WO1993014098A1

WO1993014098A1 - Antigen recognized by patients with antibody associated lambert-eaton myasthenic syndrome (lems), dna encoding same and uses thereof

Info

Publication number: WO1993014098A1
Application number: PCT/US1993/000227
Authority: WO
Inventors: Henry M. Furneaux; Jerome B. Posner
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 1992-01-10
Filing date: 1993-01-11
Publication date: 1993-07-22
Also published as: AU3582593A

Abstract

An isolated nucleic acid sequence encoding myasthenic paraneoplastic antigenic polypeptide is provided by this invention. This invention also provides a purified myasthenic antigenic polypeptide and compositions containing the purified myasthenic antigenic polypeptide. Further provided by this invention is a monoclonal antibody directed to an epitope on the myasthenic paraneoplastic antigenic polypeptide. Compositions containing this monoclonal antibody also are provided by this invention. This invention also provides methods of diagnosis and treatment using the compositions described hereinabove.

Description

ANTIGEN RECOGNIZED BY PATIENTS WITH

ANTIBODY ASSOCIATED LAMBERT-EATON MYASTHENIC SYNDROME (LEMS), DNA ENCODING SAME AND USES THEREOF

This invention was made in the course of work under Grant No. PD 359 from the American Cancer Society. The United States government has certain rights in this invention.

Background of the Invention

Throughout this application various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, immediately preceding the claims.

Lambert-Eaton myasthenic syndrome (LEMS) is a paraneoplastic disorder of the neuromuscular junction characterized by a defect in the presynaptic quantal release of acetylcholine [1]. At least seventy percent of the patients with LEMS have small cell carcinomas of the lung [2], LEMS patients develop an immune response against component (s) of a voltage-gated CA2+ channel (VGCC) expressed in small cell carcinomas of the lung [3]. It has been suggested that this immune response cross reacts with Ca++ channels in the presynaptic neuromuscular junction and is responsible for the neurological dysfunction.

Paraneoplastic neurological syndromes are disorders of nervous system function that occur in association with cancer but not as a direct effect of the tumor or metastasis [5, 6]. An immune basis has been suggested for several of these disorders [7, 8, 9, 10], but LEMS is the prototype for which direct evidence of a pathogenic antibody(s) has been demonstrated by passive transfer of the disease to animals [11]. Clinical symptoms of LEMS include: proximal muscle weakness, a reduction or absence of tendon reflexes, and cholinergic dysautonomia [2]. Electophysiological studies show low amplitude of the compound muscle action potential that increases progressively during high frequency repetitive stimulation [1]. Morphological studies show a decrease and disorganization of presynaptic active zone particles which are thought to be Ca++ channel complexes [3]. An immune etiology of LEMS is also supported by the improvement of LEMS patients with plasmapheresis or immunosuppressive therapy [2]. The IgG of LEMS patients inhibits K+-stimulated Ca++ influx in SCLC cell lines [12] and a subgroup of LEMS patients have antibodies that are able to immunoprecipitate voltage-gated calcium channels (vgcc) pre-labelled with the specific BGCC antagonist ω- conotoxin [13,14]. This suggests that the presynaptic VGCC is the target antigen but to date there is no direct biochemical analysis of the subunits that interact with the IgG. The VGCC consists of 5 subunits (α1, α2, β , γ, δ) .

[15] The α1 subunit is the central ion-channel component [16]. The function of the α2-δ subunits are not clear but may contain the ω-conotoxin binding site [17]. The δ subunit interacts with the α1 subunit and may function in a regulatory role [15]. The β subunit has been shown to accelerate both activation and inactivation of the calcium channel current and to increase the number of calcium antagonist binding sites upon transfection into LCa.11 cells and so appears to have an important regulatory function [18]. The role of the ω-conotoxin-sensitive-VGCC in the etiology of LEMS is uncertain as patients with SCLC and no detectable neurological dysfunction also harbor antibodies against ω-conotoxin labelled VGCCs [13].

Summary of the Invention

An isolated nucleic acid sequence encoding Lambert-Easton Myasthenic Syndrome (LEMS) antigenic polypeptide is provided by this invention. This invention also provides purified myasthenic antigenic polypeptides and compositions containing the purified myasthenic antigenic polypeptides.

A method of detecting an antibody associated with Lambert- Easton Myasthenic Syndrome (LEMS) is provided by this invention. This method comprises contacting a suitable sample with a purified antigenic polypeptide labelled with a detectable marker under conditions so as to form a complex between the purified myasthenic antigenic polypeptide and the antibody, detecting the presence of any complex so formed, thereby detecting an antibody associated with Lambert-Easton Myasthenic Syndrome (LEMS).

Also provided by this invention is a method of determining whether a patient exhibiting neurological symptoms harbors antigenic polypeptides associated with Lambert-Easton Myasthenic Syndrome (LEMS), which comprises contacting a suitable tissue or cell sample from the patient with a monoclonal antibody directed against a myasthenic antigen, the monoclonal antibody being labeled with a detectable marker, under suitable conditions so as to form a complex between the antibody and the antigen, detecting the presence of any complex so formed, the presence of complex being a positive determination that the patient harbors antigens associated with Lambert-Easton Myasthenic Syndrome (LEMS). Brief Description of the Figures

Figure 1: Western blot analysis of mysB fusion protein.

Preparations of E. coli protein (1-6) and mysB fusion protein (7-12) reacted with a normal human serum (1-3, 7-9) and LEMS serum (4-6, 10-12). Both sera were tested at three dilutions: 1/10,000, lanes 1, 4, 7, 10; 1/5000, lanes 2, 5, 8, 11; 1/1000, lanes 3, 6, 9, 12.

Figure 2 : Nucleotide sequence of p mys A cDNA clone. Also sequence I.D. no. 1.

Figure 3 : Nucleotide sequence of p mys B cDNA clone. Also sequence I.D. no. 2.

Figure 4: Predicted amino acid sequence of mysB cDNA clone and its homology to the β-subunit of rabbit calcium channel (RABCAC). Sequence analysis was done by the dideoxy termination method [20] using Sequenase 2.0 (United State Biochemical). Double stranded DNA was prepared using the Qiagen plasmid midi-prep system and seguenced on both strands. Single stranded DNA was prepared using a modified single strand rescue protocol (Stratagene). Internal oligonucleotide primer, SK and KS primers were used. (Sequence I.D. Nos. 3 and 4, respectively). Detailed Description of the Invention

This invention provides isolated nucleic acid sequences which each encode a myasthenic paraneoplastic antigenic polypeptide. These isolated nucleic acid sequences are useful for the recombinant production of myasthenic paraneoplastic antigenic polypeptide or as reagents to detect myasthenic paraneoplastic antibodies. As used herein, the term myasthenic antigenic polypeptide encompasses any amino acid sequence, polypeptide or protein having the biological activity of a myasthenic antigenic protein, i.e., a protein which may specifically form a complex with an antibody which is characteristic of Lambert- Eaton Myasthenic Syndrome (LEMS). This antigen, as used herein, is also called myasthenic syndrome antigen. The antibodies which may specifically form a complex with myasthenic antigenic polypeptide is characteristically found in patients with Lambert-Eaton Myasthenic Syndrome, a disorder of the brain found in association with neoplasms of lung.

In one embodiment of this invention, the isolated nucleic acid sequences described hereinabove are DNA. In other embodiments of this invention, the isolated nucleic acid sequences described hereinabove are cDNA, or RNA. In the preferred embodiment of this invention, the isolated nucleic acid sequences are cDNA sequences as shown in Figures 2 and 3. cDNA clones encoding two antigenic polypeptides of the subject invention were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The two clones, designated mys A and mys B, were accorded ATCC designation numbers 75147 and 75137, respectively. Deposited mys B has the sequence depicted in Figure 3 from nucleotide 1 to nucleotide 424.

The DNA sequences of the subject invention also include DNA sequences coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the antigen, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the polypeptide) and which share some or all the properties of naturally- occurring forms. These sequences include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors. In addition, the DNA sequences of this invention encode the full length unprocessed amino acid sequence as well as DNA sequences encoding the processed form of the antigen (a cDNA clone). Methods of making these analogs are well known to those of skill in the art, utilizing the information disclosed herein.

As noted hereinabove, the DNA sequences described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques. These sequences are useful for generating new viral and circular plasmid vectors, transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammaliain cells grown in culture), and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products.

Moreover, the nucleic acid sequences are useful for the development of probes to study the immune reaction. When labeled with detectable markers, such as a radioisotope, enzyme or dye, the nucleic acids may be employed in hybridization, histological or serological assays. A hybridization probe is useful for locating the human gene position in a chromosomal map. Nucleic acid sequences detectably labeled also are provided by this invention. Methods to label nucleic acids are well known to those skilled in the art.

Thus, vectors which comprise the isolated nucleic acid molecules described hereinabove also are provided. Suitable vectors comprise, but are not limited to, a plasmid or a virus. These vectors may be transfected into a suitable host cell to form a host cell vector system for the production of a polypeptide having the biological activity of the myasthenic antigenic polypeptide.

This invention further provides an isolated DNA or cDNA molecule described hereinabove wherein the host cell is selected from the group consisting of bacteria cells (such as E. coli), yeast cells , fungi cells, insect cells and animal cells. Suitable animal cells include, but are not limited to Vero cells, HeLa cells, Cos cells , CV1 cells and primary mouse cells.

Further provided by this invention is a method for producing a polypeptide having the biological activity of the myasthenic antigenic polypeptide comprising the steps of: a) culturing the host vector system described hereinabove under suitable conditions permitting production of the polypeptide, and b) recovering the polypeptide produced. This invention also provides the polypeptides produced by this method.

Throughout this application, references to specific nucleotides are to nucleotides present on the coding strand of the nucleic acid. The following standard abbreviations are used throughout the specification to indicate specific nucleotides:

C=cytosine A=adenosine

T=thymidine G=guanosine

U=uracil

This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of the myasthenic antigenic polypeptide, but which should not produce phenotypic changes. Alternatively, this invention also encompasses DNAs and cDNAs which hybridize to the DNA and cDNA of the subject invention. Hybridization methods are well known to those of skill in the art. This invention also encompasses cDNA and DNA molecules characterized by changes in non-coding regions that do not alter the phenotype of the polypeptide produced.

Also provided by this invention is a purified, myasthenic antigenic polypeptide. As used herein, the term "purified, myasthenic antigenic polypeptide" shall mean an isolated naturally-occurring myasthenic polypeptide or protein (purified from nature or manufactured such that the primary, secondary and tertiary conformation, and the glycosylation pattern are identical to naturally-occurring material) as well as non-naturally occurring polypeptides having a primary structural conformation (i.e., continuous sequence of amino acid residues) and glycosylation sufficiently duplicative of that of naturally-occurring myasthenic antigenic polypeptide or protein to allow possession the biological activity of naturally-occurring myasthenic antigenic polypeptides and proteins. Such polypeptides include derivatives and analogs. The purified myasthenic antigenic polypeptide may be labeled with a detectable marker. For the purposes of this invention, suitable detectable markers include, but are not limited to detectable markers selected from the group consisting of radioisotopes, dyes, enzymes and biotin. These are useful in immunological assays to detect antibody to LEMS in a patient.

For the purpose of illustration only, applicants screened a human fetal brain expression library with the serum of a patient with LEMS resulting in the isolation of two independent but related cDNA clones encoding antigens designated myasthenic syndrome antigen A and B (mysA, mysB). Fusion proteins derived from these clones were not recognized by normal human sera or by sera from patients with SCLC. The amino acid sequence of these clones show high homology to the β subunit of the rabbit skeletal muscle Ca++ channel [4].

This invention further provides a monoclonal antibody directed to an epitope on the myasthenic antigenic polypeptide. In one embodiment of this invention, the monoclonal antibody is a mouse monoclonal antibody. In another embodiment of this invention, the monoclonal antibody is a human monoclonal antibody. The monoclonal antibodies of the subject invention are useful for the immunogical detection of LEMS antigen in a patient sample.

For the isolation of mouse monoclonal antibodies, eight week old mice may be injected interperitoneally with about 50 micrograms of a synthetic, purified myasthenic antigenic polypeptide, (prepared as described above) in complete Freud's adjuvant 1:1 volume. Mice are boosted, at monthly intervals, with the polypeptide, mixed with incomplete Freund's adjuvant, and bled through the tail vein. On days 4, 3, and 2 prior to fusion, mice are boosted intravenously with 50 micrograms of the polypeptide in saline. Splenocytes are fused with non-secreting myeloma cells according to procedures which have been described and are known to those of skill in the art to which this invention pertains. Some time later, approximately two weeks later, hybridoma supernatant is screened for binding activity against the myasthenic antigenic polypeptide as described below.

Positive clones are then isolated and propagated. Isolates of human monoclonal antibodies will be similar except β cells will be isolated from patients and transformed with EBV. β cells will then be fused with non-secreting myeloma cells according to procedures which have been described and are known to those of skill in the art to which this invention pertains. Some time later, approximately two weeks later, hybridoma supernatant are then be screened for binding activity against the myasthenic antigenic polypeptide as described hereinafter. Positive clones are isolated and propagated.

Also provided by this invention are pharmaceutical compositions comprising the purified, myasthenic antigenic polypeptides described hereinabove alone, or conjugated to any one of the following, a detectable marker, a therapeutic agent, or an imaging agent, as described hereinabove and a pharmaceutically acceptable carrier. Further provided are pharmaceutical compositions comprising the monoclonal antibody described hereinabove alone, or conjugated to any one of the following, a detectable marker, a therapeutic agent, or an imaging agent.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions, such as a oil/water emulsions, and various types of wetting agents.

The pharmacuetical compositions of this invention are useful in imaging, therapy or in diagnostic assays. A method of detecting an antibody associated with Lambert- Easton Myasthenic Syndrome (LEMS) also is provided by this invention. This method comprises contacting a suitable sample with a purified, myasthenic antigenic polypeptide described hereinabove under conditions so as to form a complex between the purified myasthenic antigenic polypeptide and the antibody, detecting the presence of any complex so formed, thereby detecting an antibody associated with paraneoplastic sensory neuronopathy. Suitable samples include any sample suspected containing an antibody associated with Lambert-Eaton, such as cerberal spinal fluid and serum. In one embodiment of the invention, the synthetic, purified myasthenic antigenic polypeptide is labeled with a detectable marker selected from the group consisting of radioisotope, dye, enzyme and biotin. For the purposes of this invention, suitable radioisotopes include, but are not limited to, ³²P, ³⁵S, and ¹³¹I.

Also provided by this invention is a method of determining whether a patient exhibiting neurological symptoms harbors antibodies associated with Lambert-Easton Myasthenic

Syndrome (LEMS), which comprises contacting a suitable tissue or cell sample from the patient with a monoclonal antibody directed against a myasthenic antigen, the monoclonal antibody being labeled with a detectable marker, under suitable conditions so as to form a complex between the antibody and the antigen, detecting the presence of any complex so formed, the presence of complex being a positive determination that the patient harbors antibodies associated with Lambert-Easton Myasthenic Syndrome (LEMS).

In one embodiment of this invention, the monoclonal antibody is labeled with a detectable marker. For the purposes of this invention, suitable detectable markers include, but are not limited to a detectable marker selected from the group consisting of a radioisotope, dye, enzyme and biotin. Suitable radioisotopes have been described hereinabove. A method of determining whether a patient exhibiting neurological symptoms harbors a tumor expressing myasthenic antigen is provided by this invention. This method comprises contacting a suitable sample from the patient with the antibody of this invention under suitable conditions so as to form a complex between the antibody and the myasthenic antigen, detecting the presence of any complex so formed, the presence of complex indicating that the patient harbors a tumor expressing myasthenic antigenic polypeptide. In one embodiment of this invention, the antibody is labelled with a detectable marker, e.g., a radioisotope, dye, enzyme or biotin.

This invention further provides a method of inhibiting the proliferation of neoplastic cells in a patient having LEMS which comprises administering to the patient an amount of the antibody described hereinabove, effective to inhibit the proliferation of neoplastic cells. For the purposes of this invention, the therapeutic agent may include, but is not limited to a radioisotope, toxin, toxoid or chemotherapeutic agent.

A method of imaging neoplastic cells in a patient, wherein the neoplastic cells are associated with LEMS is provided by this invention. This invention comprises administering to the patient an amount of an antibody described hereiabove, in an amount sufficient to form a detectable complex between the antibody and the myasthenic antigen associated with LEMS, imaging any complex so formed, and thereby imaging neoplastic cells in the patient. The imaging agent may be, but is not limited to a radioisotope. Suitable radioisotopes are well known to those of skill in the art. As used herein, "administering" means a method of administering to the patient. Such methods are well known to those skilled in the art and include, but are not limited to administration orally, intravenously, or parenterally. Administration of the agent may be effected continuously or intermittently, such that the amount of the therapeutic agent in the patient is effective to inhibit proliferation of neoplastic cells. For the purposes of this invention, suitable therapeutic agents include radioisotopes, toxins, toxoids, and chemotherapeutic agents.

Materials and Methods

METHODS

A γZAP II fetal brain library (Stratagene) was screened at a density of 5 x 10⁴ pfu per 150 mm plate of E. coli XL1- Blue. After a 6 hour incubation at 37°C plates were overlaid with filters soaked in 10 mM IPTG and incubated for 12 hours at 37°C. Filters were removed, blocked with 5% Blotto, incubated with the LEMS patient's serum (10μg/ml) for 2 hours at room temperature, washed in 50mM Tris (pH 7.4), 100 mM NaCI, 0.2% Triton (TBST buffer), incubated at room temperature with 1-125 Protein A (0.1 uCi/ml) for 1 hour, washed with TBST, dried and exposed to XAR5 film at - 70°C overnight. Clones giving positive results were purified by several rounds of antibody screening until a yield of 100% positive plaques was obtained and then subcloned in XL-lBlue using the in vivo excision phage rescue protocol (Stratagene). Two clones (pMysA and pMysBO were isolated and were homologous to each other but are different gene products. Fusion protein and E. coli protein extract (pBS) were obtained by growing an individual colony to an optical density of 0.6 and inducing with 10mM IPTG for 1 hour at 37°C. Figure 1 shows the Western Blot analysis of pMys B.Cells were isolated by centrifugation and lysed by resuspension in 2% SDS, 50 mM HEPES pH 5.5. Lysates (200ug) were resolved by 10% SDS/polyacrylamide gel electrophoresis and transferred to nitrocellulose [19]. The nitrocellulose was-blocked with 5% Blotto and incubated with the indicated amount of serum for 2 hours at room temperature, washed with TBST, incubated with 1-125 Protein A as described above, dried and exposed to XAR5 film at -70°C.

Figure 4 shows the predicted amino acid sequence of mysB cDNA clone and its homology to the β-subunit of rabbit calcium channel (RABCAC). Sequence analysis was done by the dideoxy termination method [20] using Sequenase 2.0 (United Stated Biochemical). Double stranded DNA was prepared using the Qiagen plasmid midi-prep system and sequenced on both strands. Single stranded DNA was prepared using a modified single strand rescue protocol (Stratagene). Internal oligonucleotide primers, SK and KS primers were used.

REFERENCES

1. Lambert, E., Eaton, L. and Rooke, E. Am J Physiol. 187: 612-613, 1956.

2. O'Neill, J., Murray, N. and Newsom-Davis, J. The Lambert-Eaton myasthenic syndrome. A review of 50 cases. Brain. Ill: 577-96, 1988.

3. Vincent, A., Lang, B. and Newsom-Davis, J.

Autoimmunity to the voltage-gated calcium channel underlies the Lambert-Eaton myasthenic syndrome, a paraneoplastic disorder. TINS. 12(12): 496- 502, 1989.

4. Ruth, P., Rohrkasten, A., Biel, M. et al.

Primary Structure of the β subunit of the DHP- sensitive calcium channel from skeletal muscle. Science. 245: 1115-1118, 1989.

5. Brain, L. and Wilkinson, M. Subacute cerebellar degeneration associated with neoplasms. Brain 88: 465-478, 1965.

6. Furneaux, H. and Posner, J. Paraneoplastic neurological syndromes. Proc Assoc. Res. Nerv. Men. Dis. 68: 187-219, 1990. 7. Furneaux, H., Rosenblum, M., Dalmau, J., Wong, E., Woodruff, P., Graus, F. and Posner, J. Selective expression of Purkinje cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration. New Eng J Med. 322: 1844-51, 1990.

8. Szabo, A., Dalmau, J., Manley, G., Rosenfeld, M., Wong, E., Henson, J., Posner, J. and Furneaux, H. HuD a paraneoplastic encephalomyelitis antigen contains RNA binding domains and is homologous to Elav and Sex-Lethal. Cell in press: 1991.

9. Graus, F., Cordon-Cardo, C. and Posner, J.

Neuronal antinuclear antibody in sensory neuronopathy from lung cancer. Neurology. 35: 538-543, 1985.

10. Greenlee, J. and Brashear, H. Antibodies to cerebellar Purkinje cells in patients with paraneoplastic cerebellar degeneration and ovarian carcinoma. Ann Neurol. 14: 60-63, 1983.

11. Fukunaga, H., Engel, A., Osame, M. and Lambert, E. Muscle Nerv. 5: 686-697, 1982.

12. Roberts, A., Perera, S., Lang, B., Vincent, A. and Newsom-Davis, J. Paraneoplastic myasthenic syndrome IgG inhibit 45Ca2+ flux in human small carcinoma line. Nature. 317: 737-39, 1985.

13. Sher, E., Canal, N., Piccolo, G., Gotti, C., Scoppetta, C., Evoli, A. and Clementi, F.

Specificity of calcium channel autoantibodies in Lamber-Eaton myasthenic syndrome. The Lancet. :640-643, 1989. 14. Lennon, V. and Lambert, E. Autoantibodies bind solubilized calcium channel-w-conotoxin complexes from small cell lung carcinoma: A diagnostic aid for Lamber-Eaton myasthenic syndrome. May Clin. Proc. 64: 1498-1504, 1989.

15. Catterall, W., Nunoki, K., Lai, Y., De Jongh, K., Thomsen, W. and Rossie, S. "Structure and modulation of voltage-sensitive sodium and calcium channels." The Biology and Medicine of Signal Transduction. Nishizuka ed. 1990 Raven Press. New York.

16. Tanabe, T., Takeshima, H., Mikami, A. et al.

Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature. 328: 313-318, 1987.

17. Barhanin, J., Schmid, A. and Lazdunski, M.

Properties of structure and interaction of the receptor for w-conotoxin, a polypeptide active on Ca2+ channels. Biochemical and Biophysical Research Communications. 150(2): 1051-1062, 1988.

18. Varadi, G., Lory, P., Schultz, D., Varadi, M. and Schwartz, A. Acceleration of activation and inactivation by the β subunit of the skeletal muscle calcium channel. Nature. 352: 159-162,

1991.

19. Towbin, H., Staehelin, T. and Gordon, J. A procedure for the electrophoretic transfer of proteins from polyacrylamide gels to nitro cellulose sheets. Proc. Nat. Acad. Sci. 76;

4350-4352, 1979.

20. Maniatis, T., Fritsch, B. and Sambrook, J.

"Molecular cloning: a laboratory manual." 1982 Cold Spring Harbor, New York.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Furneaux,, Henry M.

Posner, Jerome B.

(ii) TITLE OF INVENTION: ANTIGEN RECOGNIZED BY PATIENTS WITH

ANTIBODY ASSOCIATED LAMBERT-EATON MYASTHENIC SYNDROME (LEMS)

(ill) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Cooper & Dunham

(B) STREET: 30 Rockefeller Plaza

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10112

(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 #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE: 10-JAN-1992

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: White, John P.

(B) REGISTRATION NUMBER: 28,67B

(C) REFERENCE/DOCKET NUMBER: 39874

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212-977-9550

(B) TELEFAX: 212-664-0525

(C) TELEX: 422523 COOP UI

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2420 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: lambda ZAP II expression library (B) CLONE: mys A (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GCCCGGCGTG AAGTAGAGAG CCAGGCTCAG CAGCAGCTCG AAAGGGCCAA GCACAAACCT 60

GTGGCATTTG CGGTGAGGAC CAATGTCAGC TACTGTGGCG TACTGGATGA GGAGTGCCCA 120

GTCCAGGGCT CTGGAGTCAA CTTTGAGGCC AAAGATTTTC TGCACATTAA AGAGAAGTAC 180

AGCAATGACT GGTGGATCGG GCGGCTAGTG AAAGAGGGCG GGGACATCGC CTTCATCCCC 240

AGCCCCCAGC GCCTGGAGAG CATCCGGCTC AAACAGGAGC AGAAGGCCAG GAGATCTGGG 300

AACCCTTCCA GCCTGAGTGA CATTGGCAAC CGACGCTCCC CTCCGCCATC TCTAGCCAAG 360

CAGAAGCAAA AGCAGGCGGA ACATGTTCCC CCATATGACG TGGTGCCCTC CATGCGGCCT 420

GTGGTGCTGG TGGGACCCTC TCTGAAAGGT TATGAGGTCA CAGACATGAT GCAGAAGGCT 480

CTCTTCGACT TCCTCAAACA CAGATTTGAT GGCAGGATCT CCATCACCCG AGTCACAGCC 540

GACCTCTCCC TGGCAAAGCG ATCTGTGCTC AACAATCCGG GCAAGAGGAC CATCATTGAG 600

CGCTCCTCTG CCCGCTCCAG CATTGCGGAA GTGCAGAGTG AGATCGAGCG CATATTTGAG 660

CTGGCCAAAT CCCTGCAGCT ACTACTCTTC GACGCTGACA CCATCAACCA CCCAGCACAG 720

CTGGCCAAGA CCTCGCTGGC CCCCATCATC GTCTTTCTCA AAGTGTCCTC ACCAAAGGTA 780

CTCCAGCGTC TCATTCGCTC CCGGGGGAAG TCACAGATGA AGCACCTGAC CGTACAGATG 840

ATGGCATATG ATAAGCTGGT TCAGTCCCCA CCCGAGTCAT TTGATGTGAT TCTGGATGAG 900

AACCAGCTGG AGGATGCCTG TGAGCACCTG GCTGAGTACC TGGAGGTTTA CTGGCGGGCC 960

ACGCACCACC CAGCCCCTGG CCCCGGACTT CTGGGTCCTC CCAGTGCCAT CCCCGGACTT 1020

CAGAACCAGC AGCTGCTGGG GGAGCGTGGC GAGGAGCACT CCCCCCTTGA GCGGGACAGC 1080

TTGATGCCCT CTGATGAGGC CAGCGAGACC TCCCGCCAAG CCTGGACAGG ATCTTCACAG 1140

CGTAGCTCCC GCCACCTGGA GGAGGACTAT GCAGATGCCT ACCAGGACCT GTACCAGCCT 1200

CACCGCCAAC ACACCTCGGG GCTGCCTAGT GCTAACGGGC ATGACCCCCA AGACCGGCTT 1260

CTAGCCCAGG ACTCAGAGCA CAACCACAGT GACCGGAACT GGCAGCGCAA CCGGCCTTGG 1320

CCCAAGGATA GCTACTGACA GCCTCCTGCT GCCCTACCCT GGCAGGCACA GGCGCAGCTG 1380

GCTGGGGGGC CCACTCCAGG CAGGGTGGCG TTAGACTGGC ATCAGGCTGG CACTAGGCTC 1440

AGCCCCCAAA ACCCCCTGCC CAGCCCCAGC TTCAGGGCTG CCTGTGGTCC CAAGGTTCTG 1500

GGAGAAACAG GGGACCCCCT CACCTCCTGG GCAGTGACCC CTACTAGGCT CCCATTCCAG 1560

GTACTAGCTG TGTGTTCTGC ACCCCTGGCA CCTTCCTCTC CTCCCACACA GGAAGCTGCC 1620

CCACTGGGCA GTGCCCTCAG GCCAGGATCC CCTTAGCAGG GTCCTTCCCA CCAGACTCAG 1680

GGAAGGGATG CCCCATTAAA GTGACAAAAG GGTGGGGTGT GGGCACCATG GCATGAGGAA 1740

GAAACAAGGT CCCTGAGCAG GCACAAGTCC TGACAGTCAA GGGACTGCTT TGGCATCCAG 1800 GGCCTCCAGT CACCTCACTG CCATACATTA GAAATGAGAC AATCAAAGCC CCCCCCAGGG 1860

TGGCACACCC ATCCGTTTGC TGGGGTGTGG CAGCCACATC CAAGACTGGA GCAGCAGGCT 1920

GGCCACGCTC GGGCCAGAGA GAGCTCACAG CTGAAGCTCT TGGAGGGAAG GGCTCTCCTC 1980

ACCCTGCCAG GAAGCTTCTT AACATGTGAC AGGACCAGGG ACCAGGAGCA TGGTGAAGCC 2040

AAGTGGCAGA TGGGAGCCAA CCTGGATGGG GGTTTGGGGA AGGAGGGCAT GTGTAGCAGA 2100

GAACTTAGGG GGGCCTCCTT GCCTTTCTCA TTCTTTTGCC CTGCATCCTG TCATTTCTGT 2160

TCTTGTCCCT CATACATCTT TGGAGAACCG GGCTCCAGAC TTTGTTCCCT GACTCATAGC 2220

TGCCGCTTGT TAGGTTAGGG TTAGATGGGG AGAGACAGGG CACAGAGGAC CTGTCTCCCC 2280

GGCTACTCTT GCCTTATGGC TCTAGTGTGT GACCTACAGA GCATGCTCCA CAAGCCCCTG 2340

CCTCACCTCA CTGTCATCAC TAATAAACAT CATGCACAGT CAAAAAAAAA AAAAAAAAAA 2400

AAAAAAAAAA AAAAAAAAAG 2420 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3471 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: mys B

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1656..1657

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GAGGAGGAGG GGACCCGCCG CCGGGGGCTG GCTGCTTCGC TCCGAGCCGA CTTTTCGCCA 60

ATGGTCCAAA GGGACATGTC CAAGTCGCCT CCCACAGCGG CGGCGGCGGT GGCGCAGGAG 120

ATCCAGATGG AACTGCTAGA GAACGTGGCT CCCGCGGGGG CGCTCGGAGC CGCCGCACAG 180

TCATATGGAA AAGGAGCCAG AAGGAAAAAC AGATTTAAAG GATCTGATGG AAGCACGTCA 240

TCTGATACTA CCTCAAATAG TTTTGTTCGC CAGGGTTCGG CAGACTCCTA CACTAGCCGT 300

CCATCCGATT CCGATGTATC TCTGGAGGAG GACCGGGAGG CAGTGCGCAG AGAAGCGGAG 360

CGGCAGGCCC AGGCACAGTT GGAAAAAGCA AAGACAAAGC CCGTTGCATT TGCGGTTCGG 420ACAAATGTCA GCTACAGTGC GGCCCATGAA GATGATGTTC CAGTGCCTGG CATGGCCATC 480 TCATTCGAAG CAAAAGATTT TCTGCATGTT AAGGAAAAAT TTAACAATGA CTGGTGGATA 540

GGGCGATTGG TAAAAGAAGG CTGTGAAATC GGATTCATTC CAAGCCCAGT CAAACTAGAA 600

AACATGAGGC TGCAGCATGA ACAGAGAGCC AAGCAAGGGA AATTCTACTC CAGTAAATCA 660

GGAGGAAATT CATCATCCAG TTTGGGTGAC ATAGTACCTA GTTCCAGAAA ATCAACACCT 720

CCATCATCTG CTATAGACAT AGATGCTACT GGCTTAGATG CAGAAGAAAA TGATATTCCA 780

GCAAACCACC GCTCCCCTAA ACCCAGTGCA AACAGTGTAA CGTCACCCCA CTCCAAAGAG 840

AAAAGAATGC CCTTCTTTAA GAAGACAGAG CACACTCCTC CGTATGATGT GGTACCTTCC 900

ATGCGACCAG TGGTCCTAGT GGGCCCTTCT CTGAAGGGCT ACGAGGTCAC AGATATGATG 960

CAAAAAGCGC TGTTTGATTT TTTAAAACAC AGATTTGAAG GGCGGATATC CATCACAAGG 1020

GTCACCGCTG ACATCTCGCT TGCCAAACGC TCGGTATTAA ACAATCCCAG TAAGCACGCA 1080

ATAATAGAAA GATCCAACAC AAGGTCAAGC TTAGCGGAAG TTCAGAGTGA AATCGAAAGG 1140

ATTTTTGAAC TTGCAAGAAC ATTGCAGTTG GTGGTCCTTG ACGCGGATAC AATTAATCAT 1200

CCAGCTCAAC TCAGTAAAAC CTCCTTGGCC CCTATTATAG TATATGTAAA GATTTCTTCT 1260

CCTAAGGTTT TACAAAGGTT AATAAAATCT CGAGGGAAAT CTCAAGCTAA ACACCTCAAC 1320

GTCCAGATGG TAGCAGCTGA TAAACTGGCT CAGTGTCCTC CAGAGCTGTT CGATGTGATC 1380

TTGGATGAGA ACCAGCTTGA GGATGCCTGT GAGCACCTTG CCGACTATCT GGAGGCCTAC 1440

TGGAAGGCCA CCCATCCTCC CAGCAGTAGC CTCCCCAACC CTCTCCTTAG CCGTACATTA 1500

GCCACTTCAA GTCTGCCTCT TAGCCCCACC CTAGCCTCTA ATTCACAGGG TTCTCAAGGT 1560

GATCAGAGGA CTGATCGCTC CGCTCCTATC CGTTCTGCTT CCAAGCTGAA GAAGAACCTA 1620

GTGTGGAACC AGTCAAGAAA TCCCAGCACC GCTCTTCCTC CTCAGCCCCA CACCACAACC 1680

ATCGCAGTGG GACAAGTCGC GGCCTCTCCA GGCAAGAGAC ATTTGACTCG GAAACCCAGG 1740

AGAGTCGAGA CTCTGCCTAC GTAGAGCCAA AGGAAGATTA TTCCCATGAC CACGTGGACC 1800

ACTATGCCTC ACACCGTGAC CACAACCACA GAGACGAGAC CCACGGGAGC AGTGACCACA 1860

GACACAGGGA GTCCCGGCAC CGTTCCCGGG ACGTGGATCG AGAGCAGGAC CACAACGAGT 1920

GCAACAAGCA GCGCACGCGT CATAAATCCA AGGATCGCTA CTGTGAAAAG GATGGAGAAG 1980

TGATATCAAA AAAACGGAAT GAGGCTGGGG AGTGGAACAG GGATGTTTAC ATCCGCCAAT 2040

GAGTTTTGCC CTTTTGTGTT TTTTTTTTTT TTTTTTTTTT GAAGTCTTGT ATAACTAACA 2100

GCATCCCCAA AACAAAAAGT CTTTGGGGTC TACACTGCAA TCATATGTGA TCTGTCTTGT 2160

AATATTTTGT ATTATTGCTG TTGCTTGAAT AGCAATAGCA TGGATAGAGT ATTGAGATAC 2220

TTTTTGTAAG TGCTACATAA ATTGGCCTGG TATGGCTGCA GTCTCCGGTT GCATACTGGA 2280

CTCTTCAAAA ACTGTTTTGG GTAGCTGCCA CTTGAACAAA ATCTGTTGCC ACCCAGGTGA 2340TGTTAGTGTT TTAAGAAATG TAGTTGATGT ATCCAACAAG CCAGAATCAG CACAGATAAA 2400 AAGTGGAATT TCTTGTTTCT CCAGATTTTT AATACGTTAA TACGCAGGCA TCTGATTTGC 2460

ATATTCATTC ATGGACCACT GTTTCTTGCT TGTACCTCTG GCTGACTAAA TTTGGGGACA 2520

GATTCAGTCT TGCCTTACAC AAAGGGGATC ATAAAGTTAG AATCTATTTT CTATGTACTA 2580

GTACTGTGTA CTGTATAGAC AGTTTGTAAA TGTTATTTCT GCAAACAAAC ACCTTCTTAT 2640

TATATATAAT ATATATATAT ATATCAGTTT GATCACACTA TTTTAGAGTC TTAATGCCAA 2700

GTCAGCAGAT TTGCTTTATG AATTACAGGG ACTAGAAATG CCCACATTCA GGAAATTTGT 2760

AATAACATTG TCTAGACACC TATCCTCATT CTAGTAGAAA GTGTGTACAT ACTGTAAATA 2820

TGTGTGATTG CTTGACTTGA AAAGGTTTGA ATTCTGAATG TTATACCATC CTTGTAAGTA 2880

AGTTTGTAAT TTCCACCATA AATTATGGTA AATATAAAAC TCCAGAGGTT GTTCTACTCC 2940

ATACAGTTCA CACTGATTGT GACACATTCT TAGTAGCTAG TGTCTGTTCT AGTCACTGCA 3000

CTGGAGTCTA CGAGCCGGAA CTCGCTATAT GCACGTGTGT GTGTCCGTAT GTAAGAAAGT 3060

GTGCACCGAG TGACTGAATG GTTGAGATGA ATTGGAATGC TGAAGACTAA CGAAGAAACT 3120

AGAGACTGAT ATCGAGCATT CTGCCCACCT CGCTCTGTAT TTAATTAATT GTGCTATATG 3180

TTGCTTTAAC AACCCATTGA GCAGTCAGGC AATGTGAGTA AGCTTGCTGC CAAAGGTAAC 3240

TAGGAAAGCA TTCATCTGCT GCCTCCTTGT TTTTGCTCCT AGAGAGTGAA AATACAGGCA 3300

ATTTTACTGT GAGTGTTTCA CTGGAAATGT ACAATCTTTG TGTGTTAGAG TATTTGTTTT 3360

AGTAAGAAAT GTTTGTTTAC ACAGCTTGTG GAATTATTTC GTGGGAAAAT AAATTTTTAT 3420

AACTTCTCCC AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A 3471 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 140 amino acids

(B) TYPE: aaino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: P MYS A

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gly Ser Ala Asp Ser Tyr Thr Ser Arg Pro Ser Asp Ser Asp Val Ser

1 5 10 15

Ley Ser Glu Asp Arg Glu Ala Val Arg AArrgg Glu Ala Glu Arg Gln Ala 20 25 30

Gln Ala Leu Glu Lys Ala Lys Thr Lys Pro Val Ala Phe Ala Val Arg

35 40 45

Thr Asn Val Ser Tyr Ser Ala Ala His Glu Asp Asp Val Pro Val Pro 50 55 60

Gly Met Ala lle Ser Phe Glu Ala Lys Asp Phe Leu His Val Lys Glu 65 70 75 80

Lys Phe Asn Asn Asp Trp Trp lle Gly Arg Leu Val Lys Glu Gly Cys

85 90 95

Glu lle Gly Phe lle Pro Ser Pro Val Lys Leu Glu Asn Met Arg Leu

100 105 110

Gln His Glu Gln Arg Ala Lys Gln Gly Lys Phe Tyr Ser Ser Lys Ser

115 120 125

Gly Gly Asn Ser Ser Ser Ser Leu Gly Asp lle Val

130 135 140

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 240 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY : Peptide

(B) LOCATION : 1. .240

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Gln Gly Lys Tyr Ser Lys Arg Lys Gly Arg Phe Lys Arg Ser Asp Gly 1 5 10 15

Ser Thr Ser Ser Asp Thr Thr Ser Asn Ser Phe Val Arg Gln Gly Ser

20 25 30

Ala Glu Ser Tyr Thr Ser Arg Pro Ser Asp Ser Asp Val Ser Leu Glu

35 40 45

Glu Asp Arg Glu Ala Leu Arg Lys Glu Ala Glu Arg Gln Ala Leu Ala 50 55 60

Gln Leu Glu Lys Ala Lys Thr Lys Pro Val Ala Phe Ala Val Arg Thr 65 70 75 80

Asn Val Gly Tyr Asn Pro Ser Pro Gly Asp Glu Val Pro Val Glu Gly

85 909 95

Val Ala lle Thr Phe Glu Pro Lys Asp Phe Leu His lle Lys Glu Lys 100 105 110

Tyr Asn Asn Asp Trp Trp lle Gly Arg Leu Val Lys Glu Gly Cys Glu

115 120 125

Val Gly Phe lle Pro Ser Pro Val Lys Leu Asp Ser Leu Arg Leu Leu 130 135 140

Gln Glu Gln Lys Leu Arg Gln Ser Arg Leu Ser Ser Ser Lys Ser Gly 145 150 155 160

Asp Asn Ser Ser Ser Ser Leu Gly Asp Val Val Thr Gly Thr Arg Arg

165 170 175

Pro Thr Pro Pro Ala Ser Gly Asn Glu Met Thr Asn Leu Ala Pro Glu

180 185 190

Leu Glu Pro Leu Asp Leu Glu Glu Asp Glu Ala Glu Leu Gly Glu Gln

195 200 205

Ser Gly Ser Ala Lys Thr Ser Val Ser Ser Val Thr Thr Pro Pro Pro 210 215 220

His Gly Thr Arg lle Pro Phe Phe Lys Lys Thr Glu His Val Pro Pro 225 230 235 240

Claims

What is claimed is:

1. An isolated nucleic acid sequence encoding myasthenic antigenic polypeptide.

2. An isolated nucleic acid sequence of claim 1, wherein the nucleic acid is DNA.

3. An isolated nucleic acid sequence of claim 2, wherein the DNA is cDNA.

4. An isolated nucleic acid sequence of claim 1, wherein the nucleic acid is RNA.

5. Purified myasthenic antigenic polypeptide.

6. Purified myasthenic antigenic polypeptide of claim 5 labelled with a detectable marker.

7. Purified myasthenic antigenic polypeptide of claim 6, wherein the detectable marker is a radioisotope, dye, enzyme or biotin.

8. An antibody directed to an epitope on the purified myasthenic antigenic polypeptide of claim 5.

9. The antibody of claim 8, wherein the antibody is a mouse antibody.

10. The antibody of claim 8, wherein the antibody is a human antibody.

11. The antibody of claim 5 conjugated to a therapeutic agent.

12. The antibody of claim 11, wherein the therapeutic agent is a radioisotope, toxin, toxoid or chemotherapeutic agent.

13. The antibody of claim 5 conjugated to an imaging agent.

14. The antibody of claim 13, wherein the imaging agent is a radioisotope.

15. A polypeptide encoded by the isolated nucleic acid sequence of claim 1.

16. The isolated nucleic acid sequence of claim 1 operatively linked to a promoter of RNA transcription.

17. A vector which comprises the isolated nucleic acid molecule of claim 1.

18. A vector of claim 17, wherein the isolated nucleic acid molecule is linked to a plasmid.

19. A vector of claim 17, wherein the isolated nucleic acid molecule is linked to a virus.

20. A host vector system for the production of a polypeptide having the biological activity of a myasthenic antigenic polypeptide which comprises the vector of claim 17 in a suitable host.

21. A host vector system of claim 20, wherein the suitable host is a bacterial cell, insect cell, or animal cell.

22. A method of producing a polypeptide having the biological activity of myasthenic antigenic polypeptide which comprises growing the host vector system of claim 21 under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

23. A method of detecting an antibody associated with paraneoplastic sensory neuronopathy which comprises contacting a suitable sample with the purified myasthenic antigenic polypeptide of claim 5 under conditions so as to form a complex between the purified myasthenic antigenic polypeptide and the antibody, detecting the presence of any complex so formed, thereby detecting the antibody associated with paraneoplastic sensory neuronopathy.

24. A method of claim 23, wherein the purified myasthenic antigenic polypeptide is labelled with a detectable marker.

25. A method of claim 24, wherein the detectable marker is a radioisotope, dye, enzyme or biotin.

26. A method of determining whether a patient exhibiting neurological symptoms harbors a tumor expressing myasthenic antigen which comprises contacting a suitable sample from the patient with the antibody of claim 8 under suitable conditions so as to form a complex between the antibody and the myasthenic antigen, detecting the presence of any complex so formed, the presence of complex indicating that the patient harbors a tumor expressing myasthenic antigenic polypeptide.

27. The method of claim 26, wherein the antibody is labelled with a detectable marker.

28. The method of claim 27, wherein the detectable marker is a radioisotope, dye, enzyme or biotin.

29. A method of inhibiting the proliferation of neoplastic cells in a patient having LEMS which comprises administering to the patient an amount of the antibody of claim 11, effective to inhibit the proliferation of neoplastic cells.

30. A method of claim 29, wherein the therapeutic agent is a radioisotope, toxin, toxoid or chemotherapeutic agent.

31. A method of imaging neoplastic cells in a patient, wherein the neoplastic cells are associated with LEMS which comprises administering to the patient an amount of the antibody of claim 13, sufficient to form a detectable complex between the antibody and the myasthenic antigen associated with LEMS, imaging any complex so formed, and thereby imaging neoplastic cells in the patient.

32. The method claim 31, wherein the imaging agent is a radioisotope.