ile Thr Pro Arg Pro Cys Pro Tyr Ser Ser Arg Thr Glu Thr Asn Tyr Ile Cys Val Lys Cys Glu Asn Gln Tyr Pro Val His Phe Ala Gly Ile Gly Arg Cys Pro (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 134 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..134 (D) OTHER INFORMATION: /note= "Human eosinophil-derived neurotoxin (EDN)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Phe Glu Thr Gln His Ile Asn Met Thr Ser Gln Gln Cys Thr Asn Ala Met Gln Val Ile Asn Asn Tyr Gln Arg Arg Cys Lys Asn Gln Asn Thr Phe Leu Leu Thr Thr Phe Ala Asn Val Val Asn Val Cys Gly Asn Pro Asn Met Thr Cys Pro Ser Asn Lys Thr Arg Lys Asn Cys His His Ser Gly Ser Gln Val Pro Leu Ile His Cys Asn Leu Thr Thr Pro Ser Pro Gln Asn Ile Ser Asn Cys Arg Tyr Ala Gln Thr Pro Ala Asn Met Phe Tyr Ile Val Ala Cys Asp Asn Arg Asp Gln Arg Arg Asp Pro Pro Gln Tyr Pro Val Val Pro Val His Leu Asp Arg Ile Ile (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..133 (D) OTHER INFORMATION: /note= "Human eosinophil cationic protein (ECP)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Arg Pro Pro Gln Phe Thr Arg Ala Gln Trp Phe Ala Ile Gln His Ile Ser Leu Asn Pro Pro Arg Cys Thr Ile Ala Met Arg Ala Ile Asn Asn Tyr Arg Trp Arg Cys Lys Asn Gln Asn Thr Phe Leu Arg Thr Thr Phe Ala Asn Val Val Asn Val Cys Gly Asn Gln Ser Ile Arg Cys Pro His Asn Arg Thr Leu Asn Asn Cys His Arg Ser Arg Phe Arg Val Pro Leu Leu His Cys Asp Leu Ile Asn Pro Gly Ala Gln Asn Ile Ser Asn Cys Arg Tyr Ala Asp Arg Pro Gly Arg Arg Phe Tyr Val Val Ala Cys Asp Asn Arg Asp Pro Arg Asp Ser Pro Arg Tyr Pro Val Val Pro Val His Leu Asp Thr Thr Ile (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 125 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..125 (D) OTHER INFORMATION: /note= "Bovine angiogenin (Ang)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ala Gln Asp Asp Tyr Arg Tyr Ile His Phe Leu Thr Gln His Tyr Asp Ala Lys Pro Lys Gly Arg Asn Asp Glu Tyr Cys Phe His Met Met Lys Asn Arg Arg Leu Thr Arg Pro Cys Lys Asp Arg Asn Thr Phe Ile His Gly Asn Lys Asn Asp Ile Lys Ala Ile Cys Glu Asp Arg Asn Gly Gln Pro Tyr Arg Gly Asp Leu Arg Ile Ser Lys Ser Glu Phe Gln Ile Thr Ile Cys Lys His Lys Gly Gly Ser Ser Arg Pro Pro Cys Arg Tyr Gly Ala Thr Glu Asp Ser Arg Val Ile Val Val Gly Cys Glu Asn Gly Leu Pro Val His Phe Asp Glu Ser Phe Ile Thr Pro Arg His (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 124 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..124 (D) OTHER INFORMATION: /note= "Bovine seminal RNase"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Lys Glu Ser Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Gly Asn Ser Pro Ser Ser Ser Ser Asn Tyr Cys Asn Leu Met Met Cys Cys Arg Lys Met Thr Gln Gly Lys Cys Lys Pro Val Asn Thr Phe Val His Glu Ser Leu Ala Asp Val Lys Ala Val Cys Ser Gln Lys Lys Val Thr Cys Lys Asn Gly Gln Thr Asn Cys Tyr Gln Ser Lys Ser Thr Met Arg Ile Thr Asp Cys Arg Glu Thr Gly Ser Ser Lys Tyr Pro Asn Cys Ala Tyr Lys Thr Thr Gln Val Glu Lys His Ile Ile Val Ala Cys Gly Gly Lys Pro Ser Val Pro Val His Phe Asp Ala Ser Val (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 124 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..124 (D) OTHER INFORMATION: /note= "Bovine pancreatic RNase A"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Ser Thr Ser Ala Ala Ser Ser Ser Asn Tyr Cys Asn Gln Met Met Lys Ser Arg Asn Leu Thr Lys Asp Arg Cys Lys Pro Val Asn Thr Phe Val His Glu Ser Leu Ala Asp Val Gln Ala Val Cys Ser Gln Lys Asn Val Ala Cys Lys Asn Gly Gln Thr Asn Cys Tyr Gln Ser Tyr Ser Thr Met Ser Ile Thr Asp Cys Arg Glu Thr Gly Ser Ser Lys Tyr Pro Asn Cys Ala Tyr Lys Thr Thr Gln Ala Asn Lys His Ile Ile Val Ala Cys Glu Gly Asn Pro Val Val Pro Val His Phe Asp Ala Ser Val (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "Xaa = Ser, Tyr or Thr"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Lys Xaa Pro (2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Xaa = Ser, Tyr or Thr"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Lys Pro Xaa (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "Xaa = Ser, Tyr or Thr"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Asn Xaa Pro (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note= "Xaa = Ser, Tyr or Thr"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Xaa Lys Pro (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note= "Xaa = Ser, Tyr or Thr"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Xaa Pro Lys (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..321 (D) OTHER INFORMATION: /note= "MetSerOnc99Ang117"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:

IleSer Asp TrpLeu ThrPhe GlnLys LysHis Ile ThrAsn ThrArg AspVal Asp CysAsp AsnIle MetSer ThrAsn Leu PheHis CysLys AspLys Asn ThrPhe IleTyr SerArg ProGlu Pro ValLys AlaIle CysLys Gly IleIle AlaSer LysAsn ValLeu Thr ThrSer GluPhe TyrLeu Ser AspCys AsnVal ThrSer ArgPro Cys LysTyr LysLeu LysLys Ser ThrAsn LysPhe CysVal ThrCys Glu AsnGln AlaPro ValHis Phe ValGln SerIle PheArg ArgPro (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Ile Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gln Ser Ile Phe Arg Arg Pro (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: pairs 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 (D) OTHER INFORMATION : ote="EDNGlyOnc"
/n (xi)SEQUENCE DESCRIPTION: SEQ ID
N0:21:

TTC

MetLys Pro Pro Gln Thr TrpAla GlnTrp Phe GluThr GlnHis Phe CAG

IleAsn Met Thr Ser Asp ValAsp CysGly Asn IleMet SerThr Gln AAG

AsnLeu Phe His Cys Asp LysAsn ThrPhe Ile TyrSer ArgPro Lys ATC

GluPro Val Lys Ala Cys LysGly IleIle Ala SerLys AsnVal Ile TTT

LeuThr Thr Ser Glu Tyr LeuSer AspCys Asn ValThr SerArg Phe Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 111 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Met Lys Pro Pro Gln Phe Thr Trp Ala Gln Trp Phe Glu Thr Gln His Ile Asn Met Thr Ser Gln Asp Val Asp Cys Gly Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 315 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..315 (D) OTHER INFORMATION: /note= "MetTyrrOnc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:

Met Tyr Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Met Tyr Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 315 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..315 (D) OTHER INFORMATION: /note= "MetSerrOnc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Thr Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:27:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH: 18 asepairs 3 b (B) TYPE: leicacid nuc (C) STRANDEDNESS:single (D) TOPOLOGY:lin ear (ii)MOLECULE TYPE:DNA

(ix)FEATURE:

(A) NAME/KEY:CDS

(B) LOCATION:1..318 (D) OTHER "MetLysTyrrOnc"
INFORMATION:
/note=

(xi)SEQUENCE DESCRIPTION: SEQ ID
N0:27:

CTT

MetLys Tyr Asp Trp Thr PheGln Lys Lys HisIleThr AsnThr Leu GAT

ArgAsp Val Asp Cys Asn IleMet Ser Thr AsnLeuPhe HisCys Asp TTT

LysAsp Lys Asn Thr Ile TyrSer Arg Pro GluProVal LysAla Phe ATA

IleCys Lys Gly Ile Ala SerLys Asn Val LeuThrThr SerGlu Ile TGC

PheTyr Leu Ser Asp Asn ValThr Ser Arg ProCysLys TyrLys Cys AAT

LeuLys Lys Ser Thr Lys PheCys Val Thr CysGluAsn GlnAla Asn GGA

ProVal His Phe Val Val GlySer Cys Gly (2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 106 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Met Lys Tyr Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..321 (D) OTHER INFORMATION: /note= "MetAlaAlaTyrrOnc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

Met Ala Ala Tyr Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys LysAsp LysAsn ThrPhe Ile TyrSer ArgPro GluPro ValLys Ala IleCys LysGly IleIle Ala SerLys AsnVal LeuThr ThrSer Glu PheTyr LeuSer AspCys Asn ValThr SerArg ProCys LysTyr 65 70 75 8p Lys LeuLys LysSer ThrAsn Lys PheCys ValThr CysGlu AsnGln 85 9p g5 Ala ProVal HisPhe ValGly Val GlySer Cys (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Met Ala Ala Tyr Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH: pairs 336 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE DNA
TYPE:

(ix)FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..336 (D) OTHER INFORMATION : ote="NL SMetSerrOnc"
/n (xi)SEQUENCE SEQ ID :
DESCRIPTION: N0:31 CGG

Pro Lys Lys Lys LysVal MetSer AspTrp Leu ThrPhe GlnLys Arg AAC

Lys His Ile Thr ThrArg AspVal AspCys Asp AsnIle MetSer Asn CAC

Thr Asn Leu Phe CysLys AspLys AsnThr Phe IleTyr SerArg His AAG

Pro Glu Pro Val AlaIle CysLys GlyIle Ile AlaSer LysAsn Lys TCT

Val Leu Thr Thr GluPhe TyrLeu SerAsp Cys AsnVal ThrSer Ser TAT

Arg Pro Cys Lys LysLeu LysLys SerThr Asn LysPhe CysVal Tyr CAG

Thr Cys Glu Asn AlaPro ValHis PheVal Gly ValGly SerCys Gln (2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Pro Lys Lys Lys Arg Lys Val Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note= "Xaa = an aliphatic amino acid, Ala, Leu, Ile, Val, or Pro"
(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "Xaa = an aliphatic amino acid, Ala, Leu, Ile, Val or Pro"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Xaa = Ser, Met, Cys, Ala or Gln"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Cys Xaa Xaa Xaa (2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Cys Val Ile Met (2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:

(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:

(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..249 (D) OTHER INFORMATION: /note= "Rana 9"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:

(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 315 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..315 (D) OTHER INFORMATION: /note= "[Met-(-1)]rOnc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:

AAA

Met GluAsp TrpLeu Thr PheGln LysLys HisIle ThrAsn ThrArg Asp ValAsp CysAsp Asn IleMet SerThr AsnLeu PheHis CysLys Asp LysAsn ThrPhe Ile TyrSer ArgPro GluPro ValLys AlaIle Cys LysGly IleIle Ala SerLys AsnVal LeuThr ThrSer GluPhe Tyr LeuSer AspCys Asn ValThr SerArg ProCys LysTyr LysLeu Lys LysSer ThrAsn Lys PheCys ValThr CysGlu AsnGln AlaPro Val HisPhe ValGly Val GlySer Cys (2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Met Glu Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1065 base rs pai (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA

(ix)FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1065 (D) OTHER INFORMATION: "sFVFBMetGluOnc"
/note=

(xi)SEQUENCE DESCRIPTION: ID
SEQ N0:40:

TCT

Asp Ile Lys Met Thr Gln Ser Pro Ser MetTyr AlaSer LeuGly Ser GCG

Glu Arg Val Thr Phe Thr Cys Lys Ser GlnAsp IleAsn AsnTyr Ala GGG

Leu Cys Trp Phe Gln Gln Lys Pro Lys SerPro LysThr LeuIle Gly GGG

Tyr Arg Ala Asn Arg Leu Val Asp Val ProSer ArgPhe SerGly Gly Ser GlySer GlyGln AspTyr Ser LeuThr IleSer SerLeu GluTyr Glu AspMet GlyIle TyrTyr Cys LeuGln TyrAsp GluPhe ProTyr Thr PheGly GlyGly ThrLys Leu GluIle LysGly GlyGly GlySer Gly GlyGly GlySer GlyGly Gly GlySer GluVal GlnLeu GlnGln Ser GlyThr ValLeu AlaArg Pro GlyAla SerVal LysMet SerCys Lys AlaSer GlyTyr ThrPhe SerSer Tyr TrpMet HisTrp IleLys Gln ArgPro GlyGln GlyLeu AspTrp Ile ValAla IleAsp ProArg Asn SerAsp ThrIle TyrAsn ProGln Phe LysHis LysAla LysLeu Thr AlaVal ThrSer ThrSer ThrAla Tyr MetGlu LeuAsn SerLeu Thr AsnGlu AspSer AlaVal TyrTyr Cys ThrPro LeuTyr TyrPhe Asp SerTrp GlyGln GlyThr ThrLeu Thr ValSer SerAla LysLys Leu AsnAsp AlaGln AlaPro LysSer Asp MetGlu AspTrp LeuThr Phe GlnLys LysHis IleThr AsnThr Arg AspVal AspCys AspAsn Ile MetSer ThrAsn LeuPhe HisCys Lys AspLys AsnThr PheIle Tyr SerArg ProGlu ProVal LysAla Ile CysLys GlyIle IleAla Ser LysAsn ValLeu ThrThr SerGlu Phe TyrLeu SerAsp CysAsn Val ThrSer ArgPro CysLys TyrLys Leu LysLys SerThr AsnLys Phe CysVal ThrCys GluAsn GlnAla Pro ValHis PheVal GlyVal Gly SerCys (2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 355 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Met Glu Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1137 (D) OTHER INFORMATION: /note= "SigPepGlnOncFBE6"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:

Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pro Leu Phe Phe Leu Leu Leu Gly Trp Val Gln Pro Ser Leu Gly Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly SerGlyGln AspTyr Ser LeuThr IleSer SerLeu Glu TyrGlu Asp MetGlyIle TyrTyr Cys LeuGln TyrAsp GluPhe Pro TyrThr Phe GlyGlyGly ThrLys Leu GluIle LysGly GlyGly Gly SerGly Gly GlyGlySer GlyGly Gly GlySer GluVal GlnLeu Gln GlnSer Gly ThrValLeu AlaArg Pro GlyAla SerVal LysMet Ser CysLys Ala SerGlyTyr ThrPhe Ser SerTyr TrpMet HisTrp Ile LysGln Arg Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 379 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pro Leu Phe Phe Leu Leu Leu Gly Trp Val Gln Pro Ser Leu Gly Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr 78a Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn 78b Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1074 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1074 (D) OTHER INFORMATION: /note= "MetSerOncA87FBE6"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:

Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Ala Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala GlnAla Pro LysSer AspAsp IleLys MetThr Gln SerPro SerSer MetTyr Ala SerLeu GlyGlu ArgVal ThrPhe Thr CysLys AlaSer GlnAsp Ile AsnAsn TyrLeu CysTrp PheGln Gln LysPro GlyLys SerPro Lys ThrLeu IleTyr ArgAla AsnArg Leu ValAsp GlyVal ProSer Arg PheSer GlySer GlySer GlyGln Asp TyrSer LeuThr IleSer Ser LeuGlu TyrGlu AspMet GlyIle Tyr TyrCys LeuGln TyrAsp Glu PhePro TyrThr PheGly GlyGly Thr LysLeu GluIle LysGly Gly GlyGly SerGly GlyGly GlySer Gly GlyGly GlySer GluVal Gln LeuGln GlnSer GlyThr ValLeu Ala ArgPro GlyAla SerVal Lys MetSer CysLys AlaSer GlyTyr Thr PheSer SerTyr TrpMet His TrpIle LysGln ArgPro GlyGln Gly LeuAsp TrpIle ValAla Ile AspPro ArgAsn SerAsp ThrIle Tyr AsnPro GlnPhe 78d Lys HisLys AlaLys LeuThr Ala ValThr SerThr SerThr AlaTyr Met GluLeu AsnSer LeuThr Asn GluAsp SerAla ValTyr TyrCys Thr ProLeu TyrTyr PheAsp Ser TrpGly GlnGly ThrThr LeuThr Val SerSer HisHis His (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 358 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Ala Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Thr Gln Ser Pro Ser Ser 78e Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser His His His (2) INFORMATION FOR SEQ ID N0:46:

78f (i) SEQUENCE
CHARACTERISTICS:

(A) LENGT H: 086 basepai rs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: lin ear (ii) DNA
MOLECULE
TYPE:

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCAT ION:1.. 1083 (D) OTHER INFORMATION : ote="MetSerOncAngsFv"
/n (xi)SEQUENCE SEQ ID :
DESCRIPTION: N0:46 CTT

Met SerAsp Trp ThrPhe GlnLys Lys HisIle ThrAsn ThrArg Leu GAT

Asp ValAsp Cys AsnIle MetSer Thr AsnLeu PheHis CysLys Asp TTT

Asp LysAsn Thr IleTyr SerArg Pro GluPro ValLys AlaIle Phe ATA

Cys LysGly Ile AlaSer LysAsn Val LeuThr ThrSer GluPhe Ile TGC

Tyr LeuSer Asp AsnVal ThrSer Arg ProCys LysTyr LysLeu Cys 65 70 75 gp AAT

Lys LysSer Thr LysPhe ValVal Ala CysGlu AsnGly LeuPro Asn CAG

Val HisLeu Asp SerIle PheArg Arg ProAla LysLys LeuAsn Gln CCG

Asp AlaGln Ala LysSer AspAsp Ile LysMet ThrGln SerPro Pro GCA

Ser SerMet Tyr SerLeu GlyGlu Arg ValThr PheThr CysLys Ala 78g Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr 78h Leu Thr Val Ser Ser His His His (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 360 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Val Val Ala Cys Glu Asn Gly Leu Pro Val His Leu Asp Gln Ser Ile Phe Arg Arg Pro Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp 78i Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser His His His (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1065 78j (D) INFORMATION: "sFvOncMetSer"
OTHER /note=

(xi) QUENCE IPTION: SEQID :
SE DESCR N0:48 Asp IleLys MetThr Gln SerPro SerSer MetTyr AlaSer Leu Gly Glu ArgVal ThrPhe Thr CysLys AlaSer GlnAsp IleAsn Asn Tyr 20 ' 25 30 Leu CysTrp PheGln Gln LysPro GlyLys SerPro LysThr Leu Ile Tyr ArgAla AsnArg Leu ValAsp GlyVal ProSer ArgPhe Ser Gly Ser GlySer GlyGln Asp TyrSer LeuThr IleSer SerLeu Glu Tyr 65 70 75 g0 Glu AspMet GlyIle Tyr TyrCys LeuGln TyrAsp GluPhe Pro Tyr Thr PheGly GlyGly Thr LysLeu GluIle LysGly GlyGly Gly Ser Gly GlyGly GlySer Gly GlyGly GlySer GluVal GlnLeu Gln Gln Ser GlyThr ValLeu Ala ArgPro GlyAla SerVal LysMet Ser Cys Lys AlaSer GlyTyr Thr PheSer SerTyr TrpMet HisTrp Ile Lys Gln ArgPro GlyGln Gly LeuAsp TrpIle ValAla IleAsp Pro Arg Asn SerAsp ThrIle Tyr AsnPro GlnPhe LysHis LysAla Lys Leu 78k Thr AlaVal ThrSer Thr SerThr AlaTyr MetGlu LeuAsn Ser Leu Thr AsnGlu AspSer Ala ValTyr TyrCys ThrPro LeuTyr Tyr Phe Asp SerTrp GlyGln Gly ThrThr LeuThr ValSer SerAla Lys Lys Leu AsnAsp AlaGln Ala ProLys SerAsp MetSer AspTrp Leu Thr Phe GlnLys LysHis Ile ThrAsn ThrArg AspVal AspCys Asp Asn Ile MetSer ThrAsn Leu PheHis CysLys AspLys AsnThr Phe Ile Tyr SerArg ProGlu Pro ValLys AlaIle CysLys GlyIle Ile Ala Ser LysAsn ValLeu Thr ThrSer GluPhe TyrLeu SerAsp Cys Asn Val ThrSer ArgPro Cys LysTyr LysLeu LysLys SerThr Asn Lys Phe CysVal ThrCys Glu AsnGln AlaPro ValHis PheVal Gly Val Gly SerCys (2) INFORMATION FOR SEQ ID
N0:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 355 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 g0 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe 78m Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1074 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1074 (D) OTHER INFORMATION: /note= "MetGluOncFBE6"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:

Met Glu Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg 78n Asp ValAsp CysAsp Asn IleMet SerThr AsnLeu PheHis Cys Lys Asp LysAsn ThrPhe Ile TyrSer ArgPro GluPro ValLys Ala Ile Cys LysGly IleIle Ala SerLys AsnVal LeuThr ThrSer Glu Phe Tyr LeuSer AspCys Asn ValThr SerArg ProCys LysTyr Lys Leu Lys LysSer ThrAsn Lys PheCys ValThr CysGlu AsnGln Ala Pro Val HisPhe ValGly Val GlySer CysAla LysLys LeuAsn Asp Ala Gln AlaPro LysSer Asp AspIle LysMet ThrGln SerPro Ser Ser Met TyrAla SerLeu Gly GluArg ValThr PheThr CysLys Ala Ser Gln AspIle AsnAsn Tyr LeuCys TrpPhe GlnGln LysPro Gly Lys Ser ProLys ThrLeu Ile TyrArg AlaAsn ArgLeu ValAsp Gly Val Pro SerArg PheSer Gly SerGly SerGly GlnAsp TyrSer Leu Thr Ile SerSer LeuGlu Tyr GluAsp MetGly IleTyr TyrCys Leu Gln Tyr AspGlu PhePro Tyr ThrPhe GlyGly GlyThr LysLeu Glu Ile Lys GlyGly GlyGly SerGly Gly GlyGly SerGly GlyGly GlySer Glu ValGln LeuGln GlnSer Gly ThrVal LeuAla ArgPro GlyAla Ser ValLys MetSer CysLys Ala SerGly TyrThr PheSer SerTyr Trp MetHis TrpIle LysGln Arg ProGly GlnGly LeuAsp TrpIle Val AlaIle AspPro ArgAsn Ser AspThr IleTyr AsnPro GlnPhe Lys HisLys AlaLys LeuThr Ala ValThr SerThr SerThr AlaTyr Met GluLeu AsnSer LeuThr Asn GluAsp SerAla ValTyr TyrCys Thr ProLeu TyrTyr PheAsp Ser TrpGly GlnGly ThrThr LeuThr Val SerSer HisHis His (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 358 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
Met Glu Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg 78p Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile 78q Val Ala Ile Asp Pro Arg Asn Ser Asp Thr Ile Tyr Asn Pro Gln Phe Lys His Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr Pro Leu Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser His His His (2)INFORMATION
FOR
SEQ
ID
N0:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1095 base rs pai (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA

(ix)FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1095 (D) OTHER INFORMATION: ote="MetSerOncA87FBMOC31"
/n (xi)SEQUENCE DESCRIPTION: ID
SEQ N0:52:

AAA

MetSer Asp Trp Leu Thr Phe Gln Lys His IleThrAsn Thr Arg Lys TCA

AspVal Asp Cys Asp Asn Ile Met Thr Asn LeuPheHis Cys Lys Ser CGT

AspLys Asn Thr Phe Ile Tyr Ser Pro Glu ProValLys Ala Ile Arg AAT

CysLys Gly Ile Ile Ala Ser Lys Val Leu ThrThrSer Glu Phe Asn AGC

78r Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu LysLysSer ThrAsn Lys PheAla ValThr CysGlu AsnGln Ala Pro ValHisPhe ValGly Val GlySer CysAla LysLys LeuAsn Asp Ala GlnAlaPro LysSer Asp GlnVal LysLeu GlnGln SerGly Pro Glu LeuLysLys ProGly Glu ThrVal LysIle SerCys LysAla Ser Gly TyrThrPhe ThrAsn Tyr GlyMet AsnTrp ValLys GlnAla Pro Gly LysGlyLeu LysTrp Met GlyTrp IleAsn ThrTyr ThrGly Glu Ser ThrTyrAla AspAsp Phe LysGly ArgPhe AlaPhe SerLeu Glu Thr SerAlaSer AlaAla Tyr LeuGln IleAsn AsnLeu LysAsn Glu Asp ThrAlaThr TyrPhe Cys AlaArg PheAla IleLys GlyAsp Tyr Trp GlyGlnGly ThrThr Val ThrVal SerSer GlyGly GlyGly Ser Gly GlyGlyGly SerGly Gly GlyGly SerAsp IleVal LeuThr Gln Ser ProPheSer AsnPro Val ThrLeu GlyThr SerAla SerIle Ser Cys ArgSerThr Lys SerLeu LeuHis SerAsn GlyIle ThrTyr Leu Tyr TrpTyrLeu Gln LysPro GlyGln SerPro GlnLeu LeuIle Tyr Gln MetSerAsn Leu AlaSer GlyVal ProAsp ArgPhe SerSer Ser Gly SerGlyThr Asp PheThr LeuArg IleSer ArgVal GluAla Glu Asp ValGlyVal Tyr TyrCys AlaGln AsnLeu GluIle ProArg Thr Phe GlyGlyGly Thr LysLeu GluIle LysArg AlaAla Ala (2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 365 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu 78t Lys Lys Ser Thr Asn Lys Phe Ala Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Phe Ser Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Thr Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Phe 78u Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala (2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1098 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1098 (D) OTHER INFORMATION: /note= "MOC3IFBMetSerOnc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:

Gly Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr Trp Gly Gln Gly Thr Thr 78v Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyGlyGly Ser AspIle ValLeu ThrGln SerPro PheSer Asn Pro ValThrLeu Gly ThrSer AlaSer IleSer CysArg SerThr Lys Ser LeuLeuHis Ser AsnGly IleThr TyrLeu TyrTrp TyrLeu Gln Lys ProGlyGln Ser ProGln LeuLeu IleTyr GlnMet SerAsn Leu Ala SerGlyVal Pro AspArg PheSer SerSer GlySer GlyThr Asp Phe ThrLeuArg Ile SerArg ValGlu AlaGlu AspVal GlyVal Tyr Tyr CysAlaGln AsnLeu Glu IlePro ArgThr PheGly GlyGly Thr Lys LeuGluIle LysArg Ala AlaAla AlaLys LysLeu AsnAsp Ala Gln AlaProLys SerAsp Met SerAsp TrpLeu ThrPhe GlnLys Lys His IleThrAsn ThrArg Asp ValAsp CysAsp AsnIle MetSer Thr Asn LeuPheHis CysLys Asp LysAsn ThrPhe IleTyr SerArg Pro Glu ProValLys AlaIle Cys LysGly IleIle AlaSer LysAsn Val Leu 78w Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 366 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
Gly Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Phe Ser Asn Pro 78x Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Thr Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 78y (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1065 (D) OTHER INFORMATION: /note= "MetSerOncFBMOC161"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:

Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Ile Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asp Tyr Trp Leu Gly Trp Val Lys His Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Ile Tyr Pro Gly Ser Asp Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Leu Lys Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Lys Ser Ile Ala Trp Tyr Gln His Lys Pro GlyLys Gly ProArg LeuLeu IleHis TyrThr Ser ThrLeu GlnPro GlyIle Pro SerArg PheSer GlySer GlySer Gly GluGlu TyrSer PheSer Ile SerAsn LeuGlu ProGlu AspIle Ala ThrTyr TyrCys GlnGln Tyr AspAsn LeuArg ThrPhe GlyGly Gly ThrLys LeuGlu LeuLys Arg 78aa (2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 355 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Ile Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asp Tyr Trp Leu Gly Trp Val Lys His Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Ile Tyr Pro Gly Ser Asp Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 78bb Ser Ala Val Tyr Phe Cys Ala Arg Gly Leu Lys Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Lys Ser Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Glu Glu Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg (2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 753 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..753 (D) OTHER INFORMATION: /note= "IL2FBMetSerOnc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:

Ala Pro Thr Ser Thr Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser 78dd Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 251 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
Ala Pro Thr Ser Thr Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 78ee Ser Asp Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys (2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 768 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..765 (D) OTHER INFORMATION: /note= "MetSerOncFBIL2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:

Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile 78ff Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Ala Pro Thr Ser Thr Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr His His His 78gg (2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 254 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
Met Ser Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp Ala Pro Thr Ser Thr Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln 78hh Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr His His His (2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..387 (D) OTHER INFORMATION: /note= "SigPepOnc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:

Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pro Leu Phe Phe Leu Leu Leu Gly Trp Val Gln Pro Ser Leu Gly Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys 78ii AsnVal LeuThr Thr SerGlu PheTyr LeuSer AspCys Asn ValThr SerArg ProCys Lys TyrLys LeuLys LysSer ThrAsn Lys PheCys ValThr CysGlu Asn GlnAla ProVal HisPhe ValGly Val GlySer Cys (2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
Met Gly Leu Glu Lys Ser Leu Ile Leu Phe Pro Leu Phe Phe Leu Leu Leu Gly Trp Val Gln Pro Ser Leu Gly Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr 85 90 g5 Ser Arg Pro Cys Lys Tyr Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys 78j7 Val Thr Cys Glu Asn Gln Ala Pro Val His Phe Val Gly Val Gly Ser Cys

Claims (32)

WHAT IS CLAIMED IS:

1. A ribonuclease molecule comprising: (a) an amino terminal end beginning with a methionine which is followed by any amino acid other than glutamic acid; (b) when aligned for maximum correspondence with SEQ ID NO:13, a cysteine at amino acid positions 26, 40, 58, 84, 95 and 110; a lysine at position 41 and a histidine at position 119, and (c) an nOnc-derived amino acid sequence; wherein said ribonuclease molecule has measurable ribonuclease activity.

2. The ribonuclease of claim 1 which has an amino terminal end selected from the group consisting of: Met-Lys; Met-Tyr;
Met-Ser; Met-Ala; Met-Arg; and Met-Asn.

3. The ribonuclease of claim 1, which has an amino terminal end selected from the group consisting of:
Met-Ala;
Met-Ala-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.

4. The ribonuclease of claim 1, which has an amino terminal end of Met-Ala.

5. The ribonuclease of claim 1, which has an amino terminal end of Met-Arg.

6. The ribonuclease of claim 1, which has an amino terminal end of Met-Lys.

7. The ribonuclease of claim 1, which has an amino terminal end of Met-Asn.

8. The ribonuclease of claim 1, which has an amino terminal end of Met-Gln.

9. The ribonuclease of claim 1, which has an amino terminal end selected from the group consisting of Met-Ser;
Met-Tyr or Met-Thr.

10. The ribonuclease of any one of claims 3 to 8, wherein aspartic acid of amino acid position 2 of the amino acid sequence of (c) or position 4 with reference to the sequence of bovine RNase is deleted or replaced by Ala or Asn.

11. The ribonuclease of claim 1 wherein the amino acid sequence comprises a sequence having the formula:
Met (-1) eosinophil derived neurotoxin (1-m) Onc (n-104) wherein Met(-1) refers to an amino terminal residue of let; wherein eosinophil derived neurotoxin (1-m) refers to a contiguous sequence of amino acids of a length beginning at amino acid position 1 of eosinophil derived neurotoxin (SEQ
ID NO:9) and continuing to and including amino acid position "m" of eosinophil derived neurotoxin; wherein Onc(n-104) refers to a sequence of contiguous amino acids beginning at amine acid position "n" and continuing to and including amino acid position 104 as set out is SEQ ID NO:1; and wherein "m" is the amino acid position of eosinophil derived neurotoxin selected from the group consisting of 5, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22; 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.

12. The ribonuclease of claim 1, comprising an amino acid sequence substantially identical to that of SEQ ID NO:28.

13. The ribonuclease of claim 1, comprising an amino acid sequence substantially identical to that of SEQ ID NO:22.

14. The ribonuclease of claim 1, comprising an amino acid sequence substantially identical to that of SEQ ID NO:24.

15. The ribonuclease of claim 1, comprising an amino acid sequence substantially identical to that of SEQ ID NO:26.

16. The ribonuclease of claim 1, comprising an amino acid sequence substantially identical to that of SEQ ID NO:30.

17. The ribonuclease of claim 1, comprising an amino acid sequence substantially identical to that of SEQ ID NO:32.

18. The ribonuclease of claim 1, which includes an amino acid sequence substantially identical to that of SEQ ID
NO:2.

19. The ribonuclease of any one of claims 1 to 18, comprising a carboxyl terminal end derived from angiogenin corresponding to the amino acid sequence of positions 101 to 107 of SEQ ID NO:20.

20. The ribonuclease of claim 19, comprising an amino acid sequence substantially identical to that of SEQ ID NO:20.

21. A fusion protein comprising the ribonuclease of any one of claims 1 to 20, joined to a ligand binding moiety or label.

22. The fusion protein of claim 21, further comprising an antibody.

23. An isolated nucleic acid sequence encoding the ribonuclease of any one of claims 1 to 20.

24. A pharmaceutical composition comprising a cytotoxic amount of a ribonuclease of any one of claims 1 to 20, and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, wherein the ribonuclease is joined to a ligand binding moiety.

26. A method of selectively killing cells in vitro comprising contacting cells to be killed with a ribonuclease of any one of claims 1 to 20, joined to a ligand binding moiety.

27. The ribonuclease molecule of any one of claims 1 to 20, which further has a nuclear localization signal.

28. The ribonuclease molecule of any one of claims 1 to 20, which further has an endoplasmic retention sequence.

29. A vector comprising a nucleic acid encoding the ribonuclease of any one of claims 1 to 20.

30. A host cell comprising a nucleic acid encoding the ribonuclease of any one of claims 1 to 20.

31. Use of a ribonuclease of any one of claims 1 to 20, joined to a ligand binding moiety for selectively killing cells.

32. Use of a ribonuclease of any one of claims 1 to 20, joined to a ligand binding moiety for preparation of a medicament for selectively killing cells.