HK1169855B - Immuno-based retargeted endopeptidase activity assays - Google Patents

Description

Immune-based retargeted endopeptidase activity assay

This patent application is in accordance with section 35 (e) of the united states code claiming priority from united states provisional patent application No. 61/160,217 filed on 3, 13, 2009, the entire contents of which are hereby incorporated by reference.

The sequences disclosed in this specification are contained in the sequence listing, which is presented together with the specification, the entire contents of which are hereby incorporated by reference.

Clostridial toxins (e.g., Botulinum neurotoxin (BoNT), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G; and Tetanus neurotoxin (TeNT)) are used in a number of therapeutic and Cosmetic Applications, see, for example, William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004). Clostridial toxins that are commercially available in the form of pharmaceutical compositions include BoNT/A preparations, e.g., BOTOX(Allergan，Inc.，Irvine，CA)、DYSPORT/RELOXIN(Ipsen Ltd.，Slough，England)、PURTOX(Mentor Corp.，Santa Barbara，CA)、XEOMIN(Merz Pharmaceuticals，GmbH.，Frankfurt，Germany)、NEURONOX(Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A (Biogen-tech Ltd., University, Yantai, Shandong, China); and BoNT/B formulations, e.g. MYOBLOC/NEUROBLOC(solvent neurosciens, Inc., South San Francisco, Calif.). For example, BOTOXAre currently approved in one or more countries for the following indications: achalasia, adult spasms, anal fissure, back pain, blepharospasm, nocturnal molars, cervical dystonia, essential tremor, glabellar or facial dynamic wrinkles, headache, facial spasm, bladder hyperactivity, hyperhidrosis, pediatric cerebral palsy, multiple sclerosis, myoclonic disorders, nasolabial lines, spasmodic dysphonia, strabismus and facial nerve disorders (VII nerve disorder).

Clostridial toxin therapy inhibits the release of neurotransmitters and neuropeptides by interrupting the exocytosis process used to secrete them into the synaptic cleft. There is an urgent need in the pharmaceutical industry to expand the use of clostridial toxin therapies in addition to the current muscle relaxant applications to treat sensory neurological disorders such as various types of chronic pain, neurogenic inflammation and genitourinary disorders, as well as other disorders such as pancreatitis. One approach currently used to extend clostridial toxin-based therapies involves modifying clostridial toxins such that the modified toxins have altered cell targeting ability for relevant neuronal or non-neuronal cells. These molecules, termed retargeted endopeptidases or Targeted Vesicle Exocytosis Modulator Proteins (TVEMPs), achieve their Exocytosis inhibitory effect through the use of target receptors present on the relevant neuronal or non-neuronal target cells. This ability to retarget is achieved by replacing the native binding domain of the clostridial toxin with a targeting domain that exhibits selective binding activity for a non-clostridial toxin receptor present in the relevant neuronal or non-neuronal target cell. This modification of the binding domain results in a molecule that is capable of selectively binding to a non-clostridial toxin receptor present on the target cell. Retargeted endopeptidases can bind to target receptors, translocate into the cytoplasm, and exert their proteolytic effect on SNARE complexes of relevant neuronal or non-neuronal target cells.

A panel of retargeted endopeptidases comprises molecules with an opioid targeting domain. These opioid retargeting endopeptidases comprise an opioid targeting domain, a clostridial toxin translocation domain, and a clostridial toxin enzyme domain. Non-limiting examples of opioid heavy-targeting endopeptidases or opioid-TVEMPs are described in the following patent documents: for example, Keith A. Foster et al, Clostridium depletion Able To modification personal sensor Functions, U.S. Pat. No. 5,989,545; oliver Dolly et al, active Recombinant neotoxins, U.S. patent 7,132,259; stephan Donovan, nasal Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,244,437; stephan Donovan, nasal Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,413,742; stephan Donovan, nasal Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,415,338; lance E.Steward et al, Multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. Pat. No. 7,514,088; keith a. foster, Fusion Proteins, U.S. patent publication 2008/0064092; keith a. foster, Fusion Proteins, U.S. patent publication 2009/0035822; lance E.Steward et al, Multi-equivalent Clos ternary Toxin Derivatives and Methods of the same Use, U.S. patent publication 2009/0048431; keith a. foster, Non-cytoxic Protein Conjugates, U.S. patent publication 2009/0162341; keith A. Foster et al, Re-targeted Toxin Conjugates, International patent publication WO 2005/023309; and Lance E.Steward, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting Capabilities for Non-cloning approach Cells, International patent application WO 2008/008805; the entire contents of each patent are hereby incorporated by reference.

One major difference between the retargeted endopeptidases and clostridial toxins is that since retargeted endopeptidases do not normally target motor neurons, overdosing, if not completely avoided, will largely overdose mammals with retargeted targetsEndopeptidase related death is minimized. For example, when opioid heavy-targeted endopeptidase is administered in an amount 10,000 times the therapeutically effective dose, signs of lethality are observed and are caused by passive diffusion of the molecule rather than a toxic process. Thus, for all practical purposes, retargeted endopeptidases are non-lethal molecules. Although this non-lethal property is of great therapeutic interest, since the standard activity assay used to make clostridial toxin-based biologies is mouse LD₅₀Bioassay (a lethal test) causes problems in manufacturing. S.s.arnon et al, JAMA 285: 1059-1070(2001). Currently, mouse LD is used by all pharmaceutical manufacturers₅₀Bioassay to show the efficacy of clostridial toxin formulations. In fact, the unit of activity of clostridial toxin is mouse LD₅₀Units. However, since the retargeted endopeptidase is essentially non-lethal, mouse LD cannot be used ₅₀Bioassays to assess the efficacy of these molecules. Therefore, a simple, reliable, validated and government agency acceptable activity assay that can assess the integrity of all the necessary steps in the re-targeted endopeptidase uptake process would be of significant value.

The present specification provides novel compositions, cells, and methods for determining the activity of a retargeted endopeptidase suitable for use in a variety of industries, such as the pharmaceutical and food industries, and also provides related benefits. These compositions, cells, and methods do not use live animals or tissues obtained from live animals, but are capable of assessing all steps necessary for the re-targeting of endopeptidases to function.

Drawings

Figure 1 shows a current schematic of an example of neurotransmitter release and clostridial toxin intoxication in central and peripheral neurons. Figure 1A shows a schematic of the neurotransmitter release mechanism in central and peripheral neurons. The release process can be described as comprising two steps: 1) vesicular docking, wherein a vesicle-binding SNARE protein of a vesicle containing a neurotransmitter molecule associates with a membrane-binding SNARE protein located at the plasma membrane; and2) neurotransmitter release, where vesicles fuse with the plasma membrane and neurotransmitter molecules are exocytosed. FIG. 1B shows a schematic of the intoxication mechanism for tetanus toxin and botulinum toxin activity in central and peripheral neurons. The poisoning process can be described as including four steps: 1) receptor binding, wherein the clostridial toxin binds to the clostridial receptor complex and initiates the intoxication process; 2) complex internalization, wherein upon toxin binding, the vesicles containing the toxin/receptor system complexes enter the cell by endocytosis; 3) translocation of the light chain, in which multiple events are thought to occur, including changes in pH inside vesicles, formation of heavy chain H comprising clostridial toxin _NA channel pore of the domain, separation of the light chain from the heavy chain of the clostridial toxin and light chain release; and 4) enzymatic target modification, wherein the light chain of a clostridial toxin proteolytically cleaves its target SNARE substrate, such as SNAP-25, VAMP, or Syntaxin (Syntaxin), thereby preventing vesicle docking and neurotransmitter release.

FIG. 2 shows the full dose response to retargeted endopeptidase Noc/A in ORL-1 clone #6 clone that overexpresses ORL-1. Specific uptake of Noc/A was observed in ORL-1 clone #6 clonal cell line overexpressing ORL-1. In the cleaved SNAP-25₁₉₇In the ECL ELISA of (1), Noc/A (LHN/A plus ligand-binding nociceptin) variant) and LH were used_NA (LC/A and H without any binding domain_N) Treatment of ORL-1 stable cell line clone #6 confirmed: Noc/A uptake is specific to this clonal cell line. The clonal cell line also exhibits enhanced sensitivity to Noc/A, with EC₅₀It was 1.2 nM.

FIG. 3 shows the full dose response to Noc/A in SK-N-DZ single cell derived clones #3 and # 22. And LH_NIn comparison, SK-N-DZ clones #3 and #22 specifically uptake Noc/a (N ═ 4 independently performed experiments). Cells were seeded in RPMI SFM + N2+ B27+ NGF onto poly D-lysine 96-well plates. The compound was treated for 22 hours. Cleaved SNAP-25 ₁₉₇The ECL ELISA of (C) confirmed that Noc/A uptake was specific to this clonal cell line. These clonal cell lines also show greater sensitivity to Noc/A, with the EC of clone #3₅₀0.3nM, and clone #22 EC₅₀It was 0.9 nM.

FIG. 4 shows the results of ECL sandwich ELISA assays from ORL1 ND7 clones 1C11, 4B7 and 4C9 treated with retargeted endopeptidase Noc/A. Parental ND7 and ORL1 ND7 clones were treated with Noc/a for 24 hours followed by 2 days of incubation. Since the parent ND7 only reaches about 50% SNAP-25₁₉₇Cracking, so that its EC cannot be calculated₅₀The value is obtained. More than 80% of SNAP-25 in clones 4B7 and 1C11₁₉₇And (4) cracking. EC for three clones was calculated₅₀The values were 5.7. + -. 0.5, 6.7. + -. 1 and 8.6. + -. 2nM, respectively.

FIG. 5 shows that anti-nociceptin polyclonal antibodies can prevent uptake of retargeted endopeptidase Noc/A in SK-N-DZ clone #3, clone #22 and AGN P33 ORL-1 clone #6 cell lines. Cells were seeded onto poly-D-lysine 96-well plates in RPMI SFM + N2+ B27+ NGF and treated in serum-free medium containing anti-nociceptin polyclonal antibodies diluted to different concentrations (0-3. mu.g/mL) at 1nM Noc/A for 22 hours.

FIG. 6 shows cells from SiMa clone AF4 and established cell line PC-12 treated with a retargeted endopeptidase Dyn/A at a concentration of 0.017nM to 1 μ M as described in Western blot (Western blot) images. Dose-dependent uptake was observed for both cell lines.

FIG. 7 shows normalized BIAcore SPR curves for the 7.8nM antibodies 2E2A6, 1D3B8, 3C1A5, and 2C9B10, and the commercially available MC-6050 and MC-6053. Figure 7A shows normalized data for association rate (on-rate) for each antibody. Fig. 7B shows normalized data for off-rate (off-rate) for each antibody.

Detailed Description

The present specification provides novel assays for determining the presence or absence of active retargeted endopeptidases in a sample and for determining the activity/potency of retargeted endopeptidases. The novel cell-based assays disclosed in this specification depend on cells, reagents and detection methods that enable the assay to detect nanomolar amounts of retargeted endopeptidases in a sample. The cell-based assays disclosed in the present specification are useful for analyzing the various functions of the retargeted endopeptidase, i.e., binding of the retargeted endopeptidase to a cell surface receptor, internalization of the endopeptidase-receptor complex, translocation of the enzyme domain into the cytoplasm, cleavage of the enzyme domain of the substrate. As discussed further below, these novel methods and compositions can be used to analyze crude and bulk samples as well as highly purified double-stranded retargeted endopeptidase and formulated retargeted endopeptidase products, and can also be determined in an automated high throughput assay format.

Accordingly, one aspect disclosed in the present specification provides immune response-inducing compositions for the production of α -SNAP-25 antibodies that selectively bind to P comprising a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An epitope of a SNAP-25 cleavage product having a carboxyl terminus at the residue. The immune response inducing composition can comprise an adjuvant and an immune response inducing composition comprising the SNAP-25 antigen, a carrier linked to the SNAP-25 antigen, or a carrier linked to a flexible spacer linked to the SNAP-25 antigen, wherein the flexible spacer is interposed between the SNAP-25 antigen and the carrier. Expected to elicit an immune response resulting in the generation of P that selectively binds to a readily cleavable bond at the BoNT/A cleavage site₁Any and all SNAP-25 antigens of the alpha-SNAP-25 antibody having a SNAP-25 epitope at the carboxyl terminus at residues can be used as SNAP-25 antigens, including, but not limited to, SNAP-25 antigens derived from native SNAP-25, SNAP-25 antigens derived from non-native SNAP-25, and SNAP-25 antigens comprising an immunoreactive fragment of SNAP-25 (i.e., SNAP-25 derived from native SNAP-25 or non-native SNAP-25). P suitable for generating a bond that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁SNAP-25 antigens of alpha-SNAP-25 antibodies having a carboxy-terminal SNAP-25 epitope at residue include, but are not limited to, SNAP-25 antigens comprising a SNAP-25 peptide having a carboxylated C-terminal glutamine linked to a carrier peptide, including, but not limited to, SEQ ID NO: 38. p suitable for preparing a bond which can be selectively bound to a cleavage site of a BoNT/A and is susceptible to cleavage₁Other immune response-inducing compositions of alpha-SNAP-25 antibodies having a SNAP-25 epitope at the carboxyl terminus of the residue include, but are not limited to, compositions comprising a peptide linked to the carboxylated C-terminal trough of the SNAP-25 antigenAn immune response inducing composition of a flexible linker linked carrier of an aminoamide, wherein the flexible linker is interposed between the SNAP-25 antigen and the carrier. It is contemplated that any and all adjuvants may be suitable for use in such immune response-inducing compositions, including, but not limited to, polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), polyvinyl alcohol (PVA), complete and incomplete Freund's adjuvant.

Another aspect disclosed in the specification provides methods for producing an alpha-SNAP-25 antibody that selectively binds to P comprising a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An epitope of a SNAP-25 cleavage product having a carboxyl terminus at the residue. Aspects of the method include the steps of: (a) administering to an animal a SNAP-25 immune response inducing composition disclosed in the specification; (b) collecting a sample containing α -SNAP-25 antibody or α -SNAP-25 antibody-producing cells from the animal; and (c) isolating the alpha-SNAP-25 antibody from the sample. The disclosed methods are useful for preparing selectively bound P containing bonds that are susceptible to cleavage at a BoNT/A cleavage site ₁An alpha-SNAP-25 monoclonal antibody having an epitope of a SNAP-25 cleavage product with a carboxyl terminus at residue, or selectively binding to P comprising a bond susceptible to cleavage at a BoNT/A cleavage site₁An alpha-SNAP-25 polyclonal antibody having an epitope of a cleavage product of SNAP-25 at the carboxyl terminus at the residue.

Another aspect disclosed in the specification provides alpha-SNAP-25 antibodies that selectively bind to P comprising a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An epitope of SNAP-25 having a carboxyl terminus at the residue. Such α -SNAP-25 antibodies include natural and non-natural antibodies, as well as monoclonal or polyclonal α -SNAP-25 antibodies. P suitable for use as a bond selectively binding to a bond susceptible to cleavage at a BoNT/A cleavage site₁Monoclonal a-SNAP-25 antibodies to a-SNAP-25 antibodies having a carboxy-terminal SNAP-25 antigen at residues include, but are not limited to, monoclonal a-SNAP-25 antibodies produced by hybridoma cell lines 1D3B8, 2C9B10, 2E2a6, 3C1a5, and 3C3E 2.

Another aspect disclosed in the present specification provides a method for detecting a heavy targeting endopeptideAn enzymatically active immune-based method. Aspects of the method include the steps of: (a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line are sensitive to retargeted endopeptidase activity of the retargeted endopeptidase; (b) isolating from the treated cells a SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; (c) contacting the SNAP-25 component with an alpha-SNAP-25 antibody disclosed in the specification; and (d) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and P susceptible to bond cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; wherein detection of the antibody-antigen complex will indicate retargeted endopeptidase activity. The α -SNAP-25 antibody in step (c) may optionally be attached to a solid support.

Yet another aspect disclosed in the present specification provides an immune-based method for detecting opioid-TVEMP activity. Aspects of the method include the steps of: (a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line can take up the retargeted endopeptidase; (b) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site₁SNAP-25 having a carboxyl terminus at residue; (c) contacting the SNAP-25 component with an alpha-SNAP-25 antibody disclosed in the specification; and (d) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and P susceptible to bond cleavage at a BoNT/A cleavage site ₁SNAP-25 having a carboxyl terminus at residue; wherein detection of the antibody-antigen complex will indicate retargeted endopeptidase activity. The α -SNAP-25 antibody in step (c) may optionally be attached to a solid support.

Another aspect disclosed in the present specification provides a method for determining retargeted endopeptidase immune resistance in a mammal. Aspects of the method include the steps of: (a) addition of retargeted endopeptidase to a mammal obtained for testing alpha-retargeted endopeptidase neutralizing antibodyIn the test sample for the presence or absence of (a); (b) treating cells from an established cell line with the test sample, wherein the cells from the established cell line are susceptible to retargeted endopeptidase activity; (c) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; (d) contacting the SNAP-25 component with an alpha-SNAP-25 antibody disclosed in the specification; (e) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and P having a bond susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; (f) repeating steps a-e by replacing the test sample with a negative control sample; and (g) comparing the amount of antibody-antigen complex detected in step (e) with the amount of antibody-antigen complex detected in step (f), wherein the presence of α -retargeted endopeptidase neutralizing antibody is indicated if the amount of antibody-antigen complex detected in step (e) is less than the amount of antibody-antigen complex detected in step (f). The α -SNAP-25 antibody in step (d) may optionally be attached to a solid support. The control sample in step (f) may comprise a positive control sample in addition to a negative control sample.

Clostridial toxins produced by Clostridium botulinum (Clostridium botulinum), Clostridium tetani (Clostridium tetani), Clostridium pasteurianum (Clostridium barati) and Clostridium butyricum (Clostridium butyricum) are widely used in the treatment and cosmetics of humans and other mammals. The Clostridium botulinum strains produce 7 antigenically distinct botulinum toxin (BoNT) serotypes identified by studying the onset of botulism in humans (BoNT/A, BoNT/B, BoNT/E and BoNT/F), animals (BoNT/C1 and BoNT/D), or isolated from soil (BoNT/G). Although all seven botulinum toxin serotypes have similar structural and biological properties, each also exhibits heterogeneous characteristics, such as different pharmacological properties. In contrast, tetanus toxin (TeNT) is produced by a homogeneous group of clostridium tetani. The other two clostridia, clostridium pasteurianum and clostridium butyricum, also produce toxins similar to BoNT/F and BoNT/E, respectively.

Each clostridial toxin is translated as a single-chain polypeptide of about 150kDa whose disulfide loop is subsequently cleaved by proteolytic cleavage by a native protease, such as an endogenous clostridial toxin protease or an environmentally produced native protease. This post-translational processing results in a double-stranded molecule comprising an about 50kDa Light Chain (LC) and an about 100kDa Heavy Chain (HC) that are held together by a single disulfide bond and non-covalent interactions. Each mature double-stranded molecule comprises three functionally distinct domains: 1) an enzyme domain located in the LC comprising a metalloprotease region containing zinc-dependent endopeptidase activity that specifically targets a core portion of a neurotransmitter-releasing organ; 2) a transpose domain contained in HC (H) _N) Within the amino-terminal half of (a), which facilitates intracellular vesicles releasing LCs into the cytoplasm of a target cell; and 3) binding domains, found in HC (H)_C) Within the carboxy-terminal half of (a), which determines the binding activity and binding specificity of the clostridial toxin to the receptor complex located on the surface of the target cell.

Binding, translocation and enzymatic activity of these three domains are all required for toxicity. While all the details of this process are not precisely known, the overall mechanism of intoxication by clostridial toxins to enter neurons and inhibit neurotransmitter release is similar, regardless of serotype or subtype. Although applicants do not wish to be limited by the following description, the poisoning mechanism may be described as comprising at least 4 steps: 1) receptor binding, 2) complex internalization, 3) light chain translocation, and 4) enzymatic target modification (fig. 1). This process begins when the HC domain of a clostridial toxin binds to a toxin-specific receptor system located on the plasma membrane surface of a target cell. The binding specificity of the receptor complexes is believed to be achieved in part by the specific combination of gangliosides and protein receptors that appear to apparently comprise the respective clostridial toxin receptor complex. Once bound, the toxin/receptor complex is internalized by endocytosis, and the internalized vesicles are classified as specific intracellular pathways. The translocation step appears to be initiated by acidification of the vesicle compartment. This process appears to initiate important pH-dependent structural rearrangements that increase hydrophobicity, promote pore formation, and facilitate separation of the toxin heavy and light chains. Once the heavy and light chains are separated, the toxin's light chain endopeptidase is released from the intracellular vesicle into the cytosol where it appears to specifically target the core of the neurotransmitter-releasing organ. These core proteins, Vesicle Associated Membrane Protein (VAMP)/synaptobrevin, 25kDa synaptosome associated protein (SNAP-25) and syntaxin, are essential for synaptic vesicle docking and fusion at the nerve terminals and constitute members of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family. BoNT/A and BoNT/E cleave the carboxy-terminal region of SNAP-25, releasing fragments of 9 or 26 amino acids, respectively, and BoNT/C1 also cleaves SNAP-25 near the carboxy terminus, releasing a fragment of 8 amino acids. Botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G and tetanus toxin act on the conserved central portion of VAMP and release the amino terminal portion of VAMP into the cytosol. BoNT/C1 cleaves syntaxin at a single site near the plasma membrane surface of the cell. Selective proteolysis of synaptic SNAREs will result in the blockade of neurotransmitter release by clostridial toxins. The SNARE protein target of clostridial toxins is common to the occurrence of exocytosis in a variety of non-neuronal types; in these cells, as in neurons, light chain peptidase activity will inhibit exocytosis, see, e.g., Yann Humeau et al, How Botulinum and tetronus Neurotoxins Block neurogransmiter Release, 82(5) Biochimie.427-446 (2000); kathryn Turton et al, Botulinum and Tetanus Neurotoxins: structure, Function and Therapeutic Utility, 27(11) Trends biochem. Sci.552-558 (2002); giovanna Lanlli et al, The journal of tetronus and Botulinum Neurotoxins in Neurons, 11(9) Trends Microbiol.431-437 (2003).

Retargeted endopeptidases typically replace the native double-stranded cyclic protease cleavage site with an exogenous protease cleavage site. See, e.g., Dolly, j.o. et al, active cloning, Toxins, U.S. patent 7,419,676, which is incorporated herein by reference. Although the overall molecular weight of the retargeted endopeptidase varies depending on the size of the targeting moiety, the activation process and its reliance on cleavage at an exogenous cleavage site to produce a di-chain molecule is essentially the same as clostridial toxins. See, e.g., Steward, l.e., et al, active cloning Toxins, U.S. patent publication 2009/0005313; steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting activities For Non-cloning approach targets Cells, U.S. patent application 11/776,075; steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Altered Targeting activities for cloning approach Cells, U.S. patent publication 2008/0241881, each of which is incorporated herein by reference.

Aspects of the disclosure include, in part, immune response-inducing compositions for the production of alpha-SNAP-25 antibodies that selectively bind P that is susceptible to bond cleavage at the BoNT/A cleavage site ₁SNAP-25 having a carboxyl terminus at the residue. The term "immune response-inducing composition" as used herein refers to a composition comprising SNAP-25 antigen that, when administered to an animal, will stimulate an immune response to the SNAP-25 antigen, thereby generating P that selectively binds to a readily cleavable bond at the BoNT/A cleavage site₁An alpha-SNAP-25 antibody to SNAP-25 having a carboxyl terminus at residue. The term "immune response" refers to any response of the immune system of an animal to an immune response-inducing composition. Exemplary immune responses include, but are not limited to: cellular and local and systemic humoral immunity, e.g., CTL responses, including antigen-specific induction responses of CD8+ CTLs, helper T cell responses (including T cell proliferation responses and cytokine release), and B cell responses (including, e.g., antibody-producing responses). The term "inducing an immune response" refers to the administration of an immune response-inducing composition or a polynucleotide encoding an immune response-inducing composition, during which the immune response is affected, i.e., stimulated, elicited or induced.

The SNAP-25 immune response-inducing composition comprises a SNAP-25 antigen. The term "antigen" as used herein refers to a molecule that elicits an immune response, and And include, but are not limited to, conjugates of peptides, polysaccharides, and lipids such as lipoproteins and glycolipids. The term "SNAP-25 antigen" as used herein refers to a P that is susceptible to bond cleavage at the BoNT/A cleavage site₁Any antigen with a carboxyl terminus at a residue that can elicit an immune response. The SNAP-25 antigen used in the immune response inducing composition must be large enough to have a significantly unique sequence, thereby reducing the likelihood of producing antibodies that cross-react with antigens other than SNAP-25. In addition, the SNAP-25 antigen used in the immune response-inducing composition must also be small enough to elicit substantially only P directed to bonds that are susceptible to cleavage at the BoNT/A cleavage site₁Immune response of SNAP-25 with a carboxy terminus at the residue, thereby increasing production of P that distinguishes bonds susceptible to cleavage at a BoNT/A cleavage site₁SNAP-25 with a carboxyl terminus at the residue and P readily cleavable at the BoNT/A cleavage site₁The possibility of an alpha-SNAP-25 antibody without a carboxy-terminal SNAP-25 at the residue. Furthermore, there is an urgent need to generate α -SNAP-25 antibodies with a single amino acid sequence that have good yields, reproducible selectivities and bind with acceptable avidity, thereby allowing highly sensitive assays to be designed.

The sequence appearing around the BoNT/A cleavage site in SNAP-25 is denoted P₅-P₄-P₃-P₂-P₁-P₁’-P₂’-P₃’-P₄’-P₅', wherein P₁-P₁' represents a frangible bond. After re-targeted endopeptidase cleavage, the cleavage product thus produced comprises P₅-P₄-P₃-P₂-P₁Fragments of the sequence and the inclusion of P₁’-P₂’-P₃’-P₄’-P₅' of (a). Thus, the term "P susceptible to bond cleavage at BoNT/A cleavage site" as used herein₁SNAP-25 "having a carboxyl terminus at the residue means that P has as its carboxyl-terminal amino acid₁Any SNAP-25 of a residue. For example, Q in human SNAP-25(SEQ ID NO: 5)₁₉₇-R₁₉₈P representing BoNT/A cleavage site₁-P₁' easy splittingA key. Thus, a "SNAP-25 having a carboxy-terminal glutamine having a bond readily cleavable from a BoNT/A cleavage site" is any SNAP-25 cleavage product having glutamine at its carboxy-terminal amino acid, where glutamine represents Q in the readily cleavable bond₁₉₇. As another example, K in skate (Torpdo marmorata) SNAP-25(SEQ ID NO: 16)₂₀₄-H₂₀₅P representing BoNT/A cleavage site₁-P₁' easily cleavable bond. Thus, a "SNAP-25 having a carboxy-terminal lysine with a bond readily cleavable from a BoNT/A cleavage site" is any SNAP-25 cleavage product having a lysine at its carboxy-terminal amino acid, where lysine represents K in a readily cleavable bond ₂₀₄。

P having bond cleavage site susceptible to BoNT/A among BoNT/A cleavage sites₁The SNAP-25 antigen having a carboxyl terminus at a residue is modified to enhance the immunogenicity of the unbound modified SNAP-25 antigen, hapten or any other immunogenic, non-immunogenic or weakly immunogenic antigenic compound. In one aspect of this embodiment, the carboxy-terminal P of the cleavable bond from the SNAP-25 antigen can be carboxylated₁And (c) a residue. Carboxylation will increase the desired immunogenicity of the SNAP-25 antigen in two ways. First, since charged amino acids can enhance immunogenicity, COO is a compound^-The addition of groups to the carboxy-terminal residue will increase the overall immunogenicity of the SNAP-25 antigen. Second, P which is susceptible to bond cleavage by BoNT/A cleavage site₁The residue is charged upon cleavage, so COO^-The addition of a group to the carboxy-terminal residue will better mimic the actual antigen to which the α -SNAP-25 antibody disclosed in this specification is designed to selectively bind.

In one aspect of this embodiment, the amino terminal residue of the SNAP-25 antigen may be modified by the addition of amino acids suitable for linking the SNAP-25 antigen to a carrier protein, such as Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Thyroglobulin (THY), Bovine Serum Albumin (BSA), Soybean Trypsin Inhibitor (STI), or Multiple Attachment Peptide (MAP). For example, a cysteine residue may be located at the amino terminus for conjugation to the carrier protein KLH.

Thus, in one embodiment, P is a bond that is susceptible to cleavage at the BoNT/A cleavage site₁The length of the SNAP-25 antigen having a carboxy terminus at a residue can be, e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acids. In another embodiment, P is one that is susceptible to bond cleavage at the BoNT/A cleavage site₁The length of the SNAP-25 antigen having a carboxy terminus at a residue can be, for example, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, or at most 30 amino acids. In yet another embodiment, P is a bond that is susceptible to cleavage at the BoNT/A cleavage site₁The number of amino acids of the SNAP-25 antigen having a carboxy terminus at a residue can be, for example, between 7 and 12, between 10 and 15, or between 13 and 18.

In another embodiment, P is one that is susceptible to bond cleavage at the BoNT/A cleavage site ₁The SNAP-25 antigen having a carboxy terminus at residue comprises SEQ ID NO: 33. in aspects of this embodiment, P is one that is susceptible to bond cleavage at the BoNT/A cleavage site₁The SNAP-25 antigen having a carboxy terminus at residue comprises SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38 or SEQ ID NO: 39. in another embodiment, P is one that is susceptible to bond cleavage at the BoNT/A cleavage site₁The SNAP-25 antigen having a carboxy terminus at residue comprises SEQ ID NO: 40.

in yet another embodiment, P is a bond that is susceptible to cleavage at the BoNT/A cleavage site₁The SNAP-25 antigen having a carboxy terminus at residue comprises SEQ ID NO: 41. in this embodimentIn aspect, P which is susceptible to bond cleavage at the BoNT/A cleavage site₁The SNAP-25 antigen having a carboxy terminus at residue comprises SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45. SEQ ID NO: 46. in another embodiment, P is one that is susceptible to bond cleavage at the BoNT/A cleavage site₁The SNAP-25 antigen having a carboxy terminus at residue comprises SEQ ID NO: 47.

expected to elicit an immune response, resulting in the generation of P that selectively binds to a bond that is susceptible to cleavage at the BoNT/A cleavage site ₁Any and all SNAP-25 antigens of the alpha-SNAP-25 antibody of SNAP-25 having a carboxyl terminus at the residue can be used as SNAP-25 antigens. Thus, a polypeptide comprising SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 or SEQ ID NO: 46 can be used as SNAP-25 antigen to elicit an immune response that results in the production of P that selectively binds to a readily cleavable bond at the BoNT/A cleavage site₁An alpha-SNAP-25 antibody to SNAP-25 having a carboxyl terminus at residue. Thus, in one embodiment, the SNAP-25 antigen may be substituted by a substitution, deletion or addition of at least 1, at least 2, at least 3, at least 4 or at least 5 amino acids to a protein comprising SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 or SEQ ID NO: 46, or a SNAP-25 antigen. In another embodiment, the SNAP-25 antigen hybridizes to a nucleic acid comprising SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 or SEQ ID NO: 46 can have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

It is contemplated that one or more carriers can be linked to the SNAP-25 antigen in order to enhance the immunogenicity of the non-carrier bound immunogenic, non-immunogenic or weakly immunogenic SNAP-25 antigen. Non-limiting examples include, for example, Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Thyroglobulin (THY), Bovine Serum Albumin (BSA), Soybean Trypsin Inhibitor (STI), or polylinker peptide (MAP). Non-antigenic or weakly antigenic antigens can be made antigenic by coupling the antigen to a carrier, as is well known in the art. Various other carriers and methods for coupling antigens to carriers are well known in the art. See, e.g., Harlow and Lane, supra, 1998 a; harlow and Lane, supra, 1998 b; and David W.Waggener, Jr. et al, oncogenerity-enhancing carriers and compositions of methods of using the same, U.S. patent publication No. 20040057958 (3/25/2004). Epitopes can also be generated by expressing the epitopes as fusion proteins. Methods for expressing polypeptide fusions are well known to those of skill in the art, as described, for example, in the following references: ausubel et al, Current Protocols in Molecular Biology (suppl 47), John Wiley &Sons, New York (1999). Since the carboxyl terminus of the SNAP-25 antigen must be P with a bond that is readily cleavable at the BoNT/A cleavage site₁Residue, the vector must be attached to the amino terminus of the SNAP-25 antigen.

It is contemplated that one or more flexible spacers can be attached to the SNAP-25 antigen in order to enhance the immunogenicity of the immunogenic, non-immunogenic or weakly immunogenic SNAP-25 antigen to which the flexible spacer is not bound. The flexible spacer can increase the overall peptide length of the SNAP-25 antigen and provide flexibility to facilitate proper presentation of the SNAP-25 antigen to immune cells. As a non-limiting example, a SNAP-25 immune response inducing composition can comprise a SNAP-25 antigen linked in tandem with one or more flexible spacers to better present the SNAP-25 antigen to immune cells, thereby facilitating an immune response.

The flexible spacer comprising the peptide is at least one amino acid in length and comprises an uncharged amino acid with a small side chain R group, such as glycine, alanine, valine, leucine or serine. Thus, in one embodiment, the flexible spacer may be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids in length. In another embodiment, the flexible spacer can be, for example, at least 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acids in length. In another embodiment, the number of amino acids of the flexible spacer can be, for example, between 1 and 3, between 2 and 4, between 3 and 5, between 4 and 6, or between 5 and 7. Non-limiting examples of flexible spacers include, for example, G spacers such as GGG, GGGG (SEQ ID NO: 57), and GGGGS (SEQ ID NO: 58); or an A spacer, such as AAA, AAAA (SEQ ID NO: 59), and AAAAV (SEQ ID NO: 60). The flexible spacer is linked in-frame to the SNAP-25 antigen as a fusion protein.

As discussed above, the use of a flexible spacer will in part increase the overall peptide length of the SNAP-25 antigen. For example, a SNAP-25 antigen having 5 to 10 amino acids can have its overall length increased by attaching a flexible spacer having 3 to 5 amino acids to the amino terminus of the SNAP-25 antigen. As another example, a SNAP-25 antigen having 5 to 10 amino acids can have its total length increased by attaching a flexible spacer having 4 to 6 amino acids to the amino terminus of the SNAP-25 antigen. As another example, a SNAP-25 antigen having 5 to 10 amino acids can have its total length increased by attaching a flexible spacer having 7 to 10 amino acids to the amino terminus of the SNAP-25 antigen. As another example, a SNAP-25 antigen having 7 to 12 amino acids can have its total length increased by attaching a flexible spacer having 1 to 3 amino acids to the amino terminus of the SNAP-25 antigen. As another example, a SNAP-25 antigen having 7 to 12 amino acids can have its total length increased by attaching a flexible spacer having 4 to 6 amino acids to the amino terminus of the SNAP-25 antigen. Increasing the length using a flexible spacer will allow selection of a smaller size SNAP-25 antigen, which in turn increases SNAP- 25 antigen elicits P only substantially against bonds that are susceptible to cleavage at BoNT/A₁The possibility of an immune response to SNAP-25 with a carboxyl terminus at the residue, thereby increasing the generation of P that can distinguish bonds susceptible to cleavage at a BoNT/A cleavage site₁SNAP-25 with a carboxyl terminus at the residue and P readily cleavable at the BoNT/A cleavage site₁The possibility of an alpha-SNAP-25 antibody without a carboxy-terminal SNAP-25 at the residue.

It is contemplated that the SNAP-25 immune response inducing compositions disclosed in the present specification may optionally comprise a SNAP-25 antigen disclosed in the present specification, and one or more adjuvants. The term "adjuvant" as used herein when used in conjunction with a SNAP-25 immune response-inducing composition refers to any substance or mixture of substances that increases or diversifies the immune response against the SNAP-25 antigen. Immune response-inducing adjuvants may be used, for example, to reduce the number of immunizations or the amount of antigen required for protective immunity. The use of immune response inducing adjuvants in immune response inducing compositions is well known. The primary purpose of using these adjuvants is to enhance the immune response. Non-limiting adjuvants include, for example, liposomes, oil phases, including, but not limited to, freund's adjuvant, such as Freund's Complete Adjuvant (FCA); freund's Incomplete Adjuvant (FIA); sapogenin glycosides (sapogenin glycosides), e.g. saponins; carbopol (carbopol); N-acetylmuramyl-L-alanyl-D-isoglutamine (commonly known as muramyl dipeptide or "MDP"); and Lipopolysaccharide (LPS). Such adjuvants are generally used in the form of an emulsion with an aqueous phase or, more commonly, they may consist of inorganic salts that are insoluble in water. These inorganic salts may consist of, for example, aluminium hydroxide, zinc sulphate, colloidal iron hydroxide, calcium phosphate or calcium chloride. Aluminum hydroxide (Al (OH) ₃) Is a common adjuvant. Currently, the only adjuvant approved by the FDA for use in humans is aluminum salt (Alum), which can "depot" the antigen by precipitating it. The adjuvants provided above are exemplary only. In fact, any immune response-inducing adjuvant may be used in the immune response-inducing compositions disclosed in the present specification, as long as such adjuvant satisfies the characteristics required to induce an immune response.

The carriers disclosed in this specification may also act as adjuvants. Specific adjuvants and methods for their preparation and use are described, for example, in the following documents: gupta et al, Vaccine, 11: 993-; arnon, R. (ed) Synthetic Vaccines 1: 83-92, CRC Press, Inc., Boca Raton, Fla., 1987; and David W.Waggener, Jr. et al, immunogenity-Enhancing Carriers and Compositions of the Methods of Using the Same, U.S. patent publication No. 20040057958 (3/25/2004). Other adjuvants include any of The compounds described in "Vaccine Design, The Subunit and Adjuvant Approach" (Powell, M.F. and Newman, M.J. ed.), Vol.6, Chapter.7 (p.141-227) published by Plenum Press (New York). Examples in this context include Muramyl Dipeptide (MDP) and Montanide 720. Molecules such as polyinosine: cytosine (Poly I: C) or plasmid DNA containing CpG motifs can also be administered as adjuvants encapsulated in microparticles with the antigen. In another example, the adjuvant is an agent that aids in the entry of the antigenic compound into the cytoplasm of the cell, such as listeriolysin, streptolysin, or a mixture thereof.

Thus, in one embodiment, a SNAP-25 immune response inducing composition comprises a SNAP-25 antigen having a carboxylated carboxy-terminal glutamine linked to a carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxy-terminal glutamine comprises the amino acid sequence of SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38 or SEQ ID NO: 39. in another aspect of this embodiment, the SNAP-25 antigen comprises the amino acid sequence of SEQ ID NO: 40. in aspects of this embodiment, the carrier peptide is Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Thyroglobulin (THY), Bovine Serum Albumin (BSA), Soybean Trypsin Inhibitor (STI), or a multiple-linker peptide (MAP).

In another embodiment, the SNAP-25 immune response inducing composition comprises a SNAP-25 antigen having a carboxylated carboxy-terminal lysine linked to a carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxy-terminal lysine comprises the amino acid sequence of SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 or SEQ ID NO: 46. in another aspect of this embodiment, the SNAP-25 antigen comprises the amino acid sequence of SEQ ID NO: 47. in aspects of this embodiment, the carrier peptide is Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Thyroglobulin (THY), Bovine Serum Albumin (BSA), Soybean Trypsin Inhibitor (STI), or a multiple-linker peptide (MAP).

In another embodiment, a SNAP-25 immune response inducing composition comprises a SNAP-25 antigen having a carboxylated C-terminal glutamine linked to one or more flexible linkers and a carrier peptide, wherein the flexible linkers are interposed between the SNAP-25 antigen and the carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxy-terminal glutamine comprises the amino acid sequence of SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38 or SEQ ID NO: 39. in another embodiment, the SNAP-25 antigen comprises SEQ ID NO: 46. in aspects of this embodiment, the carrier peptide is Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Thyroglobulin (THY), Bovine Serum Albumin (BSA), Soybean Trypsin Inhibitor (STI), or a multiple-linker peptide (MAP). In aspects of this embodiment, the flexible linker is a G-spacer or an A-spacer.

In yet another embodiment, a SNAP-25 immune response inducing composition comprises a SNAP-25 antigen having a carboxylated C-terminal lysine linked to a flexible linker and a carrier peptide, wherein the flexible linker is interposed between the SNAP-25 antigen and the carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxy-terminal lysine comprises the amino acid sequence of SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 or SEQ ID NO: 46. in another aspect of this embodiment, the SNAP-25 antigen comprises the amino acid sequence of SEQ ID NO: 47. in aspects of this embodiment, the carrier peptide is Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Thyroglobulin (THY), Bovine Serum Albumin (BSA), Soybean Trypsin Inhibitor (STI), or a multiple-linker peptide (MAP). In aspects of this embodiment, the flexible linker is a G-spacer or an A-spacer.

Aspects of the disclosure include, in part, P for generating a bond that selectively binds readily cleavable at the BoNT/A cleavage site₁Methods for producing alpha-SNAP-25 antibodies to SNAP-25 having a carboxyl terminus at the residue. A variety of methods well known in the art can be used to generate P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An alpha-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at residue. Specific protocols for making and using antibodies and detecting and measuring antibody binding specificity, binding affinity, and binding avidity are known in the art. See, e.g., ANTIBODIES: a LABORATORY MANUAL (Edward Harlow)&David Lane, eds., Cold Spring Harbor Laboratory Press, 2 nd edition, 1998 a); and the USING ANTIBODIES: a laboradry MANUAL: PORTABLE PROTOCOL No. I (Edward Harlow)&David Lane, Cold Spring Harbor Laboratory Press, 1998 b); molecular Cloning, A Laboratory Manual, 2001; and Current Protocols in Molecular Biology, 2004; david Anderson et al, Therapeutic Polypeptides, Nucleic Acids Encoding Same, and Methods of Use, U.S. Pat. No. 7,034,132 (25/4/2005); and Beatriz m. carreno et al, Antibodies Against CTLA4, U.S. patent 7,034,121 (4/25/2006).

In a non-limiting example, P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site can be generated by injecting an animal (e.g., a rabbit, goat, mouse, or other mammal) with one or more injections of an immune response-inducing composition disclosed herein₁An alpha-SNAP-25 polyclonal antibody to SNAP-25 having a carboxyl terminus at residue. In another non-limiting example, P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site can be generated by injecting an egg (e.g., hen egg) with one or more injections of an immune response-inducing composition disclosed herein₁An alpha-SNAP-25 polyclonal antibody to SNAP-25 having a carboxyl terminus at residue. Available standardTechniques such as enzyme-linked immunosorbent assays (ELISAs) or cell-based activity assays using immobilized antigens monitor antibody titers in immunized animals over time. If desired, P, which selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site, can be isolated from the mammalian body (e.g., blood)₁Polyclonal antibodies to the a-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at residues and further purified using well-known techniques (e.g., protein a affinity chromatography for obtaining IgG fractions, or affinity purification of peptides used to produce the antibody).

In another non-limiting example, a hybridoma method can be used to generate a P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl terminus at residue. See, e.g., chapter 6, Monoclonal Antibodies, pages 196-244, Harlow and Lane, supra, 1998 a; and Growing hybrids, pp 245-282, Harlow and Lane, Chapter 7, supra, 1998 a; and Goding, pages 59-103, Monoclonal Antibodies: principles and Practice, Academic Press, (1986). In this method, a host animal (e.g., a mouse, hamster, or another suitable host animal) is typically exposed to one or more injections of the SNAP-25 antigen disclosed herein to elicit lymphocytes that produce or are capable of producing alpha-SNAP-25 antibodies that will specifically bind to P readily cleavable bonds at the BoNT/A cleavage site₁SNAP-25 having a carboxyl terminus at the residue. Antibody titers in immunized animals can be monitored over time using standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized antigen or cell-based activity assays. Alternatively, lymphocytes can be immunized in vitro using a suitable cell culture line. At a suitable time after immunization, for example when the antibody titer has reached a maximum, antibody-producing cells are isolated from the animal. In general, if cells of human origin are desired, peripheral blood lymphocytes are used; whereas spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. Separated out using a suitable fluxing agent, e.g. polyethylene glycol The antibody-producing cells are fused with an immortal cell line to form hybridoma cells. Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine and human origin. Typically, murine myeloma cell lines are fused with spleen cells harvested from suitably immunized mice to produce hybridomas. Preferably, the immortal cell line is a mouse myeloma cell line sensitive to a medium containing hypoxanthine (hypoxanthine), aminopterin (aminopterin) and thymidine (thymine) (HAT). Any of a variety of myeloma cell lines may be used as fusion partners according to standard techniques, for example P3-NS1/1-Ag4-1, P3-x63-Ag8.653, or Sp2/O-Ag14 myeloma cell lines. The hybridoma cells obtained by fusion are then selected using HAT medium, which kills unfused and inefficiently fused myeloma cells (unfused splenocytes die after several days of incubation because they have not been transformed). The culture medium in which the hybridoma cells are grown can then be assayed for P selectively bound to bonds that are susceptible to cleavage at the BoNT/A cleavage site₁The presence of an alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl terminus at residue. For example, α -SNAP-25 positive media can be used to screen hybridoma supernatants in immunoprecipitation assays, in vitro binding assays (e.g., Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA)), or cell-based activity assays. Such techniques and assays are known in the art. See, e.g., chapter 11, immunopriciption, pages 421 and 470, Harlow and Lane, supra, 1998 a; chapter 12, immunolblotting, page 471-; chapter 14, Immunoassays, pages 553-612, Harlow and Lane, supra, 1998 a. Additional studies can then be performed to determine whether such antibodies also cleave P at the BoNT/A cleavage site ₁SNAP-25, which has no carboxyl terminus at the residue, is not reactive. The binding affinity of the α -SNAP-25 monoclonal antibody can also be determined, for example, by Scatchard analysis (Scatchard analysis). See, e.g., Peter j. munson and David Rodbard, Ligand: a Versatile computer formulated Approach For Characterization of Ligand-Binding Systems, 107(1) anal. biochem.220-239 (1980). In authenticationAfter the desired hybridoma cells are obtained, clones derived from the individual cells are isolated using a limiting dilution procedure until clonal cell lines expressing the desired monoclonal antibody are obtained. Selection of P for bond susceptible to cleavage at BoNT/A cleavage site₁Those antibodies with SNAP-25 having a carboxy terminus at the residue are sufficiently selective and have sufficiently high binding affinity to be further characterized and studied.

P for preparing a bond that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site₁Another alternative method for alpha-SNAP-25 monoclonal antibodies to SNAP-25 having a carboxyl terminus at residue is to screen a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the SNAP-25 peptide and isolate P in the immunoglobulin library bound to a readily cleavable bond at the BoNT/A cleavage site ₁A member of SNAP-25 having a carboxyl terminus at a residue. Kits for generating and screening Phage display libraries are commercially available, for example, the Recombinant Phage Antibody System (Amersham GE Healthcare, Piscataway, N.J.); and surfZAP^TMPhage display kit (Stratagene, La Jolla, CA). In addition, examples of methods and reagents suitable for generating and screening antibody display libraries can be found, for example, in Ladner et al, U.S. Pat. Nos. 5,223,409; borebaeck et al, U.S. Pat. No. 5,712,089; griffiths et al, U.S. Pat. No. 5,885,793; griffiths et al, U.S. Pat. No. 5,962,255; McCafferty et al, U.S. Pat. No. 5,969,108; griffiths et al, U.S. Pat. No. 6,010,884; jespers et al, U.S. Pat. No. 6,017,732; borebaeck et al, U.S. Pat. No. 6,027,930; johnson et al, U.S. Pat. No. 6,140,471; McCafferty et al, U.S. Pat. No. 6,172,197, the entire contents of each of which are hereby incorporated by reference.

Aspects of the disclosure include, in part, collecting a sample containing an alpha-SNAP-25 antibody or cells that produce an alpha-SNAP-25 antibody. As used herein, the term "sample containing an α -SNAP-25 antibody or cells that produce an α -SNAP-25 antibody" refers to a sample that contains or may contain at least one P that selectively binds to a cleavable bond that is susceptible to BoNT/A cleavage site ₁Any of the alpha-SNAP-25 antibodies having a carboxy-terminal SNAP-25 epitope at residueA biological substance. It is contemplated that P, which may contain a bond that is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site, may be used in the present method₁Any and all samples of alpha-SNAP-25 antibodies having a carboxy-terminal SNAP-25 epitope at residue, including, but not limited to, blood, plasma, serum, and lymph fluid. It is also contemplated that P can be used in the present method to produce a bond that selectively binds to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁Any cell of the a-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at residue, including, but not limited to, CD8 cells, CTL cells, helper T cells, and B cells. A variety of well known methods can be used to collect samples containing α -SNAP-25 antibodies or cells producing α -SNAP-25 antibodies from an individual, see, e.g., Harlow and Lane, supra, 1998 a; and Harlow and Lane, supra, 1998 b. Similarly, the sample can also be treated using a variety of well-known methods to isolate P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An alpha-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at residue. The procedure for collecting the sample may be selected according to the type of antibody to be isolated. In a non-limiting example, P is selectively bound to a bond susceptible to cleavage at a BoNT/A cleavage site when isolated ₁When the alpha-SNAP-25 polyclonal antibody to SNAP-25 having a carboxyl terminus at residue is used, a suitable sample may be a blood sample containing such alpha-SNAP-25 antibody, while P, which selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site, is isolated₁When the α -SNAP-25 monoclonal antibody to SNAP-25 has a carboxyl terminus at residue, a suitable sample may be cells that produce the α -SNAP-25 antibody, such as spleen cells or hybridomas.

Aspects of the disclosure include, in part, isolating from a sample a P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An alpha-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at residue. Methods for isolating such alpha-SNAP-25 antibodies, such as P, that selectively bind a bond that is readily cleavable at the BoNT/A cleavage site are well known to those skilled in the art₁alpha-SNAP-25 polyclonal antibodies to SNAP-25 having a carboxy terminus at residue or P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site₁An alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl terminus at residue. See, e.g., Harlow and Lane, supra, 1998 a; and Harlow and Lane, supra, 1998 b. For example, such a-SNAP-25 polyclonal antibodies can be isolated from a sample using well-known techniques, such as affinity chromatography using protein a or protein G, which method provides primarily the IgG fraction of the immune serum. Subsequently or alternatively, specific SNAP-25 antigens can be immobilized on a column or magnetic beads for purification of P selectively bound to a bond susceptible to cleavage at a BoNT/A cleavage site by immunoaffinity layer methods ₁An alpha-SNAP-25 polyclonal antibody to SNAP-25 having a carboxyl terminus at residue. P, which is selectively bound to a cleavable bond at the BoNT/A cleavage site, can be purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose (Sepharose) method, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography₁The alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl terminus at the residue is isolated from the culture medium or ascites fluid.

Thus, in one embodiment, P is used to generate a bond that selectively binds to a readily cleavable bond at the BoNT/A cleavage site₁A method of producing an α -SNAP-25 antibody to SNAP-25 having a carboxy terminus at a residue comprising the steps of: (a) administering to the animal a SNAP-25 immune response inducing composition comprising a SNAP-25 antigen having a carboxylated C-terminal glutamine linked to a carrier peptide; (b) collecting a sample from the animal containing the α -SNAP-25 antibody or cells producing the α -SNAP-25 antibody; and (c) isolating the alpha-SNAP-25 antibody fraction from the sample. In one aspect of this embodiment, P, which selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The alpha-SNAP-25 antibody to SNAP-25 with a carboxyl terminus at the residue is a polyclonal antibody. In another aspect of this embodiment, P, which is selectively attached to a bond susceptible to cleavage at the BoNT/A cleavage site ₁The alpha-SNAP-25 antibody to SNAP-25 having a carboxyl terminus at residue is a monoclonal antibody. In yet another aspect of this embodiment, P is produced which selectively binds to a bond susceptible to cleavage at a BoNT/A cleavage site₁The alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl terminus at residue is an IgG subtype. In this embodimentIn other aspects of (a), the SNAP-25 immune response inducing composition further comprises an adjuvant, such as polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), or polyvinyl alcohol (PVA).

In another embodiment, P is used to generate a bond that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A method of producing an α -SNAP-25 antibody to SNAP-25 having a carboxy terminus at a residue comprising the steps of: (a) administering to the animal a SNAP-25 immune response inducing composition comprising a SNAP-25 peptide having a carboxylated C-terminal glutamine linked to a flexible linker and a carrier peptide, wherein the flexible linker is interposed between the SNAP-25 peptide and the carrier peptide; (b) collecting a sample from the animal containing the α -SNAP-25 antibody or cells producing the α -SNAP-25 antibody; and (c) isolating the alpha-SNAP-25 antibody from the sample. In one aspect of this embodiment, P, which selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site ₁The alpha-SNAP-25 antibody to SNAP-25 with a carboxyl terminus at residue is a polyclonal antibody. In another aspect of this embodiment, P, which is selectively attached to a bond susceptible to cleavage at the BoNT/A cleavage site₁The alpha-SNAP-25 antibody to SNAP-25 having a carboxyl terminus at residue is a monoclonal antibody. In yet another aspect of this embodiment, P is produced which selectively binds to a bond susceptible to cleavage at a BoNT/A cleavage site₁The alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl terminus at residue is an IgG subtype. In other aspects of this embodiment, the SNAP-25 immune response inducing composition further comprises an adjuvant, such as polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), or polyvinyl alcohol (PVA).

Aspects of the disclosed invention include, in part, isolated alpha-SNAP-25 antibodies that selectively bind to P at a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 epitope having a carboxyl terminus at the residue. The term "antibody" as used herein refers to a molecule produced by the immune system in response to a particular antigen that specifically binds to that antigen and includes both natural and non-natural antibodies. The term "isolated" as used herein refers to the removal of a molecule from its natural environment using human intervention. For example, the antibody can be a polyclonal antibody Monoclonal antibodies, dimers, multimers, multispecific antibodies, humanized antibodies, chimeric antibodies, bifunctional antibodies, cell-associated antibodies (e.g., Ig receptors), linear antibodies, minibifunctional antibodies (diabodies), or minibodies, as long as the fragment exhibits the desired biological activity, and also single chain derivatives of such antibodies. The antibody may be a V-containing antibody_HField and V_LDomains and light chain constant domains (C)_L) And heavy chain constant domain C_H1、C_H2And C_H3The full-length immunoglobulin molecule of (a), or an immunologically active fragment of the full-length immunoglobulin molecule, e.g., a Fab fragment, F (ab')₂Fragments, Fc fragments, Fd fragments, Fv fragments. The antibody can be derived from any vertebrate species (e.g., human, goat, horse, donkey, murine, rat, rabbit, or chicken) and can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgA, IgD, IgE, IgG, and IgM), or subclass (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). For a general disclosure of The structure of natural Antibodies, non-natural Antibodies and antigenic compound-binding fragments thereof, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol.113, edited by Rosenburg and Moore, Springer-Verlag, New York, p.269-315 (1994); borrabeck, Antibody Engineering, 2 nd edition (Oxford University Press 1995), the entire contents of each of which are hereby incorporated by reference.

Natural antibodies are often heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at one end_H) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at one end_L) And has a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. It is known that specific amino acid residues form the light chainThe interface between the variable domain and the heavy chain variable domain.

The complete antigen recognition and antigen binding site is contained within the antibody variable domain, i.e., the Fv fragment. The fragment consists of a heavy chain variable domain (V)_H) With a light chain variable domain (V)_L) A dimer formed by close, non-covalent association. Each domain comprises four Framework Regions (FRs) connected by three hypervariable regions, predominantly in the β -sheet configuration, which form loops connecting, and in some cases forming part of, the β -sheet structure. Each hypervariable region comprises an amino acid sequence corresponding to a Complementary Determining Region (CDR). In general, the three-dimensional configuration of the 6 CDR regions defines V _H-V_LAntigen binding sites on the surface of the dimer, thereby conferring antigen binding specificity. See, e.g., Cyrus Chothia, et al, formulations of immunoglobin Hypervariable Regions, Nature 342 (6252): 877-883 (1989); elvin A.Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991), the entire contents of each of which are incorporated herein by reference. The constant domains of antibodies are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, e.g., the participation of the antibody in antibody-dependent cellular cytotoxicity processes.

The target antigen typically has one or more binding sites, also called epitopes, that are recognized by the antigen binding site formed by the CDRs. As used herein, "epitope" is synonymous with "antigenic determinant" and means a site on a target antigen, such as a peptide, polysaccharide or lipid-containing molecule, that is capable of specifically binding an immunoglobulin or T cell receptor, or otherwise interacting with the molecule. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.

Polyclonal antibodies refer to heterogeneous populations of antibody molecules containing at least two antibodies capable of binding a particular antigen. By definition, polyclonal antibodies include two different antibodies that bind at least two different epitopes. The term "monoclonal antibody" as used herein refers to a substantially homogeneous population of antibody molecules containing only one antibody capable of binding a particular antigen, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. By definition, monoclonal antibodies bind to a single epitope. Monoclonal antibodies are highly specific for a single antigenic site. In addition, each monoclonal antibody is directed against a single determinant on the antigen, as compared to polyclonal antibodies. In addition to specificity, monoclonal antibodies are also advantageous in that their synthesis is not contaminated by other antibodies. The modifier "monoclonal" indicates the nature of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use according to the present disclosure may be prepared by the hybridoma method (first described by Kohler et al (1975) in Nature 256: 495); or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,807,715). For example, Clackson et al (1991) Nature, 352: 624-: 581-597 to isolate monoclonal antibodies from phage antibody libraries.

Thus, in one embodiment, the α -SNAP-25 antibody comprises a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) That selectively bind to P that is susceptible to bond cleavage at the BoNT/A cleavage site₁SNAP-25 having a carboxyl terminus at the residue. In one aspect of this embodiment, the heavy chain variable domain (V)_H) Is SEQ ID NO: 72. SEQ ID NO: 74. SEQ ID NO: 76. SEQ ID NO: 80. SEQ ID NO: 82 or SEQ ID NO: 133. in another aspect of this embodiment, the light chain variable domain (V)_L) Is SEQ ID NO: 84. SEQ ID NO: 86. SEQ ID NO: 88. SEQ ID NO: 90 or SEQ ID NO: 92.

in another embodiment, the nucleic acid sequence encodes a polypeptide comprising a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) And selectively binds to P that is susceptible to bond cleavage at the BoNT/A cleavage site₁Having a carboxyl terminus at the residueAn alpha-SNAP-25 antibody to SNAP-25. In one aspect of this embodiment, the heavy chain variable domain (V)_H) Consisting of the nucleic acid sequence SEQ ID NO: 71. SEQ ID NO: 73. SEQ ID NO: 75. SEQ ID NO: 77. SEQ ID NO: 79. SEQ ID NO: 81 or SEQ ID NO: 132. In another aspect of this embodiment, the heavy chain variable domain (V)_H) Consists of a nucleotide sequence identical to SEQ ID NO: 71. SEQ ID NO: 73. SEQ ID NO: 75. SEQ ID NO: 77. SEQ ID NO: 79. SEQ ID NO: 81 or SEQ ID NO: 132 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. In another aspect of this embodiment, the light chain variable domain (V) _L) Consisting of SEQ ID NO: 83. SEQ ID NO: 85. SEQ ID NO: 87. SEQ ID NO: 89 or SEQ ID NO: and 91, coding. In yet another aspect of this embodiment, the light chain variable domain (V)_L) Consists of a nucleotide sequence identical to SEQ ID NO: 83. SEQ ID NO: 85. SEQ ID NO: 87. SEQ ID NO: 89 or SEQ ID NO: 91, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In another embodiment, the α -SNAP-25 antibody comprises a heavy chain variable domain (V)_H) The CDR1 region, the CDR2 region, the CDR3 region, or any combination thereof, which antibody selectively binds to P that is susceptible to a split bond at the BoNT/A cleavage site₁SNAP-25 having a carboxyl terminus at the residue. In one aspect of this embodiment, the heavy chain variable domain (V)_H) The CDR1 region of SEQ ID NO: 93. SEQ ID NO: 94. SEQ ID NO: 95. SEQ ID NO: 118. SEQ ID NO: 119 or SEQ ID NO: 120. in another aspect of this embodiment, the heavy chain variable domain (V)_H) The CDR2 region of SEQ ID NO: 96. SEQ ID NO: 97. SEQ ID NO: 98. SEQ ID NO: 99. SEQ ID NO: 121. SEQ ID NO: 122 or SEQ ID NO: 123. in yet another aspect of this embodiment, the heavy chain variable domain (V) _H) The CDR3 region of SEQ ID NO: 100. SEQ ID NO: 101. SEQ ID NO: 102. SEQ ID NO: 124. SEQ ID NO: 134 or SEQ ID NO: 135。

In another embodiment, the α -SNAP-25 antibody comprises a light chain variable domain (V)_L) The CDR1 region, the CDR2 region, the CDR3 region, or any combination thereof, which antibody selectively binds to P that is susceptible to a split bond at the BoNT/A cleavage site₁SNAP-25 having a carboxyl terminus at the residue. In one aspect of this embodiment, the light chain variable domain (V)_L) The CDR1 region of SEQ ID NO: 103. SEQ ID NO: 104. SEQ ID NO: 105. SEQ ID NO: 106. SEQ ID NO: 107. SEQ ID NO: 125. SEQ ID NO: 126. SEQ ID NO: 127. SEQ ID NO: 128 or SEQ ID NO: 129. in another aspect of this embodiment, the light chain variable domain (V)_L) The CDR2 region of SEQ ID NO: 108. SEQ ID NO: 109. SEQ ID NO: 110. SEQ ID NO: 111 or SEQ ID NO: 112. in yet another aspect of this embodiment, the light chain variable domain (V)_L) The CDR3 region of SEQ ID NO: 113. SEQ ID NO: 114. SEQ ID NO: 115. SEQ ID NO: 116 or SEQ ID NO: 117.

in another embodiment, the α -SNAP-25 antibody specifically binds to an epitope comprising P that is a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁SNAP-25 having a carboxyl terminus at the residue. In one aspect of this embodiment, the epitope comprises SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36 or SEQ ID NO: 37. in one aspect of this embodiment, the epitope comprises SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43 or SEQ ID NO: 44.

as discussed above, the sequence present around the BoNT/A cleavage site in SNAP-25 is denoted P₅-P₄-P₃-P₂-P₁-P₁’-P₂’-P₃’-P₄’-P₅', wherein P₁-P₁' represents a frangible bond. After BoNT/A cleavage, the cleavage product thus produced comprises P₅-P₄-P₃-P₂-P₁Fragments of the sequence and the inclusion of P₁’-P₂’-P₃’-P₄’-P₅' of (a). The term "P selectively bound to a bond susceptible to cleavage at BoNT/A as used herein₁An alpha-SNAP-25 antibody to SNAP-25 having a carboxy terminus at residue "refers to an antibody that selectively binds to a polypeptide comprising P₅-P₄-P₃-P₂-P₁Any SNAP-25 cleavage product fragment of sequence, but not binding to P-containing₁’-P₂’-P₃’-P₄’-P₅' any SNAP-25 cleavage product fragment of sequence or P with an intact BoNT/A cleavage site₁-P₁' any SNAP-25 antibody that easily cleaves bonds. The term "alpha-SNAP-25" as used herein₁₉₇Antibody "refers to the selective binding of the carboxy terminus P ₁Residues correspond to SEQ ID NO: 5, glutamine 197 in the presence of an antibody to SNAP-25. The term "alpha-SNAP-25" as used herein₂₀₄Antibody "refers to the selective binding of the carboxy terminus P₁Residues correspond to SEQ ID NO: 16, lysine 204 in lysine 204.

The term "selectively" as used herein means having a unique effect or influence, or reacting in only one way or with only one substance. As used herein, the term "selectively binds" when used with respect to an antibody means that the antibody binds differentially to the designated target epitope, such that the antibody does not substantially cross-react with non-target epitopes. A peptide epitope as defined herein has a minimum dimension of about 5 amino acids, and typically comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids. A peptide epitope may be non-contiguous, i.e., it comprises amino acid residues that are not contiguous in the primary structure of the peptide, but are linked together into an epitope by virtue of the secondary, tertiary, or quaternary structure of the peptide. Furthermore, it is also noted that an epitope may comprise a portion of a molecule other than an amino acid sequence, for example a carbohydrate moiety, a lipid moiety such as a lipoprotein or glycolipid, or a chemically modified amino acid moiety such as a phosphorylated amino acid. In aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site ₁Having a carboxyl terminus at residueThe alpha-SNAP-25 antibody of SNAP-25 epitope of (a) can selectively bind to P at a bond that is easily cleaved at a BoNT/A cleavage site₁A SNAP-25 epitope having a carboxy terminus at a residue, the epitope comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids. In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁alpha-SNAP-25 antibodies having a carboxy-terminal SNAP-25 epitope at residue can selectively bind to P at a bond that is readily cleavable at the BoNT/A cleavage site₁A SNAP-25 epitope having a carboxy terminus at a residue, the epitope comprising at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, or at most 20 amino acids.

Selective binding includes binding properties such as binding affinity, binding specificity and binding affinity. See David J. King, Applications and Engineering of Monoclonal Antibodies, page 240 (1998). Binding affinity refers to the length of time an antibody remains at its epitope binding site and can be viewed as the strength with which the antibody binds to its epitope. Binding affinity can be described in terms of the equilibrium constant (KD) of the antibody, which is defined as the ratio of KD/Ka at equilibrium. Where Ka is the association rate constant of the antibody and kd is the dissociation rate constant of the antibody. Binding affinity is determined by association and dissociation together, and high affinity cannot be ensured with either high association or low dissociation alone. The association rate constant (Ka), or the binding rate constant (Kon), is a measure of the number of binding events per unit time, or the propensity of an antibody to reversibly associate with an antigen to form its antibody-antigen complex. The association rate constant is in M ^-1s^-1Expressed in units and symbolically as follows: [ Ab ]]×[Ag]XKon. The greater the association rate constant, the faster the antibody binds to its antigen, or the higher the binding affinity between the antibody and the antigen. Dissociation rate constant (Kd) or dissociation rate constant (Koff) is the number of dissociation events per unit time, or antibody-antigen complexA measure of the tendency of a molecule to reversibly separate (dissociate) into its component molecules (i.e., antibody and antigen). The dissociation rate constant is in s^-1Expressed in units and symbolically as follows: [ Ab + Ag ]]Xkoff. The smaller the dissociation rate constant, the tighter the binding of the antibody to its antigen, or the higher the binding affinity between the antibody and the antigen. The equilibrium dissociation constant (KD) is a measure of the rate at which new antibody-antigen complexes are formed at equilibrium equal to the rate at which the antibody-antigen complexes dissociate. The equilibrium dissociation constant is expressed in units of M and is defined as Koff/Kon ═ Ab]×[Ag]/[Ab+Ag]Wherein [ Ab]Is the molar concentration of antibody, [ Ag ]]Is the molar concentration of the antigen, and [ Ab + Ag]Is the molar concentration of the antibody-antigen complex, where all concentrations are those of these components when the system reaches equilibrium. The smaller the equilibrium dissociation constant, the more tightly the antibody binds to its antigen, or the higher the binding affinity between the antibody and the antigen.

Thus, in one embodiment, P binds selectively to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope at a residue that is carboxy-terminal can have, for example, less than 1X 10⁵M^-1s^-1Less than 1X 10⁶M^-1s^-1Less than 1X 10⁷M^-1s^-1Or less than 1X 10⁸M^-1s^-1The association rate constant of (2). In another embodiment, P selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope at a residue that is carboxy-terminal can have, for example, greater than 1X 10⁵M^-1s^-1Greater than 1X 10⁶M^-1s^-1Greater than 1X 10⁷M^-1s^-1Or greater than 1X 10⁸M^-1s^-1The association rate constant of (2). In other aspects, P selectively binds to bonds that are susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope with a carboxyl terminus at residue can have a binding affinity of between 1X 10⁵M^-1s^-1To 1X 10⁸M^-1s^-1、1×10⁶M^-1s^-1To 1X 10⁸M^-1s^-1、1×10⁵M^-1s^-1To 1X 10⁷M^-1s^-1Or 1X 10⁶M^-1s^-1To 1X 10⁷M^-1s^-1The association rate constant therebetween.

In another embodiment, P selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope with a carboxyl terminus at residue can have less than 1X 10^-3s^-1Less than 1X 10^-4s^-1Or less than 1X 10^-5s^-1The dissociation rate constant of (2). In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site ₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope with a carboxyl terminus at residue can have, for example, less than 1.0x 10^-4s^-1Less than 2.0x 10^-4s^-1Less than 3.0x 10^-4s^-1Less than 4.0x 10^-4s^-1Less than 5.0x10^-4s^-1Less than 6.0x 10^-4s^-1Less than 7.0x 10^-4s^-1Less than 8.0x 10^-4s^-1Or less than 9.0x 10^-4s^-1The dissociation rate constant of (2). In another embodiment, P selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope at a residue that is carboxy-terminal can have, for example, greater than 1X 10^-3s^-1Greater than 1X 10^-4s^-1Or greater than 1X 10^-5s^-1The dissociation rate constant of (2). In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope with a carboxyl terminus at residue can have, for example, greater than 1.0x 10^-4s^-1Greater than 2.0x 10^-4s^-1Greater than 3.0x 10^-4s^-1Greater than 4.0x 10^-4s^-1Greater than 5.0x10^-4s^-1Greater than 6.0x 10^-4s^-1Greater than 7.0x 10^-4s^-1Greater than 8.0x 10^-4s^-1Or greater than 9.0x 10^-4s^-1The dissociation rate constant of (2).

In another embodiment, P selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an a-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue can have an equilibrium dissociation constant of less than 0.500 nM. In aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site ₁The binding affinity of an α -SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue can have an equilibrium dissociation constant of, e.g., less than 0.500nM, less than 0.450nM, less than 0.400nM, less than 0.350nM, less than 0.300nM, less than 0.250nM, less than 0.200nM, less than 0.150nM, less than 0.100nM, or less than 0.050 nM. In another embodiment, P selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an α -SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue can have an equilibrium dissociation constant of greater than 0.500 nM. In aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an α -SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue can have an equilibrium dissociation constant of, e.g., greater than 0.500nM, greater than 0.450nM, greater than 0.400nM, greater than 0.350nM, greater than 0.300nM, greater than 0.250nM, greater than 0.200nM, greater than 0.150nM, greater than 0.100nM, or greater than 0.050 nM.

In another embodiment, P selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope at a residue that is carboxy-terminal can have, for example, less than 1X 10 ⁰M^-1s^-1Less than 1X 10¹M^-1s^-1Less than 1X 10²M^-1s^-1Less than 1X 10³M^-1s^-1Or less than 1X 10⁴M^-1s^-1The association rate constant for the complete SNAP-25. In another embodiment, P selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding affinity of an alpha-SNAP-25 antibody having a SNAP-25 epitope at the residue with a carboxyl terminus can have, for example, a maximum of 1X 10⁰M^-1s^-1At most 1X 10¹M^-1s^-1At most 1X 10²M^-1s^-1At most 1X 10³M^-1s^-1Or at most 1X 10⁴M^-1s^-1The association rate constant for the complete SNAP-25.

Binding specificity is the ability of an antibody to distinguish between molecules containing its epitope and molecules not containing the epitope. One way to measure binding specificity is to compare the Kon association rate of an antibody to a molecule containing its epitope with the Kon association rate of an antibody to a molecule not containing the epitope. For example, comparison of the P-SNAP-25 antibody for bonds susceptible to cleavage at the BoNT/A cleavage site₁Association rate constant (Ka) of SNAP-25 epitope having carboxyl terminus at residue with P-25 that is susceptible to bond cleavage at BoNT/a cleavage site for SNAP-25 not comprising the epitope₁SNAP-25 epitope without carboxyl terminus at residue or intact P with BoNT/A cleavage site₁-P₁'SNAP-25 epitope of a cleavable bond)'. In aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site ₁The a-SNAP-25 antibody having a SNAP-25 epitope at the carboxyl terminus at the residue has an association rate constant (Ka) of, for example, less than 1X 10 to SNAP-25 not containing the epitope thereof⁰M^-1s^-1Less than 1X 10¹M^-1s^-1Less than 1X 10²M^-1s^-1Less than 1X 10³M^-1s^-1Or less than 1X 10⁴M^-1s^-1. In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁The alpha-SNAP-25 antibody having a SNAP-25 epitope at the residue having a carboxyl terminus has an association rate constant (Ka) of, for example, at most 1X 10 for SNAP-25 not including its epitope⁰M^-1s^-1At most 1X 10¹M^-1s^-1At most 1X 10²M^-1s^-1At most 1X 10³M^-1s^-1Or at most 1X 10⁴M^-1s^-1。

In another aspect of this embodiment, P is selectively bound to a bond susceptible to cleavage at the BoNT/A cleavage site₁An α -SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue has an association rate constant (Ka) for its epitope that is, for example, at least 2-fold or more, at least 3-fold or more, at least 4-fold or more, at least 5-fold or more, at least 6-fold or more, at least 7-fold or more, at least 8-fold or more, or at least 9-fold or more, greater than the Ka for SNAP-25 not including the epitope. In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁An α -SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue has an association rate constant (Ka) for its epitope that is, e.g., at least 10-fold more, at least 100-fold more, at least 1,000-fold more, or at least 10,000-fold more than the Ka for SNAP-25 that does not contain the epitope. In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site ₁An α -SNAP-25 antibody having a SNAP-25 epitope at the carboxyl terminal at the residue has an association rate constant (Ka) for its epitope of, for example, at most 1 time or more, at most 2 times or more, at most 3 times or more, at most 4 times or more, at most 5 times or more, at most 6 times or more, at most 7 times or more, at most 8 times or more, or at most 9 times or more of the Ka for SNAP-25 not including the epitope. In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁An α -SNAP-25 antibody having a SNAP-25 epitope at the residue with a carboxyl terminus has an association rate constant (Ka) for its epitope of, for example, at most 10 times or more, at most 100 times or more, at most 1,000 times or more, or at most 10,000 times or more, as compared to the Ka for SNAP-25 not including the epitope.

P binding selectively to a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding specificity of an α -SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue can also be characterized in a ratiometric fashion, such that such α -SNAP-25 antibodies can distinguish their SNAP-25 epitope from SNAP-25 that does not contain it (e.g., P that is susceptible to cleavage at a BoNT/A cleavage site₁SNAP-25 epitope without carboxyl terminus at residue or with BoNT/A cleavage siteIs complete P₁-P₁' SNAP-25 epitope of a cleavable bond). In aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site ₁An α -SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at a residue has a binding specificity ratio for its SNAP-25 epitope to SNAP-25 not including the epitope of, e.g., at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 64: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1, or at least 40: 1. In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁alpha-SNAP-25 antibodies having a carboxy-terminal SNAP-25 epitope at residue(s) for its SNAP-25 epitope and for P that is susceptible to cleavage at the BoNT/A cleavage site₁The binding specificity ratio of SNAP-25 without the carboxyl terminus at the residue is, e.g., at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1, or at least 40: 1. In other aspects of this embodiment, P is selectively bound to a bond that is susceptible to cleavage at the BoNT/A cleavage site₁alpha-SNAP-25 antibodies having a carboxy-terminal SNAP-25 epitope at residue to its SNAP-25 epitope and to the intact P with a BoNT/A cleavage site ₁-P₁The binding specificity ratio of SNAP-25 for a ` readily cleavable bond ` is, for example, at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 64: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1 or at least 40: 1.

Binding avidity, also called functional affinity, refers to the sum of the functional binding strengths between a multivalent antibody and its antigen. An antibody molecule can have more than one binding site (e.g., 2 for IgG and 10 for IgM), and many antigens contain more than one antigenic site. Although the binding affinity of an antibody depends on the binding affinity of the individual antibody binding sites, it must be completely dissociated at the antibody due to all antibody-antigen interactionsAnd at the same time, disrupted, so that the binding affinity is greater than the binding affinity. P expected to bind selectively to bonds susceptible to cleavage at the BoNT/A cleavage site₁An α -SNAP-25 antibody having a SNAP-25 epitope at the residue that is carboxy-terminal can selectively bind to any and all epitopes of the antibody.

Thus, in one embodiment, the α -SNAP-25 antibody is a P that selectively binds to a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁An alpha-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at residue. In aspects of this embodiment, the α -SNAP-25 antibody is an α -SNAP-25 antibody that selectively binds to an epitope of SNAP-25 having a carboxy-terminal glutamine or an α -SNAP-25 antibody that selectively binds to an epitope of SNAP-25 having a carboxy-terminal lysine. In other aspects of this embodiment, the α -SNAP-25 antibody is a heavy chain antibody that selectively binds to a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 5 Glutamine 197 carboxyl terminal P₁An α -SNAP-25 antibody that selectively binds to an epitope of SNAP-25 having residues corresponding to SEQ ID NO: 16 lysine 204 carboxy terminal P₁An alpha-SNAP-25 antibody to the SNAP-25 epitope of residues. In other aspects of this embodiment, the α -SNAP-25 antibody is a heavy chain antibody that selectively binds to a polypeptide having the amino acid sequence of SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 or SEQ ID NO: 46 at the carboxy terminal end of the amino acid sequence of SNAP-25.

Aspects of the disclosure include, in part, an immune-based method for detecting retargeted endopeptidase activity. The immune-based methods disclosed in the present specification can be assessed by several parameters including, for example, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), range, specificity, selectivity, linearity, reproducibility, and system adaptability. The accuracy of a method is a measure of the correctness of the analytical method or the consistent closeness between the measured value and the value being a recognized conventional true or recognized reference value. The accuracy of the method is the degree of agreement between individual test results when the procedure is repeated for multiple sampled specimens of the same sample. Therefore, accuracy will assess 1) intra-assay variability; 2) intra-day variability (repeatability); and 3) daytime variability (intermediate precision); and 4) inter-laboratory variability (reproducibility). Coefficient of variation (CV%) is a quantitative measure of the accuracy expressed relative to either the observed or theoretical mean.

The immune-based methods disclosed in the present specification must be able to detect in background P comprising a bond that is susceptible to cleavage at the BoNT/A cleavage site₁Presence of α -SNAP-25 antibody-antigen complex of SNAP-25 with carboxy terminus at residue. The limit of detection (LOD) of a method refers to the concentration of analyte that produces a significantly different signal than a negative control or blank, and represents the lowest concentration of analyte that is distinguishable from background.

Thus, in one embodiment, the immune-based methods disclosed in the present specification can detect LOD of a retargeted endopeptidase, wherein the amount of retargeted endopeptidase is significantly different from the amount of a negative control or blank. In one aspect of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 10ng or less, 9ng or less, 8ng or less, 7ng or less, 6ng or less, 5ng or less, 4ng or less, 3ng or less, 2ng or less, 1ng or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 900pg or less, 800pg or less, 700pg or less, 600pg or less, 500pg or less, 400pg or less, 300pg or less, 200pg or less, 100pg or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 90pg or less, 80pg or less, 70pg or less, 60pg or less, 50pg or less, 40pg or less, 30pg or less, 20pg or less, 10pg or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 9pg or less, 8pg or less, 7pg or less, 6pg or less, 5pg or less, 4pg or less, 3pg or less, 2pg or less, 1pg or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 0.9pg or less, 0.8pg or less, 0.7pg or less, 0.6pg or less, 0.5pg or less, 0.4pg or less, 0.3pg or less, 0.2pg or less, 0.1pg or less of the retargeted endopeptidase.

In another aspect of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 9nM or less, 8nM or less, 7nM or less, 6nM or less, 5nM or less, 4nM or less, 3nM or less, 2nM or less, or 1nM or less of the retargeted endopeptidase. In other aspects of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 900pM or less, 800pM or less, 700pM or less, 600pM or less, 500pM or less, 400pM or less, 300pM or less, 200pM or less, or 100pM or less of the retargeted endopeptidases. In other aspects of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 100pM or less, 90pM or less, 80pM or less, 70pM or less, 60pM or less, 50pM or less, 40pM or less, 30pM or less, 20pM or less, or 10pM or less of the retargeted endopeptidase. In other aspects of this embodiment, the LOD of the immunization-based methods disclosed in the specification is, e.g., 10pM or less of a retargeted endopeptidase, 9pM or less, 8pM or less, 7pM or less, 6pM or less, 5pM or less, 4pM or less, 3pM or less, 2pM or less, or 1pM or less of a retargeted endopeptidase.

The limit of quantitation (LOQ) is the lowest and highest concentration of an analyte in a sample or specimen that can be measured with an acceptable level of accuracy and precision. The lower limit of quantitation refers to the lowest dose that the detection method can measure from background at all times. The upper limit of quantitation is the highest concentration that the detection method can always detect before saturation of the signal occurs. The linear range of the method is the region between the lower and upper quantitative limits. The linear range is calculated by subtracting the lower limit of quantitation from the upper limit of quantitation. The term "lower asymptotic signal-to-noise ratio" as used herein refers to the ratio of the signal detected at the lower limit of detection by the present method to the background signal. The term "upper asymptote signal-to-noise ratio" as used herein refers to the ratio of the signal detected by the method at the upper limit of detection to the background signal.

Thus, in one embodiment, the immune-based methods disclosed in the present specification can detect LOQ of a retargeted endopeptidase, wherein the amount of retargeted endopeptidase is significantly different from the amount of a negative control or blank. In one aspect of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 10ng or less, 9ng or less, 8ng or less, 7ng or less, 6ng or less, 5ng or less, 4ng or less, 3ng or less, 2ng or less, 1ng or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 900pg or less, 800pg or less, 700pg or less, 600pg or less, 500pg or less, 400pg or less, 300pg or less, 200pg or less, 100pg or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 90pg or less, 80pg or less, 70pg or less, 60pg or less, 50pg or less, 40pg or less, 30pg or less, 20pg or less, 10pg or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 9pg or less, 8pg or less, 7pg or less, 6pg or less, 5pg or less, 4pg or less, 3pg or less, 2pg or less, 1pg or less of a retargeted endopeptidase. In other aspects of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 0.9pg or less, 0.8pg or less, 0.7pg or less, 0.6pg or less, 0.5pg or less, 0.4pg or less, 0.3pg or less, 0.2pg or less, 0.1pg or less of the retargeted endopeptidase.

In another aspect of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 9nM or less, 8nM or less, 7nM or less, 6nM or less, 5nM or less, 4nM or less, 3nM or less, 2nM or less, or 1nM or less of the retargeted endopeptidase. In other aspects of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., a retargeted endopeptidase of 900pM or less, 800pM or less, 700pM or less, 600pM or less, 500pM or less, 400pM or less, 300pM or less, 200pM or less, or 100pM or less. In other aspects of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 100pM or less, 90pM or less, 80pM or less, 70pM or less, 60pM or less, 50pM or less, 40pM or less, 30pM or less, 20pM or less, or 10pM or less of the retargeted endopeptidase. In other aspects of this embodiment, the LOQ of the immunization-based methods disclosed in the specification is, e.g., 10pM or less of a retargeted endopeptidase, 9pM or less, 8pM or less, 7pM or less, 6pM or less, 5pM or less, 4pM or less, 3pM or less, 2pM or less, or 1pM or less of a retargeted endopeptidase.

The accuracy of an immune-based assay suitable for use in the practice of the disclosed methods must not exceed 50%. In aspects of this embodiment, the accuracy of the immunoassay-based assay is no more than 50%, no more than 40%, no more than 30%, or no more than 20%. In other aspects of this embodiment, the accuracy of the immunoassay-based assay is no more than 15%, no more than 10%, or no more than 5%. In other aspects of this embodiment, the accuracy of the immunoassay-based assay is no more than 4%, no more than 3%, no more than 2%, or no more than 1%.

The accuracy of an immune-based assay suitable for practical aspects of the disclosed methods must be at least 50%. In aspects of this embodiment, the accuracy of the immunoassay-based assay is at least 50%, at least 60%, at least 70%, or at least 80%. In other aspects of this embodiment, the accuracy of the immunoassay-based assay is at least 85%, at least 90%, or at least 95%. In other aspects of this embodiment, the accuracy of the immunoassay-based assay is at least 96%, at least 97%, at least 98%, or at least 99%.

The immune-based method disclosed in this specification must have a statistically significant lower asymptote signal-to-noise ratio and a statistically significant upper asymptote signal-to-noise ratio. In aspects of this embodiment, the immune-based methods disclosed in the specification have a signal to noise ratio at the lower asymptote of, for example, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1, or at least 20: 1. In other aspects of this embodiment, the signal-to-noise ratio at the upper asymptote for the immune-based method is, e.g., at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1, at least 40: 1, at least 45: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, or at least 100: 1, at least 150: 1, at least 200: 1, at least 250: 1, at least 300: 1, at least 350: 1, at least 400: 1, at least 450: 1, at least 500: 1, at least 550: 1, or at least 600: 1.

Specificity of a method is defined as the ability of such a method to exclude other relevant components (e.g., partially active or inactive analytes) from measuring the relevant analytes. The selectivity of the method will describe the ability of the analytical method to distinguish between various substances in the sample. The linearity of a method is its ability to produce results that are proportional to the concentration of an analyte in a sample, either directly or through a well-defined mathematical transformation. Thus, in one embodiment, the immunity-based methods disclosed in the specification can distinguish a retargeted endopeptidase with full activity from a retargeted endopeptidase with partial activity that is, for example, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the retargeted endopeptidase with full activity.

Reproducibility of the method is the reproducibility of the test results obtained from the same sample under normal (but variable) test conditions. The robustness of a program is a measure of its ability to remain unaffected under small but intentional variations in process parameters, and provides an indication of its reliability in normal use. Thus, reproducibility evaluates unavoidable variations, while robustness evaluates intentional variations. Typical parameters evaluated with reproducibility and robustness include the effect of freeze/thaw, incubation time, incubation temperature, reagent lifetime, sample preparation, sample storage, number of cell passages, retargeted endopeptidase batches, variability between purifications, and variability between nicking reactions. Robustness parameters for cell-based assays include cell bank (freezing start, duration and end), cell passage level, cell seeding density, cell stock density (how many days incubation in culture), cell age in flask (time to wait for seeding), incubation time, different plates, excess serum and reagent source. The system flexibility of the method is the determination of assay performance (including reagent and instrument performance) over time by analysis of reference standards or reference molecules. System adaptability is emphasized when FDA guidelines mention the fact that equipment, electronics, assay performance, and the sample to be analyzed constitute a complete system. System suitability can be assessed by performing a parallel test, which means that when a log dose is plotted against response, serial dilutions of the reference and serial dilutions of the sample should produce parallel curves.

Aspects of the disclosure include, in part, cells from established cell lines. The term "cell" as used herein refers to any eukaryotic cell that is susceptible to the activity of a retargeted endopeptidase, or any eukaryotic cell that can take up the retargeted endopeptidase. The term cell encompasses cells from a variety of organisms, such as murine, rat, porcine, bovine, equine, primate, and human cells; cells from a variety of cell types, such as neuronal and non-neuronal cells; and these cells may be isolated from or part of a heterogeneous cell population, tissue or organism. The term "established cell line" as used herein is synonymous with "immortalized cell line" or "transformed cell line" and refers to a cell culture of cells selected for unlimited propagation of a population of cells derived from a biological organism, tissue or organ. By definition, cell lines are established that do not include cell cultures of primary cells. The term "primary cells" as used herein is cells that are harvested directly from fresh tissue or organs, and these cells do not have the potential to propagate indefinitely. Established cell lines may comprise heterogeneous or homogeneous cell populations. Established cell lines derived from a single cell are called clonal cell lines. It was established that a cell line could be one containing all components required for endogenous expression by the cell to cause the cell to undergo the entire cellular machinery encompassing the binding of the retargeted endopeptidase to its receptor, internalization of the endopeptidase/receptor complex, translocation of the retargeted endopeptidase light chain from the intracellular vesicle into the cytoplasm, and proteolytic cleavage of SNAP-25, thereby allowing proteolytic cleavage of the substrate SNAP-25 by the retargeted endopeptidase. Alternatively, an established cell line can be one that contains cells that have been introduced from an exogenous source into at least one component that is required to cause the cells to undergo an overall cellular mechanism that encompasses binding of the retargeted endopeptidase to its receptor, internalization of the endopeptidase/receptor complex, translocation of the retargeted endopeptidase light chain from the intracellular vesicle into the cytoplasm, and proteolytic cleavage of SNAP-25, thereby causing proteolytic cleavage of the substrate of SNAP-25 by the retargeted endopeptidase. Established cell lines, also known as genetically engineered cell lines, cells from such established cell lines can, for example, express exogenous retargeted endopeptidases, such as exogenous ORL1, exogenous DOR, exogenous KOR, exogenous MOR, exogenous galanin receptor 1, exogenous galanin receptor 2, exogenous galanin receptor 3, or any combination thereof.

Aspects of the disclosure include, in part, cells from established cell lines that are sensitive to retargeted endopeptidase activity. The terms "cell sensitive to the activity of a retargeted endopeptidase", "cell sensitive to the activity of a retargeted endopeptidase" or "cell sensitive to the activity of a retargeted endopeptidase from an established cell line" as used herein refer to a cell that can undergo the entire cellular machinery, which encompasses the binding of the retargeted endopeptidase to its receptor, the internalization of the endopeptidase/receptor complex, the translocation of the chain of retargeted endopeptidase activity from intracellular vesicles into the cytoplasm, and the proteolytic cleavage of SNAP-25, thereby proteolytically cleaving the SNAP-25, thereby causing the retargeted endopeptidase to proteolytically cleave the SNAP-25 substrate, thereby inhibiting exocytosis. By definition, cells that are sensitive to retargeted endopeptidase activity must express, or be engineered to express, at least one retargeted endopeptidase receptor and at least one SNAP-25 substrate. The term "cell that can take up the retargeted endopeptidase" or "cell that constitutes the uptakeable retargeted endopeptidase of an established cell line" as used herein refers to a cell that can undergo the entire cellular machinery that encompasses binding of a targeted endopeptidase to its receptor, internalization of an endopeptidase/receptor complex, translocation of a retargeted endopeptidase light chain from an intracellular vesicle into the cytoplasm, and proteolytic cleavage of SNAP-25, thereby causing proteolytic cleavage of the SNAP-25 substrate by the retargeted endopeptidase, thereby inhibiting exocytosis. By definition, cells that can take up the retargeted endopeptidase must express, or be engineered to express, at least one retargeted endopeptidase receptor and at least one SNAP-25 substrate.

Thus, in one embodiment, cells from established cell lines are susceptible to retargeted endopeptidase activity. In aspects of this embodiment, cells from an established cell line are sensitive to the activity of a retargeted endopeptidase, e.g., about 100nM or less, about 90nM or less, about 80nM or less, about 70nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 20nM or less, about 10nM or less. In other aspects, cells from established cell lines are sensitive to the activity of a retargeted endopeptidase enzyme, e.g., a retargeted endopeptidase enzyme of about 9nM or less, about 8nM or less, about 7nM or less, about 6nM or less, about 5nM or less, about 4nM or less, about 3nM or less, about 2nM or less, or about 1nM or less. In other aspects, cells from established cell lines are sensitive to the activity of a retargeted endopeptidase of, e.g., about 0.9nM or less, about 0.8nM or less, about 0.7nM or less, about 0.6nM or less, about 0.5nM or less, about 0.4nM or less, about 0.3nM or less, about 0.2nM or about 0.1nM or less of the retargeted endopeptidase. When a value for a specified item, number, percentage, or term is stated, the term "about" as used herein refers to a range of ± 10% of the value for the specified item, percentage, parameter, or term.

In another embodiment, the cells comprising the established cell line can take up the retargeted endopeptidase. In aspects of this embodiment, the cells comprising the established cell line can uptake, for example, about 100nM or less, about 90nM or less, about 80nM or less, about 70nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 20nM or less, about 10nM or less of the retargeted endopeptidase. In other aspects, the cells comprising the established cell line are capable of uptake of the retargeted endopeptidase of about 9nM or less, about 8nM or less, about 7nM or less, about 6nM or less, about 5nM or less, about 4nM or less, about 3nM or less, about 2nM or less, or about 1nM or less. In other aspects, the cells comprising the established cell line are capable of uptake of the retargeted endopeptidase of about 0.9nM or less, about 0.8nM or less, about 0.7nM or less, about 0.6nM or less, about 0.5nM or less, about 0.4nM or less, about 0.3nM or less, about 0.2nM or less, or about 0.1nM or less.

Aspects of the disclosure include, in part, cells from established cell lines that exhibit selective binding to the retargeted endopeptidases disclosed in the specification. The term "selectively binds" as used herein when referring to a retargeted endopeptidase refers to a retargeted endopeptidase that differentially binds to a designated target receptor, such that the retargeted endopeptidase does not substantially bind to a non-target receptor. The extent to which cells from established cell lines exhibit selective binding to a retargeted endopeptidase can be measured by the extent to which these cells exhibit non-selective uptake of molecules that do not contain the targeting domain of the retargeted endopeptidase. One way to assess non-selective uptake of molecules that do not contain a targeting domain that retards endopeptidase is to: measurement of LH _NNon-selective uptake of fragments. LH_NA fragment is a fragment that comprises the clostridial toxin translocation domain and the clostridial toxin enzyme domain, but lacks any targeting domain at all. LH_NNon-limiting examples of fragments include LH_NA fragment and LH_NFragment of/B, LH_NC fragment, LH_ND fragment, LH_NSegment of/E, LH_NThe fraction of/F and LH_N(ii) a/G fragment. Exemplary LH_NThe A fragment is SEQ ID NO: 146 consisting of the polynucleotide molecule SEQ ID NO: 147 coding.

Thus, in one embodiment, cells from established cell lines exhibit selective binding to the retargeted endopeptidase. In aspects of this embodiment, cells from the established cell line exhibit selective binding to a retargeted endopeptidase that exhibits an activity, e.g., an activity that is at least 75% of the total activity determined, at least 80% of the total activity determined, at least 85% of the total activity determined, at least 90% of the total activity determined, or at least 95% of the total activity determined. In other aspects of this embodiment, cells from an established cell line exhibit selective binding to a retargeted endopeptidase that exhibits an activity of, e.g., about 75% to about 100% of the total activity determined, about 80% to about 100% of the total activity determined, about 85% to about 100% of the total activity determined, about 90% to about 100% of the total activity determined.

In another embodiment, cells from established cell lines exhibit minimal expression of LH_NNon-selective uptake of fragments. In aspects of this embodiment, cells from established cell lines exhibit on LH_NThe non-selective uptake of a segment is, for example, at most 25% of the total uptake measured, at most 20% of the total uptake measured, at most 15% of the total uptake measured, at most 10% of the total uptake measured or at most 5% of the total uptake measured. In other aspects of this embodiment, cells from established cell lines exhibit LH_NThe non-selective uptake of a fragment is, for example, about 0% to about 25% of the total uptake measured, about 0% to about 20% of the total uptake measured, about 0% to about 15% of the total uptake measured, about 0% to about 10% of the total uptake measured, or about 0% to about 5% of the total uptake measured.

In another embodiment, cells from established cell lines exhibit minimal expression of LH_NNon-selective uptake of the A fragment. In aspects of this embodiment, cells from established cell lines exhibit on LH_NThe non-selective uptake of the/a segment is for example at most 25% of the measured total uptake, at most 20% of the measured total uptake, at most 15% of the measured total uptake, at most 10% of the measured total uptake or at most 5% of the measured total uptake. In other aspects of this embodiment, cells from established cell lines exhibit LH _NThe nonselective uptake of the/A fragment is, for example, about 0% to about 25% of the total uptake measured, about 0% to about 20% of the total uptake measured, about 0% to about 15% of the total uptake measured, about 0% to about 10% of the total uptake measured, or about 0% to about 5% of the total uptake measured.

Aspects of the disclosure include, in part, cells from established cell lines that present a sufficient number of receptor binding sites on the plasma membrane to confer sensitive and selective binding to heavy-targeted endopeptidases. Equilibrium saturation binding assays will measure the total non-specific binding of various concentrations of ligand. Equilibrium dissociation constant (K) for ligands_d) And the maximum number of receptor binding sites Bmax can be calculated from specific binding using non-linear regression analysis. Specific binding was calculated by subtracting non-specific binding of the ligand from the total binding observed. K_dThe concentration of ligand required to achieve half maximal binding, and is measured in molar concentration. Bmax is the maximum number of binding sites present on the plasma membrane and is measured in pmol/mg, pmol/cell, fmol/cell or site/cell.

Thus, in one embodiment, cells from established cell lines present a sufficient number of receptor binding sites on the plasma membrane to confer sensitive and selective binding to heavy-targeted endopeptidases. In aspects of this embodiment, cells from the established cell line exhibit a Bmax value for a targeted ligand of the heavy-targeted endopeptidase of, e.g., at least 0.1 fmol/cell, at least 0.2 fmol/cell, at least 0.3 fmol/cell, at least 0.4 fmol/cell, at least 0.5 fmol/cell, at least 0.6 fmol/cell, at least 0.7 fmol/cell, at least 0.8 fmol/cell, at least 0.9 fmol/cell, or at least 1.0 fmol/cell. In other aspects of this embodiment, cells from the established cell line exhibit a Bmax value for a targeted ligand of the retargeted endopeptidase of, e.g., at least 1 fmol/cell, at least 2 fmol/cell, at least 3 fmol/cell, at least 4 fmol/cell, at least 5 fmol/cell, at least 6 fmol/cell, at least 7 fmol/cell, at least 8 fmol/cell, at least 9 fmol/cell, or at least 10 fmol/cell.

Aspects of the disclosure include, in part, cells from established clonal cell lines that are more stable than cells from a parental cell line from which the clonal cell line is derived that are susceptible to retargeted endopeptidase activity. The term "stable" as used herein refers to the relative EC for retargeted endopeptidase activity exhibited by cells from established clonal cell lines over a particular number of passages₅₀Sensitivity, efficacy, well-defined upper asymptotes and/or well-defined dose-response curves similar to the relative ECs exhibited by cells from the parental cell line of the derivative clonal cell line through the same or similar number of passages₅₀Sensitivity, efficacy, well-defined upper asymptotes and/or well-defined dose-response curve values, wherein the same assay conditions and the same retargeted endopeptidase are used in both assays.

Thus, in one embodiment, cells from an established clonal cell line are more stable than cells from a parental cell line from which the clonal cell line was derived. In one aspect of this embodiment, cells from the established clonal cell line are more stable than the parental SK-N-DZ cell line. In another aspect of this embodiment, the cells from the established clonal cell line are more stable than the parental SK-N-DZ cell line ATCC CRL-2149. In other aspects of this embodiment, cells from an established clonal cell line are stable, e.g., for at least 5 passages or more, at least 10 passages or more, at least 15 passages or more, at least 20 passages or more, at least 25 passages or more, or at least 30 passages or more, relative to cells from a parental cell line from which the clonal cell line was obtained. In other aspects of this embodiment, cells from an established clonal cell line are stable, e.g., for at least 5 passages or more, at least 10 passages or more, at least 15 passages or more, at least 20 passages or more, at least 25 passages or more, or at least 30 passages or more, relative to cells from a parental cell line from which the clonal cell line was derived.

Aspects of the disclosure include, in part, cells from established clonal cell lines that are susceptible to retargeted endopeptidase activity that remain stable over multiple cell passages. The term "stable" as used herein refers to the relative EC for retargeted endopeptidase activity exhibited by cells from established clonal cell lines over a particular number of passages₅₀Sensitivity, efficacy, well-defined upper asymptotes and/or well-defined dose-response curves similar to the relative EC exhibited by cells from the same established clonal cell line prior to one or more passages₅₀Sensitivity, efficacy, well-defined upper asymptotes and/or well-defined dose-response curve values, wherein the same assay conditions and the same retargeted endopeptidase are used in both assays.

Cells from established cell lines disclosed in the present specification can exhibit consistent sensitivity to retargeted endopeptidase activity over multiple cell passages. The term "sensitivity to retargeted endopeptidase activity" as used herein refers to the lowest dose in the assay that can consistently be measured above the signal detected by an untreated control or background signal.

Thus, in one embodiment, cells from established clonal cell lines exhibit sensitivity to retargeted endopeptidase activity of, e.g., 100nM or less, about 80nM or less, about 70nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 20nM or less, about 10nM or less, about 1nM or less, about 0.9nM or less, about 0.8nM or less, about 0.7nM or less, about 0.6nM or less, about 0.5nM or less, about 0.4nM or less, about 0.3nM or less, about 0.2nM or less, or about 0.1nM or less, through any given passage. In aspects of this embodiment, cells from established clonal cell lines exhibit sensitivity to the activity of a retargeted endopeptidase enzyme, e.g., from about 0.01nM to about 100nM, from about 0.01nM to about 75nM, from about 0.01nM to about 50nM, from about 0.01nM to about 25nM, from about 0.01nM to about 20nM, from about 0.01nM to about 15nM, from about 0.01nM to about 10nM, from about 0.01nM to about 5nM, from about 0.001nM to about 100nM, from about 0.001nM to about 75nM, from about 0.001nM to about 50nM, from about 0.001nM to about 25nM, from about 0.001nM to about 20nM, from about 0.001nM to about 15nM, from about 0.001nM to about 10nM, or from about 0.001nM to about 5nM, through any given passage number of passages.

In another embodiment, cells from established clonal cell lines exhibit sensitivity to a retargeted endopeptidase activity of about 100nM or less, about 75nM or less, about 50nM or less, about 25nM or less, less than about 20nM or less, about 15nM or less, or about 1nM or less, for example, after 5 or more cell passages, 10 or more cell passages, 15 or more cell passages, 20 or more cell passages, 25 or more cell passages, 30 or more cell passages, 35 or more cell passages, 40 or more cell passages, 45 or more cell passages, or 50 or more cell passages. In other aspects of this embodiment, the composition is administered, for example, from about 15 to about 60 passages, from about 20 to about 60 passages, from about 25 to about 60 passages, from about 30 to about 60 passages, from about 35 to about 60 passages, from about 40 to about 60 passages, from about 45 to about 60 passages, from about 50 to about 60 passages, from about 15 to about 50 passages, from about 20 to about 50 passages, from about 25 to about 50 passages, from about 30 to about 50 passages, from about 35 to about 50 passages, from about 40 to about 50 passages, from about 15 to about 40 passages, from about 20 to about 40 passages, from about 25 to about 40 passages, or from about 30 to about 40 passages, cells from established clonal cell lines exhibit sensitivity to retargeted endopeptidase activity of about 100nM or less, about 75nM or less, about 50nM or less, about 25nM or less, less than about 20nM or less, about 15nM or less, about 10nM or less, or about 1nM or less.

Cells from established cell lines disclosed in the present specification can exhibit consistent relative efficacy for uptake of a retargeted endopeptidase or retargeted endopeptidase activity over multiple cell passages. The term "relative efficacy" as used herein refers to the degree of comparison of the upper asymptote of retargeted endopeptidase activity detected in performing the present assay to the upper asymptote of retargeted endopeptidase activity detected in the assay using a reference standard, reference molecule or reference passage number. The term "upper asymptotic signal-to-noise ratio" as used herein refers to the ratio of the signal detected at the upper limit of detection in the assay to the signal detected by the untreated control or background signal. The upper limit of detection is the maximum dose that can always be measured by the assay before saturation of the signal occurs.

Thus, in one embodiment, cells from established cell lines disclosed in the present specification can exhibit a well-defined upper asymptote over multiple cell passages and maintain a consistent and appropriate signal-to-noise ratio in the assay. In aspects of this embodiment, following, e.g., 5 or more cell passages, 10 or more cell passages, 15 or more cell passages, 20 or more cell passages, 25 or more cell passages, 30 or more cell passages, 35 or more cell passages, 40 or more cell passages, 45 or more cell passages, or 50 or more cell passages, the well-defined signal-to-noise ratio of the upper asymptote for heavy-target endopeptidase activity of cells from established cell lines disclosed in this specification must be, e.g., at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1, at least 40: 1, at least 45: 1, at least 50: 1, At least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1 or at least 100: 1, at least 150: 1, at least 200: 1, at least 250: 1, at least 300: 1, at least 350: 1, at least 400: 1, at least 450: 1, at least 500: 1, at least 550: 1 or at least 600: 1. In other aspects of this embodiment, the cells from the cell line disclosed in this specification have a well-defined signal-to-noise ratio for the upper asymptote for the activity of the heavy target endopeptidase of, e.g., at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, such as from about 15 to about 60 passages, from about 25 to about 60 passages, from about 30 to about 60 passages, from about 35 to about 60 passages, from about 40 to about 60 passages, from about 45 to about 60 passages, from about 50 to about 60 passages, from about 15 to about 50 passages, from about 20 to about 50 passages, from about 25 to about 50 passages, from about 30 to about 50 passages, from about 35 to about 50 passages, from about 40 to about 50 passages, from about 15 to about 40 passages, from about 20 to about 40 passages, from about 25 to about 40 passages, or from about 30 to about 40 passages, and have a well-defined signal-to-noise ratio for the upper asymptote for the activity of the heavy target endopeptidase of, e.g., at least 3: 1, at least 4: 1, At least 9: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1, at least 40: 1, at least 45: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1 or at least 100: 1, at least 150: 1, at least 200: 1, at least 250: 1, at least 300: 1, at least 350: 1, at least 400: 1, at least 450: 1, at least 500: 1, at least 550: 1, or at least 600: 1.

Cells from established cell lines disclosed in the present specification can exhibit well-defined dose-response curves for retargeted endopeptidase activity over multiple cell passages. The term "dose-response curve" as used herein refers to the degree of fit of the raw data to the statistical model selected for the present assay. In a non-limiting example, a sigmoidal curve obtained using a four parameter logistic fitting method (four parameter biology fit) is a dose-response curve for enzymatic activity assays such as potency assays. In another non-limiting example, ligand binding using a single site saturation fit (one site saturation fit) is a dose-response curve for a ligand/antibody binding assay.

Thus, in one embodiment, cells from established cell lines disclosed in the present specification still exhibit well-defined dose-response curves for retargeted endopeptidase activity over multiple cell passages. In aspects of this embodiment, cells from established cell lines disclosed in the specification still exhibit well-defined dose-response curves over, e.g., 5 or more cell passages, 10 or more cell passages, 15 or more cell passages, 20 or more cell passages, 25 or more cell passages, 30 or more cell passages, 35 or more cell passages, 40 or more cell passages, 45 or more cell passages, or 50 or more cell passages. In other aspects of this embodiment, the cells from the cell lines disclosed in this specification still exhibit a well-defined targeted dose-response curve for heavy endopeptidase activity over, for example, about 15 to about 60 passages, about 20 to about 60 passages, about 25 to about 60 passages, about 30 to about 60 passages, about 35 to about 60 passages, about 40 to about 60 passages, about 45 to about 60 passages, about 50 to about 60 passages, about 15 to about 50 passages, about 20 to about 50 passages, about 25 to about 50 passages, about 30 to about 50 passages, about 35 to about 50 passages, about 40 to about 50 passages, about 15 to about 40 passages, about 20 to about 40 passages, about 25 to about 40 passages, or about 30 to about 40 passages.

Cells from established cell lines disclosed in the present specification can exhibit consistent relative EC for retargeted endopeptidase activity over multiple cell passages₅₀The value is obtained. The term "relative EC" as used herein₅₀"OR" relative EC₅₀The value "means the EC for the reference standard, reference molecule or reference passage number used in the assay calculated₅₀And normalized EC of retargeted endopeptidase Activity₅₀The value is obtained.

Thus, in one embodiment, cells from established clonal cell lines still exhibit consistent relative EC for retargeted endopeptidase activity over multiple cell passages₅₀The value is obtained. In aspects of this embodiment, cells from established clonal cell lines exhibit a consistent relative EC for retargeted endopeptidase activity over, e.g., 5 or more cell passages, 10 or more cell passages, 15 or more cell passages, 20 or more cell passages, 25 or more cell passages, 30 or more cell passages, 35 or more cell passages, 40 or more cell passages, 45 or more cell passages, or 50 or more cell passages₅₀Values, i.e. relative EC for retargeted endopeptidase activity₅₀About ± 10%, about ± 20%, about ± 30%, about ± 40%, about ± 50%, about ± 60%, about ± 70%, or about ± 75% of the value. In other aspects of this embodiment, cells from established clonal cell lines have a relative EC for presentation of retargeted endopeptidase activity over, e.g., 5 or more cell passages, 10 or more cell passages, 15 or more cell passages, 20 or more cell passages, 25 or more cell passages, 30 or more cell passages, 35 or more cell passages, 40 or more cell passages, 45 or more cell passages, or 50 or more cell passages ₅₀Values are relative EC for retargeted endopeptidase activity₅₀For example, about 10% to about 75%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, or 10% to about 20% of the value.

Aspects of the invention include, in part, retargeting endopeptidases. The term "retargeted endopeptidase" as used herein is synonymous with "targeting vesicular exocytosis modulator protein" or "TVEMP". Non-limiting examples of retargeted endopeptidases are disclosed, for example, in the following documents: keith A. Foster et al, cloning Toxin Derivatives Able To modification personal sensor Functions, U.S. Pat. No. 5,989,545; clifford c. shone et al, regrombinant Toxin Fragments, U.S. patent 6,461,617; conrad P.Quinn et al, Methods and Compounds for the Treatment of the Mucus Hypersecretion, U.S. Pat. No. 6,632,440; lance E.Steward et al, Methods And Compositions For The Treatment Of Patches, U.S. Pat. No. 6,843,998; stephan Donovan, cloning Toxin Derivatives and Methods For Treating Pain, U.S. patent publication 2002/0037833; keith A. Foster et al, Inhibition of precipitation from Non-neural Cells, U.S. patent publication 2003/0180289; oliver Dolly et al, active Recombinant Neurotoxins, WO 2001/014570; keith A. Foster et al, Re-targeted Toxin Conjugates, International patent publication WO 2005/023309; lance E.Steward et al, Multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. patent application Ser. No. 11/376,696; steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting activities For Non-cloning approach targets Cells, U.S. patent application Ser. No. 11/776,075; dolly, J.O. et al, active cloning tools, U.S. patent application Ser. No. 11/829,475; foster, K.A., et al, Fusion Proteins, International patent publication WO 2006/059093; and Foster, k.a. et al, Non-cytotoxin Protein Conjugates, international patent publication WO 2006/059105, the entire contents of each of which are incorporated herein by reference. Non-limiting examples of retargeted endopeptidases include SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 130 and SEQ ID NO: 131.

Thus, in one embodiment, the detected retargeted endopeptidase activity is from a retargeted endopeptidase. In aspects of this embodiment, the detected retargeted endopeptidase activity is a retargeted endopeptidase from the following publications: keith A. Foster et al, cloning Toxin Derivatives Able To modification personal sensor Functions, U.S. Pat. No. 5,989,545; clifford c. shone et al, regrombinant Toxin Fragments, U.S. patent 6,461,617; conrad P.Quinn et al, Methods and Compounds for the Treatment of the Mucus Hypersecretion, U.S. Pat. No. 6,632,440; lance E.Steward et al, Methods And Compositions For The Treatment Of Patches, U.S. Pat. No. 6,843,998; stephan Donovan, cloning Toxin Derivatives and Methods For Treating Pain, U.S. patent publication 2002/0037833; keith A. Foster et al, Inhibition of precipitation from Non-neural Cells, U.S. patent publication 2003/0180289; oliver Dolly et al, active Recombinant Neurotoxins, WO 2001/014570; keith A. Foster et al, Re-targeted Toxin Conjugates, International patent publication WO 2005/023309; lance E.Steward et al, Multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. patent application Ser. No. 11/376,696; steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting activities For Non-cloning approach targets Cells, U.S. patent application Ser. No. 11/776,075; dolly, J.O. et al, active cloning tools, U.S. patent application Ser. No. 11/829,475; foster, K.A., et al, Fusion Proteins, International patent publication WO 2006/059093; and Foster, k.a. et al, Non-cytotoxin Protein Conjugates, international patent publication WO 2006/059105, the entire contents of each of which are incorporated herein by reference. In aspects of this embodiment, the retargeted endopeptidase is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 130 or SEQ ID NO: 131.

In another embodiment, the detected retargeted endopeptidase activity is from a retargeted endopeptidase having, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity to a retargeted endopeptidase disclosed in: keith A. Foster et al, cloning Toxin Derivatives Able To modification personal sensor Functions, U.S. Pat. No. 5,989,545; clifford c. shone et al, regrombinant Toxin Fragments, U.S. patent 6,461,617; conrad P.Quinn et al, Methods and Compounds for the Treatment of the Mucus Hypersecretion, U.S. Pat. No. 6,632,440; lance E.Steward et al, Methods And Compositions For The Treatment Of Patches, U.S. Pat. No. 6,843,998; stephan Donovan, cloning Toxin Derivatives and Methods For Treating Pain, U.S. patent publication 2002/0037833; keith A. Foster et al, Inhibition of precipitation from Non-neural Cells, U.S. patent publication 2003/0180289; oliver Dolly et al, active Recombinant Neurotoxins, WO 2001/014570; keith A. Foster et al, Re-targeted Toxin Conjugates, International patent publication WO 2005/023309; lance E.Steward et al, Multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. patent application Ser. No. 11/376,696; steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting activities For Non-cloning approach targets Cells, U.S. patent application Ser. No. 11/776,075; dolly, J.O. et al, active cloning tools, U.S. patent application Ser. No. 11/829,475; foster, K.A., et al, Fusion Proteins, International patent publication WO 2006/059093; and Foster, k.a. et al, Non-cytotoxin Protein Conjugates, international patent publication WO 2006/059105, the entire contents of each of which are incorporated herein by reference. In another embodiment, the detected retargeted endopeptidase activity is from a polypeptide that differs from the polypeptide of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 130 or SEQ ID NO: 131 have, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In other aspects of this embodiment, the detected retargeted endopeptidase activity is from a retargeted endopeptidase having, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions relative to a retargeted endopeptidase disclosed in: keith A. Foster et al, cloning Toxin Derivatives Able To modification personal sensor Functions, U.S. Pat. No. 5,989,545; clifford c. shone et al, regrombinant Toxin Fragments, U.S. patent 6,461,617; conrad P.Quinn et al, Methods and Compounds for the Treatment of the Mucus Hypersecretion, U.S. Pat. No. 6,632,440; lance E.Steward et al, Methods And Compositions For The Treatment Of Patches, U.S. Pat. No. 6,843,998; stephan Donovan, cloning Toxin Derivatives and Methods For Treating Pain, U.S. patent publication 2002/0037833; keith A. Foster et al, Inhibition of precipitation from Non-neural Cells, U.S. patent publication 2003/0180289; oliver Dolly et al, active Recombinant Neurotoxins, WO 2001/014570; keith A. Foster et al, Re-targeted Toxin Conjugates, International patent publication WO 2005/023309; lance E.Steward et al, Multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. patent application Ser. No. 11/376,696; steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting activities For Non-cloning approach targets Cells, U.S. patent application Ser. No. 11/776,075; dolly, J.O. et al, active cloning tools, U.S. patent application Ser. No. 11/829,475; foster, K.A., et al, Fusion Proteins, International patent publication WO 2006/059093; and Foster, k.a. et al, Non-cytotoxin Protein Conjugates, international patent publication WO 2006/059105, the entire contents of each of which are incorporated herein by reference. In other aspects of this embodiment, the detected retargeted endopeptidase activity is from a polypeptide sequence relative to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 130 or SEQ ID NO: 131 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions or additions.

In other aspects of this embodiment, the detected retargeted endopeptidase activity is from a non-natural retargeted endopeptidase variant having, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more consecutive amino acid substitutions, deletions, or additions relative to a retargeted endopeptidase disclosed in: keith A. Foster et al, cloning Toxin Derivatives Able To modification personal sensor Functions, U.S. Pat. No. 5,989,545; clifford c. shone et al, regrombinant Toxin Fragments, U.S. patent 6,461,617; conrad P.Quinn et al, Methods and Compounds for the Treatment of the Mucus Hypersecretion, U.S. Pat. No. 6,632,440; lance E.Steward et al, Methods And Compositions For The Treatment Of Patches, U.S. Pat. No. 6,843,998; stephan Donovan, cloning Toxin Derivatives and Methods For Treating Pain, U.S. patent publication 2002/0037833; keith A. Foster et al, Inhibition of precipitation from Non-neural Cells, U.S. patent publication 2003/0180289; oliver Dolly et al, active Recombinant Neurotoxins, WO 2001/014570; keith A. Foster et al, Re-targeted Toxin Conjugates, International patent publication WO 2005/023309; lance E.Steward et al, Multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. patent application Ser. No. 11/376,696; steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting activities For Non-cloning approach targets Cells, U.S. patent application Ser. No. 11/776,075; dolly, J.O. et al, active cloning tools, U.S. patent application Ser. No. 11/829,475; foster, K.A., et al, Fusion Proteins, International patent publication WO 2006/059093; and Foster, k.a. et al, Non-cytotoxin Protein Conjugates, international patent publication WO 2006/059105, the entire contents of each of which are incorporated herein by reference. In other aspects of this embodiment, the detected retargeted endopeptidase activity is from a polypeptide sequence relative to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 130 or SEQ ID NO: 131 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more consecutive amino acid substitutions, deletions, or additions.

In another embodiment, the detected retargeted endopeptidase activity is from an opioid retargeted endopeptidase. Non-limiting examples of opioid heavy-targeting endopeptidases or opioid-TVEMPs are described, for example, in the following documents: keith A. Foster et al, cloning Toxin Derivatives Able To modification personal sensor Functions, U.S. Pat. No. 5,989,545; oliver Dolly et al, active Recombinant neotoxins, U.S. patent 7,132,259; stephan Donovan, nasal Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,244,437; stephan Donovan, nasal Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,413,742; stephan Donovan, nasal Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,415,338; lance E.Steward et al, Multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. Pat. No. 7,514,088; keith a. foster, Fusion Proteins, U.S. patent publication 2008/0064092; keith a. foster, Fusion Proteins, U.S. patent publication 2009/0035822; lancet e.steward et al, multi-equivalent Clostridium Toxin Derivatives and Methods of the same Use, U.S. patent publication 2009/0048431; keith a. foster, Non-cytoxic Protein Conjugates, U.S. patent publication 2009/0162341; keith A. Foster et al, Re-targeted Toxin Conjugates, International patent publication WO 2005/023309; and Lance E.Steward, Modified cloning approaches with Enhanced transformation Capabilities and Modified Targeting Capabilities for Non-cloning approach Cells, International patent publication WO 2008/008805; the entire contents of each patent are incorporated herein by reference.

In another embodiment, the detected retargeted endopeptidase activity is from a galanin retargeted endopeptidase. Non-limiting examples of galanin retargeting endopeptidases or galanin-TVEMPs are described, for example, in the following documents: steward, L.E., et al, Modified cloning approaches with Enhanced transport capabilities and Enhanced Targeting Activity, U.S. patent application Ser. No. 11/776,043 (11/7/2007); steward, L.E., et al, Modified cloning approaches with Enhanced transformation Capabilities and Altered Targeting activities For cloning approach Cells, U.S. patent application Ser. No. 11/776,052 (11/7/2007); and Steward, L.E., et al, Modified cloning strategies with Enhanced transformation Capabilities and Modified Targeting activities For Non-cloning Targeting Cells, U.S. patent application Ser. No. 11/776,075 (2007, 7, 11), the entire contents of each of which are incorporated herein by reference.

Aspects of the invention include, in part, SNAP-25. The term "SNAP-25" as used herein refers to native SNAP-25 or non-native SNAP-25 that is preferentially cleaved by a retargeted endopeptidase. The term "preferential cleavage" as used herein means that the rate of re-targeted endopeptidase cleavage of SNAP-25 is at least one order of magnitude higher than the rate of targeted endopeptidase cleavage of any other substrate. In aspects of this embodiment, the rate of re-targeted endopeptidase cleavage of SNAP-25 is at least two orders of magnitude, at least three orders of magnitude, at least four orders of magnitude, or at least five orders of magnitude higher than the rate of targeted endopeptidase cleavage of any other substrate.

The term "native SNAP-25" as used herein refers to any SNAP-25 produced by a natural process, including but not limited to SNAP-25 isoforms and SNAP-25 subtypes resulting from post-translational modifications, alternatively spliced transcripts, or spontaneous mutations. Native SNAP-25 includes, but is not limited to, SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, or SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids.

The term "non-native SNAP-25" as used herein refers to any SNAP-25 whose structure is modified by human manipulation, including, but not limited to, SNAP-25 produced by genetic engineering using random mutagenesis or rational design, and SNAP-25 produced by in vitro chemical synthesis. Non-limiting examples of non-native SNAP-25 are described in the following references: for example Steward, l.e., et al, FRET Protease Assays for Clostridial Toxins, U.S. patent 7,332,567; Fernandez-Salas et al, Lipohilic Dye-based FRET Assays for compact Toxin Activity, U.S. patent publication 2008/0160561, the entire contents of each of which are incorporated herein by reference. Non-native SNAP-25 can be encoded by SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids.

Thus, in one embodiment, SNAP-25 is native SNAP-25. In aspects of this embodiment, SNAP-25 is an isoform of SNAP-25 or a subtype of SNAP-25. In aspects of this embodiment, native SNAP-25 is SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, native SNAP-25. In other aspects of this embodiment, SNAP-25 is a polypeptide that differs from SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, a native SNAP-25 having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In another embodiment, SNAP-25 is a non-native SNAP-25. In other aspects of this embodiment, SNAP-25 is a polypeptide that differs from SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity. In other aspects of this embodiment, SNAP-25 is a consensus sequence relative to SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, for example, a non-native SNAP-25 having 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions. In other aspects of this embodiment, SNAP-25 is a consensus sequence relative to SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more consecutive amino acid substitutions, deletions, or additions of non-native SNAP-25.

SNAP-25 can be endogenous SNAP-25 or exogenous SNAP-25. The term "endogenous SNAP-25" as used herein refers to SNAP-25 that is naturally present in a cell as it is naturally encoded within the genome of the cell, such that the cell inherently expresses SNAP-25 without the need for an externally derived SNAP-25 or an externally derived genetic material encoding SNAP-25. Expression of endogenous SNAP-25 may or may not utilize environmental stimuli, such as cell differentiation. Endogenous SNAP-25 can, by definition, only be native SNAP-25 or a variant thereof. For example, each of the following established cell lines will express endogenous SNAP-25: BE (2) -M17, Kelly, LA1-55N, N1E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH-SY5Y, SiMa, SK-N-DZ and SK-N-BE (2) -C.

The term "exogenous SNAP-25" as used herein refers to SNAP-25 that is expressed in a cell by introduction of an externally derived SNAP-25 or externally derived genetic material encoding SNAP-25 by human manipulation. Expression of exogenous SNAP-25 may or may not utilize environmental stimuli, such as cell differentiation. In a non-limiting example, cells from established cell lines can express exogenous SNAP-25 by transient transfection or stable transfection of SNAP-25. As another non-limiting example, cells from established cell lines can express exogenous SNAP-25 by protein transfection of SNAP-25. Exogenous SNAP-25 can be native SNAP-25 or a variant thereof, or non-native SNAP-25 or a variant thereof.

Thus, in one embodiment, cells from established cell lines express endogenous SNAP-25. In aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is native SNAP-25. In other aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24. in other aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is native SNAP-25, e.g., a SNAP-25 isoform or a SNAP-25 subtype. In other aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is a polypeptide that is identical to SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, a native SNAP-25 having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In another embodiment, cells from established cell lines are transiently or stably engineered to express exogenous SNAP-25. In one aspect of this embodiment, cells from established cell lines are transiently or stably engineered to express native SNAP-25. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, native SNAP-25. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express native SNAP-25, e.g., SNAP-25 isoforms or SNAP-25 subtypes. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express an amino acid sequence identical to SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, a native SNAP-25 having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In another aspect of this embodiment, cells from established cell lines are transiently or stably engineered to express non-native SNAP-25. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express an amino acid sequence identical to SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express an amino acid sequence relative to SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24, for example, a non-native SNAP-25 having 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express an amino acid sequence relative to SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23 or SEQ ID NO: 24 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more consecutive amino acid substitutions, deletions, or additions of non-native SNAP-25.

An assay that detects cleavage of SNAP-25 following exposure to a retargeted endopeptidase can be used to assess whether a cell expresses endogenous SNAP-25 or exogenous SNAP-25. In these assays, the production of SNAP-25 cleavage products will be detected in SNAP-25 expressing cells following re-targeting endopeptidase treatment. Non-limiting examples of specific Western blot analysis (Western blot analysis) and well characterized reagents, conditions and protocols are readily available from suppliers including, but not limited to: amersham Biosciences, Piscataway, NJ; Bio-Rad Laboratories, Hercules, Calif.; pierce Biotechnology, inc., Rockford, IL; promega Corporation, Madison, Wis., and Stratagene, Inc., La Jolla, Calif. It will be appreciated that these and similar assays directed to cleavage of SNAP-25 can be used to identify cells expressing endogenous SNAP-25 or exogenous SNAP-25.

In a non-limiting example, uptake of a re-targeted endopeptidase can be determined using western blot analysis using antibodies that recognize a cleavage product of SNAP-25 or cleaved and uncleaved forms of SNAP-25. Examples of α -SNAP-25 antibodies suitable for use in these assays include, but are not limited to, α -SNAP-25 mouse monoclonal antibody SMI-81(Sternberger Monoclonals Inc., Lutherville, Md.), mouse α -SNAP-25 monoclonal antibody CI 71.1 (synthetic Systems, Goettingen, Germany), α -SNAP-25 mouse monoclonal antibody CI 71.2 (synthetic Systems, Goettingen, Germany), α -SNAP-25 mouse monoclonal antibody SP12(Abcam, Cambridge, MA), α -SNAP-25 rabbit polyclonal antiserum (synthetic Systems, Goettingen, Germany), α -SNAP-25 rabbit polyclonal antiserum (Abcam, Cambridge, MA), and α -SNAP-25 rabbit polyclonal antiserum S9684(Sigma, St Louis, S9684).

Aspects of the disclosure include, in part, retargeting endopeptidase receptors. The term "retargeted endopeptidase receptor" as used herein refers to a naturally or non-naturally retargeted endopeptidase receptor that preferentially interacts with a retargeted endopeptidase in a manner that elicits a response in the activity of the retargeted endopeptidase. The term "preferential interaction" as used herein means that the equilibrium dissociation constant (KD) of the retargeted endopeptidase to the retargeted endopeptidase receptor is at least one order of magnitude lower than the KD of the retargeted endopeptidase to any other receptor on the cell surface. The equilibrium dissociation constant, a specific type of equilibrium constant that measures the propensity of a re-targeted endopeptidase receptor complex to reversibly separate (dissociate) into its component molecules (i.e., re-targeted endopeptidase and re-targeted endopeptidase receptor), is defined as KD ═ Ka/KD at equilibrium. The association constant (Ka) is defined as Ka ═ C/[ L ] [ R ], and the dissociation constant (Kd) is defined as Kd ═ L ] [ R ]/[ C ], where [ L ] equals the molar concentration of the retargeted endopeptidase, [ R ] is the molar concentration of the retargeted endopeptidase receptor, and [ C ] is the molar concentration of the endopeptidase-receptor complex, and where all these concentrations are the concentrations of these components when the system reaches equilibrium. The smaller the dissociation constant, the more tightly the retargeted endopeptidase binds to its receptor, or the higher the binding affinity between the retargeted endopeptidase and the retargeted endopeptidase receptor. In aspects of this embodiment, the dissociation constant of the retargeted endopeptidase for its receptor is at least two orders of magnitude, at least three orders of magnitude, at least four orders of magnitude, or at least five orders of magnitude lower than the dissociation constant of the retargeted endopeptidase for any other receptor. In other aspects of this embodiment, the equilibrium dissociation constant (KD) for the binding affinity of a retargeted endopeptidase to interact preferentially with its receptor can be, e.g., 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less. In other aspects of this embodiment, the equilibrium dissociation constant (KD) for the binding affinity of a retargeted endopeptidase to interact preferentially with its receptor can be, e.g., 90nM or less, 80nM or less, 70nM or less, 60nM, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less. The term "eliciting a retargeted endopeptidase activity response" as used herein refers to the ability of a retargeted endopeptidase receptor to interact with a retargeted endopeptidase to form an endopeptidase/receptor complex and subsequently internalize the complex into the cytoplasm of a cell.

The term "naturally retargeted endopeptidase receptor" as used herein refers to any retargeted endopeptidase receptor produced by a natural process, including but not limited to retargeted endopeptidase receptor isoforms produced by post-translational modifications, alternatively spliced transcripts, or spontaneous mutations, as well as retargeted endopeptidase receptor subtypes. Natural retargeted endopeptidase receptors include, but are not limited to: natural opioid receptors, such as opioid-like receptors (ORL 1), -opioid receptors (DOR), Kappa Opioid Receptors (KOR), and mu-opioid receptors (MOR), such as those described in: christopher Evans et al, Opioid receiver Genes, U.S. Pat. No. 6,265,563; christopher Evans et al, Methods of Screening Modulators of Optid Receptor A activity, U.S. Pat. No. 6,432,652; christopher Evans et al, Opioid Receptors and Methods of Use, U.S. Pat. No. 7,282,563; and Christopher Evans et al, Delta Opioid receiver Proteins, U.S. patent publication 2008/0219925, the entire contents of each of which are hereby incorporated by reference. Other examples of naturally retargeted endopeptidase receptors include, but are not limited to: galanin receptor 1, galanin receptor 2, and galanin receptor 3. Natural opioid receptors from other vertebrate species, e.g., from primates, cows, dogs, mice, rats, chickens, fish, and the like, are known in the art and are useful in various aspects of the present description.

Natural ORL1 includes, but is not limited to: SEQ ID NO: 25 and SEQ ID NO: 26, or in SEQ ID NO: 25 or SEQ ID NO: 26, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids, to yield native ORL 1. Natural DORs include, but are not limited to: SEQ ID NO: 27 and SEQ ID NO: 28, or in SEQ ID NO: 27 or SEQ ID NO: 28, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids. Natural KORs include, but are not limited to: SEQ ID NO: 29 and SEQ ID NO: 30, or in SEQ ID NO: 29 or SEQ ID NO: 30, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids. Natural MOR includes, but is not limited to: SEQ ID NO: 31, or in SEQ ID NO: 31, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids.

Natural galanin receptor 1 includes, but is not limited to: SEQ ID NO: 136. SEQ ID NO: 137 and SEQ ID NO: 138, or in SEQ ID NO: 136. SEQ ID NO: 137 or SEQ ID NO: 138, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids. Natural galanin receptor 2 includes, but is not limited to: SEQ ID NO: 139, or in SEQ ID NO: 139, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids. Natural galanin receptor 3 includes, but is not limited to: SEQ ID NO: 140, or in SEQ ID NO: 140, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids, to obtain the native galanin receptor 3.

The term "non-naturally targeted endopeptidase receptor variant" as used herein refers to any retargeted endopeptidase receptor produced by means of human manipulation or design, including but not limited to: retargeted endopeptidase receptors produced by genetic engineering using random mutagenesis or rational design, and retargeted endopeptidase receptors produced by chemical synthesis. Non-limiting examples of non-naturally retargeted endopeptidase receptor variants include, for example, conservative retargeted endopeptidase receptor variants, non-conservative retargeted endopeptidase receptor variants, chimeric variants of retargeted endopeptidase receptors, and active retargeted endopeptidase receptor fragments.

The term "non-natural retargeted endopeptidase receptor" as used herein refers to any retargeted endopeptidase receptor whose structure is modified by means of human manipulation, including but not limited to: retargeted endopeptidase receptors produced by genetic engineering using random mutagenesis or rational design, and retargeted endopeptidase receptors produced by in vitro chemical synthesis. The non-native retargeted endopeptidase receptor can be found in SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids.

Thus, in one embodiment, the retargeted endopeptidase receptor is a naturally retargeted endopeptidase receptor, such as ORL1, DOR, KOR, or MOR. In aspects of this embodiment, the retargeted endopeptidase receptor is a retargeted endopeptidase receptor isoform or a retargeted endopeptidase receptor subtype. In aspects of this embodiment, the natural retargeted endopeptidase receptor is SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 or SEQ ID NO: 31 to the endopeptidase receptor. In other aspects of this embodiment, the retargeted endopeptidase receptor is a peptide that hybridizes to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 or SEQ ID NO: 31, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In another embodiment, the retargeted endopeptidase receptor is a non-natural retargeted endopeptidase receptor, such as genetically engineered ORL1, genetically engineered DOR, genetically engineered KOR, or genetically engineered MOR. In other aspects of this embodiment, the retargeted endopeptidase receptor is a peptide that hybridizes to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 or SEQ ID NO: 31, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity. In other aspects of this embodiment, the retargeted endopeptidase receptor is a heavy chain variable region relative to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 or SEQ ID NO: 31, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions or additions. In other aspects of this embodiment, the retargeted endopeptidase receptor is a heavy chain variable region relative to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 or SEQ ID NO: 31, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more consecutive amino acid substitutions, deletions or additions.

In another embodiment, the retargeted endopeptidase receptor is a naturally retargeted endopeptidase receptor, such as galanin receptor 1, galanin receptor 2, or galanin receptor 3. In aspects of this embodiment, the retargeted endopeptidase receptor is a retargeted endopeptidase receptor isoform or a retargeted endopeptidase receptor subtype. In aspects of this embodiment, the natural retargeted endopeptidase receptor is SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 to the endopeptidase receptor. In other aspects of this embodiment, the retargeted endopeptidase receptor is a peptide that hybridizes to SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, for example, at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In another embodiment, the retargeted endopeptidase receptor is a non-natural retargeted endopeptidase receptor, such as genetically engineered galanin receptor 1, genetically engineered galanin receptor 2, or genetically engineered galanin receptor 3. In other aspects of this embodiment, the retargeted endopeptidase receptor is a peptide that hybridizes to SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity. In other aspects of this embodiment, the retargeted endopeptidase receptor is a heavy chain variable region relative to SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions or additions. In other aspects of this embodiment, the retargeted endopeptidase receptor is a heavy chain variable region relative to SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more consecutive amino acid substitutions, deletions or additions.

The retargeted endopeptidase receptor can be an endogenous retargeted endopeptidase receptor or an exogenous retargeted endopeptidase receptor. The term "endogenous retargeted endopeptidase receptor" as used herein refers to a naturally retargeted endopeptidase receptor that is present in a cell as it is naturally encoded in the genome of the cell, such that the cell inherently expresses the retargeted endopeptidase receptor without the need for an externally derived retargeted endopeptidase receptor or an externally derived genetic material encoding the retargeted endopeptidase receptor. Expression of endogenous retargeted endopeptidase receptors may or may not utilize environmental stimuli, such as cell differentiation or promoter activation. For example, the following established cell lines will express at least one endogenous retargeted endopeptidase receptor: AGN P33, Neuro-2a, SiMa and SK-N-DZ. Endogenous retargeted endopeptidase receptors can only be native retargeted endopeptidase receptors or native variants thereof.

The term "exogenous retargeted endopeptidase receptor" as used herein refers to a retargeted endopeptidase receptor that is expressed in a cell by introduction of an externally derived retargeted endopeptidase receptor or externally derived genetic material encoding a retargeted endopeptidase receptor using human manipulation. Expression of exogenous retargeted endopeptidase receptors may or may not utilize environmental stimuli, such as cell differentiation or promoter activation. In a non-limiting example, cells from an established cell line can express one or more exogenous retargeted endopeptidase receptors by transient or stable transfection of a polynucleotide molecule encoding a retargeted endopeptidase receptor, such as ORL1, DOR, KOR, MOR, galanin receptor 1, galanin receptor 2, or galanin receptor 3. In another non-limiting example, cells from established cell lines can express one or more exogenous retargeted endopeptidase receptors by transfection with proteins that retarget endopeptidase receptors, such as ORL1, DOR, KOR, MOR, galanin receptor 1, galanin receptor 2, or galanin receptor 3. The exogenous retargeted endopeptidase receptor can be a native retargeted endopeptidase receptor or a native variant thereof, or a non-native retargeted endopeptidase receptor or a non-native variant thereof.

Thus, in one embodiment, cells from established cell lines express endogenous retargeted endopeptidase receptors. In aspects of this embodiment, the endogenous retargeted endopeptidase receptor expressed by the cells from the established cell line is a native retargeted endopeptidase receptor. In other aspects of this embodiment, the endogenous retargeted endopeptidase receptor expressed by cells from an established cell line is SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140. in other aspects of this embodiment, the endogenous retargeted endopeptidase receptor expressed by cells from an established cell line is a naturally retargeted endopeptidase receptor, e.g., retargeted endopeptidase receptor isoform or retargeted endopeptidase receptor subtype. In other aspects of this embodiment, the endogenous retargeted endopeptidase receptor expressed by cells from an established cell line is a polypeptide that differs from SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, for example, at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In another embodiment, cells from established cell lines are transiently or stably engineered to express an exogenous retargeted endopeptidase receptor. In one aspect of this embodiment, cells from established cell lines are transiently or stably engineered to express a native retargeted endopeptidase receptor. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 to the endopeptidase receptor. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express a naturally retargeted endopeptidase receptor, e.g., retargeted endopeptidase receptor isoform or retargeted endopeptidase receptor subtype. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express an amino acid sequence identical to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, for example, at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity.

In another aspect of this embodiment, cells from established cell lines are transiently or stably engineered to express non-naturally retargeted endopeptidase receptors. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express an amino acid sequence identical to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express an amino acid sequence relative to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions or additions. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express an amino acid sequence relative to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 136. SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139 or SEQ ID NO: 140 have, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more consecutive amino acid substitutions, deletions or additions.

In another embodiment, cells from established cell lines are transiently or stably engineered to express exogenous ORL1, exogenous DOR, exogenous KOR, exogenous MOR, or any combination thereof. In aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express native ORL1, native DOR, native KOR, native MOR, or any combination thereof. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express non-native ORL1, non-native DOR, non-native KOR, non-native MOR, or any combination thereof. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express native ORL1 or non-native ORL1, native DOR or non-native DOR, native KOR or non-native KOR, native MOR or non-native MOR, or any combination thereof.

In another embodiment, cells from established cell lines are transiently or stably engineered to express exogenous galanin receptor 1, exogenous galanin receptor 2, exogenous galanin receptor 3, or any combination thereof. In aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express native galanin receptor 1, native galanin receptor 2, native galanin receptor 3, or any combination thereof. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express non-native galanin receptor 1, non-native galanin receptor 2, non-native galanin receptor 3, or any combination thereof. In other aspects of this embodiment, cells from established cell lines are transiently or stably engineered to express natural galanin receptor 1 or non-natural galanin receptor 1, natural galanin receptor 2 or non-natural galanin receptor 2, natural galanin receptor 3 or non-natural galanin receptor 3, or any combination thereof.

Cells expressing one or more endogenous or exogenous retargeted endopeptidase receptors can be identified by conventional methods including direct and indirect assays for retargeted endopeptidase uptake. Assays that determine the binding or uptake properties of a retargeted endopeptidase can be used to assess whether a cell expresses a retargeted endopeptidase receptor. These assays include, but are not limited to: crosslinking assays using labeled retargeted endopeptidases such as [125I ] retargeted endopeptidases, see, e.g., Noriko Yokosawa et al, Binding of Clostridium boutulinum type C neuroxin to differential neuroblastoma cell lines, 57(1) feed.Immun.272-277 (1989); noriko Yokoshawa et al, Binding of botulin type Cl, D and E neurotoxins to neural cell lines and syntosomes, 29(2) Toxicon261-264 (1991); and Tei-ichi Nishiki et al, Identification of protein receiver for Clostridium type B neurotoxin in rat synaptosomes, 269(14) J.biol.chem.10498-10503 (1994). Other non-limiting assays include immunocytochemical assays that use labeled or unlabeled antibodies to detect binding of heavy-target endopeptidases, see, e.g., Atsushi Nishikawa et al, The receiver and transporter for interaction of Clostridium type C promoter toxin to HT-29 cells, 319(2) biochem. Biophys. Res. Commun.327-333; and immunoprecipitation assays, see, e.g., Yukako Fujinaga et al, Molecular characterization of binding and ligands of Clostridium type C promoter toxin for intracellular epithelial cells and erythrocytes, 150(Pt 5) Microbiology 1529-1538(2004), which uses labeled or unlabeled antibodies to detect bound retargeted endopeptidases. Antibodies suitable for use in these assays include, but are not limited to: antibodies selected against a re-targeted endopeptidase and/or antibodies selected against a re-targeted endopeptidase receptor, such as ORL1, DOR, KOR, MOR, galanin receptor 1, galanin receptor 2, or galanin receptor 3. If the antibody is labeled, binding of the molecule can be detected by a variety of means, including western blot analysis, direct microscopic observation of antibody cell localization, measurement of cell or substrate bound antibody after washing steps, flow cytometry, electrophoresis, or capillary electrophoresis, using techniques well known to those skilled in the art. If the antibody is unlabeled, the bound molecule can be detected indirectly using a labeled secondary antibody, and the detection can be performed by reference to the labeled antibody. It will be appreciated that these and similar assays for determining the uptake characteristics or features of a retargeted endopeptidase can be used to identify cells that express an endogenous retargeted endopeptidase receptor or an exogenous or retargeted endopeptidase receptor.

Assays that monitor the release of molecules following exposure to a retargeted endopeptidase can also be used to assess whether a cell expresses one or more endogenous or exogenous retargeted endopeptidase receptors. In these assays, release of the molecule in cells expressing the retargeted endopeptidase receptor will be inhibited following treatment with the retargeted endopeptidase. Well known assays include methods of measuring the inhibition of radiolabeled catecholamine release (e.g., [3H ] norepinephrine or [3H ] dopamine release) in neurons, see, e.g., A Fasisio et al, Evidence for calcium-dependent vascular transmitter release sensitive to viral toxin and botulin toxin type F, 90(3) Neuroscience 893-902 (1999); and Sara Stigliani et al, The sensitivity of catheteramine release to botulin toxin C1 and E supensions selective targeting of visities set into The reactive releasable pool, 85(2) J.neurochem.409-421 (2003); or a method for measuring catecholamine release using a fluorometric procedure, see, e.g., Anton de Paiva et al, A role for the interaction of lipid derivatives or salts of particulate matters in the interaction of botulin A regenerated by a toxin derivative of lipids to ecto-receptors and inhibitors of transporter release intracellular, 268(28) J.biol.chem.20838-20844 (1993); gary W.Lawrence et al, Distingt exogenous responses of intact and transdermal collagen cells after clean of the 25-kDa synaptosomal-associated protein (SNAP-25) or synaptoblemin by boric toxin A or B, 236(3) Eur.J.biochem.877-886 (1996); and Patrick form et al, Botulinum neurotoxin C1 cleaves booth synthesis and SNAP-25 in intact and permeabilized chromaffins cells: correlation with its bits block ade of cathechoamine release, 35(8) Biochemistry 2630-2636 (1996). Other non-limiting examples include assays that measure inhibition of hormone release in endocrine cells such as anterior pituitary cells or ovarian cells. It will be appreciated that these and similar assays for the release of molecules can be used to identify cells expressing endogenous or exogenous retargeted or retargeted endopeptidase receptors.

Assays that detect cleavage of a SNAP-25 substrate following exposure to a retargeted endopeptidase can also be used to assess whether a cell expresses one or more endogenous or exogenous retargeted endopeptidase receptors. In these assays, the production of SNAP-25 cleavage products or the disappearance of intact SNAP-25 will be detected in cells expressing the retargeted endopeptidase receptor after treatment with the retargeted endopeptidase. Non-limiting examples of specific western blot analyses and well characterized reagents, conditions and protocols are readily available from commercial suppliers, including but not limited to: amersham Biosciences, Piscataway, NJ; Bio-Rad Laboratories, Hercules, Calif.; pierce Biotechnology, inc., Rockford, IL; promega Corporation, Madison, WI; and Stratagene, inc., La Jolla, CA. It will be appreciated that these and similar assays directed to cleavage of SNAP-25 can be used to identify cells expressing either an endogenous retargeted endopeptidase receptor or an exogenous retargeted endopeptidase receptor.

In a non-limiting example, uptake of a re-targeted endopeptidase can be determined using western blot analysis using antibodies that recognize the cleavage product of SNAP-25 or cleaved and uncleaved forms of SNAP-25. Examples of α -SNAP-25 antibodies suitable for use in these assays include, but are not limited to: SMI-81 α -SNAP-25 mouse monoclonal antibody (Sternberger Monoclonals Inc., Lutherville, Md.), CI 71.1 mouse α -SNAP-25 monoclonal antibody (synthetic Systems, Goettingen, Germany), CI 71.2 α -SNAP-25 mouse monoclonal antibody (synthetic Systems, Goettingen, Germany), SP12 α -SNAP-25 mouse monoclonal antibody (Abcam, Cambridge, MA), α -SNAP-25 rabbit polyclonal antiserum (synthetic Systems, Goettingen, Germany), α -SNAP-25 rabbit polyclonal antiserum S9684(Sigma, St. Louis, Mo.) and α -SNAP-25 rabbit polyclonal antiserum (Abcam, Cambridge, MA).

Aspects of the disclosure provide cells that are genetically manipulated or recombinantly engineered to express exogenous SNAP-25 and/or one or more exogenous retargeted endopeptidase receptors. Cells suitable for expression of exogenous SNAP-25 and/or one or more exogenous retargeted endopeptidase receptors by genetic manipulation or recombinant engineering include neuronal and non-neuronal cells that may or may not express endogenous SNAP-25 and/or one or more endogenous retargeted endopeptidase receptors. It will also be appreciated that these genetically or recombinantly engineered cells may express exogenous SNAP-25 and one or more exogenous retargeted endopeptidase receptors under the control of constitutive, tissue-specific, cell-specific, or inducible promoter elements, enhancer elements, or both. It will be appreciated that any cell can be used, provided that the cell is genetically or recombinantly engineered to express exogenous SNAP-25 and/or one or more exogenous retargeted endopeptidase receptors, and is capable of undergoing retargeted endopeptidase activity.

Suitable methods for introducing an exogenous polynucleotide molecule into a cell include, but are not limited to: chemical-mediated delivery methods, such as calcium phosphate-mediated delivery methods, Diethylaminoethyl (DEAE) dextran-mediated delivery methods, lipid-mediated delivery methods, Polyethyleneimine (PEI) -mediated delivery methods, polylysine-mediated delivery methods, and polybrene-mediated delivery methods; physically mediated delivery methods such as gene gun delivery, microinjection, protoplast fusion, and electroporation; and virus-mediated delivery methods, such as retrovirus-mediated transfection, see, e.g., Introducing Cloned Genes into Cultured Mammalian Cells, pages 16.1-16.62 (compiled by Sambrook and Russell, Molecular Cloning A Laboratory Manual, Vol.3, 3 rd edition, 2001); alessia Colosimo et al, Transfer and Expression of Foreign Genes in Mammalian Cells, 29(2) Biotechniques 314-318, 320-322, 324 (2000); philip Washbourne and A.Kimberley McAllist, Techniques for Gene Transfer into neurones, 12(5) curr. Opin. Neurobiol.566-573 (2002); and Current Protocols in Molecular Biology, John Wiley and Sons, pp 9.16.4-9.16.11 (2000), each of which is incorporated herein by reference in its entirety; the exogenous polynucleotide molecule in the present method will encode components necessary to subject the cell to the entire cellular machinery, thereby allowing proteolytic cleavage of a SNAP-25 substrate, such as SNAP-25, ORL1, DOR, KOR, or MOR, by a retargeted endopeptidase. Those skilled in the art will appreciate that the choice of a particular method of introducing a polynucleotide molecule into a cell will depend in part on whether the cell temporarily or stably contains components necessary to subject the cell to the entire cellular machinery, thereby allowing the retargeted endopeptidase to proteolytically cleave the SNAP-25 substrate. Non-limiting examples of polynucleotide molecules encoding components required to subject a cell to the entire cellular machinery, thereby allowing the re-targeted intein to proteolytically cleave the SNAP-25 substrate by an enzyme, are as follows: ORL1 polynucleotide molecule SEQ ID NO: 61 or SEQ ID NO: 62, a first step of mixing; DOR polynucleotide molecule SEQ ID NO: 63 or SEQ ID NO: 64; KOR polynucleotide molecule SEQ ID NO: 65 or SEQ ID NO: 66; MOR polynucleotide molecule SEQ ID NO: 67; galanin receptor 1 polynucleotide molecule SEQ ID NO: 141. SEQ ID NO: 142 or SEQ ID NO: 143; galanin receptor 2 polynucleotide molecule SEQ ID NO: 144 or galanin receptor 3 polynucleotide molecule SEQ ID NO: 145; and SNAP-25 polynucleotide molecule SEQ ID NO: 68 or SEQ ID NO: 69.

Chemical-mediated delivery Methods are well known to those skilled in the art and are described, for example, in Martin Jordan and Florian word, transformation of additive and Suspended Cells by Calcium Phosphate, 33(2) Methods 136-143 (2004); chun Zhang et al, polyethylene strands for Plasmid Delivery to Brain-delivered Cells, 33(2) Methods 144-150(2004), the entire contents of each of which are hereby incorporated by reference. These chemical-mediated delivery methods can be prepared by standard procedures and are commercially available, see, e.g., CellPhect transfection kit (Amersham Biosciences, Piscataway, N.J.); mammalian transfection kit, calcium phosphate and DEAE dextran (Stratagene, inc., La Jolla, CA); lipofectamine^TMTransfection reagents (Invitrogen, inc., Carlsbad, CA); the ExGen 500 transfection kit (Fermentas, inc., Hanover, MD); and SuperFect and efectene transfection kit (Qiagen, inc., Valencia, CA).

Physically Mediated delivery methods are well known to those of ordinary skill in the art and are described, for example, in Jeike E.Biewenga et al, Plasmid-Mediated Gene Transfer in nerves using the Biolistics Technique, 71(1) J.Neurosci.methods.67-75 (1997); john O' Brien and Sarah c.r.lummis, Biolistic and Biolistic transmission: using the Gene Gun to Deliver DNA and Lipophilic Dyes into Mammarian Cells, 33(2) Methods 121-125 (2004); golzio et al, In Vitro Electric Field-Mediated recommendation, Gene Transfer, and Expression, 33(2) Methods, 126-135 (2004); and Oliver Gresch et al, New Non-Viral Method for Gene Transfer in Primary Cells, 33(2) Methods, 151-163(2004), the entire contents of each of which are hereby incorporated by reference.

Methods of Virus-mediated delivery are well known to those of ordinary skill in the art and are described, for example, in Chooi M.Lai et al, Adenoviral and Adeno-Associated Virus Vectors, 21(12) DNA Cell biol.895-913 (2002); ilya Frolov et al, alphaviruses-Based Expression Vectors: strategies and Applications, 93(21) proc.Natl.Acad.Sci.U.S.A.11371-11377 (1996); roland Wolwowicz et al, Lentiviral Vectors for the Delivery of DNA to Mammarian Cells, 246, Methods mol. biol.391-411 (2004); huser and c.hofmann, baculoviral Vectors: novel Mammarian Cell Gene-Delivery Vehicles and Their Applications, 3(1) am. J. Pharmacogenerics 53-63 (2003); tiziana Tonnii et al, Transmission Production of Retroviral-and Lentiviral-Based Vectors for the transformation of Mammarian Cells, 285, Methods mol. biol.141-148 (2004); manfred Gossen and Hermann Bujard, light Control of Gene Expression in Eukaryotic Cells by reactive Promoters, U.S. Pat. No. 5,464,758; hermann Bujard and Manfred Gossen, Methods for Regulating Gene Expression, U.S. Pat. No. 5,814,618; david s.hogness, polynucleotide Encoding instruments gradient host receptors and Cells Transformed With Same, U.S. patent No. 5,514,578; hogness, polynuceotides Encoding instrument Ecdysone Receptor, U.S. patent 6,245,531; elisabeta Vegeto et al, Progesterone Receptor Having C.terminal Hormonebinding Domain clones, U.S. Pat. No. 5,364,791; elisabeta Vegeto et al, modified Steroid Hormone Receptors, Methods for the Use of same and Molecular Switch for Gene Therapy, U.S. Pat. No. 5,874,534, the entire contents of each of which are hereby incorporated by reference. These virus-mediated delivery methods can be prepared by standard procedures and are commercially available, see, e.g., ViraPower ^TMAdenovirus expression systems (Invitrogen, Inc., Carlsbad, Calif.) and ViraPower^TMAdenoviral expression System instructions 25-0543Version a, Invitrogen inc, (7/15/2002); and AdEasy adenovirus vector systems (Stratagene, Inc., La Jolla, Calif.) and AdEasy^TMThe adenovirus vector system was described in 064004f, Stratagene, Inc. In addition, these viral delivery systems can also be prepared by standard methods and are commercially available, see, e.g., BD^TMThe Tet-Off and Tet-On gene expression systems (BD Biosciences-Clonetech, Palo Alto, CA) and BD^TMThe use of the Tet-Off and Tet-On Gene expression systems is described by PT3001-1, BD Biosciences Clonetech (3.3.14.2003), GeneSwitch^TMSystems (Invitrogen, Inc., Carlsbad, Calif.) and GeneSwitch^TMSystem a mifepristone regulated mammalian cell expression system version D, 25-0313, invitrogen inc, (11/4/2002); ViraPower^TMLentiviral expression System (Invitrogen, Inc., Carlsbad, Calif.) and ViraPower^TMLentiviral expression System instructions 25-0501 version E, Invitrogen Inc. (12/8/2003); and Complete ControlRetrovirus induces mammalian expression System (Stratagene, La Jolla, Calif.) and Complete Control Instructions for the use of the retroviral inducible mammalian expression system, 064005 e.

Thus, in one embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity transiently contain polynucleotide molecules that encode components necessary to subject the cells to the entire cellular machinery, thereby allowing the retargeted endopeptidase to proteolytically cleave the SNAP-25 substrate. In another embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain polynucleotide molecules encoding the components required to subject the cells to the entire cellular machinery, thereby allowing proteolytic cleavage of the SNAP-25 substrate by the retargeted endopeptidase. In aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain a polynucleotide molecule encoding ORL1, DOR, KOR, MOR, or SNAP-25. In aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding ORL1 SEQ ID NO: 61 or SEQ ID NO: 62. in other aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding DOR of SEQ ID NO: 63 or SEQ ID NO: 64. in other aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding KOR of SEQ ID NO: 65 or SEQ ID NO: 66. in other aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding MOR of SEQ ID NO: 67.

In other aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding galanin receptor 1 of SEQ ID NO: 141. SEQ ID NO: 142 or SEQ ID NO: 143. in other aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding galanin receptor 2 of SEQ ID NO: 144. in other aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding galanin receptor 3 SEQ ID NO: 145. in other aspects of this embodiment, cells from established cell lines that are susceptible to retargeted endopeptidase activity transiently contain the polynucleotide molecule encoding SNAP-25 SEQ ID NO: 68 or SEQ ID NO: 69.

in another embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain polynucleotide molecules that encode components necessary to subject the cells to the entire cellular machinery, thereby allowing proteolytic cleavage of the SNAP-25 substrate by a retargeted endopeptidase. In another embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain polynucleotide molecules that encode multiple components required to subject the cells to the entire cellular machinery, thereby allowing proteolytic cleavage of the SNAP-25 substrate by a retargeted endopeptidase. In aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain a polynucleotide molecule encoding ORL1, DOR, KOR, MOR, or SNAP-25. In aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain the polynucleotide molecule encoding ORL1 SEQ ID NO: 61 or SEQ ID NO: 62. in other aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain a polynucleotide molecule encoding a DOR of SEQ ID NO: 63 or SEQ ID NO: 64. in other aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain a polynucleotide molecule encoding a KOR of SEQ ID NO: 65 or SEQ ID NO: 66. in other aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain a polynucleotide molecule encoding MOR of SEQ ID NO: 67.

In other aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain the polynucleotide molecule encoding galanin receptor 1 of SEQ ID NO: 141. SEQ ID NO: 142 or SEQ ID NO: 143. in other aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain the polynucleotide molecule encoding galanin receptor 2 of SEQ ID NO: 144. in other aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain the polynucleotide molecule encoding galanin receptor 3 of SEQ ID NO: 145. in other aspects of this embodiment, cells from established cell lines that are sensitive to retargeted endopeptidase activity stably contain the polynucleotide molecule encoding SNAP-25 SEQ ID NO: 68 or SEQ ID NO: 69.

as mentioned above, exogenous components disclosed in the present specification that are required to subject the cell to the entire cellular machinery, thereby allowing the retargeted endopeptidase to proteolytically cleave a SNAP-25 substrate such as SNAP-25, ORL1, DOR, KOR, MOR, galanin receptor 1, galanin receptor 2, or galanin receptor 3, can be introduced into the cell. Any and all methods for introducing such exogenous components into a cell population using a delivery agent can be used, provided that the method can temporarily introduce the exogenous components disclosed in this specification into at least 50% of the cells within a given cell population. Thus, aspects of this embodiment can include a population of cells wherein, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a given population of cells transiently contain the exogenous components disclosed in the present specification necessary to subject the cells to the entire cellular machinery, thereby allowing retargeted endopeptidases to proteolytically cleave a SNAP-25 substrate such as SNAP-25, ORL1, DOR, KOR, MOR, galanin receptor 1, galanin receptor 2, or galanin receptor 3. The term "delivery agent" as used herein refers to any molecule capable of effecting or increasing internalization of a covalently linked, non-covalently linked or in any other way associated polypeptide into a cell. Thus, the term "delivery agent" encompasses, but is not limited to, proteins, peptides, peptidomimetics, small molecules, polynucleotide molecules, liposomes, lipids, viruses, retroviruses, and cells that transport covalently or non-covalently linked molecules to the cell membrane, cytoplasm, or nucleus. It is also to be understood that the term "delivery agent" encompasses molecules internalized by any mechanism, including delivery agents that act by receptor-mediated endocytosis, as well as delivery agents that do not rely on receptor-mediated endocytosis.

The delivery agent may also be an agent that enables or increases cellular uptake of covalently linked components such as SNAP-25, ORL1, DOR, KOR, MOR, galanin receptor 1, galanin receptor 2, or galanin receptor 3, for example, by chemical conjugation or fusion proteins produced by genetic methods. Methods of covalently linking delivery agents and methods of using these agents are described, for example, in the following patent documents: down F, Protein Transduction System and Methods of Use Thereof, International publication No. WO 00/34308; g rard Chassaging and Alain Prochiantz, Peptides while can be Used as Vectors for the interferometric Addressing of Active Molecules, U.S. Pat. No. 6,080,724; alan Frankel et al, Fusion Protein sharing TAT-derived Transport Moiert, U.S. Pat. No. 5,674,980; alan Frankel et al, TAT-derived Transport Polypeptide Conjugates, U.S. Pat. No. 5,747,641; alan Frankel et al, TAT-derived Transport Polypeptides and Fusion Proteins, U.S. Pat. No. 5,804,604; peter F.J.O' Hare et al, Use of Transport Proteins, U.S. Pat. No. 6,734,167; Yao-Zhong Lin and Jack J.Hawiger, Method for imaging biological Active Molecules in Cells, U.S. Pat. No. 5,807,746; Yao-Zhong Lin and Jack J.Hawiger, Method for imaging biological Active Cells in Cells, U.S. Pat. No. 6,043,339; Yao-Zhong Lin et al, Sequence and Method for Genetic Engineering of Proteins with Cell Membrane relocation Activity, U.S. Pat. No. 6,248,558; Yao-Zhong Lin et al, Sequence and Method for Genetic Engineering of Proteins with Cell Membrane relocation Activity, U.S. Pat. No. 6,432,680; hawiger et al, Method for imaging biological Active Molecules in Cells, U.S. Pat. No. 6,495,518; Yao-Zhong Lin et al, Sequence and Method for Genetic Engineering of Proteins with Cell Membrane relocation Activity, U.S. Pat. No. 6,780,843; jonathan B.Rothbard and Paul A Wender, Method and Composition for Enhancing Transport Across Biological Membranes, U.S. Pat. No. 6,306,993; jonathan B.Rothbard and Paul A Wender, Method and Composition for Enhancing Transport Across Biological Membranes, U.S. Pat. No. 6,495,663; and Pamela b. davis et al, Fusion Proteins for Protein Delivery, U.S. patent No. 6,287,817, each of which is incorporated herein by reference in its entirety.

The delivery agent may also be an agent that enables or increases cellular uptake of non-covalently associated components such as SNAP-25, ORL1, DOR, KOR, MOR, galanin receptor 1, galanin receptor 2, or galanin receptor 3. Acting in the absence of covalent bondsMethods of (a) and methods of use of these agents are described, for example, in the following documents: gilles Divita et al, Peptide-Mediated transformation Agents and Methods of Use, U.S. Pat. No. 6,841,535; philip L Felgner and Olivier Zellphati, Intracellular Protein Delivery composites and Methods of Use, U.S. patent publication No. 2003/0008813; and Michael Karas, Intracellular Delivery of Small Molecules, Proteins and Nucleic Acids, U.S. patent publication 2004/0209797, the entire contents of each of which are incorporated herein by reference. These peptide delivery agents can be prepared and used by standard methods and are commercially available, see, e.g., CHARIOT^TMReagents (Active Motif, Carlsbad, CA); BIO-PORTERReagents (Gene Therapy Systems, Inc., San Diego, Calif.), BIO TREK^TMProtein delivery agent (Stratagene, La Jolla, Calif.) and PRO-JECT ^TMProtein transfection reagents (Pierce Biotechnology inc., Rockford, IL).

Aspects of the invention include, in part, a sample comprising a retargeted endopeptidase. The term "sample containing a retargeted endopeptidase" as used herein refers to any biological substance that contains or may contain an active retargeted endopeptidase. According to the methods disclosed herein, a variety of samples can be assayed, including but not limited to: purified, partially purified or unpurified retargeted endopeptidase; a recombinant single-or double-stranded retargeted endopeptidase having a native or non-native sequence; a recombinant retargeted endopeptidase with modified protease specificity; recombinant retargeted endopeptidases with altered cell specificity; a plurality of heavy-target endopeptidases; a formulated retargeted endopeptidase product; and for example cells of bacterial, yeast, insect or mammalian origin or crude, fractionated or partially purified cell lysates; blood, plasma or serum; uncooked, semi-cooked, cooked or processed foods; a beverage; animal feed; a soil sample; a water sample; pond bottom mud; a lotion; a cosmetic; and clinical preparations. It is understood that the term sample encompasses tissue samples, including but not limited to: mammalian tissue samples, livestock tissue samples, such as tissue samples of sheep, cows, and pigs; tissue samples of primates; and human tissue samples. These samples encompass, but are not limited to: intestinal samples, such as infant intestinal samples, and tissue samples obtained from wounds. In non-limiting examples, methods of detecting picomolar amounts of retargeted endopeptidase activity can be used to determine the presence or activity of a retargeted endopeptidase in a food or beverage sample, assaying a sample of a human or animal exposed to a retargeted endopeptidase or exhibiting one or more symptoms of botulism, tracking activity during production and purification of a large number of retargeted endopeptidases, assaying a formulated retargeted endopeptidase product for use in pharmaceutical or cosmetic applications, or assaying the presence or absence of neutralizing alpha-retargeted endopeptidase antibodies in the serum of a subject.

Thus, in one embodiment, the sample containing the retargeted endopeptidase is a sample containing any amount of retargeted endopeptidase. In aspects of this embodiment, the sample containing the retargeted endopeptidase comprises about 100ng or less, about 10ng or less, about 1ng or less, about 100pg or less, about 10pg or less, or about 1pg or less of the retargeted endopeptidase. In other aspects of this embodiment, the sample containing the retargeted endopeptidase comprises about 1 μ M or less, about 100nM or less, about 10nM or less, about 1nM or less of the retargeted endopeptidase.

Aspects of the invention include, in part, isolating from the treated cells a P comprising a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 component of SNAP-25 having a carboxyl terminus at the residue. The term "P comprising a bond susceptible to cleavage at BoNT/A" as used herein₁"SNAP-25 fraction of SNAP-25 having a carboxyl terminus at the residue" refers to a fraction of cells containing a cleavage product of SNAP-25. It is contemplated that any method suitable for enriching or isolating SNAP-25 components may be used, including but not limited to: cell lysis protocol, spin column purification protocol, immunoprecipitation method, affinity purification method and protein chromatography.

Aspects of the disclosure include, in part, an a-SNAP-25 antibody linked to a solid support. The term "solid phase support" is used herein synonymously with "solid phase" and means any matrix that can be used to immobilize the α -SNAP-25 antibody disclosed in the present specification. Non-limiting examples of solid supports include, for example: a test tube; a plate; a column; needle or "dipstick"; magnetic particles, beads or other spherical or fibrous chromatographic media, such as agarose, sepharose, silica and plastics; and sheets or films such as nitrocellulose and polyvinylidene fluoride (PVDF). The solid support can be constructed using a variety of materials such as glass, carbon, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, nylon, diazocellulose, or starch. The physical properties of the solid support are selected such that it is readily separable from soluble or unbound materials, and typically such that unbound materials, e.g., excess reagents, reaction by-products, or solvents, can be separated or otherwise removed (e.g., by washing, filtering, centrifuging, etc.) from the assay components bound to the solid support. Non-limiting examples of methods of making and using solid supports are described in, for example: molecular Cloning, A Laboratory Manual, supra, (2001); and Current Protocols in Molecular Biology, supra, (2004), the entire contents of each document are hereby incorporated by reference herein.

Aspects of the invention include, in part, detecting the presence of an antibody-antigen complex comprising P selectively bound to a readily cleavable bond at the BoNT/A cleavage site₁alpha-SNAP-25 antibody having a carboxy-terminal SNAP-25 epitope at residue and P having a bond readily cleavable at the BoNT/A cleavage site₁SNAP-25 cleavage product with a carboxyl terminus at the residue. It is contemplated that any detection system may be used to practice aspects of the disclosed immunity-based methods, provided that the signal-to-noise ratio is statistically significant to distinguish the signal from the antibody-antigen complex from the background signal. Non-limiting examples of immune-based detection systems include immunoassays, such as western blotting and dot-blotting (dot-blotting), immunoblottingImmunoprecipitation assays, enzyme-linked immunosorbent assays (ELISA) and sandwich ELISA. Detection of the signal may be achieved using Autoradiography (AU), Chemiluminescence (CL), Electrochemiluminescence (ECL), Bioluminescence (BL), fluorescence, resonance energy transfer, planar polarization, colorimetry, or Flow Cytometry (FC) coupled with imaging or photoimaging. Descriptions of immune-based detection systems are disclosed, for example, in the following documents: michael M. Rauhut, Chemimunescence, Kirk-Othmer convention Encyclopedia of Chemical Technology (Grayson, 3 rd edition, John Wiley and Sons, 1985); knight, A Review of Recent Trends in Analytical Applications of Electrogenetically regulated Chemicals, Trends Anal chem.18 (1): 47-62 (1999); fahnrich, et al, Recent Applications of Electrogenetically regulated Chemical Analysis, Talanta 54 (4): 531-559 (2001); common Used Techniques in Molecular Cloning, pages A8.1-A8-55 (compiled by Sambrook and Russell, Molecular Cloning A Laboratory Manual, Vol.3, 3 rd edition, 2001); detection Systems, pages A9.1-A9-49 (compiled by Sambrook and Russell, Molecular Cloning A Laboratory Manual, Vol.3, 3 rd edition, 2001); electrogenic cheminescence (Allen j. bard, Marcel Dekker inc., 2004), the entire contents of each of which are hereby incorporated by reference.

Sandwich ELISA (or sandwich immunoassay) is a method based on two antibodies that will bind different epitopes on an antigen. Capture antibodies with high binding specificity for the antigen of interest are bound to a solid surface. The antigen is then added followed by a second antibody called the detection antibody. The detection antibody binds to a different epitope on the antigen than the capture antibody. The antigen is thus "sandwiched" between two antibodies. The binding affinity of an antibody to an antigen is often a major determinant of immunoassay sensitivity. As the antigen concentration increases, the amount of detection antibody also increases, thereby producing a higher measurement response. To quantify the degree of binding, different reporter systems may be used, such as an enzyme linked to a secondary antibody, and a reporter substrate that forms a readout in the enzymatic response that is taken as the detection signal. The signal generated is proportional to the amount of target antigen present in the sample. The reporter substrate used to measure the binding event will determine the detection mode. A reader plate measuring spectral photometry is used for colorimetric detection. Chemiluminescent and electrochemiluminescent substrates have been developed that further broaden the signal and can be read out on luminescence readers. The reporter may also be a fluorescence reader, wherein the enzymatic step of the assay is replaced with a fluorophore, and the readout signal is subsequently measured using the fluorescence reader. Reagents and protocols required for performing ECL sandwich ELISA are commercially available and include, but are not limited to: MSD sandwich ELISA-ECL assay platform (Meso Scale Discovery, Gaithersburg, Md.).

Thus, in one embodiment, immunoblot analysis, immunoprecipitation analysis, ELISA, or sandwich ELISA can be used to detect the presence of an antibody-antigen complex comprising P selectively bound to a readily cleavable bond at the BoNT/A cleavage site₁alpha-SNAP-25 antibody having a carboxyl-terminal SNAP-25 epitope at residue, and P having a bond that is readily cleavable at the BoNT/A cleavage site₁SNAP-25 cleavage product with a carboxyl terminus at the residue. In aspects of this embodiment, the detecting is by AU, CL, ECL, or BL immunoblot analysis; AU, CL, ECL, BL or FC immunoprecipitation assays; AU, CL, ECL, BL or FC ELISA; or AU, CL, ECL, BL or FC sandwich ELISA.

Aspects of the disclosure may be practiced in a single or multiple manner. An immune-based method for detecting heavy-target endopeptidase activity carried out in a singleplex format is one that can only detect P that contains the alpha-SNAP-25 antibody and a bond that is readily cleavable at the BoNT/A cleavage site₁A method of presence of an antibody-antigen complex of SNAP-25 cleavage product having a carboxyl terminus at a residue. An immune-based method for detecting retargeted endopeptidase activity that is performed in a multiplex format is a method of simultaneously detecting the presence of two or more antibody-antigen complexes; wherein an antibody-antigen complex comprises an alpha-SNAP-25 antibody and P with bond cleavage site susceptible to BoNT/A₁An antibody-antigen complex of SNAP-25 cleavage product having a carboxy terminus at residue; and the other (other) antibody-antigen complex is an antibody-antigen complex directed against a second, third, fourth, etc. different protein. The second protein can, for example, serve as an internal control to minimize sample-to-sample variation by normalizing the amount of alpha-SNAP-25/SNAP-25 antibody-antigen complex detected against the amount of antibody-antigen complex detected against the second protein. Thus, the second protein is typically a protein that is consistently expressed by the cell, such as a housekeeping protein. Non-limiting examples of suitable second proteins include, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), syntaxin, cytokines. Methods for performing immune-based assays in multiplex format are described, for example, in the following documents: nielsen and b.h. geierstanger, Multiplexed Sandwich Assays in Microarray Format, j.immunol.methods.290 (1-2): 107-1202004); barry and M, Soloviev, Quantitative Protein Profiling using Antibody Arrays, Proteomics, 4 (12): 3717-3726 (2004); M.Ling et al, Multiplexing Molecular Diagnostics and Immunoassassays using engineering Microarray Technologies, Expert Rev Mol diagnostics.7 (1): 87-98 (2007); S.X.Leng et al, ELISA and Multiplex Technologies for Cytokine Measurement in infection and Aging Research, J Gerontol A Biol Sci Med Sci.63 (8): 879- > 884(2008), the entire contents of each of which are hereby incorporated by reference.

Thus, in one embodiment, an immune-based method for detecting heavy-targeted endopeptidase activity performed in a singleplex format detects only P comprising an α -SNAP-25 antibody and a bond that is readily cleavable at the BoNT/A cleavage site₁Presence of an antibody-antigen complex of SNAP-25 cleavage product with a carboxyl terminus at residue. In another embodiment, an immune-based method for detecting retargeted endopeptidase activity performed in a multiplex format simultaneously detects P comprising an alpha-SNAP-25 antibody and a bond that is readily cleavable at the BoNT/A cleavage site₁Having a carboxyl terminus at the residueSNAP-25 cleavage products and at least one other antibody-antigen complex directed against a protein other than SNAP-25 (e.g., GAPDH or syntaxin) are present.

Aspects of the disclosure provide, in part, methods for determining heavy-target endopeptidase immune resistance. The term "retargeted endopeptidase immune resistance" as used herein refers to a reduction in the efficacy of retargeted endopeptidase therapy, either directly or indirectly, due to the immune response of the mammal, such that the mammal does not respond adequately to, or exhibits a reduced beneficial effect of, retargeted endopeptidase therapy. A non-limiting example of reduced efficacy would be in a mammal having at least one neutralizing a-retargeted endopeptidase antibody that binds to a retargeted endopeptidase in a manner that reduces or prevents the specificity or activity of the retargeted endopeptidase. The term "retargeted endopeptidase therapy" as used herein refers to a drug, formulation or mixture that treats, ameliorates, cures, heals, rehabilitates or in any other way, against an undesirable event occurring in a mammal in need of neuromodulation using retargeted endopeptidases, or that administers to the mammal one or more controlled doses of retargeted endopeptidases having a medical, therapeutic, curative, cosmetic, remedial and any other beneficial effect. Retargeted endopeptidase therapy encompasses, but is not limited to: any formulation of any carrier or active ingredient is combined with any naturally occurring or modified fragment thereof and administered by any route of administration.

Aspects of the present disclosure provide, in part, a test sample obtained from a mammal for testing for the presence or absence of alpha-retargeted endopeptidase neutralizing antibodies. The term "test sample" as used herein refers to any biological substance that contains or may contain at least one alpha-retargeted endopeptidase antibody. The alpha-retargeted endopeptidase antibody can be a neutralizing alpha-retargeted endopeptidase antibody or a non-neutralizing alpha-retargeted endopeptidase antibody. The term "neutralizing a-retargeted endopeptidase antibody" as used herein refers to any a-retargeted endopeptidase antibody that will bind to a region of a retargeted endopeptidase under physiological conditions in a manner that reduces or prevents the retargeted endopeptidase from exerting its role in retargeted endopeptidase therapy. The term "non-neutralizing alpha-retargeted endopeptidase antibody" as used herein refers to any alpha-retargeted endopeptidase antibody that will bind to a region of a retargeted endopeptidase under physiological conditions, but does not prevent the retargeted endopeptidase from exerting its role in retargeted endopeptidase therapy. It is contemplated that any and all samples that may contain alpha-retargeted endopeptidase antibodies may be used in the present methods, including but not limited to: blood, plasma, serum and lymph. In addition, any and all organisms capable of producing alpha-retargeted endopeptidase antibodies to retargeted endopeptidases can be used as a source of samples, including but not limited to: birds and mammals, including mice, rats, goats, sheep, horses, donkeys, cows, primates and humans. Non-limiting examples of specific protocols for blood collection and serum preparation are described, for example, in the following documents: marjire Schaub Di Lorenzo and Susan King Strasinger, Blood Collection in Healthcare (F.A. Davis Company, 2001); and Diana Garza and Kathleen Becan-McBride, Phlebotomy Handbook: blood Collection essences (Prentice Hall, 6 th edition, 2002). These protocols are within the skill of those in the art and are routine procedures taught herein. The test sample may be obtained prior to exposure of the organism to the retargeted endopeptidase, after a single retargeted endopeptidase treatment, after multiple retargeted endopeptidase treatments, before resistance to the retargeted endopeptidase therapy occurs, or after resistance to the retargeted endopeptidase therapy occurs.

Aspects of the disclosure provide, in part, control samples. The term "control sample" as used herein refers to any sample known to be present or absent from a test sample, and includes negative control samples and positive control samples. For neutralizing alpha-retargeted endopeptidase antibodies, negative control samples may be obtained from individuals that have not been exposed to retargeted endopeptidase, and may include, but are not limited to: a sample taken from the same individual as the test sample provided prior to undergoing the retargeted endopeptidase treatment; samples taken from different individuals who have not been exposed to retargeted endopeptidases; pooled samples taken from multiple different individuals who had not been exposed to BoNT/A. For neutralizing alpha-retargeted endopeptidase antibodies, positive control samples can be obtained from individuals exhibiting resistance to retargeted endopeptidase immunity and include, but are not limited to: testing individuals that are positive in a patient-based test assay; testing individuals who are positive in an in vivo bioassay; and individuals exhibiting high immunity, such as individuals vaccinated with retargeted endopeptidases.

It is also contemplated that the alpha-retargeted endopeptidase antibody can be purified from the sample. Alpha-retargeted endopeptidase antibodies can be purified from a sample using a variety of procedures including, but not limited to: protein A/G chromatography and affinity chromatography. Non-limiting examples of specific protocols for purifying antibodies in a sample are described, for example, in the following documents: ANTIBODIES: a LABORATORY MANUAL (eds. Edward Harlow and David Lane, Cold Spring Harbor LABORATORY Press, 2 nd edition, 1998); USING ANTIBODIES: a laboradry MANUAL: PORTABLE PROTOCOL No. I (Edward Harlow and David Lane, Cold Spring Harbor Laboratory Press, 1998); and MOLECULAR CLONING, a laborary MANUAL, supra, (2001), which are hereby incorporated by reference. Furthermore, non-limiting examples of antibody purification methods and well-characterized reagents, conditions and protocols are readily available from commercial suppliers, including, but not limited to: pierce Biotechnology inc., Rockford, IL); and Zymed Laboratories inc, South San Francisco, CA. These protocols are routine procedures within the skill of those in the art.

Thus, in one embodiment, the sample comprises blood. In one aspect of this embodiment, the sample comprises mouse blood, rat blood, goat blood, sheep blood, horse blood, donkey blood, cow blood, primate blood, or human blood. In another embodiment, the sample comprises plasma. In one aspect of this embodiment, the test sample comprises mouse plasma, rat plasma, goat plasma, sheep plasma, horse plasma, donkey plasma, cow plasma, primate plasma, or human plasma. In another embodiment, the sample comprises serum. In one aspect of this embodiment, the sample comprises mouse serum, rat serum, goat serum, sheep serum, horse serum, donkey serum, cow serum, primate serum, or human serum. In another embodiment, the sample comprises lymph fluid. In one aspect of this embodiment, the sample comprises mouse lymph fluid, rat lymph fluid, goat lymph fluid, sheep lymph fluid, horse lymph fluid, donkey lymph fluid, cow lymph fluid, primate lymph fluid, or human lymph fluid. In another embodiment, the sample is a test sample. In another embodiment, the sample is a control sample. In aspects of this embodiment, the control sample is a negative control sample or a positive control sample,

Aspects of the invention provide, in part, P detected in step (d) as a bond susceptible to cleavage at a BoNT/A cleavage site₁The amount of SNAP-25 having a carboxyl terminus at the residue is related to the amount of P detected in step (e) as a bond susceptible to cleavage at a BoNT/A cleavage site₁Comparison of the amount of SNAP-25 having a carboxyl terminus at the residue. In one embodiment, the amount of SNAP-25 cleavage product in the test sample is greater than the amount of SNAP-25 cleavage product in the control sample. In one aspect of this embodiment, an amount of SNAP-25 cleavage product in the test sample that is greater than the positive control sample indicates that the mammal has less resistance or lacks resistance to retargeted endopeptidase immunity. In another aspect of this embodiment, the amount of SNAP-25 cleavage product in the test sample compared to the negative control sample indicates that the mammal has a lower or lack of resistance to retargeted endopeptidase immunity. In another embodiment, the amount of SNAP-25 cleavage product in the test sample is less than the amount of SNAP-25 cleavage product in the control sample. In one aspect of this embodiment, a lower or comparable amount of SNAP-25 cleavage product in the test sample as compared to the positive control sample will indicate that the mammal has increased or is otherwise resistant to retargeted endopeptidase immunity. In another aspect of this embodiment, a lower amount of SNAP-25 cleavage product in the test sample than in the negative control sample indicates that the mammal has a higher or present resistance to retargeted endopeptidase immunity.

It is contemplated that any and all assay conditions suitable for detecting the presence of neutralizing alpha-retargeted endopeptidase antibodies in a sample can be used in the methods disclosed herein, e.g., linear assay conditions and non-linear assay conditions. In one embodiment, these analytical determinations are linear. In one aspect of this embodiment, the measured amount of retargeted endopeptidase is in excess. In another aspect of this embodiment, the measured amount of retargeted endopeptidase is rate-limiting. In another aspect of this embodiment, the measured amount of the test sample is rate-limiting.

Aspects of the disclosure may also be described as follows:

1. a method for detecting retargeted endopeptidase activity, the method comprising the steps of: a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line are sensitive to retargeted endopeptidase activity of the retargeted endopeptidase; b) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; c) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in the SNAP-25 cleavage product that is susceptible to bond cleavage at the BoNT/A cleavage site ₁An epitope comprising the carboxy terminus at residue; and d) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; wherein detection of the antibody-antigen complex will indicate retargeted endopeptidase activity.

2. A method for detecting retargeted endopeptidase activity, the method comprising the steps of: a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line are sensitive to retargeted endopeptidase activity of the retargeted endopeptidase; b) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; c) contacting the SNAP-25 component with an alpha-SNAP-25 antibody linked to a solid support, wherein the alpha-SNAP-25 antibodyP-binding to bond susceptible to cleavage at BoNT/A cleavage site in SNAP-25 cleavage product₁An epitope comprising the carboxy terminus at residue; and d) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; wherein detection of the antibody-antigen complex will indicate retargeted endopeptidase activity.

3. A method for detecting retargeted endopeptidase activity, the method comprising the steps of: a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line are sensitive to retargeted endopeptidase activity of the retargeted endopeptidase; b) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; c) immobilizing the SNAP-25 component to a solid support; d) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in the SNAP-25 cleavage product that is susceptible to bond cleavage at the BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; and e) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; wherein detection of the antibody-antigen complex will indicate retargeted endopeptidase activity.

4. A method for detecting retargeted endopeptidase activity, the method comprising the steps of: a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line can take up the retargeted endopeptidase; b) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; c) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in the SNAP-25 cleavage product that is susceptible to bond cleavage at the BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; and d) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; wherein the detection of the antibody-antigen complex will refer toIndicating retargeted endopeptidase activity.

5. A method for detecting retargeted endopeptidase activity, the method comprising the steps of: a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line can take up the retargeted endopeptidase; b) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; c) contacting the SNAP-25 component with an alpha-SNAP-25 antibody attached to a solid support, wherein the alpha-SNAP-25 antibody binds to P in a cleavage product of SNAP-25 that is susceptible to bond cleavage at a BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; and d) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; wherein detection of the antibody-antigen complex will indicate retargeted endopeptidase activity.

6. A method for detecting retargeted endopeptidase activity, the method comprising the steps of: a) treating cells from the established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line can take up the retargeted endopeptidase; b) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site₁A SNAP-25 cleavage product having a carboxy terminus at the residue; c) immobilizing the SNAP-25 component to a solid support; d) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in the SNAP-25 cleavage product that is susceptible to bond cleavage at the BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; and e) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; wherein detection of the antibody-antigen complex will indicate retargeted endopeptidase activity.

7. A method for determining retargeted endopeptidase immune resistance in a mammal, comprising the steps of: a) addition of retargeted endopeptidase to a mammal obtained for testing alpha-retargeted endopeptidase neutralizationIn a test sample for the presence or absence of antibodies; b) treating cells from an established cell line with the test sample, wherein the cells from the established cell line are susceptible to retargeted endopeptidase activity; c) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; d) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in a SNAP-25 cleavage product that is susceptible to bond cleavage at a BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; e) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; f) repeating steps b-e by replacing the test sample with a negative control sample comprising the retargeted endopeptidase and serum known to be free of alpha-retargeted endopeptidase neutralising antibodies; and g) comparing the amount of antibody-antigen complex detected in step e with the amount of antibody-antigen complex detected in step f, wherein detection of an amount of antibody-antigen complex detected in step e that is less than the amount of antibody-antigen complex detected in step f indicates the presence of alpha-retargeted endopeptidase neutralizing antibody.

8. A method for determining retargeted endopeptidase immune resistance in a mammal, comprising the steps of: a) adding a retargeted endopeptidase to a test sample obtained from a mammal for testing for the presence or absence of an alpha-retargeted endopeptidase neutralizing antibody; b) treating cells from an established cell line with the test sample, wherein the cells from the established cell line are susceptible to retargeted endopeptidase activity; c) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; d) contacting the SNAP-25 component with an alpha-SNAP-25 antibody attached to a solid support, wherein the alpha-SNAP-25 antibody binds to P in a SNAP-25 cleavage product that is susceptible to bond cleavage at a BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; e) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleftDecomposing the product; f) repeating steps b-e by replacing the test sample with a negative control sample comprising the retargeted endopeptidase and serum known to be free of alpha-retargeted endopeptidase neutralising antibodies; and g) comparing the amount of antibody-antigen complex detected in step e with the amount of antibody-antigen complex detected in step f, wherein detection of an amount of antibody-antigen complex detected in step e that is less than the amount of antibody-antigen complex detected in step f indicates the presence of alpha-retargeted endopeptidase neutralizing antibody.

9. A method for determining retargeted endopeptidase immune resistance in a mammal, comprising the steps of: a) adding a retargeted endopeptidase to a test sample obtained from a mammal for testing for the presence or absence of an alpha-retargeted endopeptidase neutralizing antibody; b) treating cells from an established cell line with the test sample, wherein the cells from the established cell line are susceptible to retargeted endopeptidase activity; c) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; d) immobilizing the SNAP-25 component to a solid support; e) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in a SNAP-25 cleavage product that is susceptible to bond cleavage at a BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; f) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; g) repeating steps b-f by replacing the test sample with a negative control sample comprising the retargeted endopeptidase and serum known to be free of alpha-retargeted endopeptidase neutralising antibodies; and h) comparing the amount of antibody-antigen complex detected in step f with the amount of antibody-antigen complex detected in step g, wherein detection of an amount of antibody-antigen complex detected in step f that is less than the amount of antibody-antigen complex detected in step g indicates the presence of alpha-retargeted endopeptidase neutralizing antibody.

10. A method for determining retargeted endopeptidase immune resistance in a mammal comprising the followingThe method comprises the following steps: a) adding a retargeted endopeptidase to a test sample obtained from a mammal for testing for the presence or absence of an alpha-retargeted endopeptidase neutralizing antibody; b) treating cells from the established cell line with the test sample, wherein the cells from the established cell line can take up the retargeted endopeptidase; c) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; d) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in a SNAP-25 cleavage product that is susceptible to bond cleavage at a BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; e) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; f) repeating steps b-e by replacing the test sample with a negative control sample comprising the retargeted endopeptidase and serum known to be free of alpha-retargeted endopeptidase neutralising antibodies; and g) comparing the amount of antibody-antigen complex detected in step e with the amount of antibody-antigen complex detected in step f, wherein detection of an amount of antibody-antigen complex detected in step e that is less than the amount of antibody-antigen complex detected in step f indicates the presence of alpha-retargeted endopeptidase neutralizing antibody.

11. A method for determining retargeted endopeptidase immune resistance in a mammal, comprising the steps of: a) adding a retargeted endopeptidase to a test sample obtained from a mammal for testing for the presence or absence of an alpha-retargeted endopeptidase neutralizing antibody; b) treating cells from the established cell line with the test sample, wherein the cells from the established cell line can take up the retargeted endopeptidase; c) isolating from the treated cells a SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; d) contacting the SNAP-25 component with an alpha-SNAP-25 antibody attached to a solid support, wherein the alpha-SNAP-25 antibody binds to P in a SNAP-25 cleavage product that is susceptible to bond cleavage at a BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; e) detection of antibodiesThe presence of a body-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; f) repeating steps b-e by replacing the test sample with a negative control sample comprising the retargeted endopeptidase and serum known to be free of alpha-retargeted endopeptidase neutralising antibodies; and g) comparing the amount of antibody-antigen complex detected in step e with the amount of antibody-antigen complex detected in step f, wherein detection of an amount of antibody-antigen complex detected in step e that is less than the amount of antibody-antigen complex detected in step f indicates the presence of alpha-retargeted endopeptidase neutralizing antibody.

12. A method for determining retargeted endopeptidase immune resistance in a mammal, comprising the steps of: a) adding a retargeted endopeptidase to a test sample obtained from a mammal for testing for the presence or absence of an alpha-retargeted endopeptidase neutralizing antibody; b) treating cells from the established cell line with the test sample, wherein the cells from the established cell line can take up the retargeted endopeptidase; c) isolating from the treated cells a SNAP-25 fraction, said SNAP-25 fraction comprising P having a bond that is susceptible to cleavage at a BoNT/A cleavage site ₁A SNAP-25 cleavage product having a carboxy terminus at the residue; d) immobilizing the SNAP-25 component to a solid support; e) contacting the SNAP-25 component with an alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody binds to P in a SNAP-25 cleavage product that is susceptible to bond cleavage at a BoNT/A cleavage site₁An epitope comprising the carboxy terminus at residue; f) detecting the presence of an antibody-antigen complex comprising an alpha-SNAP-25 antibody and a SNAP-25 cleavage product; g) repeating steps b-f by replacing the test sample with a negative control sample comprising the retargeted endopeptidase and serum known to be free of alpha-retargeted endopeptidase neutralising antibodies; and h) comparing the amount of antibody-antigen complex detected in step f with the amount of antibody-antigen complex detected in step g, wherein detection of an amount of antibody-antigen complex detected in step f that is less than the amount of antibody-antigen complex detected in step g indicates the presence of alpha-retargeted endopeptidase neutralizing antibody.

13. The method of 1-3 and 7-9, wherein the cell is sensitive to a retargeted endopeptidase activity of about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 100nM or less of a retargeted endopeptidase.

14. The method of 4-6 and 10-12, wherein the cell can uptake about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 100nM or less of the retargeted endopeptidase.

15. The method of 1-6, wherein the sample comprises about 100ng or less, about 10ng or less, about 1ng or less, 100fg or less, 10fg or less, or 1fg or less of the retargeted endopeptidase.

16. The method of 1-6, wherein the sample comprises about 100nM or less, about 10nM or less, about 1nM or less, about 0.5nM or less, or about 0.1nM or less of the retargeted endopeptidase.

17. The method of 1 to 12, wherein the presence of the antibody-antigen complex is detected by immunoblot analysis, immunoprecipitation analysis, ELISA or sandwich ELISA.

18. The method of 1 to 12, wherein the method has a signal to noise ratio of at least 3: 1, at least 5: 1, at least 10: 1, at least 20: 1, at least 50: 1, or at least 100: 1 at the lower asymptote.

19. The method of 1 to 12, wherein the method has a signal to noise ratio at the upper asymptote of at least 10: 1, at least 20: 1, at least 50: 1, at least 100: 1, at least 200: 1, at least 300: 1, at least 400: 1, at least 500: 1, or at least 600: 1.

20. The method of claims 1-12, wherein the method can detect EC retargeted to endopeptidase of e.g., at least 100ng, at least 50ng, at least 10ng, at least 5ng, at least 100pg, at least 50pg, at least 10pg, at least 5pg, at least 100fg, at least 50fg, at least 10fg, or at least 5fg₅₀Activity of。

21. The method of 1 to 12, wherein the method can detect, e.g., at least 10nM, at least 5nM, at least 100nM, at least 50nM, at least 10nM, at least 5nM, at least 1nM, at least 0.5nM, or at least 0.1nM of EC for retargeting endopeptidase₅₀And (4) activity.

22. The method of 1-12, wherein the LOD of the method is, e.g., 10pg or less, 9pg or less, 8pg or less, 7pg or less, 6pg or less, 5pg or less, 4pg or less, 3pg or less, 2pg or less, 1pg or less of the retargeted endopeptidase.

23. The method of 1-12, wherein the LOD of the method is, e.g., 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, or 10nM or less of the retargeted endopeptidase.

24. The method of 1-12, wherein the LOQ of the method is, e.g., 10pg or less, 9pg or less, 8pg or less, 7pg or less, 6pg or less, 5pg or less, 4pg or less, 3pg or less, 2pg or less, 1pg or less of the retargeted endopeptidase.

25. The method of 1-12, wherein the LOQ of the method is, e.g., 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, or 10nM or less of the retargeted endopeptidase.

26. The method of 1-12, wherein the method can distinguish a retargeted endopeptidase with full activity from a retargeted endopeptidase with partial activity that is 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less active than retargeted endopeptidase a with full activity.

27. The method of 1 to 12, wherein the a-SNAP-25 antibody binds to BoNT/a cleft in SNAP-25 cleavage productP with cleavage site susceptible to bond cleavage₁The carboxyl terminal epitope is included at the residue.

28. The method of 27, wherein the α -SNAP-25 antibody has an association rate constant of less than 1 x 10 for an epitope of a carboxy-terminal glutamine in a SNAP-25 cleavage product that does not comprise the BoNT/A cleavage site labile bond¹M^-1s^-1(ii) a And wherein the equilibrium dissociation constant of the a-SNAP-25 antibody for the epitope is less than 0.450 nM.

29. The method of 27, wherein the isolated a-SNAP-25 antibody has an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 72. SEQ ID NO: 74. SEQ ID NO: 76. SEQ ID NO: 80 and SEQ ID NO: 82; and comprises a sequence selected from the group consisting of SEQ ID NO: 84. SEQ ID NO: 86. SEQ ID NO: 88. SEQ ID NO: 90 and SEQ ID NO: 92.

30. The method of 27, wherein the isolated a-SNAP-25 antibody comprises at least a heavy chain variable region having the amino acid sequence of SEQ ID NO: 93V of_HCDR1, having SEQ ID NO: v of 94_HCDR1, having SEQ ID NO: v of 95_HCDR1, having SEQ ID NO: 118 of V_HCDR1, having SEQ ID NO: v of 119_HCDR1 or a polypeptide having SEQ ID NO: v of 120_H CDR1。

32. The method of 27, wherein the isolated a-SNAP-25 antibody comprises at least a heavy chain variable region having the amino acid sequence of SEQ ID NO: v of 96_HCDR2, having SEQ ID NO: 97V_HCDR2, having SEQ ID NO: v of 98_HCDR2, having SEQ ID NO: v of 99_HCDR2, having SEQ ID NO: v of 121_HCDR2, having SEQ ID NO: 122V_HCDR2 or a polypeptide having SEQ ID NO: v of 123_H CDR2。

33. The method of 27, wherein the isolated a-SNAP-25 antibody comprises at least a heavy chain variable region having the amino acid sequence of SEQ ID NO: v of 100 _HCDR3, having SEQ ID NO: v of 101_HCDR3, having SEQ ID NO: 102V_HCDR3 or a polypeptide having SEQ ID NO: 124V_H CDR3。

34. The method of 27, wherein the isolated a-SNAP-25 antibody comprises at least a heavy chain variable region having the amino acid sequence of SEQ ID NO: v of 103_LCDR1, having SEQ ID NO: v of 104_LCDR1, having SEQ ID NO: v of 105_LCDR1, having SEQ ID NO: 106V_LCDR1, having SEQ ID NO: v of 107_LCDR1, having SEQ ID NO: v of 125_LCDR1, having SEQ ID NO: 126V_LCDR1, having SEQ ID NO: v of 127_LCDR1, having SEQ ID NO: v of 128_LCDR1 or a polypeptide having SEQ ID NO: v of 129_L CDR1。

35. The method of 27, wherein the isolated a-SNAP-25 antibody comprises at least a heavy chain variable region having the amino acid sequence of SEQ ID NO: v of 108_LCDR2, having SEQ ID NO: v of 109_LCDR2, having SEQ ID NO: 110V_LCDR2, having SEQ ID NO: v of 111_LCDR2 or a polypeptide having SEQ ID NO: 112V_L CDR2。

36. The method of 27, wherein the isolated a-SNAP-25 antibody comprises at least a heavy chain variable region having the amino acid sequence of SEQ ID NO: v of 113_LCDR3, having SEQ ID NO: 114V_LCDR3, having SEQ ID NO: 115V_LCDR3, having SEQ ID NO: 116V_LCDR3 or a polypeptide having SEQ ID NO: v of 117 _L CDR3。

37. The method of 27, wherein the isolated a-SNAP-25 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93. SEQ ID NO: 121 and SEQ ID NO: 100, respectively; and a light chain variable region comprising SEQ ID NO: 105. SEQ ID NO: 110 and SEQ ID NO: 115.

38. the method of 27, wherein the isolated a-SNAP-25 antibody selectively binds to a polypeptide having the amino acid sequence of SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 147 or SEQ ID NO: 148, or a SNAP-25 epitope of 148.

39. The method of 27, wherein the isolated a-SNAP-25 antibody selectively binds to a polypeptide having the amino acid sequence of SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43 or SEQ ID NO: 44, or a SNAP-25 epitope.

Examples

Example I

Screening candidate cell lines for expression of endogenous retargeted endopeptidase receptors

The following example illustrates how to identify established cell lines possessing the ability to re-target endopeptidase uptake required for the development of cell-based potency assays.

1. Growth of candidate cell line stock cultures.

To grow the cell lines, cells from the cell line tested were seeded at a suitable density at 162cm containing 30mL of suitable growth medium (see Table 1) ²Tissue culture flasks were grown in a 37 ℃ incubator at 5% or 10% carbon dioxide until the cells reached the desired density.

2. Cells expressing the target receptor on the cell surface are screened.

Cell lines are screened for the presence of the desired target receptor using flow cytometry and/or ligand binding assays. Although the following examples use reagents to identify opioid or opioid-like receptors in the plasma membrane, the methods disclosed below can also be used to identify any cognate receptor that retards endopeptidase.

a. Cell lines were identified using flow cytometry.

To identify established cell lines comprising cells expressing a retargeted endopeptidase target receptor on the cell surface, flow cytometry analysis was performed. Cells from each candidate cell line were grown, trypsinized, washed in staining buffer containing 1 x PBS, 0.5% BSA, and centrifuged at 1200rpm for 3 minutes as described in section 1. The spherical cells were resuspended in staining buffer and approximately 2.0X 10⁶Individual cells were transferred to new tubes, two for each recipient tested. To screen for the presence of opiate or opioid receptors, approximately 2.0 μ L to 5.0 μ L of α -ORL-1 RA14133 (neurosis, Edina, MN), α -DOR rabbit polyclonal antibody RA10101 (neurosis, Edina, MN), α -KOR rabbit polyclonal antibody RA10103 (neurosis, Edina, MN) or α -MOR rabbit polyclonal antibody RA10104 (neurosis, Edina, MN) was added to one tube and the mixture was incubated at 4 ℃ for 1 hour. In the absence of any antibody, the second tube was incubated at 4 ℃ for 1 hour and used as a negative control. After antibody incubation, 1.0mL of staining buffer was added to each tube and centrifuged at 1200rpm for 3 minutes. The cell pellet was washed 1 more times with 1.0mL of staining buffer. The cell pellet was resuspended in 200. mu.L staining buffer, and 2.0. mu.L goat anti-rabbit IgG FITC antibody was added to each tube and incubated at 4 ℃ in the dark for 1 hour. After incubation with secondary antibody, 1.0mL of staining buffer was added to each tube and centrifuged at 1200rpm for 3 minutes. The cell pellet was washed 1 more times with 1.0mL of staining buffer and the pellet was resuspended in 500. mu.L of staining buffer. The samples were analyzed using a flow cytometer, and the data is presented as anti-receptor antibody staining overlaid on rabbit IgG FITC staining.

The results show that, among the cell lines tested, ORL-1 is expressed on the cell surface of about 50% of the cells constituting the established cell lines SiMa, SiMa P > 33, clones H10, ND7 and SK-N-DZ; expressed on the cell surface of between about 25% to about 50% of the cells constituting established cell lines SH-SY5Y and ND 15; and expressed on the cell surface of less than about 25% of the cells constituting established cell lines ND3, ND8, N18 and Neuro-2a (Table 2). The results also showed that KOR was expressed on the cell surface of about 50% of the cells constituting established cell lines SH-SY5Y and ND 7; expressed on the cell surface of between about 25% to about 50% of the cells constituting the established cell lines SiMa clone H10, SiMa P > 33, ND15 and Neuro-2 a; and expressed on the cell surface of less than about 25% of the cells constituting established cell lines ND3, ND8 and N18 (table 2). The results also revealed that MOR is expressed on the cell surface of about 50% of the cells constituting the established cell lines ND7, ND15 and SiMa P > 33; expressed on the cell surface of between about 25% to about 50% of the cells constituting the established cell lines SH-SY5Y, SiMa clone H10, ND8 and Neuro-2 a; and expressed on the cell surface of less than about 25% of the cells constituting established cell lines ND3 and N18 (table 2). The α -DOR rabbit polyclonal antibody RA10101 did not work properly and produced unusable data.

b. Cell lines were identified using ligand binding.

To identify established cell lines comprising cells expressing a retargeted endopeptidase target receptor on the cell surface, a ligand binding assay is performed. Cells from the candidate cell line to be tested were seeded in black clear-bottomed 96-well plates and incubated for about 4 hours to promote attachment. To screen for the presence of opioid or opioid-like receptors, the culture medium was then aspirated from each well and replaced with 50 μ L of ligand solution containing 0 (untreated control), 0.001nM, 0.01nM, 0.1nM, or 1nM FAM-nociceptin (Phoenix Pharmaceuticals, inc., Burlingame, CA), or containing 0 (untreated control), 0.001nM, 0.01nM, 0.1nM, or 1nM FAM-dynorphin a (dynorphin a) (Phoenix Pharmaceuticals, inc., buingame, CA). Cells were incubated with ligand solution for 1 hour at 37 ℃ in an incubator at 5% carbon dioxide. The cells were washed by washing the cells 3 times with 100 μ L of 1 × PBS to remove unbound ligand. The plates were scanned on Typhoon (Ex 488 and Em 520nm) and the RFU signal in the plates was subsequently read on M5 plate reader (Ex 495 and Em 520 nm). The results show that the cells constituting the established cell lines SiMa clone H10, SH-SY5Y and SK-N-DZ bind nociceptin, while the cells constituting SiMa clone H10 also bind dynorphin (Table 2).

Using a similar approach, cell lines containing cells with other cognate receptors that retarget endopeptidases can be identified by FAM labeling the targeting domain of these endopeptidases and screening the cell lines as described above.

3. Candidate cell lines were screened for a single dose using a retargeted endopeptidase molecule.

To determine whether the cell line is capable of uptake the appropriate retargeted endopeptidase molecule, appropriate densities of cells from stock cultures of test cell lines were seeded into each well of a 24-well tissue culture plate containing 1mL of the appropriate serum growth medium (table 1). Cells were grown in an incubator at 37 ℃ under 5% carbon dioxide until the cells reached the desired density (about 18 to 24 hours). To assess uptake of opioid-heavy-targeted endopeptidase, growth medium was aspirated from each well and used 1) fresh growth medium without opioid-heavy-targeted endopeptidase (untreated cell line); or 2) fresh growth medium replacement with 30nM nociceptin retargeted endopeptidase (Noc/A) or 100nM dynorphin retargeted endopeptidase (Dyn/A) (treated cell line). After overnight incubation, cells were washed by aspirating growth medium and rinsing each well with 200 μ L of 1 × PBS. To harvest the cells, 1 × PBS was aspirated, the cells were lysed by adding 50 μ L of 2 × SDS Loading Buffer (Loading Buffer), the lysate was transferred to a clean tube, and the samples were heated to 95 ℃ for 5 minutes.

To detect the presence of uncleaved SNAP-25 substrate and cleaved SNAP-25 product, Western blotting was usedAn aliquot of each harvested sample was analyzed. In this analysis, NuPAGE was used under denaturing, reducing conditions by MOPS polyacrylamide gel electrophoresisNovex 12% Bis-Tris pre-made polyacrylamide gel (Invitrogen inc., Carlsbad, CA) separated 12 μ L aliquots of harvested samples. Using TRANS-BLOT by Western blottingSD semi-dry electrophoretic transfer cytometry (Bio-Rad Laboratories, Hercules, Calif.) transfers the isolated peptides from the gel onto a polyvinylidene fluoride (PVDF) membrane (Invitrogen Inc., Carlsbad, Calif.). PVDF membranes were blocked by incubation at room temperature for 2 hours in a solution containing Tris-Buffered Saline (TBS) (25mM 2-amino-2-hydroxymethyl-1, 3-propanediol hydrochloride (Tris-HCl) (pH 7.4), 137mM sodium chloride, 2.7mM potassium chloride), 0.1% TWEEN-20(polyoxyethylene (20) sorbitan monolaurate), 2% Bovine Serum Albumin (BSA), 5% skim milk powder. At 4 ℃ in TBS, 0.1% TWEEN-20Blocked membranes were incubated overnight (polyoxyethylene (20) sorbitan monolaurate), 2% BSA, and 5% skim milk powder containing 1) a 1: 5,000 dilution of the α -SNAP-25 mouse monoclonal antibody as the primary antibody (SMI-81; sternberger Monoclonals Inc., Lutherville, Md.); or 2) 1: 5,000 dilutions of S9684. alpha. -SNAP-25 rabbit polyclonal antiserum were used as primary antibodies (Sigma, St Louis, Mo.). Both the alpha-SNAP-25 mouse monoclonal antibody and the rabbit polyclonal antibody can simultaneously detect the uncleaved SNAP-25 substrate and the cleaved SNAP-25 product, thereby being capable of evaluating the overall expression condition of SNAP-25 in each cell line and the cleaved SNAP-25 percentage after the treatment of retargeted endopeptidase so as to evaluate the overall expression condition of SNAP-25 in each cell line The uptake of retargeted endopeptidase was evaluated as a parameter. In TBS, TWEEN-20Washes in (polyoxyethylene (20) sorbitan monolaurate) blot probed with primary antibody 3 times for 15 min each. At room temperature, in TBS, 0.1% TWEEN-20(polyethylene oxide (20) sorbitan monolaurate), 2% BSA and 5% skimmed milk powder containing 1) goat polyclonal anti-mouse immunoglobulin G heavy and light chain (IgG, H + L) antibody conjugated to horseradish peroxidase (Zymed, South San Francisco, Calif.) in a 1: 10,000 dilution for 2 hours on washed membranes; or 2) goat polyclonal anti-rabbit immunoglobulin G heavy and light chain (IgG, H + L) antibody conjugated to horseradish peroxidase (Zymed, South San Francisco, Calif.) in a 1: 10,000 dilution as secondary antibody. In TBS, 0.1% TWEEN-20Blots probed with secondary antibody were washed 3 times in (polyoxyethylene (20) sorbitan monolaurate) for 15 minutes each. Using ECL Plus^TMThe western blot detection system (GE Healthcare, Amersham Biosciences, Piscataway, NJ) observes signal detection of labeled SNAP-25 products and acquires membrane images and quantifies the percentage of cleavage products using a Typhoon 9410 Variable Mode Imager (Variable Mode Imager) and image Analysis software (Imager Analysis software) (GE Healthcare, Amersham Biosciences, Piscataway, NJ). The pixel size (100 to 200 pixels) and PMT voltage setting (350 to 600, typically 400) are selected based on the individual footprints.

Based on detection of SNAP-25 cleavage products, the following cell lines exhibited uptake of 30nM Noc/A: BE (2) -C, N18TG2, Neuro-2a, SiMa, SK-N-BE (2) -C and SK-N-DZ (Table 3); while the following cell lines exhibited uptake of 100nM Dyn/A: n18TG2, Neuro-2a, PC12 and SiMa. Some of these sensitive cell lines were tested with lower doses of compound and/or with full dose response.

Using similar methods, cell lines containing cells with other retargeted endopeptidase cognate receptors can be assessed for retargeted endopeptidase uptake.

Example II

Screening candidate clonal cell lines for endogenous retargeted endopeptidase receptor expression

1. Single dose retargeted endopeptidase screening of candidate clonal cell lines from the parental SiMa cell line.

Accompanying the filed patent application Zhu Hong et al, Cell Lines use mul in immune-Based Botulinum Toxin Serotype A Activity Assays, U.S. patent application Ser. No. 61/160,199 discloses clonal Cell Lines derived from parental SiMa Cell Lines that can be used in BoNT/A potency Assays, as described in Ester Fernandez-Salas et al, immune-Based Botulinum Toxin Serotype A Activity Assays, U.S. patent application Ser. No. 12/403,531, the entire contents of each patent application being hereby incorporated by reference. To determine whether these clonal cell lines are capable of uptake the appropriate retargeted endopeptidase, each cell line was screened using an ECL sandwich ELISA assay.

To prepare lysates treated with retargeted endopeptidase, cells from stock cultures of the cell lines tested were seeded at appropriate densities into each well of a 96-well tissue culture plate containing 100 μ L of appropriate serum growth medium (table 1) and cultured overnight. The medium from the seeded cells was aspirated from each well and replaced with fresh medium containing 30nM Noc/A retargeted endopeptidase or 80nM Dyn/A retargeted endopeptidase. After 24 hours of incubation, cells were washed by aspirating growth medium and rinsing each well with 200 μ L of 1 × PBS. To harvest the cells, 1 XPBS was aspirated, the cells were lysed by adding 30 μ L lysis buffer containing 20mM Tris-HCl (pH 7,5), 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100 to each well, and the plates were incubated for 30 minutes at 4 ℃ on a shaker rotating at 500 rpm. The plate was centrifuged at 4000rpm for 20 minutes at 4 ℃ to pellet the cell debris and the supernatant was transferred to a 96-well plate coated with capture antibody for the detection step.

To prepare alpha-SNAP-25₁₉₇Capture antibody solution, purification of alpha-SNAP-25 contained in ascites from hybridoma cell line 2E2A6 (example XI) Using Standard protein A purification protocol ₁₉₇A mouse monoclonal antibody.

To prepare the α -SNAP-25 detection antibody solution, α -SNAP-25 rabbit polyclonal antibody S9684(Sigma, st. louis, MO) was conjugated with ruthenium (II) -tris-bipyridine- (4-methylsulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer' S instructions (Meso Scale Discovery, Gaithersburg, MD). The conjugation reaction was carried out in the following manner: 30 μ L of distilled water resuspended (recinstituted) MSD SULFO-TAG^TMThe stock solution was added to 200. mu.L of 2 mg/mL α -SNAP-25 polyclonal antibody and the reaction was incubated at room temperature in the dark for 2 hours. The labeled antibody was purified using standard spin column protocols and the protein concentration was determined using standard colorimetric protein assays. Measurement of alpha-SNAP-25 antibody/MSD SULFO-TAG at 455nm Using a Spectrophotometer^TMAbsorbance of the conjugate, to determine concentration (in moles/liter). The detection antibody solution was stored at 4 ℃ until use. Unused aliquots were stored for long periods at-20 ℃.

For preparing a polypeptide containing alpha-SNAP-25₁₉₇Capture of antibody alpha-SNAP-25 solid phase carrier, about 5 u L appropriate alpha-SNAP-25₁₉₇The monoclonal antibody solution (20. mu.g/mL in 1 XPBS) was added to each well of a 96-well MSD High binding (MSD High Bind) plate and the solution was allowed to air dry in a biosafety cabinet for 2 to 3 hours to evaporate the solution. The closed plates were sealed and stored at 4 ℃ until use.

To detect the presence of the cleaved SNAP-25 product by ECL sandwich ELISA, the capture antibody-bound wells were then blocked by incubation at room temperature for 2 hours by adding 150 μ L of Blocking Buffer (Blocking Buffer) containing 2% Amersham Blocking reagent (GE Life Sciences, Piscataway, NJ) and 10% goat serum (VWR, West Chester, PA). The blocking buffer was aspirated, 25 μ L of lysate of cells treated with retargeted endopeptidase was added to each well, and the plate was incubated overnight at 4 ℃. By aspirating the cell lysate and applying 200. mu.L of 1 XPBS, 0.1% TWEEN-20(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times and each well in the plate was washed 3 times. After washing, 25 μ L of 2% Amersham blocking reagent in 1 XPBS, 0.1% TWEEN-20 was added to each wellA5. mu.g/mL α -SNAP-25 detection antibody solution (polyoxyethylene (20) sorbitan monolaurate) was then sealed and incubated at room temperature for 1 hour with shaking. After incubation of the alpha-SNAP-25 detection antibody, 200. mu.L of 1 XPBS, 0.1% TWEEN-20 was used(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times. After washing, 150. mu.L of 1 Xread Buffer (Read Buffer) (Meso Scale Discovery, Gaithersburg, Md.) was added to each well and SECTOR was used ^TMAn Image Reader (Image Reader) model Imager 6000 (Meso Scale Discovery, Gaithersburg, MD) reads the plate. Raw data was collected using an ECL imager.

The results show that both the parental SiMa cell line as well as the clonal cell line H10 showed good uptake of Noc/a retargeted endopeptidase (table 4). In addition, these results also revealed that many cell lines exhibited uptake of Dyn/a retargeted endopeptidases (table 4). Three clonal cell lines (1E11, AF4 and DC4) showed good uptake of Dyn/a retargeted endopeptidase; 11 clonal cell lines (1E3, 2D2, 2D6, 3D8, 5C10, 5F3, BB10, BF8, CG8, CG10 and DE7) exhibited moderate uptake of Dyn/a retargeted endopeptidases; and (3B8, 2B9, CE6, YB8, 4C8, 2F5, AC9, CD6, DD10, YF5) showed minimal uptake of Dyn/a retargeted endopeptidase. A subset of these candidate cell lines were tested in a full dose response assay with the corresponding retargeted endopeptidase.

2. Full dose response screening of candidate cell lines.

Next, established cell lines identified above were evaluated using the full dose response of the appropriate retargeted endopeptidase. Cells from different cell lines were seeded in 96-well plates and exposed to various concentrations of Noc/A (0, 0.14nM, 0.4nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM and 100nM) or Dyn/A (0.017nM, 0.05nM, 0.15nM, 0.45nM, 1.4nM, 4.1nM, 12nM, 37nM, 111nM, 333nM and 1000nM) for 24 hours. The medium containing the retargeted endopeptidase was then removed and replaced with fresh complete medium. At 37 deg.C, 5% CO ₂The plates were then incubated for an additional 24 hours to allow cleavage of SNAP-25. Cells in lysis buffer(Table 5) and the well plates were centrifuged to remove debris. Lysates were used in western blot assays or sandwich ELISA.

For Western blot analysis, samples were assayed for the presence of intact SNAP-25 and SNAP-25 cleavage products as described in example I.

For sandwich ELISA, ELISA plates coated with 2E2a6 monoclonal antibody were blocked with 150 μ L blocking buffer for 2 hours at room temperature. After removal of the blocking buffer, 25 μ L of cell lysate was added to each well and the plates were incubated at 4 ℃ for 2 hours. The plate was washed 3 times with PBS-T and 25. mu.L of the SULFO-TAG NHS-ester labeled anti-SNAP 25pAb detection antibody at a concentration of 5. mu.g/mL in 2% blocking agent in PBS-T was added to the base angle of each well. Plates were sealed and shaken at room temperature for 1 hour, followed by 3 washes with PBS-T. After washing was complete, 150. mu.L of 1 × reading buffer was added to each well and the plates were read in a SI6000 image reader. To determine the sensitivity of each cell line tested, the EC of each cell line was calculated₅₀The value is obtained. The values of Noc/a retargeted endopeptidases are summarized in table 5. Full dose response of the retargeted endopeptidase Dyn/A was only performed in PC12 and clone AF 4. In both cases, the assay did not reach the upper asymptote and EC could not be calculated ₅₀The value is obtained. The dose that gave a clonal signal for AF4 was lower for both cell lines, 12 nM.

Using similar methods, clonal cell lines containing cells with other retargeted endopeptidase cognate receptors can be screened and evaluated for retargeted endopeptidase uptake.

Example III

Assessment of the Effect of growth conditions on the uptake of retargeted endopeptidase in candidate cell lines

The following example illustrates how to determine the culture conditions (growth and differentiation) that maximize uptake of the retargeted endopeptidase in established cell lines.

1. Effect of cell differentiation and trophic factors on retargeted endopeptidase uptake by candidate cell lines.

To determine whether the presence of trophic factors in cell differentiation or growth media improved retargeted endopeptidase uptake, cell lines exhibiting good Noc/a uptake were tested using different media compositions. Cells from stock cultures of the SiMa P > 30 cell line tested at the appropriate density were seeded into wells of 96-well tissue culture plates containing 100 μ L of serum-free medium containing RPMI1640, 1% penicillin-streptomycin, 2mM L-glutamine supplemented with B27 and N2 or 100 μ L of serum-free medium containing RPMI1640, 1% penicillin-streptomycin, 2mM L-glutamine supplemented with B27, N2 and NGF (nerve growth factor, 100 ng/mL). These cells were incubated in an incubator at 37 ℃ under 5% carbon dioxide until cell differentiation was found by evaluation with standard and conventional morphological criteria (e.g., growth arrest and neurite extension) (about 1 to 2 days). As a control, cells from stock cultures of the test cell lines were seeded at appropriate densities into each well of a 96-well tissue culture plate containing 100 μ L of appropriate growth medium (table 1) with or without NGF (100 ng/mL). These undifferentiated control cells were grown in an incubator at 37 ℃ under 5% carbon dioxide until the cells reached the desired density (about 18 to 24 hours). The culture medium of the differentiated and undifferentiated control cultures was aspirated from each well and used with Noc/A (0.14 nM) at 0 (untreated samples) or various concentrations 0.4nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM and 100 nM). After 24 hours of treatment, cells were washed and incubated for 24 hours in media without retargeted endopeptidase to increase SNAP25 produced₁₉₇The amount of (c). Cells were then washed and harvested for use in ECL sandwich ELISA assays as described in example II.

Trophic factors were also tested for their effect on the SK-N-DZ cell line. SK-N-DZ cells were seeded at 25,000 cells per well in 8 different SM media on poly-D-lysine coated 96-well plates (Table 6) for 72 hours. Cells were treated in the same eight media containing Noc/A at 0, 0.3nM, 3nM and 30nM doses. After 24 hours of treatment, cells were washed and incubated for 24 hours in medium without retargeted endopeptidase to increase SNAP25 produced₁₉₇Amount of cleavage product. Cells were then washed and harvested for use in western blot assays as described in example I.

Differentiation had no effect on Noc/A uptake in the SiMa > P30 cell line, but appeared to improve uptake in the SK-N-DZ cell line. Basal medium had a significant effect on Noc/a uptake in SK-N-DZ cell line, with RPMI1640 containing trophic factors N2 and B27 being the best combination. The presence of NGF in the culture medium did not appear to improve uptake in the two cell lines tested.

Using similar methods, clonal cell lines containing cells with other retargeted endopeptidase cognate receptors can be evaluated for growth conditions and differentiation conditions.

Example IV

Development of established cell lines expressing exogenous retargeted endopeptidase receptors

The following example illustrates how established cell lines expressing exogenous receptors for retargeted endopeptidases can be prepared.

1. The target receptor is transfected into cells that constitute a candidate cell line.

The retargeted endopeptidase Noc/A contains a nociceptin targeting domain, which is a natural ligand for opioid receptor-1 (ORL-1). To obtain an expression construct comprising the open reading frame of ORL-1, the expression construct pReceiver-M02/ORL-1 was obtained from GeneCopoeia (GeneCopoeia, Germantown, Md.).

Alternatively, standard procedures (Blueheron) may be usedBiotechnology, Bothell, WA), according to the amino acid sequence of ORL-1 (e.g. the amino acid sequence SEQ ID NO: 25 or SEQ ID NO: 26) synthesizing a polynucleotide molecule. Oligonucleotides of 20 to 50 bases in length were synthesized using standard phosphoramidite synthesis. These oligonucleotides are hybridized to double-stranded duplexes, which are ligated together to assemble a full-length polynucleotide molecule. This polynucleotide molecule was cloned into the pUCBHB1 vector at the SmaI site using standard molecular biology methods to generate pUCBHB 1/ORL-1. By using Big Dye Terminator ^TMChemistry 3.1(Applied Biosystems, Foster City, Calif.) and ABI model 3100 (Applied Biosystems, Foster City, Calif.) were sequenced to verify the synthesized polynucleotide molecules. If desired, expression-optimized polynucleotide molecules can be synthesized based on the ORL-1 amino acid sequence (e.g., amino acid sequence SEQ ID NO: 25 or SEQ ID NO: 26) in order to improve its expression in E.coli strains. The polynucleotide molecule encoding ORL-1 can be modified to 1) contain synonymous codons that are typically found in the native polynucleotide molecule of an e.coli strain; 2) containing G + C in amounts equivalent to those of the native polynucleotide molecules found in E.coli strainsThe G + C contents are matched extremely; 3) reducing poly-mononucleotide regions found within a polynucleotide molecule; and/or 4) elimination of internal regulatory or structural sites found within polynucleotide molecules, see, e.g., Lance E.Steward et al, Optimizing Expression of Active Botulinum Toxin Type A, U.S. patent publication 2008/0057575(2008, 3/6); and Lance E.Steward et al, Optimizing Expression of Active Botulinum Toxin Type E, U.S. patent publication 2008/0138893 (12.6.2008). Once sequence optimization is complete, oligonucleotides of 20 to 50 bases in length are synthesized using standard phosphoramidite synthesis methods. These oligonucleotides are hybridized to double-stranded duplexes, which are ligated together to assemble a full-length polynucleotide molecule. This polynucleotide molecule was cloned into the pUCBHB1 vector at the SmaI site using standard molecular biology methods to generate pUCBHB 1/ORL-1. The synthesized polynucleotide molecules were verified by DNA sequencing. Expression optimization can be performed, if desired, for different organisms, such as yeast strains, insect cell lines, or mammalian cell lines, see, e.g., Steward, U.S. patent publication 2008/0057575, supra, (2008); and Steward, U.S. patent publication 2008/0138893, supra, (2008). Exemplary polynucleotide molecules encoding ORL-1 include SEQ ID NO: 61 and SEQ ID NO: 62.

To construct an expression construct encoding ORL-1, the pUCBHB1/ORL-1 construct was digested with a restriction endonuclease to 1) excise the polynucleotide molecule encoding the open reading frame of ORL-1; and 2) operably linking the polynucleotide molecule to pcDNA3 vector (Invitrogen Inc., Carlsbad, Calif.). The insert was subcloned into pcDNA3 vector using the T4DNA ligase procedure and digested with the appropriate restriction endonucleases to yield pcDNA 3/ORL-1. Using electroporation, the ligation mixture was transformed into shock-transformation-competent E.coli BL21(DE3) cells (Edge Biosystems, Gaitherburg, Md.) and the cells were seeded on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50. mu.g/mL ampicillin and grown overnight in a 37 ℃ incubator. Bacteria containing the expression construct were identified as ampicillin resistant colonies. Candidate constructs were isolated using the alkaline lysis plasmid miniprep procedure and analyzed by restriction endonuclease digestion patterns to determine the presence and orientation of the insert. This cloning strategy will result in a pcDNA3 expression construct comprising a polynucleotide molecule encoding ORL-1.

The retargeted endopeptidase Dyn/a contains a dynorphin targeting domain, which is a natural ligand for the kappa-opioid receptor (KOR). To obtain an expression construct comprising the open reading frame of ORL-1, the expression construct pReceiver-M02/KOR-1 was obtained from GeneCopoeia (GeneCopoeia, Germantown, Md.). Alternatively, an expression construct encoding the KOR may be synthesized and subcloned using methods similar to those described above to produce the expression construct pcDNA3.1/KOR. An exemplary KOR amino acid sequence includes SEQ ID NO: 29 and SEQ ID NO: 30, of a nitrogen-containing gas; exemplary polynucleotide molecules encoding KORs include SEQ ID NO: 65 and SEQ ID NO: 66.

Similar cloning strategies can be used to prepare expression constructs encoding other retargeted endopeptidase receptors, such as pcDNA3.1/DOR or pcDNA3.1/MOR, pcDNA3.1/galanin receptor 1, pcDNA3.1/galanin receptor 2, or pcDNA3.1/galanin receptor 3. An exemplary DOR amino acid sequence includes SEQ ID NO: 27 and SEQ ID NO: 28; an exemplary amino acid sequence of MOR includes SEQ ID NO: 31; the amino acid sequence of exemplary galanin receptor 1 includes SEQ ID NO: 136. SEQ ID NO: 137 and SEQ ID NO: 138; an exemplary galanin receptor 2 amino acid sequence includes SEQ ID NO: 139; and the amino acid sequence of exemplary galanin receptor 3 includes SEQ ID NO: 140. exemplary DOR-encoding polynucleotide molecules include SEQ ID NO: 63 and SEQ ID NO: 64; exemplary polynucleotide molecules encoding MOR include SEQ ID NO: 67; exemplary polynucleotide molecules encoding galanin receptor 1 include SEQ ID NO: 141. SEQ ID NO: 142 and SEQ ID NO: 143; exemplary polynucleotide molecules encoding galanin receptor 2 include SEQ ID NO: 144, 144; and exemplary polynucleotide molecules encoding galanin receptor 3 include SEQ ID NO: 145.

for introducing expression constructs encoding heavy-target endopeptidase receptors, expression of the encoded heavy-target endopeptidase receptors is used Constructs transfected cell lines. To transfect cell lines with opioid or opioid receptors, cells from candidate cell lines are plated at 1X 10⁷The density of individual cells is seeded on the T-coated cell₁₇₅Collagen IV (T)₁₇₅Collagen IV) and grown in an incubator at 37 ℃ under 5% carbon dioxide until the cells reached the desired density. 4.2mL of transfection solution was prepared by adding 4mL of OPTI-MEM reduced serum medium containing 200. mu.L LipofectAmine 2000(Invitrogen, Carlsbad, Calif.) incubated at room temperature for 5 minutes to 4mL of OPTI-MEM reduced serum medium containing 20. mu.g of pRecever-M02/ORL-1 or 20. mu.g of pRecever-M02/KOR-1. The transfection solution was incubated at room temperature for about 20 minutes. The medium was replaced with 8mL of fresh medium without serum and antibiotics, and the transfection solution was added to the cells. The cells were then incubated at 37 ℃ in an incubator at 5% carbon dioxide for about 16-18 hours. The transfection medium was replaced with fresh growth medium and the cells were incubated at 37 ℃ in an incubator with 5% carbon dioxide. After 24 hours, the growth medium was replaced with fresh growth medium containing the antibiotic G418 at a concentration of 1mg/mL in the medium (selection medium) and the cells were incubated for 7 days. Selection medium was changed weekly for a total of 4 weeks (during each week medium was changed, about 90% of cells were dead and removed).

Candidate cell lines transfected with the ORL-1 receptor include SiMa > P30, ND15, ND7, NG108-T15, and SK-N-DZ cell lines. Candidate cell lines transfected with KOR-1 receptor include SiMa, SiMa > P30, ND15, ND7, NG108-T15, and SK-N-DZ cell lines. Transfected NG108-T15 cells failed to survive selection in G418.

2. Single dose and dose response screens of stably transfected cell lines using retargeted endopeptidase molecules.

Cells from the candidate cell line transfected and selected in the previous section were plated at 1X 10⁵Cells/well were seeded into poly-D-lysine or collagen IV coated 96-well plates in RPMI1640 medium containing N2 and B27 supplements and NGF (50-100ng/ml) for 20. + -.4 hours, followed by treatment with compounds. Then using the same species to cultureCells stably transfected with ORL-1 receptor were treated with 30nM of the retargeted endopeptidase Noc/A in panels for 24. + -.2 hours, except for the SK-N-DZ cell line, which was treated with 10nM of the retargeted endopeptidase Noc/A. Cells were lysed in 120 μ L lysis buffer and 20 μ L of lysate was mixed with 2 x SDS buffer for western blot assay as detailed in example I. All cell lines exhibited increased uptake of the retargeted Noc/a compound when transfected with the ORL-1 receptor (table 7).

Cells from the candidate cell line transfected and selected in the previous section were plated at 1X 10⁵Cells/well were seeded into RPMI1640 medium containing 10% FBS and N2 and B27 supplements on poly D-lysine or collagen IV coated 96-well plates for 20 ± 4 hours, followed by treatment with compounds. Cells stably transfected with KOR-1 receptor were treated with 100nM of the retargeted endopeptidase Dyn/A in the same medium for 24 + -2 hours. Cells were lysed in 120 μ L lysis buffer and 20 μ L of lysate was mixed with 2 x SDS buffer for western blot assay as detailed in example I. All cell lines exhibited increased uptake of the retargeted Dyn/A compound when transfected with human KOR-1 receptor.

3. Selection of stably transfected clonal cell lines exhibiting high sensitivity by serial dilution

The following example illustrates how clonal cells affected by the action of a retargeted endopeptidase or having the ability to retargete endopeptidase uptake can be identified in stably transfected established cell lines.

For single cell cloning of the selected cells described above, a limiting dilution cell line cloning method was used. Cells were treated with trypsin, counted, diluted to achieve 0.5-1 cells per 100 μ L, and seeded at 100 μ L per well in selective medium in 5 poly D-lysine coated 96-well plates. Cells were incubated for more than 2 weeks until colonies formed at the bottom of the wells. Positive colonies derived from a single cell were labeled. Photographs of single cell-derived clones were taken using a microscope camera. Cells from wells containing a single clone were grown for an additional 1 week approximately 4 weeks after cloning began and transferred to 24-well plates.

For selection of clones, the main parameter for screening positive clones is the highest amount of cleavage product of SNAP-25 obtained after Noc/A or Dyn/A treatment, measured with antibodies recognizing intact and cleaved SNAP-25 using Western blot analysis. Once enough cells were available, ORL-1 overexpressing clones were tested overnight with 10nM and 30nM of retargeted endopeptidase Noc/A (Table 8). Clones overexpressing KOR-1 were tested overnight with 100nM of retargeted endopeptidase Dyn/A (Table 9). Furthermore, clones overexpressing KOR-1 were tested according to the dynorphin binding assay described in example I.

4. Dose response screening of stably transfected clonal cell lines using retargeted endopeptidase.

Candidate stably transfected clones from section 3 that showed good uptake of retargeted endopeptidase Noc/A were tested in a full dose response experiment to determine their sensitivity and efficacy to retargeted endopeptidase Noc/A. The cells were arranged at 1X 10⁵The cells/well were seeded in poly-D-lysine or collagen IV coated 96-well plates in RPMI1640 medium containing N2 and B27 supplements and NGF (50-100ng/ml) for 20 + -4 hours, followed byTreatment with the compound. Cells from parental AGN P33 and ND7 clone were treated with 0, 0.14nM, 0.4nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM, and 100nM Noc/A in the same medium for 24 hours and incubated in medium without retargeting endopeptidase for 24 hours to lyse SNAP-25. In addition, cells from the parental AGN P33 cell line were treated with Noc/A at 0, 0.03nM, 0.08nM, 0.24nM, 0.74nM, 2.22nM, 6.67nM and 20nM in the same medium for 24 hours and incubated in medium without the retargeted endopeptidase for 24 hours to lyse SNAP-25. The medium was removed and the cells were washed and lysed for ECL sandwich ELISA assay as detailed in example II. Data obtained from parental and clonal AGN P33 cell lines stably transfected with the ORL-1 receptor are summarized in Table 10. Clone #2 and clone #6 exhibited better sensitivity and efficacy than the parental cell line to the retargeted endopeptidase Noc/A. Furthermore, the new clonal cell lines with higher sensitivity allow dose response using lower concentrations, thus demonstrating that the new clonal cell lines are more sensitive.

Data obtained from ND7 parental and clonal cell lines stably transfected with ORL-1 receptor are summarized in table 11. All clones tested exhibited improved sensitivity and efficacy to the retargeted endopeptidase Noc/A over the parental cell line ND 7. Clone 4B7, clone 1E6, and clone 1C11 were most sensitive, and EC₅₀The values were all below 10 pM.

Table 12 summarizes the results obtained from the generation and testing of clonal cell lines that overexpress the ORL-1 receptor in different cellular backgrounds.

Example V

Development of clonal cell lines derived from the parental SK-N-DZ cell line.

The following example illustrates how clonal cells that are sensitive to exocytosis inhibition by a retargeted endopeptidase or have the ability to retargete endopeptidase uptake in a parental established cell line can be identified.

1. Isolation of clonal cell lines.

During characterization of the SK-N-DZ cell line, the cells that make up the established cell line were found to comprise at least 5 different cell phenotypes. To determine whether any of these phenotypically distinct cell types sensitize the cell line to inhibition of retargeted endopeptidase exocytosis, two different limiting dilution screens were performed to obtain single colony isolates of each phenotypically distinct cell type.

Cells from SK-N-DZ stock solution at appropriate densities were grown in DMEM, 10% fetal bovine serum (heat inactivated), 0.1mM non-essential amino acids, 10mM HEPES, 1mM sodium pyruvate, 100U/ml penicillin, 100 μ g/ml streptomycin contained in collagen IV coated T175 flasks. After the second passage, the cells were treated with trypsin to produce a cell suspension, and the cell concentration was determined. About 4.0X 10 of the cell suspension ⁶Individual cells were transferred to 50mL tubes and dissociated into single cells by repeated forceful expelling through an 18.5 gauge needle using a 10mL syringe. Cells from this dissociated single cell suspension were then diluted to 0.2X 10 by adding 15mL of fresh growth medium⁶One cell/mL concentration and 2.5 μ L of this dilution was added to 50mL of fresh growth medium to reach a concentration of 10 cells/mL. For this final diluted stock solution, 100 μ L of growth medium was added to each well of a collagen IV coated 96-well plate and the cells were incubated at 37 ℃ in an incubatorResting growth for 4 weeks in medium 5% carbon dioxide. 4 96-well plates were set up for analysis. After 4 weeks, the wells were observed microscopically to identify single colonies that were growing, and for each colony identified, 100 μ L of fresh growth medium was added to each well and the cells were allowed to grow quietly in an incubator at 37 ℃ under 5% carbon dioxide for 2 weeks. After 2 more weeks of growth, the growing single colonies were trypsinized and transferred to new 96-well plates for further growth. Once colonies reached approximately 1,000 cells growth according to visual observation, the cells were trypsinized and each cell suspension was transferred to each well of a new collagen IV-coated 24-well plate. These cells were grown in an incubator at 37 ℃ under 5% carbon dioxide, with fresh growth medium being replenished every 2 to 3 days if necessary. The cells were grown until the cultures reached about 60% or more confluence, at which time, the cells were treated with trypsin according to the confluence of the cells in the 24-well plate, and each cell suspension was transferred to 25cm coated with collagen IV ²In a flask. These cells were grown in an incubator at 37 ℃ under 5% carbon dioxide, with fresh growth medium being replenished every 2 to 3 days if necessary. Once the cells in the flask reached 70-80% confluence, they were frozen and stored in liquid nitrogen until clonal testing was performed to determine their sensitivity to exocytosis inhibition by Noc/A. From the 384 colony isolates initially established by the two screenings, 24 clonal cell lines were selected and expanded for use in subsequent screening procedures, according to viability and growth criteria. Of these cell lines, 12 fast growing cell lines were identified.

2. Preliminary screening for retargeted endopeptidase activity sensitivity of cells from clonal cell lines using retargeted endopeptidase.

To determine whether cells from clonal cell lines are susceptible to retargeted endopeptidase Noc/A activity, a preliminary screen was performed using an immune-based method for determining endopeptidase activity.

13 SK-N-DZ clones (#3, #4, #5, #8, #9, #10, #13, #15, #16, #17, #18, #22 and #23) plus SK-N-DZ parental cells were seeded in EMEM, 10% FBS, 1 XB 27 and 1 XN 2 on 96-well plates (cell number per well unknown) and incubated overnight. Cells were treated with 1nM Noc/A for 24 h. Cells were lysed with 100. mu.L of lysis buffer for 20 minutes and centrifuged at 4000rpm for 20 minutes. mu.L of 2 XSDS sample buffer was added to 50. mu.L of cell lysate and heated at 95 ℃ for 5 minutes. A 10 μ L protein sample was loaded onto each lane of a 12% NuPage gel and subjected to western blot assay as described in example I. Evaluation against total SNAP-25 and cleaved SNAP-25 confirmed that clone #3, clone #8, clone #15 and clone #22 were at least as good as the parental cells in terms of Noc/A uptake. After scaling up the cells, full dose response treatment and analysis using ECL sandwich ELISA assay were performed.

3. Secondary response screens were performed on clonal cell lines using retargeted endopeptidase molecules.

To determine whether cells from clonal cell lines are susceptible to retargeted endopeptidase Noc/A activity, a secondary screen is performed using an immunization-based method for determining endopeptidase activity.

To further compare the cell lines of these SK-N-DZ clones, ECL sandwich ELISA assays were performed. 5 clones (#3, #9, #15, #16, #22) plus SK-N-DZ parent cells were seeded at 25,000 cells/well per cell line in poly-D-lysine coated 96-well plates in RPMI 1640, 10% FBS, 1 XB 27 and 1 XN 2 medium (without NGF) over the weekend. Cells were treated with Noc/A at doses of 0 to 20nM (0nM, 0.03nM, 0.08nM, 0.24nM, 0.74nM, 2.22nM, 6.67nM, 20nM) for 24 hours. Quantification of cleaved SNAP-25 by ECL ELISA assay as detailed in example I₁₉₇。

Table 13 shows the EC of 5 clones and their parental cell lines₅₀Value and signal-to-noise ratio. The 3 clones named #3, #9 and #15 produced lower EC than the parental cell line (about 2nM)₅₀Values (< 1nM) and clone #16 and clone #22 produced similar EC₅₀The value is obtained. However, the total signal from the lysed SNAP25 was higher than in clone #3, clone #22 and parental cells. And it Clone #9, clone #16 and clone #15 had lower total signal compared to the remaining cell lines.

Noc/a treatment conditions for SK-N-DZ clones were optimized and assays were performed to compare clone #3, clone #15 and clone #22 to parental heterogeneous SK-N-DZ cell lines. Table 14 shows the results of the comparison and demonstrates that assay optimization greatly improved the signal-to-noise ratio of the assay. Clone #3 and clone #22 were selected for further assay development because both clones possessed superior sensitivity and efficacy.

Example V

Characterization and comparison of clonal cell lines for retargeted endopeptidase uptake

The following examples illustrate how clonal cell lines derived from established cell lines comprising heterogeneous cell populations or by transfection of target receptors and subsequent cloning of cell lines can be characterized and compared.

To assess the specificity or selectivity of uptake of the retargeted endopeptidase, non-specific uptake assays were performed using retargeted endopeptidases lacking a targeting domain. For opioid retargeted endopeptidase, cells from clone #6 AGN P33 (containing cells stably transformed with an expression construct encoding the ORL-1 receptor) and clone #3 and #22 SK-N-DZ (containing cells expressing the endogenous ORL-1 receptor) were expressed as follows 150,000 cells/well were seeded in poly-D-lysine coated 96-well plates in RPMI 1640 serum-free medium containing N2 and B27 supplements and NGF (50ng/mL) in 5% CO₂Was incubated at 37 ℃ for 20. + -.4 hours and subsequently treated with the compound. 8 doses of Noc/A in the range of 0nM to 20nM or 0nM to 40nM or 8 doses of LH in the range of 0nM to 400nM or 0nM to 40nM in the same medium_NCells were treated for 22 hours. The medium was removed and the cells were washed, lysed and centrifuged to remove debris from the preparation for use in a sandwich ELISA assay. The ELISA plate coated with the 2E2a6 monoclonal antibody was blocked with 150 μ L of blocking buffer for 1 hour at room temperature. After removal of the blocking buffer, 30 μ L of cell lysate was added to each well and the plates were incubated for 2 hours at 4 ℃. Plates were washed 3 times with PBS-T and 30. mu.L of a detection alpha-SNAP 25 polyclonal antibody labeled SULFO-TAG NHS-ester at a concentration of 5. mu.g/mL in 2% blocking agent in PBS-T was added to the bottom corner of each well. Plates were sealed and shaken at room temperature for 1 hour, followed by 3 washes with PBS-T. After washing was complete, 150. mu.L of 1 × reading buffer was added to each well and the plates were read in a SI6000 image reader. Noc/A uptake and negative control LH _NThe results of the comparison/A are shown in tables 15 and 16. These results indicate that Noc/A and LH were present in both cell lines_NThere was a clear difference between/A uptake, confirming the specificity of Noc/A uptake.

Table 17 summarizes the results of characterizing and comparing 3 cell lines. The SK-N-DZ clone #3 and #22 possessed the same sensitivity as primary eDRG and had excellent signal-to-noise ratio, from which robust assays could be developed for the retargeted endopeptidase Noc/a. AGN P33 clone #6 also became an excellent candidate due to low non-specific uptake and appropriate sensitivity.

To assess the sensitivity of retargeted endopeptidase uptake, a ligand saturation binding assay was performed. The interaction of most ligands with their binding sites can be characterized in terms of binding affinity (NIH assay guidelines). In general, high affinity binding involves a longer residence time of the ligand at its receptor binding site than in the case of low affinity binding. Dissociation constants are often used to describeLigands(L) (e.g., a drug) and protein (P), i.e., how tightly the ligand binds to a particular protein. Equilibrium saturation binding experiments can measure total binding and non-specific binding (NSB) at various radioligand concentrations. Equilibrium dissociation constant or affinity for radioligand K _dAnd the maximum number of receptor binding sites Bmax can be calculated from specific binding (total binding-NSB) using non-linear regression analysis. For specific binding of K_dCan be calculated using a single-site binding hyperbolic nonlinear regression analysis (i.e., GraphPad Prism) as shown in the following equation, where Bmax is the maximum number of binding sites (pmol/mg or pmol/cell or site/cell), and K is_d(nM, pM, etc.) is the concentration of radioligand required to achieve half-maximal binding:

for opioid retargeted endopeptidase, cells from the AGN P33 clone #6 cell line (containing cells stably transformed with an expression construct encoding the ORL-1 receptor), the SK-N-DZ parent cell line, and the SK-N-DZ clone #3, #15, and #22 (containing cells expressing the endogenous ORL-1 receptor) were seeded at 200,000 cells/well in RPMI1640 serum-free medium containing 1 XN 2 and 1 XB 27 supplements on 48-well plates coated with poly-D-lysine and at 5% CO₂Incubate overnight in an incubator at 37 ℃. The medium was removed and cells and 150 μ L of Tris binding buffer were added to each well to assess total binding and 100 μ L of Tris binding buffer was added to each well to assess non-specific binding well. Approximately 50 μ L of cold pain sensitivity peptide (2.5 μ M in SK-N-DZ cell line, and 1 μ M in AGN P33 clone # 6) at 4 Xfinal concentration was added to the non-specific binding wells, and 50 μ L of cold pain sensitivity peptide at 4 Xfinal concentration was added to the non-specific binding wells ³H-nociceptin (0nM, 0.05nM, 0.1nM, 0.2nM, 0.4nM, 0.8nM, 1.6nM, 3.1nM, 6.3nM, 12.5nM, 25nM and 50nM were added to SK-N-DZ cell line and 0, 0.01nM, 0.02nM, 0.039nM, 0.078nM, 0.156nM, 0.313nM, 0.625nM, 1.25nM, 2.5nM, 5.0nM and 10nM were added to AGN P33 clone # 6) was added to both total and non-specific binding wells to a final volume of 200 μ L. After incubation at 37 ℃ for 30 min, cells were washed 2 times with 0.5mL cold wash buffer. Cells were then denatured in 200 μ L2N NaOH and transferred to 20mL scintillation vials containing 5mL scintillation fluid. Dose-response plots were plotted using raw data and K was calculated for each sample_dThe value is obtained. The resulting raw data were transmitted into SigmaPlot v10.0 and dose-response curves were defined from the equation classes for ligand binding using single point saturation fitting. Generating a graphical report and containing the following parameters: r²(correlation coefficient), Bmax and K_dSE (coefficients. + -. standard error). Plots of total binding, specific binding and non-specific binding were obtained from assays performed on SK-N-DZ clonal cell lines #3, #15 and #22 and the AGNP33 clonal cell line #6 cells. SK-N-DZ clone #3 and #22 pair ³Concentration of H-nociceptin produced is dependent onDependent and saturable binding. Under the same experimental conditions, the SK-N-DZ clone #15 pair³H-nociceptin produces a dose-dependent response, but is also not saturated at the highest dose of 50 nM. Cells from the AGN P33 clone #6 bound with significantly higher affinity than SK-N-DZ cell line expressing endogenous ORL-1³H-nociceptin (highest dose of 10nM vs 50nM in SK-N-DZ) and has low non-specific binding.

K was estimated from 3 independent binding experiments performed on 3 different days for each cell line using the saturated binding curves of the SK-N-DZ clone #3, #22, #15 and the AGN P33 clone #6_dAnd a Bmax value. The rank order of these 4 cell lines was: AGN P33 clone #6 (K)_dClone #3 SK-N-DZ at 1.86nM and Bmax at 2.9 fmol/cell (K)_dClone #22 SK-N-DZ (K) 14nM and Bmax 0.6 fmol/cell ≧ SK-N-DZ_d17nM and Bmax 0.6 fmol/cell > SK-N-DZ clone #15 (K)_d> 50 nM). To achieve a saturating dose response for the SK-N-DZ clone #15, a higher dose range is required³H-nociceptin. Table 16 summarizes data on the characteristics of specific plasma membrane nociceptin binding sites in 3 SK-N-DZ clone #3, #15, and #22 and AGN P33 clone #6 stable cell lines. And (3) displaying data: 1) high affinity sites and very low non-specific binding (K) in clone #6 of AGN P33 _d1.8nM, Bmax 2.9 fmol/cell); 2) nociceptin binding can be performed on SK-N-DZ native cells expressing endogenous receptors; 3) the affinity of the AGN P33 clone #6 for nociceptin was about 10-fold higher than that of the SK-N-DZ cell line; 4) SK-N-DZ clonal cell lines #3 and #22 (K) as can be seen in cell-based potency assays_d14-17nM, Bmax 0.6 fmol/cell) more acceptor sites per cell than SK-N-DZ clone #15 (saturation was not achieved at the same dose range).

To assess the sensitivity of retargeted endopeptidase uptake, the amount of expressed retargeted endopeptidase receptor was assessed at the mRNA level using RT-PCR. The amount of receptor expressed in the cells is an important aspect of characterizing the cell line for testing and is related to the sensitivity to heavy targeted endopeptidases. The amount of expressed retargeted endopeptidase receptor can also be a tool for screening other potential cell lines and eliminating cell lines that do not express the target receptor. One method of measuring receptor expression is to quantify the amount of retargeted endopeptidase receptor mRNA using real-time PCR (RT-PCR).

For opioid re-targeted endopeptidases, RNA was isolated from cells of the untransfected parental SiMa cell line, from AGN P33 clone #6, from the parental SK-N-DZ cell line, and from SK-N-DZ clone #3 and #22 grown in serum-free or serum-containing medium. mRNA was converted to cDNA and ORL-1 was amplified using the following oligonucleotide primers for ORL-1, with real-time measurements performed to determine the relative amounts present in each cell line: forward primer 5'-CACTCGGCTGGTGCTGGTGG-3' (SEQ ID NO: 148) and reverse primer 5'-AATGGCCACGGCAGTCTCGC-3' (SEQ ID NO: 149). By using SYBR The DNA is quantified by a green fluorescent dye, which fluoresces with respect to the amount of double stranded DNA (PCR product) present in the reaction. The amount of fluorescence versus cycle number was plotted to obtain a logistic curve (logistic curve) for each reaction. The faster the reaction reaches the linear stage of the curve, the more ORL-1 receptor cDNA is present in the reaction. Control RT reactions with no added enzyme were used to determine the presence of contamination. Since no RT enzyme was present in this reaction, no cDNA was produced. The use of RNA templates does not result in PCR products, so if a PCR curve is generated in the-RT reaction, only one possibility is the presence of genomic DNA contamination. In the-RT reaction, if no PCR curve was present, it was determined that there was little genomic DNA contamination (data not shown). Table 18 lists the cell lines and their CT values. CT is higher thanThe number of PCR cycles of the corresponding PCR reaction at which the signal is set to the threshold value. The amount of ORL-1 receptor mRNA in one cell line can be compared to another by observing the corresponding CT values. According to CT values, cells from the AGN P33 clone #6 produced ORL-1mRNA in serum-free medium (average CT: 28.6vs.17.3) and serum-containing medium (average CT: 26.1vs.16.5) much higher than the parental SiMa cell line. The mRNA obtained from cells passaged 6 and 16 of the AGN P33 clone #6 appeared to have very little difference. The CT values and curves for the parental SK-N-DZ cell line and the clonal cell lines #3 and #22 also appear to have minimal differences. This conclusion is consistent with cells grown in serum-containing and serum-free media and reflects the similarity of these cell lines observed in cell-based potency assays against Noc/a.

Example VII

P binding selectively to a bond susceptible to cleavage at the BoNT/A cleavage site₁Development of an alpha-SNAP-25 monoclonal antibody having a SNAP-25 epitope at the residue with a free carboxyl terminus

The following example illustrates how to prepare P which selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site₁An alpha-SNAP-25 monoclonal antibody having a carboxy-terminal SNAP-25 epitope at residue.

1. Production of the alpha-SNAP-25 monoclonal antibody.

For the development of P which selectively binds to bonds which are easily cleavable at the BoNT/A cleavage site₁SN having a carboxyl terminus at residueMonoclonal alpha-SNAP-25 antibody to AP-25, peptide CDSNKTRIDEANQ having 13 residues_COOH(SEQ ID NO: 38) is designed as a SNAP-25 cleavage product antigen. The peptide comprises a flexible linker region and an N-terminal cysteine residue for binding to KLH and amino acids 186-197 of human SNAP-25(SEQ ID NO: 5) with a carboxylated C-terminal glutamine (SEQ ID NO: 38). Monoclonal antibodies raised against appropriately selected unique peptide sequences will control epitope specificity, allowing the identification of specific protein subpopulations from a pool of closely related isoforms. Blast searches showed that this peptide has only high homology to SNAP-25 and is nearly impossible to cross-react with other proteins in neuronal cells. At the same time, the sequence is carefully scrutinized by using computer algorithms to determine the hydrophilicity index, protein surface probability, flexibility and favorable secondary structure, followed by appropriate localization and presentation of the selected peptide sequence. Peptides were synthesized and conjugated to Keyhole Limpet Hemocyanin (KLH) to increase immunogenicity. 6 Balb/c mice were immunized with this peptide and 3 immunizations were performed in about 8 weeks, after which the mice were bled for testing. The blood was incubated at 4 ℃ for 60 minutes to clot it. The coagulated blood was centrifuged at 10,000Xg for 10 minutes at 4 ℃ to pellet the cell debris. The resulting serum samples were divided into 50 μ l aliquots and stored at-20 ℃ until use.

Similar strategies based on other SNAP-25 antigens disclosed in the specification were used to develop P that selectively binds to a readily cleavable bond at the BoNT/A cleavage site₁An alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl terminus at residue. For example, SNAP-25 antigen SEQ ID NO: 45 may be conjugated to KLH, rather than SNAP-25 antigen SEQ ID NO: 38. as another example, the amino acid sequence of SEQ ID NO: 38 of human SNAP-25 can be replaced with the amino acid 186-197 of SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43 or SEQ ID NO: 44.

2. screening for the presence of an alpha-SNAP-25 monoclonal antibody.

To determine P which selectively binds a bond susceptible to cleavage at the BoNT/A cleavage site₁The presence of an alpha-SNAP-25 monoclonal antibody to SNAP-25 antigen with a carboxyl terminus at residue was determined by comparative ELISA and cell-based lysis using extracted mouse serum. For comparative ELISA, two fusion proteins were constructed: BirA-HisTag-SNAP-25_134-197(SEQ ID NO: 48) and BirA-HisTag-SNAP-25_134-206(SEQ ID NO：49)。BirA-HisTag-SNAP-25_134-197Comprising a naturally biotinylated 16-amino acid BirA peptide (SEQ ID NO: 50) having the amino acid sequence of SEQ ID NO: the 50 amino terminus was linked to the SNAP-25 peptide comprising amino acids 134-197(SEQ ID NO: 5). BirA-HisTag -SNAP-25_134-206Comprising a naturally biotinylated 16-amino acid BirA peptide (SEQ ID NO: 50) having the amino acid sequence of SEQ ID NO: the 50 amino terminus is linked to the SNAP-25 peptide comprising amino acids 134-206(SEQ ID NO: 5). The two substrates were dosed at 10. mu.g/mL BirA-HisTag-SNAP-25_134-197And BirA-HisTag-SNAP-25_134-206Was suspended in 1 × PBS. BirA-HisTag was prepared by adding about 100. mu.l of the appropriate substrate solution and incubating the plates at room temperature for 1 hour-SNAP-25_134-197And BirA-HisTag-SNAP-25_134-206Coated on separate plates. The washed plates were incubated for 1 hour at 37 ℃ in 0.5% BSA in 1: 10 to 1: 100 dilutions in 1: 10 to 1: 100 serum containing antibodies from one of the 6 immunized mice (mouse 1, mouse 2, mouse 3, mouse 4, mouse 5 and mouse 6). In 200. mu.l TBS, 0.1% TWEEN-20Wash the probed primary plate 4 times for 5 minutes (polyoxyethylene (20) sorbitan monolaurate). The washed plates were incubated for 1 hour at 37 ℃ in 1: 10,000 dilution of goat polyclonal anti-mouse IgG antibody conjugated to horseradish peroxidase (Pierce Biotechnology, Rockford, Ill.) as secondary antibody in 1 × TBS. At 200. mu.l TBS, 0.1% TWEEN-20The plates probed with secondary antibody were washed 4 times in (polyoxyethylene (20) sorbitan monolaurate). The results of the chromogenic detection of the labeled SNAP-25 product were observed by chromogenic detection using the ImmunoPure TMB substrate kit (Pierce Biotechnology, Rockford, Ill.). Coated with BirA-HisTag -SNAP-25_134-197The plates showed yellow colour but were coated with BirA-HisTag-SNAP-25_134-206The plates showed no yellow color, indicating that the alpha-SNAP-25 antibody preferentially recognizes SNAP-25₁₉₇And (3) cracking the product. The results showed that of 6 mice used for immunization, 3 mice (mouse 2, mouse 3 and mouse 4) were paired with P which is susceptible to bond cleavage at the BoNT/A cleavage site₁SNAP-25 antigen with a carboxyl terminal at the residue has higher titer and higher specificity.

Determination of this using ELISA light chain Activity assayThese results were obtained. A96-well plate coated with React-Bind streptavidin was prepared by adding about 100. mu.l of the following substrate solutions (Pierce Biotechnology, Rockford, IL): 100 mu L of 12 BirA-HisTag different concentrations were used-SNAP-25_134-197Coating lines A-C; using 100. mu.L of BirA-HisTag with a concentration of 10. mu.g/mL-SNAP-25_134-206D-H lines were coated. By aspirating the substrate solution and applying 200. mu.l TBS, 0.1% TWEEN-20The plates were washed by rinsing each well 3 times (polyoxyethylene (20) sorbitan monolaurate). Incubation of buffer (50mM HEPES, pH 7.4, 1% fetal bovine serum, 10. mu.M ZnCl) at 37 ℃ in BoNT/A₂10mM dithiothreitol) for 20 minutes and 100. mu.l of the pre-reduced BoNT/A are added to the substrate-coated plate and incubated at 37 ℃ for 90 minutes. Incubation buffer was incubated by aspiration of BoNT/A and incubated with 200. mu.l TBS, 0.1% TWEEN-20 The BoNT/A treated plates were washed by rinsing each plate 3 times (polyethylene oxide (20) sorbitan monolaurate). The washed plates were incubated for 1 hour at 37 ℃ in 0.5% BSA in 1 × TBS, which 1 × TBS contained 1: 10 to 1: 100 dilutions of the antibody-containing sera tested. At 200. mu.l TBS, 0.1% TWEEN-20Wash the probed primary plate 4 times for 5 minutes (polyoxyethylene (20) sorbitan monolaurate). The washed plates were incubated for 1 hour at 37 ℃ in 1 × TBS containing a 1: 10,000 dilution of goat polyclonal anti-mouse IgG antibody conjugated to horseradish peroxidase (Pierce Biotechnology, Rockford, Ill.) as a secondary antibody. In 200. mu.l TBS, 0.1％ TWEEN-20The plates probed with secondary antibody were washed 4 times in (polyoxyethylene (20) sorbitan monolaurate). The results of the chromogenic detection of the labeled SNAP-25 product were observed by chromogenic detection using the ImmunoPure TMB substrate kit (Pierce Biotechnology, Rockford, Ill.). Using antibody-containing sera from all 6 immunized mice (mouse 1, mouse 2, mouse 3, mouse 4, mouse 5, and mouse 6), detection of SNAP-25 in samples treated with BoNT/A was determined₁₉₇The presence of cleavage products correlated with yellow development, but was not observed in the untreated control. Thus, comparative ELISA analysis showed that of 6 mice used for immunization, 3 mice (mouse 2, mouse 3, and mouse 4) had higher titers and higher specificities for SNAP-25 antigen with a carboxy terminus at residue P1 of the bond that is readily cleavable at the BoNT/a cleavage site.

For cell-based lysis assays, PC12 cells were seeded at a suitable density in 60mm containing 3mL of suitable serum medium (Table 1)²In tissue culture plates. Cells were grown in an incubator at 37 ℃ under 5% carbon dioxide until the cells reached the appropriate density. 500 μ L of transfection solution was prepared by: mu.L of OPTI-MEM reduced serum medium containing 15. mu.L of LipofectAmine 2000(Invitrogen Inc., Carlsbad, Calif.) incubated for 5 minutes at room temperature was added to 250. mu.L of OPTI-MEM reduced serum medium containing 10. mu.g of pQBI-25/GFP-BoNT/A-LC expression construct (SEQ ID NO: 51). The pQBI-25/GFP-BoNT/A-LC expression construct comprises a pQBI-25 expression vector (Qbiogene Inc., Carlsbad, Calif.) having its promoter element functionally linked to a polynucleotide encoding the GFP-BoNT/A light chain (SEQ ID NO: 52). The transfection mixture was incubated at room temperature for about 20 minutes. The medium was replaced with fresh, unsupplemented medium and 500 μ Ι _ of transfection solution was added to the cells. The cells were then incubated at 37 ℃ in an incubator at 5% carbon dioxide for about 6 to 18 hours. Cells were washed and harvested as described in example II. For detection of cleaved SNAP-25₁₉₇The presence of the product, as described in example II, by means of the protein Aliquots of each harvested sample were analyzed by blotting, but the primary antibody used was a 1: 1,000 dilution of antibody-containing serum and the secondary antibody used was a 1: 20,000 dilution of mouse alpha-IgG horseradish peroxidase (Pierce Biotechnology, Rockford, IL). Using antibody-containing sera from 3 mice (mouse 2, mouse 3 and mouse 4), detection corresponding to SNAP-25 was detected in samples treated with BoNT/A₁₉₇A single band of lysate, but not in the untreated control. Thus, cell-based lysis assays showed that of the mice used for immunization, 3 mice (mouse 2, mouse 3 and mouse 4) were paired with P, which is susceptible to bond cleavage at the BoNT/A cleavage site₁SNAP-25 antigen with a carboxyl terminal at the residue has higher titer and higher specificity.

3. Generation of hybridomas.

For the preparation of P which produces a bond susceptible to cleavage selectively bound at the BoNT/A cleavage site₁Hybridomas of alpha-SNAP-25 monoclonal antibody with the carboxy-terminal SNAP-25 antigen at residue, spleens of mouse 2 were harvested 3 days after the last "boost" immunization and splenocytes were fused with myeloma cells P3-X63 Ag8.653 using standard hybridoma protocols. These cells were seeded in 5 96-well plates and hybrids were selected using HAT medium. Within 8 to 14 days after fusion, BirA-HisTag coated in two separate plates using comparative ELISA -SNAP-25_134-197Peptides and BirA-HisTag-SNAP-25_134-206Peptides were screened for the first time for approximately 480 parental clones. Comparative ELISA is a method for identifying SNAP-25 that produces cleavage₁₉₇A method for rapidly screening hybridomas for specific antibodies. The top 18 clones were further screened using the cell-based lysis assay described above and immunostaining of LC/a transfected cells (table 20).

Clones 1D3, 1G10, 2E2, 3C1, 3C3 and 3E8 were further cloned by limiting dilution because the conditioned media produced by these clones contained alpha-SNAP-25 antibody with preferential binding specificity, i.e., SNAP-25₁₉₇Cleavage product vs. SNAP-25₂₀₆Ratio of uncleaved substrate_197/206At least 4: 1, and detection of SNAP-25 using a cell-based lysis assay and immunostaining of PC12 cells transfected with GFP-LC/A₁₉₇And (3) cracking the product. Similarly, clones 2C9, 2F11, 3G2, 4D1 and 4G6 were further cloned by limiting dilution because the conditioned media produced by these clones contained alpha-SNAP-25 antibody with preferential binding specificity, i.e., SNAP-25₂₀₆Uncleaved substrate vs. SNAP-25₁₉₇Ratio of cleavage products_206/197At least 1.5: 1, and using a cell-based lysis assay to detect SNAP-25 ₂₀₆The substrate was not cleaved. These single cell derived clones were screened again using comparative ELISA, cell-based lysis and immunostaining to determine their affinity and specificity, and the antibodies were isotype-classified using standard procedures. Ascites were produced by clones 1D3B8(igm.k), 1G10a12(igg3.k), 2C9B10(igg3.k), 2E2a6(igg3.k), 2F11B6(igm.k), 3C1a5(igg2a.k) and 3C3E2(igg 2a.k). During cloning, clone 3E8 ceased antibody production and was not able to be evaluated further.

4. Evaluation of the binding specificity of the α -SNAP-25 monoclonal antibody.

To evaluate P selectively binding a bond susceptible to cleavage at the BoNT/A cleavage site₁The binding specificity of the α -SNAP-25 monoclonal antibody to SNAP-25 antigen having a carboxyl terminus at residue was determined for SNAP-25 lysates using ascites fluid produced by clones 1D3B8, 1G10A12, 2C9B10, 2E2A6, 2F11B6, 3C1A5, and 3C3E2 using cell-based activity assays, immunocytochemistry, and immunoprecipitation.

For cell-based activity assays, uncleaved SNAP-25 was detected by analyzing ascites fluid containing alpha-SNAP-25 antibody using Western blot analysis₂₀₆Substrate and cleaved SNAP-25₁₉₇The ability of the product to determine binding specificity. PC12 cells were seeded at a suitable density in 60mm containing 3mL of suitable serum medium as described above ²Tissue culture plates were grown in an incubator at 37 ℃ under 5% carbon dioxide until the appropriate cell density was reached and transfected with transfection solutions containing expression constructs not containing pQBI-25/GFP-BoNT/A-LC (untransfected cells) or pQBI-25/GFP-BoNT/A-LC (transfected cells). Cells were washed and harvested as described in example I. For detection of uncleaved SNAP-25₂₀₆Substrate and cleaved SNAP-25₁₉₇Aliquots of each harvested sample were analyzed by western blotting for the presence of product, as described in example I, except that the primary antibody used was a 1: 100 dilution of ascites fluid containing the α -SNAP-25 monoclonal antibody, and the secondary antibody used was a 1: 20,000 dilution of horseradish peroxidase-conjugated α -mouse IgG (Pierce Biotechnology, Rockford, IL). In addition, 3 commercially available mouse α -SNAP-25 monoclonal antibodies were also tested. SMI-81(Sternberger Monoclonals Inc., Lutherville, Md.), an α -SNAP-25 antibody, indicated by the manufacturer for detection of uncleaved SNAP-25₂₀₆Substrate and cleaved SNAP-25₁₉₇The product was used at 15,000 dilutions according to the manufacturer's recommendations. MC-6050 (Research) &Diagnostic Antibodies, Las Vegas, NV), an α -SNAP-25 antibody, indicated by the manufacturer to detect uncleaved SNAP-25₂₀₆Substrate and cleaved SNAP-25₁₉₇The product was used in a 1: 100 dilution according to the manufacturer's recommendations. MC-6053 (Research)&Diagnostic Antibodies, Las Vegas, NV), an alpha-SNAP-25 antibody, manufacturer's instructions to detect cleaved SNAP-25 only₁₉₇The product was used in a 1: 100 dilution according to the manufacturer's recommendations.

Table 21 indicates that ascites fluid containing the alpha-SNAP-25 antibody only detects SNAP-25₁₉₇And (3) cracking the product. Cell-based lysisThe assay indicated that ascites produced by clones 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 would synthesize pairs of SNAP-25₁₉₇alpha-SNAP-25 monoclonal antibody with high binding specificity for the cleavage product, thereby allowing for relative SNAP-25₂₀₆The uncleaved substrate selectively recognizes the cleavage product. Detection of SNAP-25 by commercial antibody SMI-81₂₀₆Not cleaving the substrate but recognizing SNAP-25₁₉₇The ability to cleave the product was poor (Table 21). Surprisingly, the commercial antibody MC-6050 only detected SNAP-25₂₀₆The substrate was not cleaved and SNAP-25 could not be recognized₁₉₇Cleavage products (table 21). More surprisingly, the commercial antibody MC-6050 only detected SNAP-25₂₀₆The substrate was not cleaved and SNAP-25 could not be recognized ₁₉₇Cleavage products, even if the manufacturer claims that such antibodies can selectively detect SNAP-25₁₉₇Cleavage products (table 21). Thus, this analysis shows that although 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 are on SNAP-25₁₉₇The cleavage products exhibited suitable selectivity, but 1G10a12 and 2F11B6 did not. Furthermore, none of the commercial antibodies SMI-81, MC-6050 and MC-6053 are suitable for use in the immuno-based methods disclosed in the present application, as none of them is capable of selectively detecting SNAP-25₁₉₇And (3) cracking the product.

For immunocytochemical analysis, uncleaved SNAP-25 was detected by analyzing ascites fluid containing alpha-SNAP-25 antibody by immunostaining₂₀₆Substrate and cleaved SNAP-25₁₉₇The ability of the product to determine binding specificity. See, for example, Ester Fernandez-Salas et al, Plasma Membrane Localization Signals in the Light Chain of Botulinum Neurotoxin, Proc. Natl. Acad. Sci., U.S. A.101 (9): 3208-3213(2004). PC12 cells were seeded at an appropriate density, grown, and transfected with a transfection solution containing no pQBI-25/GFP-BoNT/a-LC expression construct (untransfected cells) or a transfection solution containing pQBI-25/GFP-BoNT/a-LC expression construct (transfected cells) as described above. Cells were washed with 1 × PBS and fixed in 5mL PAF for 30 min at room temperature. The fixed cells were washed in phosphate buffered saline and 5ml of 0.5% Triton in 1 XPBS X-100 (polyethylene glycol octyl phenyl ether), washed with 1 XPBS, and permeabilized in 5mL of methanol at-20 ℃ for 6 minutes. Permeabilized cells were blocked in 5mL 100mM glycine for 30 min at room temperature, washed with 1 XPBS, and blocked in 5mL 0.5% BSA in 1 XPBS for 30 min at room temperature. The blocked cells were washed in 1 XPBS, and incubated for 2 hours at room temperature in 0.5% BSA in 1 XPBS containing a 1: 10 dilution of ascites produced by the clonal hybridoma cell line tested. Cells probed with primary antibody were washed 3 times in 1 × PBS for 5 minutes each. The washed cells were incubated at room temperature for 2 hours in 1 XPBS containing conjugated ALEXAGoat polyclonal anti-mouse immunoglobulin G heavy and light chain (IgG, H + L) antibodies (Invitrogen, Carlsbad, CA) from FLUOR 568 were diluted 1: 200 as secondary antibodies. Cells probed with secondary antibody were washed 3 times in 1 × PBS for 5 min each. Mounting the washed cells to VECTASHIELDIn a coverslip (Vector Laboratories, Burlingame, CA) and covered with a coverslip in preparation for microscopic examination. Images of signal detection were obtained using a Leica confocal microscope, using a suitable laser setup. Table 21 shows specific detection of SNAP-25 in ascites Using the alpha-SNAP-25 antibody ₁₉₇And (3) cracking the product. Immunocytochemistry analysis of the surface, ascites fluid produced by clones 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 will synthesize the pair SNAP-25₁₉₇alpha-SNAP-25 monoclonal antibody with high binding specificity for the cleavage product, allowing for comparison with SNAP-25₂₀₆Uncleaved substrate preferentially recognizes the cleavage product.

For immunoprecipitation analysis, protein A (HiTrap) was analyzed^TMProtein A HP Columns, GE Healthcare, Amersham, Piscataway, NJ), purified alpha-SNAP-25 monoclonal antibody to uncleaved SNAP-25₂₀₆Substrate and cleavageSNAP-25 of₁₉₇The ability of the product to precipitate determines the binding specificity. See, e.g., chapter 8, Storing and Purifying additives, pages 309-311, Harlow and Lane, supra, 1998 a. PC12 cells were seeded at an appropriate density, grown, and transfected with a transfection solution containing the pQBI-25/GFP expression construct (control cells; SEQ ID NO: 53) or the pQBI-25/GFP-BoNT/A-LC expression construct (test cells) as described above. The pQBI-25/GFP expression construct comprises an expression vector whose promoter element is functionally linked to a polynucleotide encoding GFP (SEQ ID NO: 54). After overnight incubation, cells were washed by aspirating growth medium and rinsing each well with 200 μ L of 1 × PBS. To harvest the cells, the PBS was aspirated, the cells were lysed by adding immunoprecipitation lysis buffer containing 50mM HEPES, 150mM NaCl, 1.5mM MgCl, and incubating at 4 ℃ for 1 hour ₂1mM EGDT, 10% glycerol, 1% TritonX-100 (octyl phenyl ether polyethylene glycol) and 1 × COMPLETE^TMProtease inhibitor cocktail (Roche Applied Biosciences, Indianapolis, IN). The lysed cells were centrifuged at 3,000 Xg for 10 minutes at 4 ℃ to remove cell debris, and the supernatant was transferred to a clean tube and diluted to a protein concentration of about 1 mg/mL. About 5. mu.g of purified monoclonal antibody was added to 0.5mL of diluted supernatant and incubated at 4 ℃ for 2 hours. After primary antibody incubation, approximately 50 μ L of immobilized protein G (Pierce Biotechnology, Rockford, IL) was added to the diluted supernatant and incubated at 4 ℃ for 1 hour. The incubated supernatant was washed 3 times for 30 minutes each by adding 0.5mL immunoprecipitation lysis buffer, centrifuged at 300 Xg for 1 minute at 4 ℃ to pellet the immobilized protein G, and the supernatant was decanted. After washing, the particles were resuspended in 30. mu.l of 1 xSDS loading buffer and the samples were heated to 95 ℃ for 5 minutes. For detection of uncleaved SNAP-25₂₀₆Substrate and cleaved SNAP-25₁₉₇The presence of the product, as described in example I, by means of the protein Aliquots of each harvested sample were analyzed by blotting, but the primary antibody used was a 1: 1,000 dilution of α -SNAP-25 polyclonal antibody serum (see example V) and the secondary antibody used was a 1: 20,000 dilution of rabbit α -IgG horseradish peroxidase (Pierce Biotechnology, Rockford, Ill.). Table 21 shows specific precipitation of SNAP-25 by immunoprecipitation analysis₁₉₇Ascites fluid of the lysate containing the alpha-SNAP-25 antibody. Immunoprecipitation analysis showed that ascites fluid produced by clones 2E2A6 and 3C1A5 synthesized the couple SNAP-25₁₉₇alpha-SNAP-25 monoclonal antibody with high binding specificity of the cleavage product, which allows to compare with SNAP-25₂₀₆Uncleaved substrate preferentially recognizes the cleavage product.

5. alpha-SNAP-25 monoclonal antibody binding affinity assessment.

For determining the SNAP-25₁₉₇Cleavage product or SNAP-25₂₀₆Uncleaved substrate showed binding affinity of alpha-SNAP-25 monoclonal antibody of high binding specificity, binding affinity assay was performed on BIAcore 3000 instrument using carboxymethyl dextran (CM5) sensor chip (BIAcore, Piscataway, NJ). The sagging was performed at 25 ℃ with HBS-EP buffer containing 10mM HEPES (pH 7.4), 150mM sodium chloride, 3mM EDTA, 0.005% (v/v) surfactant P20 at a flow rate of 10. mu.L/min. Using standard amino coupling, a peptide comprising amino acids 134-197(SEQ ID NO: 5) (SNAP-25) _134-197) Or amino acids 134-206(SEQ ID NO: 5) (SNAP-25)_134-206) Is covalently attached to the CM5 sensor chip surface. Briefly, C was activated by injecting a mixture of 0.2M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.05M N-hydroxysuccinimide over a period of 7 minutesAn M5 chip; the SNAP-25 peptide was then injected into 10mM sodium acetate (pH 4.0) at a flow rate of 10. mu.L/min, taking 20 minutes; and unreacted succinimidyl ester was blocked by injection of 1M ethanolamine hydrochloride (pH 8.5) over a period of 7 minutes. Increases in response units of 100-150 (about 0.10-0.15 ng/mm)²) To reflect on-chip SNAP-25_134-197Or SNAP-25_134-206A fixed amount of (c). Samples containing antibodies to ascites or purified monoclonal antibodies produced by clones 1D3B8, 2C9B10, 2E2a6, 3C1a5, and 3C3E2, and commercially available a-SNAP-25 antibodies were passed over the surface of CM5 chips, allowing for an association time of 10 minutes and a dissociation time of 20 minutes. Between each round of analysis, the surface was regenerated between sags by injecting 10mM glycine-HCl (pH 2.5) at a flow rate of 15. mu.L/min for 1 minute. The sensorgram curves were fitted to a 1: 1 kinetic binding model using BIAevaluation 3.0 software.

The results indicate that 2E2A6 and 3C1A5 are paired with cleaved SNAP-25₁₉₇The product specificity was higher than that of the substrate not cleaved by SNAP-25 (Table 22). The equilibrium dissociation constant of 1D3B6 for SNAP-25 cleavage product was about 10-fold higher for these commercial antibodies when compared to the binding affinities of MC-6050 and MC-6053 (Table 22). Interestingly, the equilibrium dissociation constant of 2E2a6 for SNAP-25 cleavage products was only slightly lower than those of the commercial antibodies (0.405nM versus 0.497 and 0.508) (table 22). Since none of these commercial α -SNAP-25 antibodies selectively recognized SNAP-25 cleavage products (Table 21), an equilibrium dissociation constant of less than about 0.5nM appears to be somewhat critical to achieving this selection. Similarly, the dissociation rate/dissociation constant of 2E2A6 was approximately at least 1-fold slower (6.74X 10) when compared to the binding affinities of MC-6050 and MC-6053^-5For 8.82X 10^-4s^-1And 1.18X 10^-3s^-1) (Table 22). This further indicates that less than about 8.82X 10^-4Seems to be somewhat critical to achieving selective binding of SNAP-25 cleavage products. The results are consistent with 1D3B8, with a decomposition rate/dissociation constant of 5.78X 10^-5s^-1(Table 22).

To compare 6 different antibodies, the binding rate (ka) and the dissociation rate (kd) were normalized separately using the program of the BIA evaluation 4.1 software. To compare the binding rates, the data were first individually collated by deleting the regeneration fraction and injection peak, and then normalized to a scale of 0 to 100. For comparison of the dissolution rates, data were normalized to the injection stop/top point. This analysis showed that the binding rate of 2C9B10 was much slower than the other antibodies (fig. 7A), and the dissociation rate (dissociation) of MC-6053 was much faster than the other antibodies (fig. 7B). The faster rate of decomposition of MC-6053 indicates that this antibody does not work well in the methods disclosed in the present specification because the antibody is difficult to maintain in substrate antigen binding during the washing step.

6. The epitopes of the isolated α -SNAP-25 monoclonal antibody were sequenced.

To determine P which selectively binds a bond susceptible to cleavage at the BoNT/A cleavage site₁Epitopes of an isolated alpha-SNAP-25 monoclonal antibody having a carboxy-terminal SNAP-25 antigen at residue to the variable heavy chain (V) encoding the alpha-SNAP-25 monoclonal antibody produced by hybridomas 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2_H) And variable light chain (V)_L) Sequencing the polynucleotide molecule of (a). mRNA was extracted from each hybridoma and purified using standard protocols, and mRNA was reverse transcribed to cDNA using oligo dT antisense or gene specific (murine IgG1CH and κ CL) antisense primers. After cDNA was generated, cDNA was amplified by PCR using specific murine and human constant domain primers to determine the isotype of the antibody. Using degenerate V_HAnd V_LThe primers amplify the variable domains in the cDNA. For 5 'RACE, a homopolymer dCTP tail was added to the 3' end of the cDNA. Subsequently, the process of the present invention,heavy and light chains were amplified with oligo dG sense primers and gene-specific (CH/KC) antisense primers. The PCR product included a signal peptide, variable domain and constant domain to antisense primer sequences. The PCR product was gel purified to remove small fragments and cloned into blunt-ended or TA vectors for sequencing. 5 independent clones of each strand were sequenced and V was determined _HAnd alignment of the VL chain and consensus sequences. For determining V_HAnd V_LMethods for amino acid sequences are described, for example, in the following documents: roger A.Sabbadini et al, Novel Bioactive Lipid Derivatives and Methods of Making and Using Same, U.S. patent publication 2007/0281320; and Peter Amerersdorfer, et al, Molecular Characterization of Murine Humoral Immune Response to boron neuron Type A Binding domains as isolated by Using phase Antibody library, 65(9) feed. Immun.3743-3752, the entire contents of each of which are incorporated herein by reference. In addition, commercial services are available for variable heavy chain (V) to antibodies_H) And variable light chain (V)_L) Sequencing was performed and the CDR regions identified, see, e.g., Fusion Antibodies ltd., Northern Ireland. In one case, V for 3C1A5_LThe amino acid sequence can also be determined by separating affinity-purified antibodies by high resolution 2DE electrophoresis, followed by peptide fingerprinting (peptide fragmentation fingerprinting) of this protein using high resolution nano LC-MSMS after proteolytic digestion.

V constituting the alpha-SNAP-25 monoclonal antibody produced by the hybridoma disclosed in the present specification_HChain and V_LThe polynucleotide sequence of the strand is as follows: 1D3B 8V_H(SEQ ID NO：71)、2C9B10 V_H(SEQ ID NO：73)、2E2A6 V_H(SEQ ID NO：75)、3C1A5 V_H(SEQ ID NO：77)、3C3E2 V_HVariant 1(SEQ ID NO: 79), 3C3E 2V_HVariant 2(SEQ ID NO: 81), 3C3E 2V_HVariant 3(SEQ ID NO: 132), 1D3B 8V_L(SEQ ID NO：83)、2C9B10 V_L(SEQ ID NO：85)、2E2A6 V_L(SEQ ID NO：87)、3C1A5 V_L(SEQ ID NO: 89) and 3C3E 2V_L(SEQ ID NO: 91). V constituting the alpha-SNAP-25 monoclonal antibody produced by the hybridoma disclosed in the present specification_HChain and V_LThe amino acid sequence of the chain is as follows: 1D3B 8V_H(SEQ ID NO：72)、2C9B10 V_H(SEQ ID NO：74)、2E2A6 V_H(SEQ ID NO：76)、3C1A5 V_H(SEQ ID NO：78)、3C3E2 V_HVariant 1(SEQ ID NO: 80), 3C3E 2V_H variant 2(SEQ ID NO：82)；3C3E2 V_HVariant 2(SEQ ID NO: 133), 1D3B 8V_L(SEQ ID NO：84)、2C9B10 V_L(SEQ ID NO：86)、2E2A6 V_L(SEQ ID NO：88)、3C1A5 V_L(SEQ ID NO: 90) and 3C3E 2V_L(SEQ ID NO: 92). V comprising the alpha-SNAP-25 monoclonal antibodies produced by hybridomas 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2_HAnd V_LThe amino acid sequences of the CDR domains are provided in table 23.

V comprising an alpha-SNAP-25 monoclonal antibody produced by a hybridoma as disclosed in the specification_HNon-limiting examples of amino acid sequences of CDR domain variants include: v of 1D3B8_HCDR1 variant SEQ ID NO: 118; 2C9B10, 2E2A6, and 3C1A 5V_HV of_HCDR1 variant SEQ ID NO: 119; 3C1A 5V_HAnd V of variant 3 of 3C3E2_HCDR1 variant SEQ ID NO: 120 of a solvent; v of 1D3B8 and 2E2A6 _H CDR2Variant SEQ ID NO: 121, a carrier; 2C9B10 and 3C1A 5V_HV of_HCDR2 variant SEQ ID NO: 122; 3C1A 5V_HAnd V of variant 3 of 3C3E2_HCDR2 variant SEQ ID NO: 123; v of 1D3B8 and 2C9B10_HCDR3 variant MDY; 2E2A6 and 3C1A 5V_HV of_HCDR3 variant MGY; and 3C1A 5V_HAnd V of variant 3 of 3C3E2_HCDR3 variant SEQ ID NO: 124. v comprising an alpha-SNAP-25 monoclonal antibody produced by a hybridoma as disclosed in the specification_LNon-limiting examples of amino acid sequences of CDR domain variants include: v of 1D3B8_LCDR1 variant SEQ ID NO: 125; v of 2C9B10_LCDR1 variant SEQ ID NO: 126; v of 2E2A6_LCDR1 variant SEQ ID NO: 127; v of 3C1A5_LCDR1 variant SEQ ID NO: 128; v of 3C3E2_LCDR1 variant SEQ ID NO: 129; v of 1D3B8_LCDR2 variant KVS; v of 2C9B10_LCDR2 variant NAK; v of 2E2A6_LCDR2 variant LVS; v of 3C1A5_LCDR2 variant YAT; and V of 3C3E2_LCDR2 variant YAS.

Example VIII

P binding selectively to a bond susceptible to cleavage at the BoNT/A cleavage site₁Development of an alpha-SNAP-25 polyclonal antibody having a SNAP-25 epitope at the residue with a free carboxyl terminus

The following example illustrates how to prepare P which selectively binds to a bond susceptible to cleavage at the BoNT/A cleavage site ₁An alpha-SNAP-25 polyclonal antibody having a SNAP-25 epitope at the carboxyl terminus at residue.

For the development of P which selectively binds to bonds which are easily cleavable at the BoNT/A cleavage site₁alpha-SNAP-25 polyclonal antibody to SNAP-25 having a carboxyl terminus at residue peptide CGGGRIDEANQ (SEQ ID NO: 46) having 10 residues was designed as a SNAP-25 cleavage product antigen. This peptide comprises an N-terminal cysteine residue conjugated to KLH, a G-spacer flexible spacer (GGG) linked to amino acids 191-197(SEQ ID NO: 5) of human SNAP-25, and having a carboxyl groupA glycosylated C-terminal glutamine. Blast searches showed that this peptide has only high homology to SNAP-25 and is nearly impossible to cross-react with other proteins in neuronal cells. The sequence can also be carefully scrutinized using computer algorithms to determine the hydropathic index, protein surface probability, flexibility and favorable secondary structure, followed by appropriate localization and presentation of the selected peptide sequence. Peptides were synthesized and conjugated to Keyhole Limpet Hemocyanin (KLH) to increase immunogenicity. Prior to immunization of animals, local rabbits were first screened in western blots against cell lysates from candidate cell lines to identify animals that were not immunoreactive to the proteins present in the cell lysates. Two pre-screened rabbits were immunized with this peptide and 3 immunizations were performed in about 8 weeks, after which the blood of the rabbits was drawn for testing. The blood was incubated at 4 ℃ for 60 minutes to clot the blood. The clot blood was centrifuged at 10,000Xg for 10 minutes at 4 ℃ to pellet the cell debris. The resulting serum samples were divided into 50 μ l aliquots and stored at-20 ℃ until use.

Similar strategies based on other SNAP-25 antigens disclosed in the specification were used to develop P that selectively binds to a readily cleavable bond at the BoNT/A cleavage site₁An alpha-SNAP-25 polyclonal antibody to SNAP-25 having a carboxyl terminus at residue. For example, SNAP-25 antigen SEQ ID NO: 47 may be conjugated to KLH, rather than SNAP-25 antigen SEQ ID NO: 46. as another example, the amino acid sequence of SEQ ID NO: 38 can be replaced with amino acids 191-197 of the human SNAP-25 of SEQ ID NO: 33. SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43 or SEQ ID NO: 44.

2. screening for the presence of alpha-SNAP-25 polyclonal antibodies.

To determine P which selectively binds a bond susceptible to cleavage at the BoNT/A cleavage site₁The presence of alpha-SNAP-25 polyclonal antibodies to SNAP-25 antigen with a carboxyl terminus at the residue, as described in example III, using extracted rabbit serumComparative ELISA and cell-based lysis assays were performed. Sera from two rabbits contained P selectively bound to a bond susceptible to cleavage at BoNT/A₁An alpha-SNAP-25 polyclonal antibody to SNAP-25 antigen having a carboxyl terminus at the residue. The alpha-SNAP-25 rabbit polyclonal antibodies are named NTP 22 and NTP 23.

3. Purification of the alpha-SNAP-25 polyclonal antibody.

For purification of P selectively bound to a bond susceptible to cleavage at the BoNT/A cleavage site₁An alpha-SNAP-25 polyclonal antibody to SNAP-25 antigen having a carboxyl terminus at residue, using a monoclonal antibody comprising SNAP-25 antigen SEQ ID NO: 46 to purify NTP 22 and NTP 23 antibodies from rabbit serum.

4. Evaluation of the binding specificity of the α -SNAP-25 polyclonal antibody.

To evaluate P selectively binding a bond susceptible to cleavage at the BoNT/A cleavage site₁Binding specificity of alpha-SNAP-25 polyclonal antibodies to SNAP-25 antigen having a carboxyl terminus at residue cleavage products were detected by cell-based activity assays, immunocytochemistry and immunoprecipitation using purified NTP 22 and NTP 23 alpha-SNAP-25 polyclonal antibodies as described in example III. Cell-based lysis assays, immunocytochemistry assays, and immunoprecipitation assays all showed that NTP 22 and NTP 23 α -SNAP-25 polyclonal antibodies do not cross-react with uncleaved SNAP-25. Thus, both NTP 22 and NTP 23 are directed towards cleaved SNAP-25₁₉₇The product has high binding specificity, allowing for SNAP-25 versus₂₀₆This cleavage product is preferentially recognized by uncleaved substrate. The affinity of the antigen can be determined using SPR in BiAcore as described in example III.

Example IX

Component and Condition preparation for Sandwich ELISA

The following example illustrates how to identify and prepare the components and conditions required for performing a sandwich ELISA, such asSandwich ELISA is performed by using P which is susceptible to bond cleavage at the BoNT/A cleavage site₁An alpha-SNAP-25 monoclonal antibody specific for SNAP-25 having a carboxyl terminus at residue detects SNAP-25 cleavage products to perform an immune-based method for detecting retargeted endopeptidase activity.

1. Preparation of cell lysates of cells treated with retargeted endopeptidase.

To obtain cell lysates treated with retargeted endopeptidase for analysis, cells from stock cultures of Neuro-2a at appropriate densities were seeded in T175 flasks containing 50mL of serum-free Medium containing MEM Medium (Minimu Essential Medium), 2mM GlutaMAX containing early's salts (Earle's salt)^TMI. 1 XB 27 supplement, 1 XN 2 supplement, 0.1mM nonessential amino acids, 10mM HEPES. These cells were incubated in an incubator at 37 ℃ under 5% carbon dioxide until cell differentiation, as assessed by standard and conventional morphological criteria, e.g., growth arrest and neurite extension (about 2 to 3 days). As a control, cells from stock cultures of Neuro-2a were seeded at appropriate densities in T175 flasks containing 50mL of the appropriate growth medium (Table 1). These undifferentiated control cells were grown in an incubator at 37 ℃ under 5% carbon dioxide until 50% confluence was reached (about 18 hours). The culture medium of the differentiated and undifferentiated control cultures was aspirated from each well and replaced with fresh medium containing 0 (untreated sample) or 10nM of the retargeted endopeptidase. After overnight incubation, the cells were washed and lysed by incubation in fresh Triton X-100 lysis buffer (50mM HEPES, 150mM NaCl, 1.5mM MgCl) at 4 ℃ under constant agitation ₂1mM EGTA, 1% Triton X-100) for 30 minutes to harvest the cells. The lysed cells were centrifuged at 4000rpm for 20 minutes at 4 ℃ using a bench centrifuge to remove debris. The protein concentration of the cell lysate was measured by Bradford assay.

2. Preparation and identification of sandwich ELISA Components.

To identify suitable capture antibody-detection antibody pairs, ECL sandwich ELISA assays were performed on 26 different combinations of capture and detection antibody pairs, comprising 11 different α -SNAP-25 capture antibodies and 7 different α -SNAP-25 detection antibodies (table 12). The alpha-SNAP-25 antibody used is 2E2A6 and 3C1A5 alpha-SNAP-25 mouse monoclonal antibody disclosed in the specification; SMI-81, MC-6050 and MC-6053 alpha-SNAP-25 mouse monoclonal antibodies disclosed in the specification; NTP 23 alpha-SNAP-25 rabbit polyclonal antibody disclosed in the specification; s9684 α -SNAP-25 rabbit polyclonal antibody (Sigma, st. louis, MO); h-50 α -SNAP-25 Rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.); c-18 α -SNAP-25 goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.); n-19 α -SNAP-25 goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.); and SP12 α -SNAP-25 mouse polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

To prepare the capture antibody solution, the α -SNAP-25 monoclonal antibody, as well as the α -SNAP-25MC-6050 and MC-6053 monoclonal antibodies contained in ascites fluid from hybridoma cell lines 2E2A6 and 3C1A5, were purified using standard protein A purification protocols. All other α -SNAP-25 antibodies were purchased as purified antibodies.

To prepare the detection antibody solutions, the appropriate α -SNAP-25 antibody was conjugated to ruthenium (II) -tris-bipyridine- (4-methylsulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, MD). The conjugation reaction was carried out in the following manner: MSD SULFO-TAG resuspended in 30. mu.L of distilled water^TMThe stock was added to 200. mu.L of 2mg/mL alpha-SNAP-25 polyclonal antibody and the reaction was incubated at room temperature in the dark for 2 hours. The labeled antibody was purified using a standard spin column and the protein concentration was determined using a standard colorimetric protein assay. Measurement of alpha-SNAP-25 antibody/MSD SULFO-TAG at 455nm Using a Spectrophotometer^TMAbsorbance of the conjugate, to determine concentration (in moles/liter). The detection antibody solution was stored at 4 ℃ until use.

To prepare a solid support containing a capture antibody specific for SNAP-25 lysate, approximately 5 μ L of an appropriate α -SNAP-25 monoclonal antibody solution (20 μ g/mL of a 1 XPBS solution) was added to each well of a 96-well MSD High Bind plate and the solution was allowed to air dry in a biosafety cabinet for 2 to 3 hours in order to subject the solution to liquid evaporation. Subsequently, the wells bound to the capture antibody were blocked at room temperature for 2 hours by adding 150 μ L of blocking buffer containing 2% Amersham blocking agent (GE Life Sciences, Piscataway, NJ) and 10% goat serum (VWR, West Chester, PA). The closed plates were sealed and stored at 4 ℃ until use.

To detect the presence of cleaved SNAP-25 cleavage products by ECL sandwich ELISA assay, blocking buffer was aspirated from each well of the storage plate, 25 μ L of cell lysate treated with retargeted endopeptidase was added to each well, and the plate was incubated overnight at 4 ℃, as described above. By aspirating the cell lysate and applying 200. mu.L of 1 XPBS, 0.1% TWEEN-20(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times and each well in the plate was washed 3 times. After washing, 25 μ L of 2% Amersham blocking reagent in 1 XPBS, 0.1% TWEEN-20 was added to each wellA5. mu.g/mL solution of detection antibody (polyoxyethylene (20) sorbitan monolaurate), the plate was sealed, and the sealed plate was incubated at room temperature for 1 hour with shaking. After incubation of the detection antibody, 200. mu.L of 1 XPBS, 0.1% TWEEN-20 was used(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times. After washing, 150. mu.L of 1 Xread buffer (Meso Scale Discovery, Gaithersburg, Md.) was added to each well and SECTOR was used^TMAn Imager model 6000 image reader (Meso Scale Discovery, Gaithersburg, MD) reads the plates. By dividing the signal obtained by each antibody pair at a dose of 10nM by 0n Ratios were calculated for each antibody at M doses to the signals obtained (table 24). These results indicate that of the 26 different antibody pair combinations tested, only 3 antibody pairs had a signal to noise ratio above 10: 1 at the higher doses tested: pair No. 1 (2E2a6 mouse monoclonal antibody and S9684 rabbit polyclonal antibody), pair No. 4 (3C1a5 mouse monoclonal antibody and S9684 rabbit polyclonal antibody), and pair No. 18 (S9684 rabbit polyclonal antibody and 2E2a6 mouse monoclonal antibody). Antibodies were selected for further assay development for 1.

Example X

Immunity-based method for detecting retargeted endopeptidase with BoNT/A light chain enzyme activity using ECL sandwich ELISA

The following example illustrates an immune-based method for detecting retargeted endopeptidase activity by ECL sandwich ELISA using P for bonds susceptible to cleavage at the BoNT/A cleavage site₁The cleavage product of SNAP-25 having a carboxyl terminus at the residue is detected by an alpha-SNAP-25 monoclonal antibody specific for the cleavage product of SNAP-25.

To prepare lysates of cells treated with the retargeted endopeptidase with BoNT/a light chain enzyme activity, cells from established cell lines were seeded at appropriate densities into each well of a 96-well tissue culture plate containing 100 μ L of the appropriate medium. These cells were incubated at 37 ℃ in an incubator at 5% carbon dioxide for about 24 hours. The culture medium of the cells is aspirated from each well and replaced with fresh medium containing 0 (untreated sample) or one of a plurality of doses of the retargeted endopeptidase as determined by a dose response experiment for this retargeted endopeptidase. After 24 hours of incubation, the cells were washed and harvested.

To prepare the alpha-SNAP-25 capture antibody solution, the alpha-SNAP-25 monoclonal antibody contained in ascites fluid from hybridoma cell line 2E2A6 was purified using standard protein A purification protocols. To prepare the α -SNAP-25 detection antibody solution, α -SNAP-25 rabbit polyclonal antibody S9684(Sigma, st. louis, MO) was conjugated with ruthenium (II) -tris-bipyridine- (4-methylsulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer' S instructions (Meso Scale Discovery, Gaithersburg, MD). Conjugation reactions, purification of labeled α -SNAP-25 antibody, concentration determination, and storage were all as described in example VI.

To prepare a solid support containing a capture antibody specific for SNAP-25 lysate, approximately 5. mu.L of a solution of the α -SNAP-25 monoclonal antibody 2E2A6 (20. mu.g/mL of a 1 XPBS solution) was added to each well of a 96-well MSD High Bind plate and the solution was allowed to air dry in a biosafety cabinet for 2 to 3 hours in order to subject the solution to liquid evaporation. The wells bound to the capture antibody are then blocked and used directly to detect the re-targeting endopeptidase activity.

To detect the presence of cleaved SNAP-25 product by ECL sandwich ELISA assay, blocking buffer was aspirated from each well of the storage plate, 25 μ L of cell lysate treated with retargeted endopeptidase was added to each well, and the plate was incubated overnight at 4 ℃. By aspirating the cell lysate and applying 200. mu.L of 1 XPBS, 0.1% TWEEN-20 (polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times and each well in the plate was washed 3 times. After washing, 25. mu.L of a 2% Amersham blocking reagent in 1 XPBS, was added to each well,0.1％TWEEN-20A5. mu.g/mL solution of detection antibody (polyoxyethylene (20) sorbitan monolaurate), the plate was sealed, and the sealed plate was incubated at room temperature for 1 hour with shaking. After incubation of the detection antibody, 200. mu.L of 1 XPBS, 0.1% TWEEN-20 was used(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times. After washing, 150. mu.L of 1 Xread buffer (Meso Scale Discovery, Gaithersburg, Md.) was added to each well and SECTOR was used^TMAn Imager model 6000 image reader (Meso Scale Discovery, Gaithersburg, MD) reads the plates. The collected data were analyzed and EC calculated as described in example VI₅₀The value is obtained. For opioid retargeted endopeptidases, these results indicate that, at EC₅₀An average Noc/A of 1.0nM (in the range of about 0.3nM to about 2.0 nM) was detected, where the signal-to-noise ratio of the lower asymptote is about 15: 1 to about 20: 1; while the signal-to-noise ratio of the upper asymptote is about 180: 1 to about 300: 1.

Example XI

Immunity-based method for detecting retargeted endopeptidase activity using CL sandwich ELISA

The following example illustrates an immune-based method for detecting retargeted endopeptidase activity by CL sandwich ELISA using P for bonds that are susceptible to cleavage at the BoNT/A cleavage site₁The C-terminal SNAP-25 at residue has a specific alpha-SNAP-25 monoclonal antibody to detect SNAP-25 cleavage products.

Lysates and alpha-SNAP-25 capture antibody solutions were prepared as described in example VII for cells treated with retargeted endopeptidase.

To prepare the α -SNAP-25 detection antibody solution, α -SNAP-25 polyclonal antibody S9684(Sigma, St. Louis, Mo.) was conjugated to horseradish peroxidase (HRP) according to the manufacturer' S instructions (Pierce Biotechnology, Rockford, Ill.). Conjugation reactions were performed in the following manner: add 500. mu.L of 1mg/mL α -SNAP-25 polyclonal antibody to a vial containing lyophilized active peroxidase, mix the components, and then add 10. mu.L sodium cyanoborohydride. The reaction mixture was incubated in a fume hood at room temperature for 1 hour. Following quenching of the reaction, the labeled antibody is purified using standard spin column protocols and the protein concentration is determined using standard colorimetric protein analysis. The absorbance of the α -SNAP-25 polyclonal antibody/HRP conjugate was measured at 455nm using a spectrophotometer to determine the concentration (in moles/liter). The alpha-SNAP-25 detection antibody solution was stored at 4 ℃ until use.

To prepare a solid support comprising an α -SNAP-25 capture antibody specific for the cleavage product of SNAP-25, approximately 100 μ L of α -SNAP-25 monoclonal antibody 2E2a6 solution (1mg/mL of 1 × PBS solution) was added to each well of a 96-well Greiner white plate (Greiner white plate) and the plate was incubated overnight at 4 ℃, and any excess antibody solution was discarded. Wells bound to capture antibody were then blocked at room temperature for 1 hour by adding 150 μ L of blocking buffer containing 2% Amersham blocking reagent (GE Life Sciences, Piscataway, NJ) and 10% goat serum (VWR, West Chester, PA). The blocking buffer was discarded and blotted onto paper towels by inverting the plate over the paper towel and tapping. The wells bound to the capture antibody are then blocked and used directly to detect the re-targeting endopeptidase activity.

To detect the presence of cleaved SNAP-25 product by CL sandwich ELISA assay, 50 μ L of cell lysate treated with retargeted endopeptidase was added to each well, the plate was sealed, and the sealed plate was incubated on a shaker rotating at 500rpm for 2-4 hours to overnight at 4 ℃. By aspirating the cell lysate and using 200. mu.l of 1 XPBS, 0.05% TWEEN-20(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times and each well in the plate was washed 3 times. After the washing, the water is washed, To each well was added 100. mu.L of 2% Amersham blocking reagent in 1 XPBS, 0.1% TWEEN-201mg/mL of alpha-SNAP-25 polyclonal antibody/HRP detection antibody solution (polyoxyethylene (20) sorbitan monolaurate), plates were sealed, and the sealed plates were incubated for 1 hour at room temperature on a shaker rotating at 650 rpm. After incubation of the detection antibody, 200. mu.L of 1 XPBS, 0.05% TWEEN-20 was used(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times. After washing, 100. mu.l SuperSignal ELISA Pico 1: 1 mix (Pierce Biotechnology, Rockford, IL) was added to each well and the plates were read using a luminometer (Molecular Devices, Sunnyvale, Calif.) at 395 nm. The collected data were analyzed and EC calculated as described in example VI₅₀The value is obtained.

Example XII

Immune-based method for detecting retargeted endopeptidase activity using multiple ECL sandwich ELISA

The following example illustrates a multiplex immunity-based method for detecting retargeted endopeptidase activity using an alpha-SNAP-25 monoclonal antibody specific for SNAP-25 cleavage products and a second antibody pair directed against a different protein to detect SNAP-25 cleavage products.

The retargeted endopeptidase potency assay can be performed using a multiple ECL sandwich ELISA. This analysis is described in co-pending patent application Ser. No. 12/403,531, issued to Ester Fernandez-Salas et al, Immuno-Based Botulinum Toxin type A Activity Assays, U.S. patent application No. 12/403,531 (the entire contents of which are incorporated herein by reference), and can be performed using the cell lines and retargeted endopeptidases and corresponding cell lines disclosed in this specification.

Example XIII

Immune-based method for detecting retargeted endopeptidase activity using multiple ECL sandwich ELISA

The following example illustrates a multiplex immunity-based method for detecting retargeted endopeptidase activity using an alpha-SNAP-25 monoclonal antibody specific for SNAP-25 cleavage products and a second antibody pair directed against a different protein to detect SNAP-25 cleavage products.

A multiple EC sandwich ELISA can be used for the heavy-target endopeptidase potency assay. This assay is described in co-pending patent application, issued to Ester Fernandez-Salas et al, Immuno-Based Botulinum Toxin type a Activity Assays, U.S. patent application No. 12/403,531, the entire contents of which are incorporated herein by reference, and which can be used with the cell lines and retargeted endopeptidases and corresponding cell lines disclosed in this specification.

Example XIV

Immune-based method for detecting nanomolar concentrations of retargeted endopeptidases

The following example illustrates how an immune-based method for detecting the activity of retargeted endopeptidase in nanomolar concentrations can be performed.

1. An immune-based method for detecting heavy-targeted endopeptidases using ECL sandwich ELISA.

To prepare lysates of cells treated with retargeted endopeptidase, approximately 50,000 to 150,000 cells from an established cell line suitable for the assay were seeded into each well of a 96-well tissue culture plate coated with poly-D-lysine containing 100 μ L of the appropriate medium (see examples I and II). The cells were incubated at 37 ℃ in an incubator with 5% carbon dioxide for 24 hours. By sucking cells from the wellsThe medium was replaced with fresh medium containing 0 (untreated sample) and appropriate dose-responsive retargeted endopeptidases as described herein. After 24 hours of incubation, the cells were washed and harvested or, prior to harvest, incubated for an additional 2 days without retargeting endopeptidase. To harvest the cells, the medium was aspirated and washed with 1 XPBS, and the cells were harvested by adding 30 μ L of a medium containing 50mM HEPES, 150mM NaCl, 1.5mM MgCl ₂1mM EGTA, 1% Triton X-100, and incubating the plates at 4 ℃ for 30 minutes on a shaker rotating at 500 rpm. The plates were centrifuged at 4000rpm for 20 minutes at 4 ℃ to pellet the cell debris and the supernatant was transferred to a 96-well plate coated with capture antibody for the detection step.

The preparation of the alpha-SNAP-25 capture antibody solution, the alpha-SNAP-25 detection antibody solution, and the solid support comprising the capture antibody specific for the product of SNAP-25 cleavage was performed as described in example VII.

To detect the presence of SNAP-25 lysate by ECL sandwich ELISA assay, blocking buffer was aspirated from the storage plate, 25-30 μ L of cell lysate treated with retargeted endopeptidase was added to each well, and the plate was incubated at 4 ℃ for 2 hours or 24 hours. By aspirating the cell lysate and applying 200. mu.L of 1 XPBS, 0.1% TWEEN-20(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times, while each well in the plate was washed 3 times. After washing, 25 μ L of 2% Amersham blocking reagent in 1 XPBS, 0.1% TWEEN-20 was added to each wellalpha-SNAP-25 detection antibody solution (polyoxyethylene (20) sorbitan monolaurate) at 5. mu.g/mL, plates were sealed, and sealed plates were incubated with shaking at room temperature for 1 hour. After incubation of the alpha-SNAP-25 detection antibody, 200. mu.L of 1 XPBS, 0.1% TWEEN-20 was used (polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times. After washing, the plates were processed, the collected data analyzed, and EC calculated as described in example VI₅₀The value is obtained. These results indicate that when SK-N-DZ clonal cell line #3 cells are used, at EC₅₀An average Noc/A of 1.0nM (in the range of about 0.3nM to about 2.0 nM) was detected in the lower, where the signal-to-noise ratio of the upper asymptote was about 20: 1 to about 300: 1. Furthermore, when AGN P33 clone #6 cells were used, in EC₅₀An average of 3.7nM Noc/A (in the range of about 2.0nM to about 5.5 nM) was detected in the lower, where the signal-to-noise ratio of the upper asymptote was about 20: 1 to about 500: 1. For SK12 cells specific for retargeted endopeptidases containing dynorphin a ligands, when SK12 cells were used, at EC₅₀An average of 8.4nM Dyn/A (in the range of about 4.5nM to about 10.0 nM) was detected in the lower, where the signal-to-noise ratio of the upper asymptote was about 10: 1 to about 20: 1. Furthermore, when the Neuro-2a clone #7 cell line was used, it was found that the cell line was stable in EC₅₀An average of 8.8nM TVEMP-galanin (in the range of about 5.0nM to about 15.5 nM) was detected in the lower, where the signal-to-noise ratio of the upper asymptote was about 20: 1 to about 200: 1. The process can also be carried out in a multiplex manner as described in example IX.

2. An immune-based method for detecting a heavy-targeted endopeptidase using a CL sandwich ELISA.

Preparation of lysates and alpha-SNAP-25 capture antibody solutions of cells treated with retargeted endopeptidase were performed as described in example VII. The preparation of the alpha-SNAP-25 detection antibody solution and solid support comprising capture antibodies specific for the cleavage product of SNAP-25 was performed as described in example VIII.

To detect the presence of SNAP-25 lysate by CL sandwich ELISA assay, 100 μ L of cell lysate treated with retargeted endopeptidase was added to each well, plates were sealed, and sealed plates were incubated for 2 hours or 24 hours at 4 ℃ on a shaker rotating at 500 rpm. By aspirating the cell lysate and using 200. mu.l of 1 XPBS, 0.05% TWEEN-20(polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times and each well in the plate was washed 3 times. After washing, 100. mu.L of 1 XPBS containing 2% Amersham blocking reagent, 0.1% TWEEN-20 was added to each well1mg/mL of alpha-SNAP-25 polyclonal antibody/HRP detection antibody solution (polyoxyethylene (20) sorbitan monolaurate), plates were sealed, and the sealed plates were incubated for 1 hour at room temperature on a shaker rotating at 650 rpm. After incubation of the detection antibody, 200. mu.L of 1 XPBS, 0.05% TWEEN-20 was used (polyethylene oxide (20) sorbitan monolaurate) each well was washed 3 times. After washing, 100. mu.l SuperSignal ELISA Pico 1: 1 mix (Pierce Biotechnology, Rockford, IL) was added to each well and the plates were read using a luminometer (Molecular Devices, Sunnyvale, Calif.) at 395 nm. The collected data were analyzed and EC calculated as described in example VI₅₀The value is obtained. The process can also be carried out in a multiplex manner as described in example IX.

Example XV

Immunity-based method for detecting neutralizing alpha-retargeted-endopeptidase antibodies

The following example illustrates how an immune-based method of detectably neutralizing the presence of α -Noc/a antibodies can be performed.

Noc/a is an antibody currently being evaluated for the treatment of pain conditions, some of which are chronic. Repeated long-term treatment with Noc/A, patients develop neutralizing alpha-Noc/A antibodies against the retargeted endopeptidase, resulting in immune resistance. Neutralizing the alpha-Noc/A antibody will re-target the target ligand and/or translocation domain (H) of the endopeptidase by binding_N) Prevention of neuronal and other target cellsRetargeting the uptake of endopeptidase to inhibit retargeted endopeptidase activity. There is currently no established assay for determining the presence of neutralizing alpha-Noc/a antibodies in the blood of a patient. Cost and time would be saved if a cell-based assay could be developed that detects neutralizing antibodies in patients treated with retargeted endopeptidases.

To detect the presence or absence of neutralizing alpha-Noc/A antibodies, the immune-based methods disclosed in the present specification for determining retargeted endopeptidase activity can be used. One way is to use a western blot assay to determine the amount of SNAP-25 cleavage product present after treatment with various concentrations of Noc/a, and the other way is to use an ECL sandwich ELISA assay.

To prepare samples containing neutralizing α -Noc/A antibodies, sera were isolated from blood of monkeys immunized with Noc/A and subjected to affinity purification for the antibodies. Rabbits were also immunized with the nociceptin variant peptide (target ligand present in the Noc/a molecule), sera were collected, and antibodies (anti-nociceptin polyclonal antibodies) were affinity purified.

To prepare lysates of cells treated with samples containing Noc/a, SK-N-DZ clone #3 cells and AGN P33 clone #6 cells were seeded in poly D-lysine coated 96-well plates for 16-18 hours. Anti-nociceptin polyclonal antibodies were diluted 0-3 μ g/mL in RPMI SFM (with N2, B27, and NGF supplements) containing 1nM Noc/A and the mixture was pre-incubated at room temperature for 1 hour. The solution was then added to the cells and incubated for 24 hours before ECL ELISA assay. This anti-nociceptin variant antibody completely blocked 1nmNoc/A uptake (> 90% inhibition) on both cell lines at 1. mu.g/mL. anti-Noc/a monkey polyclonal antibodies were also assayed on these cell lines. Cells were seeded at 100,000 cells/well in poly-D-lysine coated 96-well plates in RPMI growth medium supplemented with N2, B27, and NGF for 24 hours. anti-Noc/A polyclonal antibodies at 0-20. mu.g/mL were diluted in medium containing 1nM Noc/A and the mixture was preincubated for 1 hour at room temperature. The mixture was then added to the cells and incubated for 24 hours before ECL ELISA assay. With higher concentrations of anti-Noc/a polyclonal antibody (6-20 μ g/mL), up to 60% inhibition was observed on SK-N-DZ cell line, and about 30% inhibition was observed on AGN P33 clone #6 cell line. This may be due to the fact that the anti-Noc/a polyclonal antibody is not specific for the binding site and contains additional antibodies that bind to other parts of the molecule, resulting in only partial blocking at the concentrations tested. Higher concentrations may be required to achieve complete occlusion.

To detect the presence of SNAP-25 lysate by Western blot analysis, the medium was aspirated from each well, and the cells were suspended in 50. mu.L of SDS-PAGE loading buffer and then heated to 95 ℃ for 5 minutes. Aliquots of each harvested sample were analyzed by Western blotting as described in example I, but the harvested samples were separated by SDS-PAGE using 12% 26-well Criterion gel (Bio-Rad Laboratories, Hercules, Calif.) and rabbit polyclonal α -SNAP-25 was used₁₉₇Antibody serum was used as the primary antibody (see example V). The results reveal that the lowest concentration of retargeted endopeptidase can produce a detectable SNAP-25 cleavage product band in western blots.

To detect the presence of SNAP-25 lysate by ECL sandwich ELISA, the media was removed from each well and the cells lysed as described in example VI. The alpha-SNAP-25 capture antibody solution, alpha-SNAP-25 detection antibody solution, and alpha-SNAP-25 solid support were prepared as described in example VIII. The supernatant was transferred to an α -SNAP-25 solid support and an ECL sandwich ELISA assay was performed as described in example VI. The data collected was analyzed and EC calculated as described in example VI₅₀Value, but EC ₅₀Is the dilution of serum required to inhibit the activity of the retargeted endopeptidase to 1/2 of its maximum inhibition, and the maximum signal (signal)_Max) And minimum signal (signal)_Min) Is obtained by dividing the SNAP-25 cleavage product signal obtained at the maximum antibody dilution by the signal obtained at the lowest antibody dilution.

The results indicate that neutralizing alpha-Noc/a antibodies are detectable in monkey serum and alpha-nociceptin variant antibodies from rabbits are present. As the antibody dilution is reduced, the activity of the Noc/a molecule incubated in the affinity-purified antibody from the immunized animal is also reduced. The same assay was performed with Dyn/A and TVEMP-galanin compounds using cell lines specific for each test compound.

Example XV

Development of a cell-based assay for retargeting endopeptidase to galanin

The following example illustrates how to identify established cell lines with the ability to re-target endopeptidase uptake required to develop cell-based potency assays.

1. Growth of stock cultures of candidate cell lines.

To grow the cell lines, cells from the test cell line were seeded at a suitable density at 162cm in 30mL of suitable growth medium (see Table 25) ²Tissue culture flasks were grown in an incubator at 37 ℃ under 5% or 10% carbon dioxide until the cells reached the desired density.

2. Screening of commercial cell lines for sensitivity to galanin TVEMP-galanin compounds

Commercial cell lines were screened for sensitivity to TVEMP-galanin compounds as measured by cleavage of SNAP25 after treatment with the corresponding compound. Various TVEMP-galanin compounds were used for screening and testing. PC-12, Neuro-2a, SiMa and P19 cells were seeded in serum-free medium for 3 days, or in CM for 1 day. These differentiated and native cells were treated with TVEMP-galanin batch A at concentrations of 0 and 75nM for 18 hours. Increased presence of cleaved SNAP25 was observed, indicating that TVEMP-galanin batch a showed activity in PC-12 and Neuro-2a cells, and that Neuro-2a cells were more sensitive to TVEMP compounds with galanin ligands than native cells in the differentiated state. The ranking of cellular activities showed that PC-12 cells were the most active, followed by Neuro-2a cells and finally SiMa cells. If necessary, it is important to determine whether such uptake is specific for these galanin-retargeting compounds, and therefore it is important to test cells with other compounds that do not contain galanin ligands. Noc/A is a retargeting compound containing a nociceptin variant ligand, and LH _NA (negative control) is a compound without a binding domain. LH_NUptake of/A is not specific, whereas if the cell line has specific uptake of the heavy targeting compound, LH is_NThe activity of/A should be significantly lower than that of TVEMP-galanin compounds. Noc/a compounds have previously been shown to have specific uptake in SiMa cells and serve as a baseline for testing cell lines. Favorable cell lines for LH_Nthe/A compound and the Noc/A compound have low uptake, while having high uptake for the TVEMP-galanin compound. Table 26 shows the results from this experiment.

The results show that TVEMP-galanin batches A and B for the cell lines testedTVEMP-galanin batch B has a curve or EC with activity similar to or only 1 to 2 times higher than that of the negative control₅₀The value is obtained. This data indicates that native cells are not sufficiently sensitive and that these cells must be transfected with plasmids encoding the galanin receptor proteins GalR1 or GalR2 receptor.

3. PC-12, Neuro-2a and SiMa cells were stably transfected with GalR.

One day before transfection, cells were plated at 0.5X 10⁶The density of individual cells/well was seeded in collagen IV coated 6-well plates (Cat # 354554: BD Biosciences) (SiMa, PC-12) or 6-well Costar plates (Cat # 3516: Corning) (Neuro-2 a). By mixing 12. mu.l Lipofectamine ^TM2000(Cat # 52758, Invitrogen) in 250. mu.l Opti-MEMTransfection was performed in reduced serum medium (Cat # 3195, Invitrogen) followed by 5 min incubation at room temperature. Mu.g of GalR plasmid DNA and 0.4. mu.g of pAdVantage^TMVector (1mg/ml, Cat # E1711, Promega) was mixed in 250. mu.l of Opti-MEMI in reduced serum medium, for 5 minutes. After 5 minutes incubation, diluted Lipofectamine was added^TM2000 were mixed with the diluted plasmid DNA and incubated at room temperature for an additional 20 minutes to form a complex. At the same time, OPTI-MEM is usedThe cells were washed, and 0.5ml of OPTI-MEM was addedAdded to each well. After 20 min incubation, 0.5ml containing diluted Lipofectamine^TM2000 complexes with diluted plasmid DNA 0.5ml of OPTI-MEM was carefully addedIn the well containing the cell. At 37 deg.CPlates were incubated for 5 hours, after which 1mL of complete medium was added. The next day, the medium was replaced with growth medium and incubated for 48 hours. On day 4, transfected cells were recovered, and the growth medium was subsequently diluted with a medium containing 0.5mg/ml (1: 100 dilution) GeneticinFresh long medium of (Cat # 10131: Invitrogen) was replaced and incubated for an additional 3 days. On day 7 post-transfection, cells were transferred to 75cm collagen IV flasks (Cat # 35423: BD Biosciences) containing growth medium and geneticin (0.5mg/ml, 1: 100 dilution). When this transfer was performed, about 90% of the cells died and were removed during the medium change. Growth medium containing geneticin (0.5mg/ml, 1: 100 dilution) was changed every 2 days until day 21.

To select stable cells capable of absorbing galanin TVEMP compounds, each parameter should screen for clones that produced the highest percentage of SNAP25 lysate when treated with TVEMP-galanin in ECL sandwich ELISA using monoclonal 2E2a6 coated plates for capture and a sulfo-labeled polyclonal SNAP25(Sigma, Cat # S9684) antibody for detection. EC in Table 27₅₀The values show that TVEMP-galanin batch D exhibited at least 10-fold uptake over the negative control in SiMa and Neuro-2a cells transfected with GalR1 and GalR2, and only 2 to 4-fold in transfected PC-12 cells. Since the sensitivity and specificity of transfected PC-12 cells appear to be lower than those of SiMa and Neuro-2a cells, they will not be cloned. In addition, since galanin 1 to 16 monomer ligands in the TVEMP-galanin compounds bind to GALR1 receptor with higher affinity than GALR2, only cells transfected with GALR1 were cloned. It is also shown that in Neuro-2a GalR1, TVEMP-galanin batches C and D exhibit an uptake of LH_N9 to 10 times of/A and the retargeted nociceptin compound TVEMP-nociceptin.

Conventionally, the selected non-clonal cell populations are not good cell populations for use because these contain a mixture of cells that express different levels of receptor and may change over time. To obtain stable cell lines derived from single cells, a dilution cloning method was initiated. On day 21, the transfected cells were treated with trypsin, separated with a needle, and counted. The remaining transfected cell lines were frozen until use. Cells were serially diluted to 10 cells/well in growth medium containing geneticin (0.5mg/ml, 1: 100 dilution). Cells were seeded at 100. mu.l/well into 2 collagen IV coated 96-well plates (SiMa, PC-12) or 2 96-well Costar plates (Neuro-2a) to a density of 1 cell/well. Plates were returned to the incubator and incubated for 2 weeks without contact to form colonies. After 2 weeks (day 35), wells were carefully checked for the presence of a single colony formed at the bottom of the well (for multiple colonies, the entire well was carefully checked). When a single cell pellet is identified in a well, the entire well is carefully scrutinized to ensure that there is and only one cell pellet present. A photograph of the cell pellet was taken. If a clump of cells is suspected to be present, the well is not selected. On day 36, selected clones were detached with TrypLE and 0.5mL of complete medium containing geneticin (0.5mg/mL, 1: 100 dilution) was added to stop the trypsin reaction. The entire volume was transferred to a 6-well plate and further diluted with 3.0ml of complete medium containing geneticin (0.5mg/ml, 1: 100 dilution). Clones were grown to 90% confluence, then trypsinized and transferred to 75cm collagen IV coated flasks or Costar flasks with 10.0ml complete medium containing geneticin (0.5mg/ml, 1: 100 dilution). Once the cells reached 90% confluence again, these cells were used to fill 3 cryovials for cryopreservation or for screening of galanin retargeted compounds in ELISA assays.

These clones were tested using the reference compound TVEMP-galanin batch C, independently tested by two operators. The SiMa GalR1 clone grew slowly and was not available for testing at this time. Fortunately, Neuro-2a clones grew faster and 8 out of 12 clones soon reached sufficient numbers for testing. These Neuro-2a GalR1 clonal cells were tested with the full dose range of TVEMP-galanin compounds (0-300nM), and the results for 9 of these clones are shown below. The remaining 4 clones grew very slowly and were not tested. Selected non-clonal parental cells are inoculated along with these clones to serve as a benchmark. Table 28 shows the activity of each of the 8 clones, as well as selected non-clonal Neuro-2aGalR1 cells, when tested with TVEMP-galanin compounds. Of the 8 clones tested, only clone #4, clone #7 and clone #12 showed good uptake of the TVEMP-galanin compound with acceptable EC₅₀The value is obtained. Neuro-2a GalR1 clone #1, clone #3 and clone #10 did not take up TVEMP-galanin compounds, while clone #5, clone #11 and clone #13 and the non-clonal cell population produced very high EC₅₀Values, and no further testing was performed using these cells.

4. Characterization of GalR1 expression in clonal cell lines

Screening for clones showed that only clone #4, clone #7 and clone #12 were more sensitive than non-clonal cells. Messenger RNA (mRNA) was extracted from these 3 clones as well as from untransfected parental Neuro-2a cells and stably transfected non-clonal Neuro-2a cells and characterized by RT-PCR using the RT-PCR conditions described in example V and the primers described in Table 29.

The results in Table 30 show that the transfected non-clonal cells and clones had much higher amounts of GALR1mRNA than the parental cells. Clone #7 showed the highest sensitivity to TVEMP-galanin in the TVEMP-galanin cell screen. From table 30, clone #7 was also shown to have the highest GALR1mRNA quantity. Neuro-2a GalR1 clone 7(Neuro-2a #7) had the lowest CT value, followed by clone 4, followed by clone 12. At this point, the non-clonal cells tested provided CT values very close to clone 12, but these cells contained ever-changing populations of cells containing varying concentrations of GalR1 receptor and were therefore not considered suitable for further study. At 3 EC₅₀Of the lower clones, the Neuro-2a clone GalR1 clone #12(Neuro-2a #12) grew fastest, followed by Neuro-2a clone #7 and finally Neuro-2a clone # 4. In addition to its slower growth rate, Neuro-2a clone #4 was not tested because Neuro-2a clone #4 was much less sensitive than clone # 7.

5. Comparison of sensitivity and specificity of Neuro-2a clone #7 to clone #12 using TVEMP-galanin compounds

Two clones were tested simultaneously in an attempt to identify the one of the two which is the most sensitive and selective, and thus data could be collected positively from the best performing clone. Table 31 shows the batches C and LH when TVEMP-galanin is used_NResults of sensitivity and selectivity of each of these two clones upon treatment with/A. Both clones exhibited high signal-to-noise ratios. EC of Neuro-2a clone #7₅₀The value was 5.5nM, while EC of Neuro-2a clone #12₅₀The value was 68.4 nM. Neuro-2a clone #12 was tested with a dose range of 0nM to 300nMWhereas Neuro-2a clone #7 could be tested with a dose range of 0nM to 30nM to induce a steady state at the highest concentration used. Both clones showed LH_NThe clear difference between/A and TVEMP-galanin batch C, Neuro-2a clone #12 showed some non-specific uptake at high concentration, while Neuro-2a clone #7 did not. As can be seen from the results in the table, the range tested with Neuro-2a #7 cells was 10 times lower than that tested with Neuro-2a #12 cells, resulting in Neuro-2a #7 using 10 times lower amount of compound than Neuro-2a # 12. When LH is required _NWhen used as a comparison, Neuro-2a #7 was 8-fold more selective than Neuro-2a clone # 12. The signal to noise ratio of both clones exceeded 100, but a ratio of 10 was sufficient for cell-based potency assays. Clone EC of Neuro-2a #7₅₀A value of 5.5nM, which is about 12-fold lower than that of Neuro-2a #12, EC of Neuro-2a #12₅₀The value was 68.4 nM. Lower test dose range for LH_N24 times selectivity,/A, high signal-to-noise ratio, low EC production₅₀The superior sensitivity of the values and the low protein quality required for each test indicate that Neuro-2a clone #7 will be the clone that will be subjected to a cell-based potency assay for determining the potency ratio against the TVEMP-galanin compound.

Example XVI

Generation of KOR-1 receptor overexpressing clonal cell lines for uptake of dynorphin a retargeted endopeptidase

The following example illustrates how to characterize and compare several clonal cell lines derived from the establishment of a cell line transfected with a target receptor and subsequent cloning of the cell line. This specific example will mention the identification and characterization of the hKOR-1 transfected clonal cell lines first described in Table 9 of example III.

4 AGN P33-KOR clones (example III clones in Table 9) were selectedNumbers 8, 9, 10 and 12) and tested by full dose response from 0nM to 150nM using Dyn/a. Meanwhile, 2 SiMa-KOR clones (clone numbers 12 and 16 in Table 9 of example III) were selected and tested by full dose response of 0nM to 150nM using Dyn/A. In this experiment, AGN P33-KOR clone 8, clone 9 and clone 12 produced very low uptake and were therefore discarded; AGN P33-KOR clone 10 showed good uptake and its EC ₅₀The value was 30.3 nM. The two SiMa-KOR clones tested exhibited good uptake, and the EC of clone 16₅₀Value of 26.6nM and EC for clone 12₅₀The value was 11.8 nM. Then by mixing the target Dyn/A compound with a negative control LH lacking the target ligand_NThe sensitivity and selectivity of these three clones was tested by comparing the uptake of the/A and Noc/A controls. A comparison of the three clones and the parental SiMa cells with a full dose response of 0nM to 150nM is summarized in table 32.

The Dyn/A uptake of KOR-1 transfected clones treated with Dyn/A was significantly increased, whereas parental SiMa cells showed very low uptake of the compound (uptake was similar to that of the negative control LH)_NA). An amount of Noc/a is present in all cell lines, including the parental SiMa cells. This is also unexpected because during the assay studies for the retargeted compound, SiMa cells were observed to take up Noc/a. Furthermore, Noc/a uptake was highest in the cell line AGN P33 derived specifically for this retargeted endopeptidase. There was a clear difference in Noc/a uptake and Dyn/a compound uptake in asexual SiMa-KOR clone 12(SK12) cells. In all graphs, the activity of the negative control LHN/A was minimal, indicating that no specific uptake occurred in these cell lines in the absence of the binding domain, and minimal uptake in SK12 cells, indicating that uptake of the Dyn/A compound was highly specific. From these results, SK12 clone should be selected for subsequent optimization and characterization.

An optimization study was performed using SK12 cells to develop a stable assay with specificity and sensitivity. Several parameters to be measured include inoculation medium and inoculation density, treatment medium and treatment time. A summary of the data obtained during the optimization is provided in table 33.

Table B shows the EC for cells seeded at 100,000 cells/well in CM and treated with compounds from CM in each experiment₅₀The values varied greatly (4.6nM, 1.2nM and 13.72nM) while cells seeded at 100,000 cells/well in SFM and treated with compounds diluted in SFM provided excellent curves and consistent EC₅₀Values (9.0nM and 8.4 nM). In the future, cells will be seeded at 100,000 cells/well in SFM and also treated with compounds in SFM.

SK12 was seeded at 100,000 cells/well in SFM on PDL plates for 24 hours followed by 16 hours of treatment in SFM to give the lowest EC₅₀A value of 8.4+/-1.1nM and a signal-to-noise ratio of 12. These two values are acceptable for future use of the cell in CBPA.

Characterization of SK12 cells using a saturation binding assay

The saturation binding assay utilized herein is described in detail in example V. Use of KOR-1 antagonists³Saturation binding studies were performed with H-diproporphine (diprenorphine) to assess binding. Total binding, specific binding and non-specific binding were measured in several experiments. Generated by two independent experiments ³Saturation binding curves of H-diproporphine to the receptor. As can be seen, about 25% of the binding is non-specific binding, and 75% is the specificity of the molecule for the receptorAnd (4) sexual combination. The affinity of the molecule for the receptor is suitably 6.5 nM. Bmax showed that on SK12 cells, each cell had 23fmol KOR-1 receptor.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (e.g., molecular weight), reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention or the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members of the group or other elements found herein. For convenience and/or patentability, it is contemplated that one or more members may be included in a group or deleted from a reorganization. When any such inclusion or deletion occurs, the specification is considered herein to contain a modified group so as to satisfy the written statement of all Markush groups (Markush groups) used in the appended claims.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Particular embodiments disclosed herein may be further limited to the use of a claim that "consists of or" consists essentially of. The conjunction "consisting of" when used in a claim does not include any element, step, or ingredient not specifically recited in the claim (whether submitted or added per modification). The conjunction "consisting essentially of limits the scope of the claims to the specifically described materials or steps, as well as materials or steps that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are described herein, either inherently or explicitly, and may be practiced.

In addition, throughout this specification, many references are made to patents and printed publications. Each of the references and printed publications cited above are individually incorporated by reference herein in their entirety.

Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, for example (but not limited to), alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to what has been particularly shown and described.

Claims (15)

1. A method of detecting retargeted endopeptidase activity, the method comprising the steps of:

a. treating cells from an established cell line with a sample comprising a retargeted endopeptidase, wherein the cells from the established cell line are sensitive to retargeted endopeptidase activity of a retargeted endopeptidase;

b. isolating from said treated cells a SNAP-25 fraction comprising the carboxy-terminal SNAP-25 cleavage product of residue P1 having a BoNT/a cleavage site labile bond;

c. contacting the SNAP-25 component with an alpha-SNAP-25 antibody attached to a solid support,

wherein the α -SNAP-25 antibody binds to an epitope at the carboxy terminus of the P1 residue in the SNAP-25 cleavage product comprising the readily cleavable bond of the BoNT/A cleavage site;

d. detecting the presence of an antibody-antigen complex comprising the a-SNAP-25 antibody and the SNAP-25 cleavage product;

wherein detection by the antibody-antigen complex is indicative of retargeted endopeptidase activity;

wherein the α -SNAP-25 antibody binds to an epitope at the carboxy terminus of the P1 residue in the SNAP-25 cleavage product comprising the readily cleavable bond of the BoNT/A cleavage site;

wherein the α -SNAP-25 antibody has a heavy chain variable region of an amino acid sequence encoded by the nucleic acid sequence SEQ ID NO 71, SEQ ID NO 75, SEQ ID NO 77, SEQ ID NO 79, or SEQ ID NO 81;

And wherein the α -SNAP-25 antibody has a light chain variable region of an amino acid sequence encoded by the nucleic acid sequence SEQ ID NO 83, SEQ ID NO 87, SEQ ID NO 89 or SEQ ID NO 91.

2. The method of claim 1, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 72, SEQ ID NO 76, SEQ ID NO 78, SEQ ID NO 80, and SEQ ID NO 82; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:90, and SEQ ID NO: 92.

3. The method of claim 1 or 2, wherein the SNAP-25 cleavage product is SNAP-25₁₉₇。

4. The method of claim 1 or 2, wherein the presence of antibody-antigen complexes is detected using a sandwich ELISA.

5. The method of claim 1 or 2, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84.

6. The method of claim 1 or 2, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88.

7. The method of claim 1 or 2, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 90.

8. The method of claim 1 or 2, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:80 or SEQ ID NO: 82; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 92.

9. An α -SNAP-25 antibody that binds to an epitope in the SNAP-25 cleavage product that is carboxy-terminal to residue P1 that comprises a BoNT/a cleavage site labile bond;

wherein the α -SNAP-25 antibody has a heavy chain variable region of an amino acid sequence encoded by the nucleic acid sequence SEQ ID NO 71, SEQ ID NO 75, SEQ ID NO 77, SEQ ID NO 79, or SEQ ID NO 81;

and wherein the α -SNAP-25 antibody has a light chain variable region of an amino acid sequence encoded by the nucleic acid sequence SEQ ID NO 83, SEQ ID NO 87, SEQ ID NO 89 or SEQ ID NO 91.

10. The antibody of claim 9, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 72, SEQ ID NO 76, SEQ ID NO 78, SEQ ID NO 80, and SEQ ID NO 82; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:90, and SEQ ID NO: 92.

11. The antibody of claim 9 or 10, wherein the SNAP-25 cleavage product is SNAP-25₁₉₇。

12. The antibody of claim 9 or 10, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84.

13. The antibody of claim 9 or 10, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88.

14. The antibody of claim 9 or 10, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 90.

15. The antibody of claim 9 or 10, wherein the a-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:80 or SEQ ID NO: 82; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 92.