WO2002046422A1 - Single-chain variable fragment antibodies specific for the synaptic variant of acetylcholinesterase (ache-s), and their use in the diagnosis of progressive neuromuscular disorders - Google Patents

Abstract

The invention relates to a nucleic acid sequence coding for single-chain variable fragment antibody that has specific affinity for synaptic variant of acetylcholinesterase (AChE-S). This single-chain variable fragment antibody consists essentially of a polypeptide comprising the binding portion of the heavy chain variable region of an antibody. The invention further relates to expression vehicle comprising said nucleic acid sequence coding for the anti AChE-S single-chain variable fragment antibody. Moreover, the invention relates to methods for the diagnosis of a progressive neuromuscular disorder in a mammal, preferably in humans and particularly ∫i⊃Myasthenia gravis∫/i⊃, by using the single-chain variable fragment antibody of the invention.

Description

SINGLE-CHAIN VARIABLE FRAGMENT ANTIBODIES SPECIFIC FOR THE SYNAPTIC VARIANT OF ACETYLCHOLINESTERASE (ACHE-S) , AND THEIR USE IN THE DIAGNOSIS OF PROGRESSIVE NEUROMUSCULAR DISORDERS

Field of the Invention

The present invention relates to the diagnosis of a progressive neuromuscular disorder. Specifically, the invention relates to the development of a specific scFv antibody that recognizes and binds to the "synaptic" acetylcholinesterase variant AChE-S, and uses thereof in a novel method for the diagnosis of progressive neuromuscular disorders.

Background of the Invention

Neuromuscular junctions (NMJs) are highly specialized, morphologically distinct, and well-characterized cholinergic synapses [Hall and Sanes, Cell 72(Suppl): 99-121, (1993)]. Chronic impairments in NMJ activity induce neuromuscular . disorders characterized by progressive deterioration of muscle structure and function. The molecular and cellular mechanisms leading from compromised -NMJ activity to muscle wasting have not been elucidated.

One of these disorders is Myasthenia gravis (MG), which is caused by a defect in neuromuscular transmission due to autoantibody-mediated attack upon the muscle nicotinic acetylcholine receptors (AChR). MG is characterized by fluctuating weakness that may be transiently improved by inhibitors of acetylcholinesterase (AChE). The clinical severity of Myasthenia graυis is usually graded functionally and regionally, according to an adaptation of a scale devised by Osserman: Grade I involves focal disease (e.g., restricted to ocular muscles); Grade II is generalized disease that is either mild or moderate; Grade III is severe generalized disease; and Grade IN, a crisis, with life-threatening impairment of respiration [Drachman, D.B., et al., Ν. Engl. J. Med. 330:1797- 810, (1994); Osserman, K., et al, Myasthenia Gravis, G. Stratton, ed. New York,. 80, 1958]. The basic abnormality in MG is a decrease in the density of nicotinic AChRs (nAChRs) at neuromuscular junctions.

The characteristic electrodiagnostic abnormality is a progressive, rapid decrement in the amplitude of muscle action potentials evoked by repetitive nerve stimulation at 3 or 5 Hz. This myasthenic fatigue is caused by decrease in the number of AChR molecules available at the post-synaptic site. Inhibiting anti-AChR antibodies are present in^" 85% to 90% of patients.

Neuromuscular junctions from MG patients show morphological changes, in particular simplification of the post-synaptic membrane folding and end-plate potentials which fail to trigger action potential in some fibers [Drachman, (1994) ibid.]. The diagnosis is routinely made by searching for the anti-AChR antibody titer, which is positive in 80% to 90% of patients [Drachman, (1994) -.bid.; Vincent (1998) ibid.].

However, the clinical characteristics of seronegative patients do not differ substantially fro those of patients with high antibody titers. Moreover, lack of serum antibodies does not preclude the chnical phenotype of muscle fatigue or favorable response to therapies like thymectomy or plasmapheresis [Soliven et al., Neurology 38:514-7 (1988)].

Actually, these patients have circulating AChR antibodies that are not detected by radio-immunoassay. Passive transfer of their ----nmunoglobulin to mice caused the loss of junctional AChRs [Drachman D., et al., Neurology 37:214 (1987)]. Together with results from cultured-muscle-cell assay systems, these findings suggest that the antibodies may be directed at epitopes not present in soluble AChR extracts or may present affinity too low for detection in soluble assay systems [Drachman et al., (1994) ibid.].

Three other sensitive tests are being used to diagnose MG: AntichoHnesterases test, the repetitive nerve stimulation (RNS) test and the single fiber electromyography (SFEMG) [Drachman et al, (1994) ibid.; Oh et al, Muscle Nerve 15:720-4 (1992)]. Edrophoniu (Tensilon) is commonly used for the anticholinesterase test, because of the rapid onset (30 seconds) and short duration (about 5 minutes) of its effect. This drug, which inhibits the enzyme acetylcholinesterase, allows acetylcholine (ACh) that is released from the nerve to interact repeatedly with the limited number of junctional AChRs, resulting in enhanced strength of myasthenic muscle functioning. If there is unequivocal improvement in an objectively weak muscle, the test is considered positive. In RNS, electric shock is delivered to the nerve and action potentials are recorded from surface electrodes over the muscle. A rapid reduction in the amplitude of the evoked muscle action potential (decremental response of 15 percent) is considered a positive response. SFEMG detects delayed or failed neuromuscular transmission in pairs of muscle fibers supplied by branches of a single nerve fiber. The antichohnesterase and RNS tests are the least sensitive and specific of the tests, perhaps indicating that excess AChE also occurs in other diseases. A positive assay for AChR antibodies is specific for Myasthenia graυis but detectable in only about 85 percent of all patients. SFEMG is sometimes helpful in difficult diagnostic situations but its specificity is limited, with positive findings in other disorders of nerves, muscles or neuromuscular junctions [Drachman, D., (1994) ibid.]. This may reflect electrophysiological failure due to AChE excess that is unrelated to anti-AChR antibodies, similar to the situation in AChE transgenic mice [Andres, C, et al, Proc. Natl. Acad. Sci. USA 94:8173-8 (1997)]. Altogether, this raises the need for alternative diagnostic assay, preferably using the serum phase. While the neuromuscular malfunctioning associated with MG can be transiently alleviated by chronic systemic administration of AChE inhibitors, e.g. pyridostigmine, it was recently found that pyridostigmine induces a feedback response leading to excess AChE accumulation [Friedman, A., et al, Nat Med 2:1382-1385 (1996); Kaufer, D., et al, Nature 393:373-7 (1998)]. In transgenic mice, chronic neuronal AChE excess was found to cause progressive neuromotor deterioration [Andres et al, (1997) ibid.; Sternfeld et al, J Neurosci 18:1240-9 (1998)]. More recently, it was demonstrated that transgenic expression of neuronal AChE alters pre-synaptic properties and intensifies antichohnesterase responses in mouse NMJs [Farchi et al, unpublished data].

AChE is commonly known for its role in terminating cholinergic neurotransmission by hydrolyzing the neurotransmitter acetylcholine. In all mammals, AChE is encoded by one gene but alternative splicing at its 3'-end yields three different mRNA transcripts, which encode proteins with distinct carboxyl termini [Ben Aziz-Aloya et al, Prog., Brain Res. 98:147-53 (1993); Karpel, R., et al, Exp. Cell Res. 210:268-77 (1994) (Fig. 1)]. The three proteins are all catalytically active: they include the "synaptic" form, AChE-S (S), encoded by the transcript that ends with exon 6, the hematopoietic form bound to the erythrocyte membrane, AChE-E (E), encoded by the transcript ending with exon 5 and the readthrough form, AChE-R, encoded by the transcript containing pseudointron 4. This transcript accumulates under multiple stress insults through the feedback response described above in brain [Kaufer, D., et al, Chem. Biol. Interact. 119-120:349-60 (1999)], muscle [Lev-Lehman, E., et al, J. Mol. Neurosci. 14:93-105 (2000)] and intestine [Shapira, E., et al, Hum. Mol. Genet. 9:1273-1281 (2000)]. Beside its catalytic function, AChE has morphogenic, non-catalytic capacities [Grisaru et al, Molecular Medicine. In press, (2000), Grisaru et al, Eur. J. Biochem. 264, 672-686, (1999) and also reviewed by Soreq & Seidman, Nature Reviews Neuroscience 2:8-16 (2001)]. Transgenic mice overexpressing human AChEs in spinal cord motoneurons, but not in muscle, displayed progressive neuromotor impairments that were associated with changes in NMJ ultrastructure [Andres et al. (1997) ibid.]. However, it was not clear whether the moderate extent of overexpressed AChE in muscle was itself sufficient to mediate this severe myopathology. In rodent brain, the inventors found previously that both traumatic stress and cholinesterase inhibitors induce dramatic calcium-dependent overexpression of AChE-R [Kaufer, et al, (1998) ibid.].

Chronic AChE excess was found to cause in transgenic mice and amphibian embryos, progressive neuromotor deterioration [Ben Aziz-Aloya et al, Proc. Natl. Acad. Sci. USA, 90:2471-2475 (1993); Seidman, S. et al, J. Neurochem. 62:1670-1681 (1994); Seidman, S., et al, Mol. Cell. Biol. 15:2993-3002 (1995); Andres, C, et al, (1997) ibid.; Ste nfeld, M.., et al., J. Neurosci. 18:1240-1249 (1998)]. Also, myasthenic patients suffer acute crisis events [average annual incidence: 2.5%, see Berrouschot et al, Crit. Care Med. 25:1228-35 (1997)] associated with respiratory failure reminiscent of anti-AChE intoxications.

In an immuno-assay using a novel antibody specifically directed to the synaptic AChE isoform, elevated levels of AChE-S were surprisingly detected in myasthenic serum, suggesting generally intensified AChE gene expression in MG patients.

These findings point at the causal involvement of AChE variants in the cholinergic imbalance initiated by autoimmune responses and suggest, and this is an object of the invention, the use of isoform-specific anti-AChE antibodies to detect AChE secreted to the blood, which may serve as a surrogate marker for MG. Summary of the Invention

In a first aspect, the present invention relates to a nucleic acid sequence coding for single-chain variable fragment antibody that has specific affinity for synaptic variant of acetylcholinesterase (AChE-S). This single-chain variable fragment antibody consists essentially of a polypeptide comprising the binding portion of the heavy chain variable region of an antibody.

In a preferred embodiment, the nucleic acid of the invention is a nucleic acid sequence substantially as denoted by any one of SEQ ID NOs:l, 2, and 3, coding for the binding portion of the heavy chain variable region of antibodies substantially as denoted by the amino acid sequence of any one of SEQ ID NO:4, 5 and 6, respectively.

A further aspect of the present invention relates to an expression vehicle comprising a nucleic acid sequence coding for a single-chain variable fragment antibody that has specific affinity for the synaptic variant of acetylcholinesterase (AChE-S). In one preferred embodiment, the vehicle is a phagemid designated pHENl.

In another preferred embodiment, the expression vehicle according to the invention comprises the nucleic acid sequence substantially as denoted by any one of SEQ ID NOs:l, 2 and 3, coding for the amino acid sequence as denoted by any one of SEQ ID NOs:4, 5 and 6, respectively, of the single-chain variable fragment antibody. More specifically, this single-chain variable fragment antibody may be any one of the antibodies designated PD-anti_hASP 1, PD-anti_hASP 2 and PD-antihASP 3, most preferably PD-antihASP 1.

Another specifically preferred embodiment relates to an expression vehicle of the invention, further comprising an amber stop codon for the expression of any one of PD-antihASP 1, PD-antihASP 2 and PD-antihASP 3 single-chain variable fragment antibody, as soluble fragments.

A further aspect of the invention relates to a single-chain Fv antibody specifically recognizing and binding to the synaptic variant of acetylcholinesterase (AChE-S). More specifically, this antibody may be any one of the antibodies designated PD-antihASP 1, PD-antihASP 2 and PD-antihASP 3, having the amino acid sequence of any one of SEQ ID NOs:4, 5 and 6. The PD-antihASP 1 antibody having the amino acid sequence of SEQ ID NO:4 is particularly preferred. The single-chain variable fragment antibodies according to a specifically preferred aspect of the present invention, comprise as the antigen recognition region the amino acid sequences of any one of SEQ ID NOs:10, 11 and 12, respectively.

The single-chain Fv antibody that specifically recognizes and binds to the synaptic variant of acetylcholinesterase (AChE-S) according to the invention is used, and this is a further aspect of the invention, for the diagnosis of a progressive neuromuscular disorder in a mammal, particularly for the diagnosis of progressive neuromuscular disorder involving muscle distortion, muscle re-innervation or neuromuscular junction (NMJ) abnormalities. More specifically, said disorder is selected from Myasthenia graυis, Eaton-Lambert disease, Muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD), multiple sclerosis, Dystonia, post-stroke sclerosis, post-injury muscle damage, excessive re-innervation, and post-exposure to AChE inhibitors. Preferably, the diagnostic use of the antibodies of the invention is for the detection of Myasthenia graυis.

A further aspect the invention relates to a method for the diagnosis of a progressive neuromuscular disorder in a mammal, preferably in humans. This method comprises the steps of: obtaining a sample from the subject and detecting intensified expression of at least one AChE variant in said sample, preferably, intensified expression of the AChE-S synaptic variant. The diagnostic method of the invention is preferably intended for the diagnosis of a progressive neuromuscular disorder that involves muscle distortion, muscle re-innervation or neuromuscular junction (NMJ) abnormalities. The neuromuscular disorder may be selected from Myasthenia graυis, Eaton-Lambert disease, muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD), multiple sclerosis, Dystonia, post-stroke sclerosis, post-injury muscle damage, excessive re-innervation, and post-exposure to AChE inhibitors. Particularly, the method is intended for diagnosing Myasthenia graυis.

In a specifically preferred embodiment, the sample used by the diagnostic method of the invention is one of serum, bone marrow and cerebrospinal fluid sample. More preferably, the sample may be a serum sample.

Detection of the intensified expression of different AChE variants in the serum sample may be carried out by different techniques such as immunoassay, RT-PCR and nondenaturing activity gel electrophoresis which is the Ellman's assay for AChE activity. Briefly, this technique comprises the steps of preparing a protein extract from a sample, separating the protein extract on poly aery la ide gel using nondenaturing conditions and detecting catalytically active AChE variants by the Karnovsky staining technique.

In a preferred embodiment, detection of intensified expression of different AChE variants in the serum sample may carried out by immunoassay. Briefly, this technique comprises the steps of preparation of a protein extract from the said serum sample, and immunoprecipitation followed by Western blot using the scFv antibody of the present invention. Brief Description of the Drawings Figure 1: AChE: from gene to protein

Schematic representations of the human AChE gene and alternative mRNA transcripts as well as their protein products. In the DNA scheme, the colored boxes represent exons while introns are designated by white boxes. AChE pre-mRNA is susceptible to alternative splicing which generates, at the post-transcriptional level, three species of AChEmRNA. The corresponding AChE translation products are three alternative protein isoforms carrying distinctly different peptides at the C-terminus. Shown are three model structures of the core AChE protein. The C-terminal peptides are drawn as wide strips at the top left of each model. The active site serine is shown by an asterisk. Abbreviations: h (human), ge (gene), Br (brain) Mus (muscles), Rea (readthrough), Syn (synaptic), Ery (erythrocyte), pep (peptide).

Figure 2: Selection of human scFυ antibodies to ASP Schematic illustration of the phage display screening method used. The display of repertoires of antibody fragments on the surface of filamentous bacteriophage offers an advantageous approach for making antibodies with predefined binding specificities. A phage display library of > 10° scFv antibodies was constructed in vitro from human V gene segments as in [Nissim et al. (1994) supra]. Screening of the library was performed using biotinylated ASP peptide. Blocked streptavidin- coated paramagnetic beads were used for retrieval of ASP-bound phages. Abbreviations: Dis (displayed), Ab (antibody), frag (fragment), φ (phage), coated (coa), bea (bead), surf (surface). Figure 3A-3B: Double selection enrichment of anti-ASP phage antibody

Culture supernatants of single bacterial clones (cl) producing phage antibodies were incubated with biotinylated ASP, and then added to streptavidin coated wells of a microtiter plate (Fig. 3B). Supernatants preincubated without the biotinylated antigen were used as negative controls (Fig. 3A). Binding of phages was detected via an anti-phage peroxidase conjugate visualized as absorption at 450 nm [for details see Harrinson et al, (1996) supra]. PD-antihASP = Phage Display anti-human AChE Synaptic Peptide.

Figure 4: Specificity analysis of monoclonal phage antibodies by ELISA ELISA assay was performed using indirectly coated ASP or ARP. For the coating, BSA-biotin, streptavidin and biotinylated antigen (ASP or ARP) were applied in consecutive steps to a microtiter plate, mainly as described [Henderikx et al (1998) supra]. Detection of bound phages was as in Figure 3. Inset: a schematic representation of the ELISA. Abbreviations: Detec (detecting) Ab (antibody), φ (phage), coatin (coa), α (anti).

Figure 5: AChE-S specificity of PD-antihASP 1 clone analysis by competitiυe ELISA

Competitive ELISA was carried out by indirect immobilization of ASP -biotin to microtiter plates, and incubated with the PD-antihASP 1 phage in the presence of increasing amounts of either human recombinant AChE-S (Sigma;) or ARP. Binding was detected via an anti-phage peroxidase conjugate visualized as absorption at 450 nm.

Figure 6A-6B: Specificity analysis of soluble monoclonal scFυ antibody fragments by ELISA

Fig. 6A: -Shows schematic map of the pHENl vector (phagemid type) used for the fusion of antibodies to the minor coat phage protein pill [taken from Winter et al, (1994) supra]. Phagemid vectors comprise the PHI fusion, plasmid and phage origins of replication (ori) and antibiotic resistance markers; a helper phage provides the other functions for replication and packing. pHENl can also be used directly for expression of the antibodies as soluble fragments, as an amber stop codon is encoded at the junction of the antibody gene and gill. Thus, the antibody fragments are displayed on phage after rescue with helper phage from an E. coli suppressor strain, or secreted as (tagged) soluble fragments from non-suppressor strains. Abbreviations: AMP¹ = ampicillin resistance gene; L = leader peptide sequence; tag = ta; Dis (displayed), Ab (antibody), frag (fragment), hφ (helper phage), sol (soluble).

Fig. 6B: Shows results of ELISA for the antibody fragments. 9E10 and a secondary anti-mouse peroxidase antibodies were used to detect nxyc-tagged antibody fragments. Abbreviations: coating (coa).

Figure 7A-7B: Immunodetection of AChE-S in PC 12 cell lysates and human CSF

Fig. 7A: Shows immunoblot analysis performed in cell lysates from AChE-S expressed in PC12 transfected cells (lane 1) and AChE-R transfected PC12 cells (lane 2). Protein extracts were fractionated on a polyacrylamide gel and transferred onto nitrocellulose. The immunoblot was blotted using PD-c-hASP (PD-antihASP 1) phage (10 t.u./ml) and a secondary anti-phage peroxidase antibodies.

Fig. 7B: shows a positive control for PC12 transfected cells expressing human AChE-S with the 190-2 monoclonal antibody obtained by hybridoma technology against the 10 C-terminal amino acids of human AChE-S [Boschetti et al, (1996) supra; gift of Dr. Brodbeck, University of Bern]. Figure 8A-8B BIAcore analysis of soluble antihASP 1 scFυ antibody Fig. 8A: Interaction between antihASP 1 scFv antibody and the short-ASP.

Fig. 8B: Interaction between antihASP 1 scFv antibody and the

AChE-S.

Abbreviations: T (time), Resp Diff (respective difference).

Figure 9A-9C Expression analysis of ASP on the surface of υarious population of cells in cord blood - FACS analysis

Fig. 9A: Cord blood cells were divided in 4 populations depending on their expression of CD45 (granulocytes, Imphocytes, monocytes and red blood cells).

Fig. 9B: Expression of ASP was studied using antiMYC-FITC secondary labeling only as negative control.

Fig. 9C: Expression of ASP was studied using antiMYC-FITC secondary labeling after labeling with the antihASP 1 scFv antibody.

Abbreviations: sid scat (side scatter), Surf (surface)

Figure 10A-10C

Cytoplasmic expression of ASP on various population of cells in cord blood

- FACS analysis

Fig. 10A: Cord blood cells were divided in 4 populations depending on their expression of CD45 (granulocytes, Imphocytes, monocytes and red blood cells).

Fig. 10B: Expression of ASP was studied using antiMYC-FITC secondary labeling only as negative control.

Fig. IOC: Expression of ASP was studied using antiMYC-FITC secondary labeling after labeling with the antihASP 1 scFv antibody.

Abbreviations: Mat (mature) sid scat (side scatter), α (anti). Figure 11A-8B Immunodetection of AChE-S in MG vs. normal serum Fig. HA: Serum samples were immunoprecipitated with antibody against the N-terminus of AChE using μMACS columns. The immunoprecipitants were electrophoresed on denaturing gels and immunoblotted using the PD-antihASP 1 phage antibody. COS cells (ce) homogenates were immunoprecipitated as a negative control as well as samples of the antibody itself (anti N-terminus).

Fig. 11B: The bar graph (B) shows a statistically significant (P value=0.05) difference between the MG serum and controls. Abbreviations: cells (ce), -N-term (anti N-terminus), n (non), ctrl (control), hea (healthy), Ba (bands), inten (intensity), Arb Un (arbitrary units).

Detailed description of the invention

A number of terms as used herein are defined herein-below:

-AChE, Acetylcholinesterase;

-ARP, acetylcholinesterase "readthrough" peptide;

-ASP, acetylcholinesterase "synaptic" peptide;

-CNS, central nervous system;

-common domain, the region of AChE which is common to all splice variants, includes exons 2-4;

-CSF, cerebrospinal fluid;

-ORF, open reading frame;

-ORI, origin of replication;

-RT, room temperature;

-UTR, untranslated terminal region;

-scFv, single chain variable fragment antibody.

Progressive neuromuscular disorder as used herein: A disorder or condition associated with excess AChE mRNA or protein production, characterized by changes in the morphology of the NMJ and impairment in neuromuscular transmission. The neuromuscular disorder may involve muscle distortion, muscle re-innervation or neuromuscular junction (NMJ) abnormalities. More preferably, the progressive neuromuscular disorder is Myasthenia gravis, Muscular Dystrophy, Multiple Sclerosis, Amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD), or Dystonia.

A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art. Such methods include site-directed mutagenesis, PCR cloning, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of bacterial and yeast cells, transfection of mammalian cells, and the like. Textbooks describing such methods are e.g., Sambrook et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology ,by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3rd ed. John Wiley & Sons; ISBN: 0471137812, 1995. These publications are incorporated herein in their entirety by reference. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art. See e.g., Current Protocols in Immunology, Coligan et al. (eds), John Wiley & Sons. Inc., New York, NY.

As described in Example 1 of the present application, a phage display library was screened using a biotinylated ASP peptide in order to isolate antibodies against a peptide of the invention, the peptide may be produced by recombinant DNA technology in mammalian cells, as described in the above general references for molecular biology. Alternatively, as mentioned in Example 1, the peptide may be synthetically produced by organic chemistry methods. The peptide may also be produced in bacterial or insect cells, as detailed in the above-noted Current Protocols in Molecular Biology, Chapter 16.

The peptide may be purified from the cells in which it has been produced. Peptide purification methods are known to the person of skill in the art and are detailed e.g., in the above-noted Current Protocols in Molecular Biology, Chapter 16, and in Current Protocols in Protein Science, Wiley and Sons Inc. Chapters 5 and 6. Advantageously, the peptide may be produced as a fusion with a second protein, such as Glutathione-S-transferase or the like, or a sequence tag, such as the histidine tag sequence. The use of fusion or tagged proteins simplifies the purification procedure, as detailed in the above-noted Current Protocols in Molecular Biology, Chapter 16, and in the instructions for the his-tag (six histidine tag) protein expression and purification kit, as available from Qiagen GmbH, 40724 Hilden, Germany.

If the protein or peptide has been expressed as a fusion protein, it may be desirable to cleave the fusion partner before using the protein for the generation of antibodies, in order to avoid isolation of antibodies against the fusion partner. The cleavage of fusion partners and the purification of the desired peptide is described in the above-noted Current Protocols in molecular Biology, Chapter 16. Vectors, protocols and reagents for expressing and purifying maltose-binding or glutathion binding protein fused recombinant proteins are also available commercially.

As noted further above, the peptide may also be synthesized by chemical methods known in the art of chemistry, and preferably biotinylated. Two peptides were used according to the present invention, the ASP peptide was used for screening the phage display library and the ARP peptide was used as control. ASP: l-DTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL-40, also denoted as SEQ ID NO: 13.

ARP: l-GMQGPAGSGWEEGSGSPPGVTPLFSP-26, also denoted as SEQ ID NO: 14.

Examples 1, 2, and 3 describe the isolation and characterization of a specific scFv antibody that has specific affinity to the AChE-S variant and does not recognizes the core of the AChE protein or the ARP peptide. Isolation of ASP highly specific antibodies was performed by using the biotinylated- ASP peptide. It is to be mentioned that the 40 amino acid ASP peptide has predicted helical conformation (not shown).

Thus, as a first aspect, the present invention relates to a nucleic acid sequence coding for single-chain variable fragment antibody. This scFv antibody has specific affinity for the synaptic variant of acetylcholinesterase (AChE-S), and consists essentially of a polypeptide comprising the binding portion of the heavy chain variable region of an antibody.

A preferred embodiment relates to the nucleic acid sequence of the invention substantially as denoted by any one of SEQ ID NOs:l, 2 and 3. The nucleic acid sequences of the invention code for the amino sequences as denoted by SEQ ID NOs: 4, 5 and 6, defining the heavy chain variable region of an antibody.

The terms "single chain variable fragment antibody", "single chain antibody" or "ScFv" are use herein interchangeably. They are genetically engineered molecules structurally defined as comprising the binding portion of a first polypeptide from the variable region of an antibody (Tight chain), associated ^"with the binding portion of a second polypeptide from the variable region of an antibody (heavy chain), the two polypeptides being joined by a peptide linker hnking the first and the second polypeptides into a single polypeptide chain. The single polypeptide chain thus comprises a pair of variable regions connected by a polypeptide linker. These regions may associate to form a functional antigen- binding or antigen recognition site. However, the single polypeptide chain fragment used in the present invention comprises the binding portion of a polypeptide from the variable region of the heavy chain of an antibody.

The possibility of multivalent single chain antibody can be also employed in the present invention. This term means two or more single chain antibody fragments covalently linked by a peptide hnker. The antibody fragments can be joined to form bivalent or trivalent and greater have one or more antibody fragments joined by an additional interpeptide linker.

The single chain antibody fragments for use in the present invention can be also derived from the light and/or heavy chain variable domains of any antibody. Preferably, the light and the heavy chain variable domains are specific for the same antigen or can be directed against different antigens.

The variable regions of both heavy and light chains show considerable variability in structure and amino acid composition from one antibody molecule to another, whereas the constant regions show little variability. The term "variable" as used herein refers to the diverse nature of the amino acid sequences of the antibody heavy and light chain variable regions. Each antibody recognizes and binds antigen through the binding site defined by the association of the heavy and light chain variable region into an Fv area. The light-chain variable region VL and the heavy-chain variable region VH of a particular antibody molecule have specific amino acid sequences that allow the antigen-binding site to assume a conformation that binds to the antigen epitope recognized by that antibody. Nevertheless, the use of scFv comprising only the heavy-chain variable region VH of an antibody molecule allow the functional properties of antigen-binding, as shown in the present invention.

In another preferred aspect, the present invention relates to an expression vehicle comprising a nucleic acid sequence coding for a single-chain variable fragment antibody and more specifically, a nucleic acid sequence substantially as denoted by any one of SEQ ID NOs: 1, 2 and 3. This scFv antibody has specific affinity for the synaptic variant of acetylcholinesterase (AChE-S).

In a specifically preferred embodiment, the expression vehicle used in the present invention is the phagemid designated pHENl.

In yet another preferred embodiment, the expression vehicle according to the invention comprises a nucleic acid sequence coding for the amino acid sequence substantially as denoted by any one of SEQ ID NOs: 4, 5 and 6, which are the binding portion of the heavy chain variable region of the single-chain variable fragment antibody. This scFv antibody has specific affinity for the synaptic variant of acetylcholinesterase (AChE-S). In a most specifically preferred embodiment, the invention relates to an expression vehicle comprising the nucleic acid sequence coding for the amino acid sequence substantially as denoted by SEQ ID NO: 4.

In a specifically preferred embodiment, the expression vehicle according to the invention comprises any one of the single-chain variable fragment antibodies designated PD-antihASP 1, PD-anti_hASP 2 and PD-antihASP 3. Example 2 describes the isolation of three different single-chain variable fragment antibodies, the PD-antihASP 1, PD-antihASP 2 and the PD-antihASP 3, that specifically bind to ASP. It is to be appreciated that all three antibodies are within the scope of the present invention.

In another specifically preferred embodiment, the invention relates to the expression vehicle further comprising an amber stop codon. Insertion of such stop codon, as described in Example 2, enables the expression of any one of PD-antihASP 1, PD-antihASP 2 and the PD-anti_hASP 3 single-chain variable fragment antibodies as soluble fragments.

Expression vehicles for production of the molecules of the invention include plasmids, phagemids or other vectors. "Vectors", as used herein, encompass plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. Such system typically includes a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of RNA expression, a sequence that encodes a suitable ribosome binding site, RNA splice junctions, sequences that terminate transcription and translation and so forth. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell.

A vector may additionally include appropriate restriction sites, antibiotic resistance or other markers for selection of vector containing cells. Plasmids are the most commonly used form of vector but other forms of vectors which serves an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y.; and Rodriquez, et al. (eds.) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988), which are incorporated herein by reference.

In general, such vectors contain in addition specific genes, which are capable of providing phenotypic selection in transformed cells. The use of prokaryotic and eukaryotic viral expression vectors to express the genes coding for the polypeptides of the present invention are also contemplated. These vectors may further contain tagging sequences which are capable of providing convenient isolation of the desired phagemid. Such tagging sequences are well known in the art and include for example FLAG, HA, His-6 (six histidine) and preferably as used herein both the myc tag and the His-6 tag.

As disclosed herein, recombinant phagemids have been prepared which, when used to transform non-suppressor bacterial host cells, permit the secretion of foreign protein outside the cytoplasmic membrane of the host cell.

The vector is introduced into a host cell by methods known to those of skilled in the art. Introduction of the vector into the host cell can be accomplished by any method that introduces the construct into the cell, including, for example, calcium phosphate precipitation, microinjection, electroporation or transformation. See, e.g., Current Protocols in Molecular Biology, Ausuble, F. M., ed., John Wiley & Sons, N.Y. (1989).

In a further preferred embodiment, the invention relates to a single-chain Fv antibody specifically recognizing and binding to the synaptic variant of acetylcholinesterase (AChE-S). Preferably, this single-chain Fv antibody may be any one of the antibodies designated PD-antihASP 1, PD-antihASP 2 and the PD-antihASP 3. Most preferably, PD-anti_hASP 1.

The ScFv antibody is said to "have specific binding affinity" to molecule or peptide, if it is capable of specifically reacting with a that molecule or peptide. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by a ScFv antibody that can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody or fragments thereof (e.g., ScFv), which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

Therefore, as a preferred embodiment, the ScFv antibody of the invention comprises as the recognition region between the antigen (ASP peptide) and the scFv antibody any one of the amino acid sequences substantially as denoted by SEQ ID NOs: 10, 11 and 12.

Preferred embodiment relates to the recognition region having the amino sequence SRPSI, also denoted by SEQ ID NO: 10, which defines the recognition region between the antigen (ASP peptide) and the scFv antibody. During the phage-display screening procedure, two other sequences that define the recognition region between the antigen (ASP peptide) have been revealed in 1 out of 10 of the phages isolated. It is to be appreciated that these sequences, SRPSH and GARFKE, which are also denoted by SEQ ID NOs: 11 and 12, respectively, as well as the DNA sequence coding for these recognition regions, are within the scope of the present invention.

In another specifically preferred aspect of the invention, the single-chain Fv antibody which specifically recognizes and binds to the synaptic variant of acetylcholinesterase (AChE-S) according to the invention, may be used for the diagnosis of a progressive neuromuscular disorder in a mammal.

In yet another specifically preferred embodiment, the progressive neuromuscular disorder involves muscle distortion, muscle re-innervation or neuromuscular junction (NMJ) abnormalities. More specifically, said disorder may be Myasthenia graυis, Eaton-Lambert disease, muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD), multiple sclerosis, Dystonia, post-stroke sclerosis, post-injury muscle damage, excessive re-innervation, and post-exposure to AChE inhibitors. Preferably, the antibody of the invention may be used for diagnosing Myasthenia graυis.

The inventors have previously found high variability of AChE specific activity in serum samples from myasthenics, as compared to healthy individuals, alterations that appeared to be non-associated to anti-nAChR antibody levels and hence independent of the intensity of the autoimmune response. In non- denaturing gel electrophoresis, rapidly migrating "readthrough" AChE was also observed in higher levels in myasthenics as compared with control serum.

Moreover, it has been previously shown by the inventors, that overexpression of AChE-S in the neuromuscular junction, induces myasthenic-like features of simplified post-synaptic folds [Andres et al, (1997) ibid.]. Furthermore, as described in Example 4, immunoprecipitation, followed by immunochemical detection using the novel scFv antibody of the invention, demonstrated elevated levels of the synaptic AChE variant in MG serum.

Altogether, this cumulative information suggesting generally intensified AChE gene expression in MG patients and calling for testing the hypothesis that over-expressed AChE, due to the autoimmune insult and/or the antichohnesterase treatment, contributes to the disease symptoms and may provide useful serum marker(s) for MG diagnosis.

The inventors hypothesize that in the presence of the nAChRs as part of the cholinergic system it is possible to find mechanism which produces more of the AChE variants. In muscle, cholinergic insults hke the autoimmune response will disturb the cholinergic balance and initiate a transcriptional process, producing selectively more AChE-R, the stress related variant. In the case of AChE accumulation in myasthenic serum, the inventors presume that the protein is secreted from endothelial cells of blood vessels. This may be the reason for the accumulation of both variants: AChE-R and AChE-S in the tested serum. Therefore, the use of specific antibodies to detect the different variants of AChE, may be a promising new method for the diagnosis of MG disease.

Thus, as a further aspect the invention relates to a method for the diagnosis of a progressive neuromuscular disorder in a mammal. This method comprises the steps of: obtaining a sample from said mammal and detecting intensified expression of at least one of AChE variants in said sample.

In a preferred embodiment, the method of the invention is intended for the diagnosis of a progressive neuromuscular disorder that involves muscle distortion, muscle re-innervation or neuromuscular junction (NMJ) abnormalities. The neuromuscular disorder may be Myasthenia graυis, Eaton-Lambert disease, muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD), multiple sclerosis, Dystonia, post-stroke sclerosis, post-injury muscle damage, excessive re-innervation, and post-exposure to AChE inhibitors. Preferably, said disorder is Myasthenia graυis.

In a specifically preferred embodiment, the method of the present invention involves detection of intensified expression of at least one of AChE variants in the sample, more preferably, the AChE variant is the synaptic variant AChE-S.

Detection of the intensified expression of different AChE variants in the serum sample may be carried out by different techniques that are well known to the man of skill in the art. For example immunoassay, RT-PCR and nondenaturing activity gel electrophoresis.

In a preferred immunoassay, the sample obtained from said mammal is contacted with the single-chain Fv antibody of the invention, any unbound antibody is then removed, and the extent of reaction between said antibody and the AChE-S isoform present in the sample is detected.

Such assays for the AChE-S variant protein of the invention typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells or cells which have been incubated in tissue culture, in the presence of a labeled antibody capable of identifying the AChE-S, and detecting the antibody by any of a number of techniques well known in the art.

The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with a detectably labeled antibody in accordance with the present invention, as noted above. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means as described in Example 1.

By "solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" is intended any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of antibody, of the invention as noted above, may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation. One of the ways in which an scFv antibody in accordance with the present invention can be detectably labeled is by linking the same to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Alternatively, the scFv antibody of the invention can be detected as described in Example 3, using antibody that recognizes and binds to the tag molecule expressed in the scFv antibody, such as the myc or HIS 6 tags used in the present invention. The antibody used for binding to the tag may be detected using the ways described herein above. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomphshed using any of a variety of other immunoassays. For example, by radioactive labeling the antibodies or antibody fragments, it is possible to detect receptor tyrosine phosphatase (R-PTPase) through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al, North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a g counter or a scintillation counter or by autoradiography.

It is also possible to label an scFv antibody in accordance with the present invention with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can be then detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²E, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the scFv antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. An scFv antibody molecule of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the scFv antibody bound to the solid support or carrier and labeled scFv antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled scFv antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay. In the "reverse" assay, stepwise addition first of a solution of labeled scFv antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of un-reacted labeled scFv antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.

The present invention provides an immunoassay for the detection and quantification of the AChE-S variant. Immunoassays, such as RIA or ELISA, has been described in many articles, textbooks, and other publications. Reference is made to WO 97/03998, p. 48, fine 4 to p. 52, line 27. Immunoassays of the invention may be of two general types: Firstly, immunoassays using an immobilized peptide of the invention, ma be used. Secondly, immunoassays using immobilized antibodies directed against an epitope of a peptide of the invention may be used to quantify the AChE-S. variant according to the invention.

In a preferred embodiment of the invention, the assay is an immunoblot assay. The sample, e.g., a serum or a cerebrospinal fluid (CSF) sample, is optionally diluted, in order to avoid overloading. Then the sample is preferably immuno-precipitated followed by western-blot. Briefly, this technique comprising the steps of preparing a protein extract from said sample, immuno-precipitating said protein extracts using antibodies common to all AChE variants, then loaded onto a polyacrylamide gel, optionally a gradient gel, and electrophoresed. The gel is then blotted, preferably onto a Nitrocellulose or Nylon membrane. The blot is reacted with antibodies against the AChE-S variant, preferably any one of the scFv antibodies of the invention, the PD-antihASP 1, PD-antihASP 2 and PD-antihASP 3, and most preferably, PD-antihASP 1 as described herein. Bound antibody may then be detected by antibodies reactive with the antibody of the invention, e.g., anti-myc or anti- His 6 antibodies. These immunoglobuhns are preferably labeled, e.g., by Peroxidase conjugation. The detection of the label is then carried out according to methods known in then art. Preferably, peroxidase-conjugated immunoglobuhns are detected using the ECL™ detection system (Amersham Pharmacia Biotech, UK).

In a specifically preferred embodiment, the sample used in the method of the invention is one of serum, bone marrow or cerebrospinal fluid sample. As described above, a preferred sample is serum. However, other body fluids may be used, including cerebrospinal fluid, saliva, and the like. Also liquid extracts of body tissue may be analyzed. Alternatively, body tissue may be analyzed without extraction using cytochemical staining or immunostaining as described herein.

Such assays as described above may find use in diagnostics, as the level of the AChE-S according to the invention may need to be evaluated in a number of conditions. For instance, such assays may be useful for monitoring the effect of treatment of a patient.

Thus, in a preferred embodiment, the invention provides a method for the diagnosis of progressive neuromuscular disorder, comprising obtaining a sample from said mammal, preferably a serum sample, contacting said sample with any one of the scFv antibodies of the invention, removing unbound antibody, and detecting the extent of reaction between said scFv antibody and the synaptic acetylcholinesterase variant or a fragment thereof present in said sample.

In a specifically preferred embodiment the invention relates to a method for the diagnosis of MG in a subject comprising obtaining a sample from said subject, contacting said sample with an scFv antibody of the invention, removing unbound antibody, and detecting the extent of reaction between said antibody and acetylcholinesterase or a fragment thereof present in said sample.

As described in the Examples, AChE accumulation has several important implications: it was shown that transgenic overexpression of neuronal AChE-S causes progressive neuromotor deterioration and simplification of the post-synaptic fold, similarly to myasthenic symptoms [Andres. C, et al, (1997) ibid.; Sternfeld. M., et al, J Neurosci 18:1240-9 (1998)]. The similarities to the myasthenic features raised the assumption that AChE-S itself contributes to the myasthenic process and/or symptoms. The results shown in the present specification support this assumption: The cholinergic imbalance induces overproduction of AChE-S which is mainly secreted from endothelial cells hning blood vessels. This secreted enzyme initiates the known myasthenic symptoms.

Therefore, as a specifically preferred embodiment, detection of enhanced ; . expression of the AChE synaptic variant (AChE-S) may be a promising . method for the diagnosis of MG.

It is to be appreciated that in addition to the described diagnostic use for the antibodies of the invention, these specific anti ASP antibodies may serve as a tool for identification of different blood cells subgroups. As described in Example 3, cytoplasmic labeling of ASP was found specifically in monocytes. Thus the anti ASP antibodies of the invention may be also useful for monocytes identification.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present - disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Examples

Experimental Procedures

Serum samples:

A total of 28 serum samples were investigated. Of these, 18 were taken from myasthenic patients hospitahzed in Bulgaria and 5 from myasthenic patients at the Hadassa Hospital, Jerusalem, Israel. Serum was also taken from 5 healthy control individuals or controls with other diseases at the Hadassa Hospital. Assays were performed on the supernatant removed from those serum samples. Tissue culture:

All cells were grown in a fully humidified atmosphere at 37°C and 5% CO₂. All tissue culture reagents were purchased from Biological Industries (Beth Ha-Emek, Israel).

PC12 rat pheochromocytoma cells were grown in Dulbecco modified Eagle medium (DMEM) containing 8% fetal calf serum (FCS) and 8% horse serum (HS). For the induction of differentiation, 50 ng/ l NGF (Nerve growth factor) (Alomone, Jerusalem) was added to the medium with 1% FCS and 1% HS. Tissue culture plates or cover shps were coated with 10 μg/ml collagen type IV (Sigma, St. Louis, MO).

Preparation of an AChE-S specific antibody:

A synthetic biotinylated peptide, which represents the C-terminal sequence unique for "synaptic" AChE-S (ASP, AChE Synaptic Peptide), was used as target for selection from a synthetic phage display library of human single-chain Fv (scFv) antibodies (diversity >10⁸). This 40 amino acid peptide displays a predicted helical conformation. Three different specific anti-ASP antibody fragments were isolated, displayed on phage or secreted from infected bacteria. Two of them have closely related third complementary determining region (CDR3) sequences and were highly specific for ASP as judged by ELISA, since they did not interact with the distinct C-terminal peptide unique to the stress-associated "readthrough" AChE-R isoform. In protein blots the monoclonal phage antibody with the best binding activity further detected the recombinant full length human AChE-S expressed in PC 12 transfected pheochromocytoma cells, and the native AChE-S in human cerebrospinal fluid.

ELISA:

For the coating, BSA-biotin, streptavidin and biotinylated antigen (ASP or ARP) were applied in consecutive steps to a microtiter plate, mainly as described [Henderikx, P., et al, Cancer Res. 58:4324-4332 (1998)]. After last wash, 10 t.u. of phages were added to different wells. Detection of bound phages was via an anti-phage peroxidase conjugate visualized as absorption at 450 nm [for details see Harrinson J.L., et al, Methods Enzymol. 267:83-109 (1996)]. For the detection of a soluble monoclonal scFv antibody fragments, the 9E10 and a secondary anti- mo use peroxidase antibodies were used.

Magnetic immunoprecipitation and subsequent analysis by SDS-PAGE: 600μg of total serum proteins were incubated overnight with 5μl of goat polyclonal antibody (1:100) directed against the N-terminal domain of AChE (Santa Cruz). 70μl of Protein G Microbeads were added to samples. Serum was applied onto μMACS columns which were placed in magnetic field of the μMACS separator and washed once with lysis buffer (1% Triton x-100,. 50mM Tris-HCl pH=8, 150mM NaCI) and twice with 0.05% Tween-20 TBS (TBST). After loading the samples, μMACS columns were washed 4 times with 0.05% TBST, 20 μl of pre-heated 95°C 1XSDS gel loading buffer were applied onto the μMACS columns and incubated 5 min at room temperature. Elution was provided by 50μl of pre-heated 95°C 1XSDS gel loading buffer. Immunoprecipitation products were loaded on linear 4-20% gradient denaturing gels, run for 1.5hr and immunoblotted using an antibody-carrying phage interacting with the C-terminus of AChE-S, selected from a phage display library. For negative control, the same immunoprecipitation procedure was applied on homogenates prepared from COS cells, not expressing AChE.

Bia core analysis:

An informative approach to measure the binding of an antibody to its antigen, by which an antigen is bound to the matrix and the antibody is free in the buffer flow; binding is recorded in real time so that on and off rates can be measured. Example 1

Preparation of human scFv antibodies to ASP

Selection of human scFv antibodies to ASP

The display of repertoires of antibody fragments on the surface of filamentous bacteriophage offers an advantageous approach for making antibodies with predefined binding specificities. A phage display library of > 10⁸ scFv antibodies was constructed in vitro from human V gene segments as in Nissim et al [Nissim, A., et al, EMBO J. 13;692-698 (1994)]. ASP was prepared by using a solid-phase peptide synthesis (4334 peptide synthesizer, Perkin-Elmer), and biotinylation was performed on the protected peptide before cleavage from the resin. The purity of ASP-biotin was assessed by HPLC.

As demonstrated in the scheme in Fig. 2A, Phages (10¹¹ t.u., transforming units) were incubated for 1 h at room temperature with 4 μM biotinylated ASP in PBS, containing 2 mg/ml BSA and 0.1% Tween 20. Blocked streptavidin-coated paramagnetic beads were used for retrieval of ASP-bound phages. After washes, the captured phages were eluted by incubating the beads with 4 M NaCI, 50mM Tris-HCl, pH 7.5 for 30 min. at roo temperature, and the eluates were then diluted four times with dd H₂0.

The recovered phage particles were amplified in the E. coli "amber" suppressor strain TGI and used in a further round of selection. Before this second round, streptavidin binders were depleted by pre-absorption on a streptavidin-coated tube. The selection was then repeated as above; to increase the specificity and the stringency of selection, two rounds of antigen binding using half-concentration of ASP-biotin (2 μM) were performed before phage growth in bacteria ("double round"). Specificity of selected phage antibodies was assessed by ELISA (Fig. 3). Culture supernatants of single bacterial clones producing phage antibodies were incubated with biotinylated ASP, in the same concentration as used for first round of selection, and then added to streptavidin coated wells of a microtiter plate. Supernatants preincubated without the biotinylated antigen were used as negative controls. Binding of phages was detected via an anti-phage peroxidase conjugate visualized as absorption at 450 nm [for details see Harrinson J.L., et al, (1996) ibid.].

An extraordinarily high frequency (46/96, 48%) of specific anti-ASP monoclonal phage antibodies was found following two rounds of selection.

The diversity of selected clones was determined by amplification of scFv inserts by PCR and DNA sequencing with an automated AB1377 sequencer (Perkin-Elmer). Deduced amino acid sequences of VH-CD3, the third complementary determining region of the antibody heavy chain, allowed to identify three different specific anti-ASP clones after two rounds (PD-antiiASP 1, 2 and 3 - the nucleic acid sequences are denoted by SEQ ID NO: 1-3, respectively, and the amino acid sequences are denoted by SEQ ID NO: 4-6, respectively. One of the three clones (PD-antihASP 1) was dominating the population (67% of specific sequenced clones). Sequence analysis and determination of the exact antigen-binding region showed that the NH-CDR3 sequences of PD-antihASP 1 and 2 are closely related (compare SEQ ID ΝOs:10 and 11, respectively).

Example 2

Specificity analysis of monoclonal phage antibodies

Specificity analysis of monoclonal phage antibodies by ELISA To exclude the possibility that the three different isolated anti-ASP monoclonal phages would also interact with distinct C-terminal peptide unique for "readthrough" AChE-R (ARP), an ELISA assay was performed using indirectly coated ASP or ARP. The biotinylated peptides were synthesized as described herein-above. For the coating, BSA-biotin, streptavidin and biotinylated antigen (ASP or ARP) were applied in consecutive steps to a microtiter plate, mainly as described [Henderikx. P., et άl. (1998) ibid.]. After last wash, 10 t.u. of phages were added to different wells. Detection of bound phages was via an anti-phage peroxidase as mentioned above. Schematic representation of the ELISA is represented in the inset of Fig. 4. The highest specific signal was observed with the PD-antihAWP 1 clone (Fig. 4), which is dominant in the population (40% of the sequenced clones after two rounds of selection).

AChE-S specificity of PD-antihASP 1 clone analyzed by comparatiυe ELISA To test whether the PD-antihASP 1 monoclonal phage antibody selected against the C-terminal sequence (40 amino acids) unique for AChE-S reacts with the whole protein as well, a competitive ELISA was carried out. As shown in Fig. 5, 100 ng of ASP-biotin (19 pmol) was indirectly immobilized as described herein before and incubated together with 10 t.u. of PD-antihASP 1 phage in the presence of increasing amounts of either human recombinant AChE-S or ARP. The selected phage clone interacted specifically with soluble AChE-S, since the protein acts as competitor diminishing the binding of phage to the wells. No significant effects were observed in the case of soluble ARP.

Specificity analysis of soluble monoclonal scFv antibody fragments by ELISA

Schematic map of the pHENl vector (phagemid type) used for the fusion of antibodies to the minor coat phage protein pill [taken from Winter, G., et al, Annu. Rev. Immunol. 12:433-455 (1994)] is shown in Fig. 6A. Phagemid vectors comprise the PHI fusion, plasmid and phage origins of replication and antibiotic resistance markers; a helper phage provides the other functions for replication and packing. pHENl can also be used directly for expression of the antibodies as soluble fragments, as an amber stop codon is encoded at the junction of the antibody gene and gill. Therefore, the antibody fragments are displayed on phage after rescue with helper phage from an E. coli suppressor strain, or secreted as (tagged) soluble fragments from non-suppressor strains.

The specificity of the soluble monoclonal scFv antibody fragments, was next analyzed. The selected phages were used to infect the E. coli nonsuppressor strain HB2151, which was then induced as described above to produce soluble scFv fragments.

ELISA for the antibody fragments was carried out using indirectly coated ASP or ARP. For the coating, BSA-biotin, streptavidin and biotinylated antigen (ASP or ARP) were applied in consecutive steps to a microtiter plate. After last wash, 10 t.u, of phages were added to different wells. Detection of the bound myc-ta.gged antibody fragments was by using of 9E10 and a secondary anti-mouse peroxidase antibodies. Average +/- stdev values are shown from three different measurements (Fig. 6B).

PD-antiiιASP monoclonal phage antibody detects AChE-S in PC12 cell lysates and in human CSF

To test whether the PD-antihASP antibody can detect AChE-S in PC12 cell lysates and in human CSF, immunoblots were performed. Cell lysates and 2.5 μg total protein amount from human cerebrospinal fluid were fractionated on a poly aery lamide gel and transferred onto nitrocellulose. As shown in Fig. 7, immunoblot analysis using PD-antihASP 1 phage (10 t.u./ml) and a secondary anti-phage peroxidase antibodies allowed to detect the human AChE-S expressed in PC12 transfected cells (lane 1, Fig. 7A). In human AChE-R transfected PC12 cells (lane 2, Fig. 7A) a weaker band was observed at the same position (~ 97 kDa), probably corresponding to the endogenous rat AChE-S which possesses a high degree of homology with the human isoform at the C-terminus. The lowest band around 14kDA (lane 1, left blot) could be degradation products. The blot in Fig. 7B shows a positive control for PC 12 transfected cells expressing human AChE-S with the 190-2 monoclonal antibody obtained by hybridoma technology against the 10 C-terminal amino acids of human AChE-S [Boschetti. N., et al, Clinical Chemistry 42:19-23 (1996); gift of Dr. Brodbeck, University of Bern].

It may be noted that the ASP peptide used for screening of the phage library, displays helical conformation, probably endowed by its N- terminal amino acids.

BIAcore analysis of soluble antihASP! scFυ antibody In order to further characterize the antihASP 1 scFv antibody, determination of the affinity constant for the interaction between this antibody and its epitope was next performed using the technique of surface plasmon resonance, as implemented on the BIAcore instrument (BIACORE AB).

A peptide which represents the 23 C-terminal amino acids of ASP (short ASP), human recombinant AChE-S (Sigma) and ARP were covalently bound to the different flow-cells of a carboxymethyl dextran coated CM5 sensor chip. Fig. 8 shows the plots (sensorgrams) of the real-time interaction in resonance units (RU) between antihASPl and the short-ASP (Fig. 8A) or the AChE-S (Fig. 8B). The purified anti_hASPl scFv antibody was passed through the different flow cells on the sensor-chip at several concentrations (from lμM to 125nM), and the kinetic constants were calculated from the sensograms by the evaluation program of the BIAcore using the 1:1 Langmuir model. The dissociation constants are 4.7xlO'⁷M for the interaction between short-ASP and antihASPl, and 7.7xlO-⁷M for the interaction between AChE-S and antihASPl. No interaction was found with ARP (not shown). Example 3

Specific expression of ACh-S on various populations of cord blood cells

To test whether the PD-antihASP antibody can detect AChE-S in human developing blood cells, FACS analysis of different blood cell populations was next performed using the antihASPl scFv antibody. Cord blood cells were first sorted by their affinity to the CD45 antigen, which is common to variety of hematopoietic cell types. Position of the different cell populations along the two axis enabled their subtype identification as lymphocytes, monocytes, granulocytes or red blood cells (Figs. 9A and 10A). Each of these enriched populations was thereafter analyzed by itself, aiming to define its specific surface or cytoplasmic ASP expression, by using the antihASPl scFv antibody. To detect binding of the antihASPl to ceUs an antiMYC-FITC or antiHIS-FITC (the latter not shown) were used as secondary labeling. As shown in Table 1 and Figs. 9B and 9C, ASP is expressed on , the surface of monocytes, granulocytes and lymphocytes. Expression of cytoplasmic ASP was detected only in the monocyte population whereas all the other populations were negative for cytoplasmic expression of ASP (Figs. 10A and 10B). When the labeling pattern of ASP was compared with the labeling pattern of ARP (not shown), several interesting differences were found. The cell subtype distribution of ASP and ARP is unique to each peptide, suggesting distinct pattern of alternative splicing in specific hematopoietic lineages. ASP signals primarily reside on the cell surface, whereas ARP signal tend to be cytoplasmic. Without being bound to the theory, this may relate to the amphipathic ASP structure and the hydrop hylic, soluble nature of the ARP peptide. None of these peptides exists in red blood cells where AChE-E is apparently the only isoform. Both of these peptides appear on granulocytes.

Thus, as further aspect the antibodies of the present invention may be used for identifying different blood cell populations. Table 1

FACS analysis of ASP expression on different populations of cord blood cells

Example 4

Involvement of AChE variants in the cholinergic imbalance, and the use of anti-AChE antibodies as a surrogate marker for MG

Myasthenia graυis (MG) is an antibody-mediated autoimmune attack directed against the nicotinic acetylchohne receptor, nAChR at neuromuscular junctions. The primary characteristics of MG include decreases in the density of nAChRs at neuromuscular junctions, morphological changes at the postsynaptic membrane and a failure to trigger action potentials in part of the fibers. Current diagnosis is based on anti-AChR antibody titers, which are positive in 80% to 90% of patients. However, seronegative patients display similar clinical symptoms and response to therapies. Three other tests are being used to diagnose MG, yet none of them selectively detects the autoimmune response against the acetylchohne receptor. It was found that the antichohnesterase carbamate pyridostigmine, the first line of drugs used for MG treatment, induces a feedback response leading to excess AChE accumulation. Moreover, both transgenic overexpression of AChE in neuromuscular junctions and antichohnesterase exposure, inducing such overexpression, were shown to cause progressive neuromuscular dysfunctioning in laboratory animals. Based on these findings, the inventors hypothesized that AChE may serve as a potential modulator of MG and hence a promising surrogate marker in MG diagnosis. To this end, a study aimed at characterizing the AChE variants in blood samples from MG patients was initiated.

Elevated leυels of AChE-S in MG serum

Detection of the AChE-S chains with higher sensitivity and selectivity, was enabled due to development of the new anti "synaptic" AChE-S antibody. The soluble Fab fragment of this antibody carried by the phage, was used to probe nitrocellulose membrane with serum proteins immunoprecipitated with antibody targeted against the N-terminal domain common to all AChE isoforms. The resultant immunoblots revealed that the main bands appeared at 45Kda display higher intensity in serum samples from MG patients as compared to healthy individuals or patients with other diseases (Ctrl, Figs. HA and B). These results suggest the accumulation of intact AChE-S molecules in the MG serum (Fig. 11).

These findings point at the causal involvement of AChE variants in the cholinergic imbalance initiated by autoimmune responses and suggest the use of isoform-specific anti-AChE antibodies to detect AChE secreted to the blood, which may serve as a surrogate marker for MG.

Claims

Claims:

1. A nucleic acid sequence coding for a single-chain variable fragment antibody that has specific affinity for the synaptic variant of acetylcholinesterase (AChE-S), said single-chain variable fragment antibody consisting essentially of a polypeptide comprising the binding portion of the heavy chain variable region of an antibody.

2. The nucleic acid sequence of claim 1, being the DNA sequence substantially as denoted by any one of SEQ ID NOs:l, 2, and 3, coding for the amino acid sequence substantially as denoted by any one of SEQ ID NOs:4, 5 and 6, respectively, or any fragment thereof.

3. The nucleic acid sequence of claim 2, being the DNA sequence substantially as denoted by SEQ ID NO:l, coding for the amino acid sequence substantially as denoted by SEQ ID NO:4, or any fragment thereof.

4. An expression vehicle comprising a nucleic acid sequence coding for a single-chain variable fragment antibody that has specific affinity for the synaptic variant of acetylcholinesterase (AChE-S).

5. The vehicle of claim 4, which is a phagemid designated pHENl.

6. The expression vehicle of any one of claims 3 to 4, wherein said nucleic acid sequence is the DNA sequence substantially as denoted by any one of SEQ ID NOs:l, 2 and 3, coding for the amino acid sequence substantially as denoted by any one of SEQ ID NOs:4, 5 and 6, respectively.

7. The expression vehicle of any one of claims 4 to 6, wherein said single-chain variable fragment antibody is any one of the antibodies designated PD-antihASP 1, PD-anti_hASP 2 and PD-antihASP 3.

8. The expression vehicle of claim 7, wherein said single-chain variable fragment antibody is the antibody designated PD-antihASP 1.

9. The expression vehicle of claim 7, further comprising an amber stop codon for expression of any one of PD-antihASP 1, PD-antihASP 2 and PD-antihASP 3 single-chain variable fragment antibodies, as soluble fragments.

10. A single-chain Fv antibody specifically recognizing and binding to the synaptic variant of acetylcholinesterase (AChE-S).

11. The single-chain Fv antibody of claim 10, wherein said antibody is any one of the antibodies designated PD-antihASP 1, PD-antihASP 2 and PD-antihASP 3, having the amino acid sequence substantially as denoted by any one of SEQ ID NOs:4, 5 and 6, respectively.

12. The single-chain Fv antibody of claim 11, wherein said antibody is the antibody designated PD-antihASP 1, having the amino acid sequence substantially as denoted by SEQ ID NO:4.

13. The single-chain Fv antibody of claim 11, having an antigen recognition region substantially as denoted by any one of SEQ ID NOs:10, 11 and 12.

14. The single-chain Fv antibody of any one of claims 11 to 13, specifically recognizing and binding to the synaptic variant of acetylchohne sterase (AChE-S), for the diagnosis of a progressive neuromuscular disorder in a mammal.

15. The single-chain Fv antibody of claim 14, wherein said progressive neuromuscular disorder involves any one of muscle distortion, muscle re-innervation and neuromuscular junction (NMJ) abnormalities.

16. The single-chain Fv antibody of claim 15, wherein said disorder is selected from Myasthenia graυis, Eaton-Lambert disease, muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD), multiple sclerosis, Dystonia, post-stroke sclerosis, post-injury muscle . damage, excessive re-innervation, and post-exposure to AChE inhibitors.

17. The single-chain Fv antibody of claim 16, wherein said disorder is Myasthenia gravis.

18. A method for the diagnosis of a progressive neuromuscular disorder in a mammal, comprising the steps of: obtaining a sample from said mammal and detecting intensified expression of at least one of AChE variants in said sample.

19. The method of claim 18, wherein said progressive neuromuscular disorder involves any one of muscle distortion, muscle re-innervation and neuromuscular junction (NMJ) abnormalities.

20. The method of claim 19, wherein said disorder is selected from Myasthenia gravis, Eaton-Lambert disease, muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD), multiple sclerosis, Dystonia, post-stroke sclerosis, post-injury muscle damage, excessive re-innervation, and post-exposure to AChE inhibitors.

21. The method of claim 20, wherein said disorder is Myasthenia gravis.

22. The method of any one of claims 18 to 21, wherein detecting the intensified expression of different AChE variants in said sample is by any one of immunoassay, RT-PCR and nondenaturing catalytic activity gel electrophoresis.

23. The method of claim 22, wherein said AChE variant is the synaptic acetylchohne sterase variant (AChE-S).

24. The method of claim 23, wherein said immunoassay comprises obtaining a sample from said mammal, contacting said sample with the single-chain Fv antibody of any one of claims 10 to 17, removing any unbound antibody, and detecting the extent of reaction between said antibody and the AChE-S isoform present in said sample.

25. The method of any one claims 18 to 24, wherein said sample is one of serum, bone marrow and cerebrospinal fluid sample.

26. The method of claim 25, wherein said sample is serum.