Antibodies that bind cell-associated ca 125/0722p and methods of use thereof

The present invention relates to the use of poly-α2,8-sialic acid (APS) mimetic peptides to specifically regulate APS-dependent MACN functions in vitro and in vivo and their application for the treatment of neurodegenerative diseases, brain injuries. and spinal cord, age- related learning and memory problems, and cancer.

The ability of the cell to modify its cell surface interactions with other cells, including neurons and glial cells, is a critical component of nerve tissue development, remodeling and repair, as well as tumor formation and metastasis. Among many candidate molecules that are potentially involved in such a process are of particular interest are nerve cell adhesion molecule (MACN) isoforms, a member of the IgG superfamily, comprising an unconventional carbohydrate polymer, the poly-2,8 sialic acid (APS).

APS is a negatively charged Nacetylneuraminic acid (sialic acid) residue polymer with an alpha-2.8 bond. The single-chain APS supported by MACN generally consists of at least 30 repeating units with chain length varying widely in MACNs isolated from various sources (von Der Ohe et al. Glycobiology, 2002, 12, 47-63; Rougon et Eur. J. Cell. Biol., 1993a, 61, 197-207). By comparison, polyia2,8-sialic acid, which is found in the capsule of bacteria such as

as Neisseria meningitidis group B and E. coli K1 form longer polymers with about 200 repeating units. Studies using NMR microscopy indicate that APS has a solution helical structure composed of eight or more contiguous sialic acid units (Rougon et al., 1993a, cited above; Yamasaki et al 1991). Hydrated APS has a large volume and high negative charge density and is therefore well placed to attenuate adhesion forces and to negatively regulate cell surface interactions (Rutishauser et al. Science, 1988, 240, 53- 57). All known alternatively combined MACN isoforms can be polylysized to the fifth Ig-like domain (Rougon et al. 1993a, cited above) and MACN is the only clearly identified carrier of polysalic acid in the nervous system (Rougon et al. J. Cell. Biol., 1986, 103, 2429-2437). Although there is a report describing the association of APS with the Na channel α chain (Zuber et al. J. Biol. Chem., 1992, 267, 9965-9971), the absence of APS immunoreactivity in mouse MACNs. knockout (Cremer et al. Nature, 1994, 367, 455-457) suggests that MACN is a major if not the only APS vehicle in the brain of vertebrates.

Linking PHC to MACN is a regulated development process; high APS MACN is associated with morphogenetic changes during development, such as cell migration, synaptogenesis and axonal growth and ramifications, while in the adult brain few sialylated forms of MACN are dominant (Rougon et al, Polysialic Acid, 1993b, Roth Rutishauser, U. and Troy FA (eds), Birkhauser-Verlag: Basel, 323-333, Rutishauser et al, 1998, cited above, Edelman et al. Rev. Cell. Biol., 1986, 2, 81-116). However,

APS-MACN persists in brain structures of adults with a high degree of plasticity (Rougon et al. 1993a, cited above). For example, APS-MACN is required for two essential forms of activity-induced synaptic plasticity, long-term potentiation (PLP) and long-term depression (DLP), which are believed to be fundamental for learning and memory, as well as formation of activity-dependent patterns during development. (Muller et al Neuron, 1996, 17, 413-422). Indeed, hippocampal tissue prepared from MACN from mutant mice exhibited a very low capacity of PLP as well as DLP, and this defect may be mimicked by enzymatic destruction of the MACN APS portion. These observations indicate that APS rather than MACN protein is required for plasticity. The morphological plasticity that occurs in the hypothalamic-neurohypophyseal system (Theodosis et al. J. Neurosci. 1999, 19, 1022810236) is also dependent on the presence of APS as in vivo injection of endoneuraminidase prevents it.

APS-MACN is again expressed in various pathological situations, such as muscle regeneration, axonal regeneration, and brain tumors (FigarellaBranger et al. Cancer Res. 1990, 50, 6364-6370; Dubois et al. Neuromuscul. Disord., 1994 , 4, 171-182; Aubert et al., Comp. Neurol., 1998, 399, 1-19; Muller et al. Neuroscience, 1994, 61, 441-445) or in neurodegenerative brain diseases (Le Gal La Salle et al. J. Neurosci., 1992, 12, 872-882). Based on these observations, APS-MACN has emerged as an important permissive factor for dynamic changes in cell surface interactions required for tissue morphogenesis and remodeling (Rougon et al, 1993b, cited above; Figarella-Branger, 1993; Rutishauser, Development, 1992, 99-104).

Many tumors with neural and endocrine characteristics expressed APS-MACN. For example, APS-MACN had been detected in neuroblastomas and medulloblastomas (Figarella-Branger et al., Cited above), small cell lung carcinoma (Patel et al. Int. J. Cancer, 1989, 44, 573-578). and rhabdomyosarcomas and is possibly related to the invasive and metastatic potential of these tumors (Rougon et al. 1993b, cited above).

Recently, injection of neuraminidase in a nude mouse model for metastasis has shown that APS clearance in the primary tumor delayed metastasis. (Daniel et al Oncogene, 2001, 20, 997-1004).

Thus, the APS-MACN molecule and, more precisely, APS carbohydrate represents one of the potential targets of future therapeutic approaches to promote plasticity and functional recovery after brain damage or to prevent metastasis formation.

Thus, several strategies have been developed to regulate PHC functions:

- genetic manipulations: MACN or polylsalyltransferase of knockout mice (Cremer et al, cited earlier): this strategy does not open therapeutic perspectives, enzymatic digestion: endoneuraminidase (al. Theodosis al, cited earlier, Daniel et al cited earlier): its therapeutic potential is quite limited due to their large size and restricted diffusion in vivo and the possibility of inducing an immune response, anti-APS monoclonal antibodies (Monnier et al. Developmental Biology, 2001, 229, 1-14); Its therapeutic potential is quite limited due to its nature as antibody, cholinic acid, an APS analogue isolated from the bacterial capsule; its therapeutic potential is quite limited due to its instability at acidic pH and the inability to control its exact composition in terms of purity and homogeneity (sialic acid chain length calibration from one batch to another),

N-butanoyl mannosamine (ManBut), a small molecule capable of inhibiting APS biosynthesis in vitro (Mahal et al. Science, 2001, 294, 380-382); its activity has not been demonstrated in vivo.

However, to date, these strategies that have been used to unravel the mechanisms of action or functions of APS have not led to the discovery of new drugs capable of regulating APS functions in vivo.

Therefore, there is a need for new molecules capable of specifically regulating APS-dependent in vivo MACN functions that can be used as pharmaceutical compositions to promote plasticity and functional recovery after brain or spinal cord injury or for prevention. of metastases.

Peptides representing carbohydrate epitope mimetic molecules of microorganisms, including Neisseria meningitidis group B-specific DPA epitopes have been described for the safe and effective development of vaccine candidates against such microorganisms (Shin et al. Infection and Immunity, 2001, 69, 3335-3342); Neisseria meningitidis group B-specific APS peptides described in Shin et. al represent epitopes that are different from ASP epitopes of vertebrate cells (neurons) and therefore do not induce antibodies that bind to said neuron ASP (MACN-linked APS) and may cause neurological damage. In addition, Shin et al. describe only two peptides (DHQRFFV (SEQ ID NO: 31) and AHQASFV (SEQ ID NO: 32) representing APS-linked MACN epitope mimetics, which are used as a control to demonstrate the specificity of group B APS mimetic peptides). Neisseria meningitidis.

Applicants have isolated other peptides, which are molecular mimetics of APS-linked MACN epitopes, and have demonstrated that these APS mimetic peptides are capable of regulating (increasing or inhibiting), in vivo, APS-dependent cellular processes that are commonly affected by MACN polysilylation. For example, we have shown a significant effect of APS mimetic peptides in vivo on axon growth, orientation and fasciculation as well as on neuron migration. In addition, administration of APS mimetic peptides results in functional recovery following spinal cord injury and is also accompanied by reduced reactive gliosis at the injury site itself.

In the context of the present invention, poly-α2,8 sialic acid or APS stands for MACN-bound APS, as opposed to other APS such as bacterial capsule APS.

These APS mimetic peptides, which are active in vivo at low doses (μΜ concentration) and show no cytotoxicity, are useful for:

Treatment of neurodegenerative diseases (Parkinson's, Huntington's chorea, Alzheimer's, multiple sclerosis):

they are useful as adjuvants for cell therapy; neuronal progenitor (APS expressing) graft in combination with APS mimetic peptides would significantly increase progenitor migration and axonal growth in damaged brain areas, treatment of brain and spinal cord injury: would promote functional recovery after brain or spinal cord injury by enhancing survival and axonal regeneration, the prevention and treatment of age-related learning and memory problems: would promote nervous system plasticity by enhancing neurogenesis and / or synaptic plasticity, treating cancer: would prevent metastasis formation by inhibiting migration of cells from APS expressing tumors, for example neuroectodermal tumors.

In addition to the reported therapeutic use, APS mimetic peptides are useful as complementary tools for unraveling the unknown mechanisms of action and functions of ASP carbohydrate.

The present invention relates to a peptide for use as a medicament for regulating MACN functions, said peptide comprising an epitope B of a MACN-linked poly-α2,8-sialic acid, which is recognized by an anti-human antibody. (poly-α2,8-sialic acid) and said peptide being selected from the group consisting of:

linear peptides having up to 30 amino acid residues, preferably about 12 amino acid residues, including at least one of the sequences of SEQ ID NO:

<td>1 to 12, 14 and 15,</td><td>corresponding</td><td>respectively to:</td>

<td>DSPLVPFIDFHP,</td><td>LWQPPLIPGIDF,</td><td>QIEPWFTPEDFP,</td>

<td>TRLAPLVFPLDY,</td><td>SWLQMPWALVRT,</td><td>EIHLRMIKQITI,</td>

<td>WHLEYMWRWPRL,</td><td>LIEQRLPKHILT,</td><td>YETSSSRLLAYA,</td>

<td>TLASQLSNTSAY,</td><td>SDQGVNGSWSNP,</td><td>WHNWNLWAPASPT,</td>

IKSPLTWLVPPD and SHLDLSTGHRTS and cyclic peptides having up to 30 amino acid residues, preferably about 12 amino acid residues, including at least one of the sequences of SEQ ID NO: 16 to 26, corresponding respectively to: CYPLNPEVYHCG, CWPLSHSVIVCG, CSSVTAGTG CYC , CWPLGPSTYICG, CSLIASMETGCG, CSKIASMETGCG,

CYIGDPPFNPCG, CWPLGDSTVICG CPLRLAFTFGCG and

CTRMSHGYWICG.

The epitope B recognized by an anti-APS antibody refers to a peptide that specifically and selectively reacts in vitro and in vivo to the paratope of an antibody produced by lymphoid cells in response to poly-a2 acid stimulation. , 8-sialic as an immunogen. Anti-APS antibodies prepared by standard techniques following the protocols described by

Antibodies: A Laboratory Manual, Howell E

Cold Spring Harbor Laboratory, 1988) are well known in the art; include, without limitation, monoclonal antibodies 735 (Frosch et al, PNAS; 1985, 82, 1194-1198), 30H12 (Coquillat et al. Infect. Immun. 2001, 69, 7130-7139) or

MenB (ABCYS AbC0019; Rougon et al. J. Cell. Biol., 1986, 103, 2429-2437).

example in Lane and D of the

The present invention includes the use of cyclic and linear peptides. Preferred cyclic peptides as defined in the present invention comprise peptides wherein the side chain of one amino acid in the peptide chain is covalently linked to the side chain of another amino acid in the peptide chain via the formation of a covalent bond such as a disulfide bond between two cysteine ​​residues.

Peptides as defined in the present invention refer to peptides that have the following activities:

antibody binding activity: are specifically recognized by an anti-APS monoclonal or polyclonal antibody and biological activity: have the activity of regulating (increasing or inhibiting) APS-dependent MACN functions.

Peptides as defined in the present invention are hereinafter referred to as APS mimetic peptides, mimetic peptides or peptides. Unless otherwise specified, said peptides are either linear or cyclic.

Antibody binding activity of mimetic peptides is verified by standard immunoassays which are well known to one skilled in the art; for example, 100% peptide binding is observed at concentrations of 10⁴ M and above in an ELISA assay with an anti-ASP monoclonal antibody at a concentration of 2.5 pg / well and this antibody binding activity is specifically inhibited by an APS analog such as cholinic acid.

The biological activity of the mimetic peptides is verified by standard cell growth and in vitro or in vivo cell migration assays, which are well known to those skilled in the art; for example, tests on primary neurons from different sources (dorsal root ganglion, cerebellar neurons, retina, ...) show the following effects:

a significant dose-dependent increase in the length of neuritis in vivo and in vitro, a significant effect on axonal orientation in vivo and in vitro (axon derasciculation) and a significant increase or inhibition of cell migration.

In addition, functional assays, including the study of functional recovery of spinal cord hemi-section by well known assays (BBB test: Basso, Beattie and Bresnahan test; Basso et al. Restor. Neurol. Neurosci, 2002, 5,189-218; route-rod assay) show significant locomotor recovery and fine motor coordination recovery in animals treated with mimetic peptides compared to animals treated with a non-relevant peptide.

The biological activity of the mimetic peptide is specifically inhibited by enzymatic digestion with endoneuraminidase and is absent in knockout rat MACN.

Preferably, said peptide consists of a sequence selected from the group consisting of SEQ ID NO: 1 to 12 and SEQ ID NO: 14 to 26.

More preferably, said peptide is selected from the group consisting of:

a linear peptide having SEQ ID NO: 1 (DSPLVPFIDFHP) hereafter referred to as p21 and a cyclic peptide wherein the side chain cysteine ​​residue at position 1 of SEQ ID NO: 18 (CSSVTAWTTGCG) or SEQ ID NO: 22 (CSKIASMETGCG ) is covalently linked to the cysteine ​​side chain residue at position 11 of SEQ ID NO: 18 or SEQ ID NO: 22 via a disulfide bond; said peptides are hereinafter referred to respectively as p65 and p66.

According to an advantageous embodiment of the present invention, said peptide is associated with another peptide and / or non-peptide molecule incorporated into a suitable support including, for example, polymers, lipid vesicles, microspheres, proteins and the like. Preferably, the other peptide and / or non-peptide molecule and / or the support as defined above allow the mimetic peptide to cross the blood-brain barrier.

This association, which can improve the solubility, absorption, bioavailability, and biological half-life of the peptide, is formed using techniques well known in the art; although not limiting, it may be by covalent bonding (eg, amide bonding, disulfide bonding ...) or by chelation, electrostatic interactions, hydrophobic interactions, hydrogen bridges, ion-dipole interactions, dipole interactions -dipole or any combination of the above.

According to another advantageous embodiment of the present invention, said peptide is incorporated into a complex formed by a plurality of identical or different peptides according to the present invention linked by covalent or non-covalent bonds.

A peptide as defined in the present invention may be associated with a label such as a fluorescent label to facilitate detection of said peptide or may be included in a fusion protein to allow expression of said peptide.

According to another advantageous embodiment of the present invention, said peptide is for the prevention and / or treatment of a pathological condition selected from the group consisting of: neurodegenerative diseases, brain and spinal cord injuries, learning and learning disorders. age-related memory.

According to another advantageous embodiment of the present invention, said peptide is for the prevention and / or treatment of cancer.

Peptides as defined in the present invention may be prepared by any suitable process. Preferably, they are obtained by chemical synthesis in liquid or solid phase by successive bonding of the different amino acid residues to be incorporated (from the end of the N-terminal to the end of the C-terminal in liquid phase or from the end of the C-terminal). to the solid-phase N-terminal end) wherein the N-terminal ends and reactive side chains are previously blocked by conventional groups. For solid phase synthesis the technique described by Merrifield (J. Am. Chem. Soc. 1964, 85, 2149-2154) is used.

Peptides as defined in the present invention may also be obtained by genetic engineering technology. Nucleic acids encoding the peptides of the present invention may be obtained by methods well known in recombinant DNA technology and / or chemical DNA synthesis. A typical example comprises culturing a host cell containing an expression vector comprising a nucleic acid sequence encoding said peptide under conditions suitable for peptide expression and recovering the peptide from the host cell culture. The peptide may be included in a fusion protein by cloning a cDNA into a frame expression vector with a polynucleotide encoding the peptide of the present invention. Alternatively, multimers of identical or different peptides may also be produced by expression of a polynucleotide encoding multiple copies of a monomer or encoding different monomers.

The medicament according to the present invention comprises an effective amount of the peptide or peptide complex as defined above in combination with a pharmaceutically acceptable carrier.

The carriers of the medicament of the present invention may be any carrier for parenteral, intrathecal, oral, aerosol, nasal or ocular administration of drugs acting on the nervous system. For example, the medicament according to the present invention is administered intrathecally, which allows the composition to penetrate directly into the central nervous system. Alternatively it is administered through the nose, allowing the aerosolized composition to penetrate the central nervous system via the olfactory nerve or ocular route or any other suitable administration process as described in WM Pardridge Peptide drug Delivery, Raven Press, NY, 1991.

The amount of peptide in the medicine is in a concentration range of about 0.1 μΜ to 10 μΜ. Preferred frequency of administration and effective dosage will vary from one individual to another.

Peptides as defined in the present invention have the following advantages:

- are active in vivo at low doses (0.5 μΜ concentration),

- are stable in vivo,

- are very efficient in vivo as long as they act extracellularly; therefore, its activity is not limited by its ability to penetrate into the cell,

- are non-toxic, and

- can be easily produced in large quantities.

The present invention will be further illustrated by the following further description and drawings, which refer to examples illustrating the isolation, binding specificity and biological effects of APS mimetic peptides according to the present invention. It is to be understood, however, that these examples are given by way of illustration only of the present invention and in no way constitute a limitation thereof.

Figure 1 illustrates the structure of APS-MACN.

Figure 2 illustrates the effect of the mimetic peptides of

APS p65 and p66 in neurite growth in vitro: mouse dorsal root ganglion transplantation (E13,5) was cultured in the absence (A, D) or in the presence of p65 and p66 coated peptides on microplates as conjugate of ASB (B, E) or in soluble form (C, F). The quantification (G) of the effects of mimetic peptides on the average length of the longest neuritis. *** P &lt;0.001 compared to Student's t-test of control. (H) Graph of cumulative frequency distribution of mean length of longest neuritis.

Figure 3 illustrates the effect of the APS p65 mimetic peptide on fasciculation and in vivo orientation: The assembled preparation (A) of the whole chick retina E9 and its schematic drawing demonstrating the position of the crystal Dil (B). The dotted rectangle indicates the area from which the photographs were taken. The arrows point to the optical fissure. E9 chick retinal axons injected into E3 with the inverse peptide (C, F) or the p65 peptide (D, G, E, H). The arrowheads show examples of axons that leave their fascicles and run perpendicular to them.

Figures 4-7 illustrate the effect of APS mimetic peptides on in vitro cell migration analyzed from subventricular (Pl) zone transplants of normal mice (MACN + / +), heterozygous mice (MACN +/-) or mice Knockout (MACN - / -), cultured in the presence of mimetic peptides (p65, p21, p66), different from said peptides (cyclic, linear and acetylated linear), control peptides (inverse p65 and inverse p66 and p22) or endosialidase N: Figure 4 illustrates the effect of p65 inverse peptide (A, D), N (B, E) endosialidase or p65 and p66 (C, F) mimetic peptides. (G) the quantification of the effects of mimetic peptides on the average distance of cell migration. *** P &lt;0.001 compared to control with Student's t-test. (H) graph of cumulative frequency distribution of average migration distance.

(I) Dose-dependent effect of p65 peptide on mean cell migration distance, Figure 5 illustrates the effect of different forms of p65 mimetic peptide (cyclic, linear, linear and acetylated) on mean cell migration distance, compared with the inverse control peptide p65. *** P &lt;0.001 compared to Student's t-test control, Figure 6 illustrates the effect of p65 mimetic peptide on mean cell migration distance in knockout (MACN - / -) or heterozygous mice (MACN + / - ). Rat MACN +/- endo N treated cells and treated p65 inverse cells are included for comparison. *** P &lt;0.001 compared to Student's t-test control and Figure 7 illustrates the effect of p65 and p21 mimetic peptides on the average distance of cell migration. Endo N treated cells and control peptide treated cells (reverse p65 and p22) were included for comparison. *** P &lt;0.001 compared to control with Student's t-test.

Figure 8 illustrates the effect of APS p65 mimetic peptide on in vivo cell migration: (A) Schematic drawing of the transplant. (B) Confocal microscopy photograph of a section showing CMT (thalamic migration current) of grafted mice in the presence of the p65 peptide. Arrowheads show examples of GFP and APS positive cells. ZSV (PI) transplants were grafted in the presence of inverse peptide (C, D) or p65 peptide (E, F) and brains were analyzed three days after grafting (C, E) or four days after grafting ( D, F). (G) Quantifying the effect of p65 peptide on the number of GFP-positive cells reaching the olfactory bulb three days after grafting. * P &lt;0.05 compared to control by Student's t-test.

control (BBB test). B en = 8 for Inv.

Figure 9 illustrates the protocol used to analyze functional recovery of spinal cord injury following injection of the p65 peptide or inverse p65 peptide taken as a control.

Figure 10 illustrates the functional recovery of spinal cord injury following p65 (p65) or inverse p65 (Inv) peptide injection

A: East of Basso, Beattie route-rod test. n = 11 for p65 *** P &lt;0.01, ** P &lt;0.01, * P &lt;0.05, compared with control by Student's t-test.

Figure 11 illustrates the decrease in reactive gliosis following spinal cord injury in rats treated with p65 peptide or inverse p65 peptide taken as a control. A: Immunofluorescence analysis using anti-GFAP and anti-APS antibodies alone or together (double labeling). B: quantification of GFAP staining. * P &lt;0.05, compared to control with test taken as and Bresnahan's Student.

Figure 12 illustrates the binding specificity of cyclic mimetic peptides:

Figure 12a: ELISA using plates coated with anti-APS 30H12 monoclonal antibody. Numbers 1 to 16 correspond to peptide sequences as shown in Table IV.

Figures 12B, 12C and 12D: Competitive binding for APS-MACN expressing cells. B: preincubation of p65 (1 mM) or p66 (1 mm) with anti-APS antibody

MenB. C: anti-APS 30H12 antibody without peptide. D: Preincubation of p65 (1 mM) or p66 (1 mm) with anti-APS 30H12 antibody.

EXAMPLE 1: TRACKING THE PEPTID LIBRARY WITH

OC-APS MONOCLONAL ANTIBODY

1) Materials and Processes

1.1) Materials

12 Peptide Monomer Phage Display Library

Two libraries were screened. The first library (12 Phage Display peptide Library, New England Biolabs) comprises 12 linear peptide monomers present on the surface of M13-like phage particles as the N-terminal fusion protein of the minor pIII coat protein (5 copies / phage particle). Library variance differs from 10⁸ to 10⁹ peptides with a constant sequence length.

The second library, prepared as described in Felici et al. (J. Mol. Biol., 1991, 222, 301-301), comprises 12 cyclic peptide monomers, including 2

<td>waste</td><td>in</td><td>cysteine</td><td>in the positions</td><td>1 and</td><td> 11</td><td>linked by</td><td>an</td>

<td>Link</td><td>in</td><td colspan="3">disulfide gifts</td><td>at</td><td>surface</td><td>in</td>

<td colspan="2">particles</td><td>of phages</td><td>similar to</td><td>M13</td><td colspan="2">like protein</td><td>in</td>

fusion to the N-terminus of the largest pVIII coated protein (100 copies / phage). The library has about 10⁸peptides with constant sequence length.

- Anti-APS Monoclonal Antibody (Acm)

Anti-APS monoclonal antibodies prepared by standard techniques are used as described in Antibodies: A Laboratory Manual, E. Howell and D. Lane, Cold

Spring Harbor Laboratory, 1988. For example, monoclonal antibodies 735 (Frosch et al, PNAS; 1985, 82, 1194-1198), 30H12 (IgG 2a; Coquillat et al. Infect. Immun. 2001, 69 may be used). , 7130-7139) or MenB (IgM;

AbCYS AbC0019).

- Plates (Maxisorp ™ NUNC)

- tubes (Maxisorp ™, NEVER)

- E. coli strain ER2537 (New England Biolabs)

- 96 glll sequencing primer (New England

Biolabs):

'CCCTCATAGTTAGCGTAACG-3' (SEQ ID NO: 27)

1.2) Tampons

Blocking solution: 0.5% ASB in SBF

Coating solution: 25 pg / ml anti-APS mAb in

SBF

TBS: 50 mM Tris-HCl pH 8.6, 150 mM NaCl.

TBST: TBS containing 0.1% or 0.5% Tween 20.

PEG / NaCl: 20% (w / v) polyethylene glycol 8000, NaCl

2.5 M.

Iodide Buffer: 10mM Tris-HCl, pH 8.0, 1mM EDTA, 4M NaCl.

1.3) Process

Maxisorp ™ tubes were incubated overnight with 2 ml of the coating solution at +4 ° C with gentle shaking according to the Ph.D. 12 ™ Phage Display peptide Library kit (New England Biolabs) manual. The coating solution was withdrawn and used for the lining of new tubes for a further passage of the biopanning process. The coated tubes were incubated with 2 ml blocking solution for 1 hour and washed 6 times with TBST. The tubes were filled with 2 ml of phage solution (7.5x10¹⁰ pfu / ml in TBST containing 0.1% Tween 20) and incubated with gentle shaking at room temperature for 1 h. After removal of the phage solution, the tubes were washed 10 times with TBST. Phage bound or eluted either specifically with 1 mM cholinic acid in TBS for 1 h or not specifically with 0.2 M HCl glycine (at pH 2.2) for 10 min with immediate neutralization with 1 M Tris-HCl Eluted products were amplified in 20 ml of an E. coli ER2537 culture. coli (from an OD 100: 0.03) for 4.5 hours at 37 ° C with vigorous stirring. The cultures were centrifuged for 10 min at 10,000 rpm at 4 ° C. The supernatant was transferred to a new tube and centrifuged for more than 10 minutes. A PEG / NaCl solution was added to the supernatant (1 volume PEG / NaCl to 6 volumes supernatant) and the phage precipitated overnight at 4 ° C. The solution containing the precipitate was centrifuged for 15 min at 10,000 rpm at 4 ° C. The supernatant was decanted and the pellet was suspended in 1 ml TBS and precipitated again with 1/6 volume PEG / NaCl for 1 hour on ice. After centrifugation, the pellet was finally suspended in 200 ml TBS, 0.02% NaN 3.

This amplified eluted product was dissolved in TBST and a second and third biopanning was performed as described above; for the second cycle, the TBST used for phage washing and incubation contained 0.1% Tween 20; in the third cycle the content was 0.5%.

Third cycle unamplified eluted product was further titrated on LB / IPTG / X-gal plates. Blue plates were harvested and the phage clone enlarged by 2 ml of E. coli ER2537 culture for 4.5 hours at 37 ° C with vigorous shaking. After centrifugation for 10 min at 10,000 rpm at 4 ° C, the supernatant was mixed with 1/6 volume PEG / NaCl and the phage precipitated at 4 ° C overnight. The precipitate was centrifuged for 15 min at 10,000 rpm at 4 ° C. The pellet was suspended in 100 μΐ TBS. 10 μΐ of this solution was mixed with 100 μΐ iodide buffer and 250 μΐ ethanol for precipitation of single stranded phage DNA. After incubation for 10 minutes at room temperature, the solution was centrifuged for 10 min at 15,000 rpm. The supernatant was discarded and the pellet washed in 70% ethanol and dried rapidly in vacuo. The pellet was suspended in 10 μΐ of distilled water containing the sequencing primer for automatic sequencing of the peptide insert (Terminator BigDye sequencing cycle with standard Applied Biosystem primer M13-40 num 877/377). The remaining single phage solution was used for ELISA experiments.

2) Results

After three rounds of selection, the phages were isolated with the following sequences (Tables I, II, III and IV).

Table I: Linear peptide sequences isolated from 17 different phages eluted with 1 mM cholinic acid

<td>SEQ ID NO:</td><td>Sequence</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td> 4</td><td>TRLAPLVFPLDY</td>

<td> 6</td><td>EIHLRMIKQITI</td>

<td> 7</td><td>WHLEYMWRWPRL</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td>s</td><td>LIEQRLPKHILT</td>

<td> 9</td><td>YETSSSRLLAYA</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td> 10</td><td>TLASQLSNTSAY</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td> 11</td><td>SDQGVNGSWSNP</td>

<td> 4</td><td>TRLAPLVFPLDY</td>

<td> 5</td><td>TRLAPLVFPLDY</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td> 4</td><td>TRLAPLVFPLDY</td>

Table II: Linear peptide sequences isolated from 20 different phages eluted with 0.2 M glycine.

<td>SEQ ID NO:</td><td>Sequence</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td> 2</td><td>LWQPPLIPGIDF</td>

<td> 2</td><td>LWQPPLIPGIDF</td>

<td> 12</td><td>WHNWNLWAPASPT</td>

<td> 3</td><td>QIEPWFTPEDFP</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td> 3</td><td>QIEPWFTPEDFP</td>

<td> 13 ²)</td><td>WHWQWTPWSIQP</td>

<td> 2</td><td>LWQPPLIPGIDF</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td>SEQ ID NO:</td><td>Sequence</td>

<td> 2</td><td>LWQPPLIPGIDF</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td> 15</td><td>SHLDLSTGHRTS</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td> 5</td><td>SWLQMPWALVRT</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td> 14</td><td>IKSPLTWLVPPD</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td> 1</td><td>DSPLVPFIDFHP</td>

<td colspan="2">³⁾ not part of the present invention</td>

Linear peptides have a high occurrence in isolated phages after three rounds of selection (Table III).

Table III: Alignment of the five most common linear sequences

<td>Frequency</td><td>SEQ ID NO:</td><td colspan="12">Sequence</td>

<td> 9/40</td><td> 5</td><td>s</td><td>W</td><td>L</td><td>Q</td><td>M</td><td>P</td><td>W</td><td>THE</td><td>L</td><td>V</td><td>R</td><td>T</td>

<td> 8/40</td><td> 1</td><td>D</td><td>s</td><td>P</td><td>L</td><td>V</td><td>P</td><td>F</td><td>I</td><td>D</td><td>F</td><td>H</td><td>P</td>

<td> 4/40</td><td> 4</td><td>T</td><td>R</td><td>L</td><td>THE</td><td>P</td><td>L</td><td>V</td><td>F</td><td>P</td><td>L</td><td>D</td><td>Y</td>

<td> 4/40</td><td> 2</td><td>L</td><td>W</td><td>Q</td><td>P</td><td>P</td><td>L</td><td>I</td><td>I</td><td>G</td><td>I</td><td>D</td><td>F</td>

<td> 2/40</td><td> 3</td><td>Q</td><td>I</td><td>AND</td><td>P</td><td>W</td><td>F</td><td>T</td><td>P</td><td>AND</td><td>D</td><td>F</td><td>P</td>

An alignment of the gene stream homepage with the sequence SEQ ID NO: 1 showed that sequence similarity ranges from 42.9% for SEQ ID NO: 2, 30.8% for SEQ ID NO: 3, 28.6% for SEQ ID NO: 4, up to 8.3% for SEQ ID NO: 5.

Phage clones displaying cyclic peptides bound to antibodies in a dose-dependent manner after three rounds of selection and did not bind to an irrelevant antibody of the same isotype. DNAs from 16 of these clones that showed the highest value in the ELISA were prepared and sequenced (Table IV). 3 Clones had the same sequence (SEQ ID NO: 17) and the dimer WP was found in 5 clones.

Table IV: Cyclic peptide sequences isolated from phage

<td>SEQ ID NO:</td><td>Peptide n^The</td><td>Sequence</td>

<td>SEQ ID NO: 16</td><td> 2</td><td>CYPLNPEVYHCG</td>

<td>SEQ ID NO: 17</td><td> 3</td><td>C WP LSHSVIVCG</td>

<td>SEQ ID NO: 17</td><td> 5</td><td>C WP LSHSVIVCG</td>

<td>SEQ ID NO: 18</td><td> 6 (_P65)</td><td>CSSVTAWTTGCG</td>

<td>SEQ ID NO: 19</td><td> 8</td><td>CYMASGVFLCG</td>

<td>SEQ ID NO: 17</td><td> 9</td><td>C WP LSHSVIVCG</td>

<td>SEQ ID NO: 20</td><td> 10</td><td>C WP LGPSTYICG</td>

<td>SEQ ID NO: 21</td><td>11 (p66)</td><td>CSLIASMETGCG</td>

<td>SEQ ID NO: 22</td><td></td><td>CSKIASMETGCG</td>

<td>SEQ ID NO: 16</td><td> 12</td><td>CYPLNPEVYHCG</td>

<td>SEQ ID NO: 23</td><td> 13</td><td>CYIGDPPFNPCG</td>

<td>SEQ ID NO: 24</td><td> 14</td><td>CWPLGDSTVICG</td>

<td>SEQ ID NO: 25</td><td> 15</td><td>CPLRLAFTFGCG</td>

<td>SEQ ID NO: 26</td><td> 16</td><td>CTRMSHGYWICG</td>

EXAMPLE 2: ANALYSIS OF PEPTIDE SPECIFICITY BY COMPARATIVE ELISA ANALYSIS OF PIGS

1) Materials and Processes

1.1) Materials

Anti-APS 735 or 30H Monoclonal Antibody 12

Maxisorp ™ Plates (NUNC) for Antibody Coating: ELISA Plates Microtiter Plates (NUNC) for Phage Dilution: Dilution Plates

- M 13 bacteriophages displaying peptides of tables III and IV of example 1

- PRS conjugated anti-M13 antibody (PHARMACIA 279411-01)

- ABTS [2,2'-azino-bis- (3-ethylbenzothiazoline-6sulfonic acid), SIGMA]

- Cholinic acid (SIGMA)

Dextran (SIGMA)

1.2) Tampons

SBF at pH 7.4

Blocking Solution: 0.5% ASB in SBF TBS

TBST: TBS containing 0.05% Tween 20

Horseradish peroxidase conjugated anti-M13 antibody solution: 1/5000 in TBST

Horseradish peroxidase substrate solution (PRS): 22 mg ABTS in 100 μΐ 50 mM sodium citrate at pH 4,0. Prior to the enzymatic reaction, add 36 ml of 30% H2O2 to 21 ml of ABTS solution.

concurrent solution: 1 mM cholinic acid in TBST control solution: 1 mM dextran in TBST

1.3) Process

Maxisorp ™ plate wells were coated with 100 μΐ of an Acm solution (25 pg / ml) for 2 h at room temperature. Control wells were coated with the blocking solution only. In parallel, the phage dilution plates were blocked with 200 μΐ blocking solution for 2 h. Antibody coated wells and ELISA plate control wells were blocked with 200 μΐ blocking solution for 1 h. In parallel, phage dilution plates were washed 6 times with TBST and 120 μΐ TBST was added to the wells. An appropriate volume of a phage solution was added to the first well and the volume adjusted to 140 μΐ with TBST. The phage solution was diluted in well 1 1/7 by removing 20 μΐ from the first well and transferring to the second well to reach the total volume of 140 μΐ. This procedure was repeated for the remaining wells. Phage dilutions for control wells were made in the same manner. The blocked ELISA plate was washed 6 times with TBST and phage dilutions or comparative solution added. After incubation for 1h, the plates were washed 10 times with TBST. After incubation for 1 h, the wells were washed 10 times with TBST. 100 μ 100 of PRS-conjugated M13 antibody solution was added to the wells. After 1 h incubation, the wells were washed 10 times with TBST and 100 μΐ PRS substrate solution (with H₂O₂). The plates were read at 405 nm using a microplate reader.

2) Results

The binding specificity of the peptides of SEQ ID NO: 1 to 5 of Example I was analyzed in a comparative ELISA using cholinic acid as a comparison. Results shown in Tables V to IX are expressed as percent binding compared to phage showing SEQ ID NO: 5 at a concentration of 710 ng / well (100%).

Table V: Binding of phage showing SEQ ID NO: 5 to Acm 735

<td></td><td colspan="7">Phage Concentration (ng / well)</td>

<td></td><td> 710</td><td> 109</td><td> 18</td><td> 3</td><td> 1</td><td> 0</td><td> 0</td>

<td>phage</td><td> 100 ± 3</td><td> 102 ± 0</td><td> 98 ± 0</td><td> 73 ± 4</td><td> 18 ± 2</td><td> 4 ± 3</td><td> 2 ± 1</td>

<td>phage + cholinic acid</td><td> 106 ± 0</td><td> 103 ± 5</td><td> 90 ± 0</td><td> 36 ± 0</td><td> 5 ± 0</td><td> 1 ± 0</td><td> 1 ± 0</td>

<td>phage + dextran</td><td> 102 ± 2</td><td> 103 ± 1</td><td> 97 ± 1</td><td> 62 ± 1</td><td> 14 ± 0</td><td> 5 ± 0</td><td> 1 ± 0</td>

<td>phage + ASB</td><td> 5 ± 1</td><td> 3 ± 1</td><td> 1 ± 0</td><td> 1 ± 0</td><td> 0 ± 0</td><td> 0 ± 0</td><td> 0 ± 0</td>

Table VI: Binding of phage showing SEQ ID NO: 1 to mAb

<td></td><td colspan="7">Phage Concentration (ng / well)</td>

<td></td><td> 1003</td><td> 154</td><td> 26</td><td> 4</td><td> 1</td><td> 0</td><td> 0</td>

<td>phage + Acm735</td><td> 108 ± 2</td><td> 111 ± 7</td><td> 111 ± 3</td><td> 112 ± 12</td><td> 86 ± 15</td><td> 28 + 2</td><td> 7 + 0</td>

<td>phage + cholinic acid + Acm735</td><td> 108 ± 0</td><td> 109 ± 12</td><td> 90 + 3</td><td> 42 + 3</td><td> 12 + 1</td><td> 2 + 1</td><td> 0 + 0</td>

<td>phage + dextran Acm735</td><td> 109 ± 6</td><td> 110 ± 6</td><td> 112 ± 5</td><td> 107 ± 1</td><td> 77 + 4</td><td> 24 + 4</td><td> 8 + 0</td>

<td>phage + ASB</td><td> 10 ± 1</td><td> 4 ± 1</td><td> 3 + 0</td><td> 1 + 1</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td>

Table VII: Binding of phage showing SEQ ID NO: 4 to mAb

<td></td><td colspan="7">Phage Concentration (ng / well)</td>

<td></td><td> 870</td><td> 134</td><td> 22</td><td> 4</td><td> 1</td><td> 0</td><td> 0</td>

<td>phage + Acm735</td><td> 107 ± 7</td><td> 112 ± 10</td><td> 109 ± 1</td><td> 108 ± 2</td><td> 67 ± 2</td><td> 17 ± 0</td><td> 4 ± 0</td>

<td>phage + cholinic acid + Acm735</td><td> 96 ± 7</td><td> 106 ± 3</td><td> 89 ± 1</td><td> 29 ± 1</td><td> 5 ± 0</td><td> 1 ± 0</td><td> 0 ± 0</td>

<td>phage + dextran + Acm735</td><td> 96 ± 12</td><td> 108 ± 7</td><td> 113 ± 0</td><td> 105 ± 0</td><td> 66 ± 3</td><td> 16 ± 1</td><td> 5 ± 0</td>

<td>phage + ASB</td><td> 10 ± 1</td><td> 4 ± 1</td><td> 1 ± 1</td><td> 0 ± 0</td><td> 0 ± 0</td><td> 0 ± 0</td><td> 0 ± 0</td>

Table VIII: Binding of phage showing SEQ ID NO: 2 to mAb

<td></td><td colspan="7">Phage Concentration (ng / well)</td>

<td></td><td> 796</td><td> 122</td><td> 20</td><td> 3</td><td> 1</td><td> 0</td><td> 0</td>

<td>phage + Acm735</td><td> 108 ± 5</td><td> 109 ± 3</td><td> 112 ± 10</td><td> 89 ± 11</td><td> 27 + 4</td><td> 4 + 2</td><td> 2 + 1</td>

<td>phage + cholinic acid + Acm735</td><td> 109 ± 6</td><td> 114 ± 12</td><td> 100 ± 6</td><td> 48 + 6</td><td> 10 + 1</td><td> 1 + 0</td><td> 1 + 0</td>

<td>Dextran + Phage + Acm735</td><td> 109 ± 8</td><td> 111 ± 6</td><td> 110 ± 5</td><td> 99 + 1</td><td> 27 + 1</td><td> 5 + 1</td><td> 1 + 0</td>

<td>phage + ASB</td><td> 12 + 1</td><td> 9 + 1</td><td> 4 + 3</td><td> 2 + 1</td><td> 1 + 0</td><td> 0 + 0</td><td> 0 + 0</td>

Table IX: Binding of phage showing SEQ ID NO: 3 to mAb

<td></td><td colspan="7">Phage Concentration (ng / well)</td>

<td></td><td> 859</td><td> 132</td><td> 22</td><td> 4</td><td> 1</td><td> 0</td><td> 0</td>

<td>phage + Acm735</td><td> 111 ± 8</td><td> 109 ± 16</td><td> 114 ± 8</td><td> 99 ± 10</td><td> 50 ± 1</td><td> 13 ± 1</td><td> 6 ± 0</td>

<td>phage + cholinic acid + Acm735</td><td> 104 ± 2</td><td> 107 ± 2</td><td> 85 ± 7</td><td> 27 ± 3</td><td> 4 ± 1</td><td> 1 ± 1</td><td> 0 ± 0</td>

<td>dextran phage + Acm735</td><td> 109 ± 2</td><td> 108 ± 0</td><td> 108 ± 3</td><td> 107 ± 8</td><td> 57 ± 3</td><td> 13 ± 1</td><td> 6 ± 0</td>

<td>phage + ASB</td><td> 10 ± 0</td><td> 5 ± 1</td><td> 2 ± 1</td><td> 1 ± 0</td><td> 0 ± 0</td><td> 0 ± 0</td><td> 0 ± 0</td>

phage displaying sequence SEQ ID NO: 1 (DSPLVPFIDFHP) showed the best binding to mAb 735 compared to the other phages. This binding competed with cholinic acid, while dextran showed no competitive effect. The phage displaying the sequence SEQ ID NO: 4 showed similar values. Due to the low occurrence of the sequence SEQ ID NO: 4, it was decided to synthesize the sequence SEQ ID NO: 1 (peptide p21) and a random variant of the sequence SEQ ID NO: 1 (peptide p22: PDHIFVFSPDLP, SEQ ID NO: 28) as a control.

Phages displaying cyclic peptides corresponding to the sequence CSSVTAWTTGCG (SEQ ID NO: 18) and CSKIASMETGCG (SEQ ID NO: 22), respectively, where the two cysteine ​​residues are linked via a disulfide bridge, showed the best binding. mAb 30H12 compared to the other phages. Thus, it was decided to synthesize the corresponding cyclic peptides (p65 and p66).

EXAMPLE 3: ANALYSIS OF p21, p65, and p66 SPECIFICITY BY COMPARATIVE ELISA, ELISA, AND COMPETITIVE LINKING WITH APS-MACN EXPRESSING CELLS

1) Preparation of Biotin-ASB Peptide Conjugates

1.1) Materials m-maleimidobenzoyl-N-hydroxysuccimide ester (MBS, SIGMA M2786, Pierce 22311) biotinamidocaproate-N-hydroxysuccimide ester (NHSbiotin, SIGMA 02643, Pierce 20217) dimethylformamide (DMF, SIGMA)

ASB (Calbiochem 122605) synthetic cysteine-containing peptides from p21 (DSPLVPFIDFHPC, SEQ ID NO: 29), p65 and p66 and corresponding to p22-derived cysteine ​​control peptides (PDHIFVFSPDLPC, SEQ ID NO: 30), p65 inverse peptides and p66, respectively. PD-10 size exclusion column (AMERSHAMPHARMACIA 17-0851-01)

Ultrafree-4 Centrifuge Filters and Biomax 50K NMWL Tube Membrane, 4ml Volume (MILLIPORE UFV4BQK25)

L-cysteine ​​(SIGMA)

1.2) Tampons

- MBS concentrated solution: 13 mg / ml in DMF

- concentrated NHS-biotin solution: 2.5 mg / ml in DMF (these concentrations carry highly activated ASB molecules with about 5 biotin molecules / ASB molecule)

- conjugation buffer: 0.083 M NaLQPO? 0.9 M NaCl at pH

7,2.

SBF at pH 7.4

- cysteine ​​solution: 100 mg / ml L-cysteine ​​in conjugation buffer

1.3) Process

10 mg of ASB was dissolved in 2 ml of conjugation buffer and 140 μΐ of concentrated MBS / NHS-biotin solution was added. The solution was incubated for 1h at room temperature with gentle shaking. Column PD-10 was equilibrated with 50 ml conjugation buffer. After addition of 2.14 ml of the solution to the column, the activated ASB was eluted with 0.5 ml aliquots of conjugation buffer. Protein elution was monitored at 280 nm. An appropriate amount of the cysteine-containing peptide was dissolved in 1 ml of conjugation buffer. For one of 5 isomers (PM 1500, 1.14 mg of 10 mg ASB peptide) was added. This peptide solution was added to the set of activated maleimide / biotinylated ASB containing fractions. After 2 h incubation at room temperature, 100 ml of a cysteine ​​solution was added to block unreacted maleimide groups. After 1 h, the reaction solution was dialyzed 5 times with 1 ml of PBS in an ultrafiltration unit. The biotinylated conjugate was dissolved in PBS and aliquots were stored at -20 ° C.

2) Comparative ELISA Assay for Biotinylated Peptide - ASB Conjugates in Anti-APS mAb

2.1) Materials, buffers and process

2.1.1) Materials

Acm 735 and 30H12

Maxisorp ™ plates (NUNC) for antibody coating: ELISA plates microtiter plates (NUNC) for peptide dilution: dilution plates

Biotinylated Peptide - ASB Conjugates Prepared As Previously

Avidin Extravidin Alkaline Phosphatase Conjugate (SIGMA E2636) p-nitrophenyl alkaline phosphatase substrate (SIGMA 104-105)

2.1.2) Tampons

SBF at pH 7.4

Blocking solution: ASB in 0.5% SBF

TBS

TBST: TBS containing 0.05% Tween 20

Extravidin-alkaline phosphatase conjugate solution - avidin: 1/5000 in TBST

Alkaline phosphatase substrate solution; 1 tablet in 5 ml 50 mM NaHCCt, MgCl solution₂ 1 mM at pH 9.6.

Mixed comparison solution: 1 mM cholinic acid with an ASB-peptide conjugate gradient in TBST

2.1.3) Process

Wells of the Maxisorp ™ plate were coated with a 100 μΐ mAb (25 pg / ml) solution for 2 h at room temperature. Control wells of ELISA plates were coated with blocking solution only. In parallel, peptide - ASB conjugate dilution plates were blocked with 200 µl blocking solution for 2 h. Antibody coated wells and control wells were blocked with 200 μΐ blocking solution for 1 h. In parallel, peptide-ASB conjugate dilution plates were washed 6 times with TBST and 120 μΐ TBST was added to the wells. An appropriate volume of ASB peptide conjugate solution was added to the first well and the volume adjusted to 140 μ with TBST. The ASB - peptide conjugate was diluted in the wells by 1/7, removing 20 μΐ from the first well and transferring to the second well to reach the total volume of 140 μΐ. This procedure was repeated for the remaining wells. Dilutions of ASB-peptide conjugate to control wells were made in the same manner. Blocked ELISA plates were washed 6 times with TBST and dilutions of the ASB-peptide conjugate or 100 μΐ of the comparative mixed solution were added to the wells. After incubation for 1 h, the wells were washed 10 times with TBST and 100 μΐ of the alkaline phosphatase substrate solution was added to the wells. The plates were read at 405 nm after 10 to 60 minutes using a microplate reader.

2.2) Results

The specificity of p21 peptide (SEQ ID NO: 1) was investigated in a comparative ELISA assay using biotinylated p21 conjugate - ASB and a randomized variant of p21 conjugate (p22) as a control. Results, shown in tables X and XI, are expressed as percent binding compared to the highest concentration p21 peptide (9.45 1CT⁵ M), which corresponds to 100% binding.

Table X: Binding of p21 to Acm 735 in a comparative assay by

<td colspan="10">Peptide concentration (M)</td>

<td></td><td> 9,45</td><td> 1, 67</td><td> 2, 94</td><td> 5,20</td><td> 9,27</td><td> 1, 62</td><td> 2,86</td><td> 5,04</td><td> 8,89</td>

<td></td><td> 10“³</td><td> 10“³</td><td> 10“</td><td> 10'</td><td>yo-</td><td>io-</td><td>yo-</td><td>ion ^</td><td> 10 ¹¹</td>

<td>Peptide + Acm 735</td><td> 100+11</td><td> 93 + 10</td><td> 95 + 1</td><td> 91 ± 2</td><td> 93 ± 9</td><td> 56 ± 4</td><td> 13 ± 1</td><td> 2 ± 0</td><td> 0 ± 0</td>

<td>Peptide + Cholinic Acid + Acm 735</td><td> 43 ± 3</td><td> 34 ± 3</td><td> 23 ± 1</td><td> 15 ± 3</td><td> 5 ± 2</td><td> 0 ± 1</td><td> 0 ± 0</td><td> 0 ± 0</td><td> 0 ± 0</td>

<td>Peptide + Chondroitin Sulphate + Acm 735</td><td> 96 + 8</td><td> 99 ± 4</td><td> 99 ± 8</td><td> 94 ± 7</td><td> 92 ± 2</td><td> 50 ± 1</td><td> 11 ± 2</td><td> 3 ± 0</td><td> 2 ± 0</td>

<td>Peptide + ASB</td><td> 3 + 1</td><td> 1 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td>

Table XI: Binding of p22 to mAb 735 in a comparative assay by

<td></td><td colspan="9">Peptide concentration (M)</td>

<td></td><td> 9, 45</td><td> 1, 67</td><td> 2, 94</td><td> 5,20</td><td> 9, 27</td><td> 1, 62</td><td> 2,86</td><td> 5, 04</td><td> 8, 89</td>

<td></td><td> 10⁵</td><td> 10⁵</td><td>Itr⁵</td><td>IO⁷</td><td> 10“</td><td> 10^y</td><td> 10^y</td><td> 10^_1“</td><td> 10^-11</td>

<td>Peptide + Acm 735</td><td> 32 + 1</td><td> 9 + 0</td><td> 1 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td>

<td>Peptide + Cholinic Acid + Acm 735</td><td> 27 + 0</td><td> 5 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td>

<td>Peptide + Chondroitin Sulphate + Acm 735</td><td> 29 + 0</td><td> 8 + 1</td><td> 3 + 1</td><td> 2 + 0</td><td> 2 + 1</td><td> 2 + 0</td><td> 2 + 0</td><td> 2 + 0</td><td> 2 + 0</td>

<td>Peptide + ASB</td><td> 3 ± 1</td><td> 1 ± 1</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 + 0</td><td> 0 ± 0</td>

Table X shows a clear inhibition of p21 binding in the presence of cholinic acid. By comparison, chondroitin sulfate C had no influence on the binding of peptide B. Table XI shows no binding with mAb 735 for the p21 conjugate random variant (p22); no differences were observed when cholinic acid or chondroitic sulfate C were present. These results lead to the conclusion that the sequence SEQ ID NO: 1 (peptide p21) specifically binds to mAb 735 in a concentration dependent manner.

3) Peptide Conjugate ELISA Assay - ASB

The specificity of the cyclic peptides was investigated in an ELISA assay using 30H12 antibody coated plates following the protocol described above for the comparative ELISA, with the exception that the competitor was omitted. The results, shown in Figure 12a, demonstrated that cyclic peptides bind to the antibody in an antigen specific manner. The p65 and p66 peptides that exhibited the highest binding were chosen for further study.

4) Comparative binding with cells expressing APS-MACN

The specificity of p65 and p66 peptides was analyzed in a comparative assay using anti-APS and APS-MACN antibodies. Preincubation of the 30H12 monoclonal antibody with 0.1 mM of each p65 and p66 peptide prevented its binding to APS-MACN expressing cells (Figure 12D versus 12B). Binding specificity was examined in more detail by peptide recognition assays using another anti-APS mononclonal antibody (MenB). The results, shown in Figure 12C, demonstrated that p65 and p66 peptides only bind to 30H12; Pre-incubation of the peptides with MenB did not prevent APS-MACN recognition (Figure 12 C). Thus, mimotopes (molecules that simulate the structure of epitopes) of p65 and p66 appear to be specific for a single (idiotypic) determinant.

EXAMPLE 8: BIOACTIVITY ANALYSIS OF MIMETIC PEPTITES

From APS

1) Materials and processes

1.1) Animals

GFP transgenic mice have been previously described in Hadjantonakis et al. (Biotechnol., 2002, 2, 11-) and all analyzes were performed on mice with a Swiss background. Knockout mouse MACN (MACN - / -) have been previously described in Cremer et al, cited above.

1.3) Culture of dorsal root ganglion (GRD) transplants

E13.5 GRDs from mouse embryos were dissected in HBSS medium and seeded on polylysine coated or ASB-linked peptide glass slides. Transplants were cultured in the presence or absence of soluble peptides (40 μΜ) in two ml neurobasal medium (Ham DMEM / F12 3: 1 (V / V), GIBCO, buffered with 20mM Hepes), completed as described in FaivreSarrailh et al. J. Cell. Sci., 1999, 18, 3015-3027 and Chazal et al. J. Neurosci. 2000, 20, 1446-1457.

1.4) Subventricular zone transplantation culture (ZSV).

ZSV transplant cultures were performed as described in Chazal et al., Cited above. Briefly, 1 day old mice were killed by rapid decapitation. The brains were dissected and sectioned with a

Vibratome (Leica). ZSV of the lateral wall of the anterior horn of the lateral ventricle was dissected in HBSS medium (LIFE TECHNOLOGIES) and cut into explants with a diameter of 200-300 pm. The transplants were mixed with Matrigel (BECKTON DICKINSON) and cultured in four well plates. After polymerization, the gel was covered with 400 μΐ serum-free medium containing supplement B-27 (LIFE TECHNOLOGIES) in the presence or absence of 40 pm peptides (p65, p66, inverse p65, p21 or p22) and 70 U from Endo N per milliliter.

1.5) Immunohistochemistry)

GRD (dorsal root ganglion) transplants were fixed and sections were incubated at 4 ° C, 2 h, respectively, with an anti-neurofilament antibody (SMI31, 1: 800 dilution, STERNBERGER MONOCLONALS) and during overnight with an anti-APS antibody (1: 200 dilution, Rougon et al, J. Cell. Biol., 1986, 103, 2429-2437). Developing was by one hour incubation with the corresponding fluorescently labeled secondary antibody (Texas red conjugated goat anti-mouse IgM, IMMUNOTECH)

1.6) Migration distance of cells (transplants of the

ZSV) and neuronal growth (GRD transplants).

After 48 h of culture, transplants were examined either directly (ZSV) or after overnight fixation with a 4% paraformaldehyde solution in FBS and immunostaining (GRDs). Observation was made using 2.5X, 5X, 10X and 32X lenses (Axiovert 35M, ZEISS).

Images were taken with a camcorder (Cool View, PHOTONIC SCIENCE) and analyzed using the image processing software (Visiolab 1000, BIOCOM). The average migration distance (calculated in five different experiments, including at least five transplants per condition) or the longest average neuritis length (calculated in two different experiments, including at least eight transplants per condition) was the distance, in micrometers, between the transplant edge and the front edge of the cell migration. Four measurements were taken for each transplant. The significance of the differences between the control and the different experimental conditions was calculated by Student's t-test.

1.7) Transplant

ZSV 100 µm diameter transplants of 1 day old GFP mice were incubated for fifteen minutes in DMEM supplemented with 10% fetal bovine serum in the presence of 0.065 M p65 or inverse peptide and stereotaxically grafted (0.5 μΐ) into ZSV of six week old mice as described in Lois and Alvarez-Buylla (Science, 1994, 264, 1145-1148). Three or four days after the graft, the animals were perfused intracardially with a 4% paraformaldehyde solution in FBS. The brains were dissected, fixed, cryoprotected and frozen in isopentane. Serial sagittal sections (12 pm) were cut with a Leica microtome and stained as described above. GFP cells that reached the olfactory bulb after three days were observed using a 40X objective UV fluorescence (Axioscope, ZEISS) and counted in two different experiments (four animals per condition). The significance of the difference between the two conditions was calculated by Student's t-test.

1.8) Intravitreal injections and overall retinal support.

Injections were given as described in Monnier et al. (Developmental Biology, 2001, 229, 1-14). Briefly, a 2 x 2 cm window was cut, cut outside on the chicken E3 embryos. A μΐ of green Fast with 10 mM p65 or inverse peptide was injected into the vitreous body of the right eye using a capillary. After a five day incubation at 37 ° C, the retinas (E8) were dissected, distributed through nitrocellulose filters (MILLIPORE) and fixed with a 4% paraformaldehyde solution in SBF. Two small crystals of Dil (1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine perchlorate) were applied dorsally to the optical fissure. The retinas were stored in the dark at 37 ° C for 10 days until the dye reached the axonal growth cones in the optical fissure mounted on glycerol: SBF (9: 1, v: v) and analyzed by confocal microscopy.

2) Results

2.1) Effect of APS mimetic peptides on axonal growth and in vitro fasciculation (Figure 2)

Mouse dorsal root ganglion (E13.5) transplants were cultured in the presence of the cyclic mimetic peptides p65 and p66 both in soluble form and coated on the microplates as conjugated with ASB. Culture cells, in the absence of peptides or in the presence of inverse peptides (coated or in soluble form), were used as controls.

The effect of peptides on neurite growth and fasciculation was analyzed qualitatively and quantitatively (Figure 2A to H).

p65 and p66, in a soluble form, induced a clear axon beam defasciculation (C versus A and F versus D) and a significant increase in axonal growth (C versus A) compared to controls.

Interestingly, p65 and p66, in a coated form, induced an opposite effect on fasciculation (B versus A and E versus D) and no effect on neurite development.

These results were confirmed by quantitative analysis showing that p65 and p66, in soluble form, increased neurite development by 34% and 21% respectively, compared to controls, whereas the same peptides in coated form did not. induced any significant increase in the development of neuritis (Figures 2G and 2H).

These results demonstrate that APS mimetic peptides are able to regulate axonal growth and fasciculation in vitro.

2.2) Effect of APS mimetic peptides on in vivo fasciculation and axonal orientation (Figure 3)

P65 and p66 were injected into chicken embryo (E3) eyes and the retina was observed at E9. The results, shown in Figure 3, show that the presence of APS mimetic peptides during retinal development induces axon orientation and fasciculation defects; the axons leave their fascicles and run perpendicular to it (arrows on D, G, E and H). In comparison, no defect in axon orientation was observed and fasciculation was observed after injection of control peptides.

These results demonstrate that APS mimetic peptides are able to regulate axonal growth and orientation in vivo.

2.3) Effect of APS mimetic peptides on in vitro cell migration (Figures 4 to 7) The effect of APS mimetic peptides on in vitro cell migration was analyzed from subventricular transplants of normal 1-day-old rats (MACN + / + ), heterozygous rats (MACN + / -) or knockout mice (MACN - / -) grown in Matrigel, in the presence of mimetic peptides (p65, p21, p66), different from p65 peptide (cyclic, linear, linear and acetylated) ), control peptides (reverse p65 and reverse p66 and p22) or N endosialidase. Results are shown in figures 4 to 7.

Figure 4 (A to I) shows that the addition of p65 and p66 peptides to the culture increases the migration rate of neuronal precursors and a modification of their arrangement as similar to chains (C and F). These effects were not

D) were found with inverse peptides (APS-dependent, since they were abolished by treatment with endo N (B and E).

These results were confirmed quantitatively (G and H), demonstrating that p65 and p66 analysis induce a significant increase in the migration rate of neuronal precursors (+ 40% and + 26%, respectively, at 0.4 mM, compared to control without the peptide), while endo N will decrease it (-21% compared to control without the peptide).

The dose response curve of p65 (I) shows that the optimal effect on cell migration is observed from the previous 0.4 pm peptide.

Figure 5 shows that p65 cyclization is a prerequisite for p65 to promote the effect as long as the corresponding amino acid sequences, in linear form, N-acetylated or unable to stimulate cell migration.

Figure 6 shows that the effect of p65 is dependent on APS expression as a significant reduction in migration precursors in mouse MACN- compared to mouse MACN +/- was observed in the presence of invert peptide or peptide. p65; the effect was comparable in endo N treated mouse MACN +/- and mouse MACN- and p65 was not able to reverse impaired migration in mouse MACN - / -.

Figure 7 shows that p65 induces a significant increase in the migration rate of neuronal precursors compared to the corresponding control peptide (inverse p65).

In contrast, p21 induces a significant reduction in the migration rate of neuronal precursors compared to the corresponding control peptide (p22 peptide); the reduction is comparable to that observed in endo N. treated cells.

These results demonstrate that mimetic peptides are capable of stimulating (p65) or inhibiting (p21) cell migration in an APS-dependent manner.

2.4) Effect of APS mimetic peptides on in vivo cell migration (Figure 8) The effect of APS mimetic peptides on in vivo cell migration was analyzed by tissue grafting and evaluation of ZSV cell migration. Small pieces of ZSV tissue (100 µm in diameter ) from 1 day old GFP mice were grafted into the ZSV area of ​​adult mice in the presence of p65, inverse p65 peptides or in the absence of peptide. The results are illustrated in figure 8.

Figure 8 shows that the presence of p65 peptide significantly increases the number of GFP-positive cells that migrate to the olfactory bulb (via the thalamic migration stream or CMT) compared to the control. This effect was observed early in the first 3 days after grafting (Figure 8E). These results were confirmed by quantitative analysis showing that the number of GFP-positive cells present in the olfactory bulb 3 days after graft is 17 times higher in the presence of p65 compared to the control (Figure 8G).

These effects were dependent on APS as they were abolished in MACN knockout mice.

These results demonstrate that mimetic peptides are capable of enhancing migration of APS positive cells.

Example 9: Analysis of Functional Recovery of Spinal Cord Injury After Injection of APS Mimetic Peptides

1) Material and processes

1.1) Spinal cord surgery and peptide administration

Male Swiss-CD1 mice (8-10 weeks old) were anesthetized with a mixture of ketamine and xylazine. The spinal cord was exposed through a midline skin incision and retraction of the paravertebral musculature. T7-T8 laminectomy was performed and the spinal cord was exposed. With iridectomy scissors, a bilateral dorsal semi-section was prepared, transecting the right and left dorsal funicular, the dorsal horns, but sparing most of the ventral funicular, resulting in a complete transection of the dorsal cortico-medullary tract (TCM). medium. For the series of mice receiving the peptides, a 10 μΐ saturated Surgicoll dressing of either p65 or inverse p65 peptide (Μμpt) was applied over the transection site and covered with vaseline to prevent diffusion. The entire inner muscle layer was sutured with thin thread. The skin was sutured with surgical staples. After surgery, 1 ml of saline was administered subcutaneously to prevent dehydration and the rats were placed under a heat lamp until fully recovered from narcosis. The mice then returned to their cages and received a daily subcutaneous injection of Baitryl ™ antibiotic to prevent infections. Manual bladder emptying was performed until complete autonomous bladder function recovered.

1.2) Functional test (Figure 9)

Functional evaluation of the animals was performed on (D) D1, D4, D7, D14, D21, D28, D35, during the first week after spinal cord injury and then on a weekly basis until D35, by two observers who were unaware of the group's identity. Locomotor recovery was assessed using the BBB test (Basso, Beattie and Bresnahan test; Basso et al. Restor. Neurol. Neurosci, 2002, 5, 189-218). The scale ranges from 0 (no hind limb movement observed) to 21 (normal gait) and can be subdivided into three intervals. Scores from 0 to 7 correspond to poor recovery (joint movements, no weight support, no paw placement). Scores 8 through 13 may be related to an intermediate recovery (paw placement, coordination of the forearm hind limbs). Scores 14 to 21 may be related to a very good recovery. Finally, on the last test day (D35), the rats were subjected to the rota-rod test to assess fine motor coordination.

1.2) Histology (figure 9)

At D5, injured animals receiving either p65 (n = 3) or inverted p65 (n = 3) were perfused transcardiacally. Spinal cord were sectioned in the sagittal plane at 20 pm intervals in 10 mm length blocks at the site of injury. To examine the extent of the lesion, Nissl staining was performed in sets of 1 out of 6 sections in all animals. Series of 1 in 3 sections were stained with MenB anti-APS antibody (mouse IgM) and / or anti-GFAP antibodies (mouse IgG). Bound antibodies were revealed by appropriate fluorescently labeled secondary antibodies.

2) Results

After several injuries, the axons of the brain and spinal cord do not advance through the adult CNS. Instead, these fibers become trapped at the site of injury and remain detached from synaptic targets, leading to deep and persistent deficits in many clinical cases. Spinal cord injury (SCI) is the clearest example of a clinical condition in which axonal disconnection leads to significant disability despite minimal neuronal death.

Thus, the effect of APS mimetic peptides, p65 (and its inverse counterpart, taken as control) on functional recovery from spinal cord injury was analyzed in mice.

2.1) Improvement of locomotor recovery after spinal cord injury in rats receiving p65 peptide at injury site

The results shown in Figure 10 (A and B) demonstrate that treatment with APS mimetic peptide correlates with functional recovery following injury of the mid-thorax dorsal half-section. More precisely, recovery was assessed by a standardized open-field measure of locomotor function after spinal cord injury, the BBB score. On this scale, 21 is normal function and 0 is total bilateral hind limb paralysis. All mice scored 0 on Dl after injury. The p65-treated mice gradually recovered partial function over a 45-day observation period (Figure 10A). The p65 treated rats' scores were significantly higher than those of the reverse p65 controls since D14 after injury and throughout the following calendar. It is considered that the time period (14 days) in which this improvement was observed is compatible with some long-distance growth of the TCM fibers extending from the injury site to the lumbar motor assembly. Local development in the lumbar spine as well as rearrangements of other descending tracts such as the redspinal system or intrinsic distal spinal cord circuits may also contribute. Regardless of mechanism, locomotor recovery in p65-treated rats was significantly higher than in control animals. The beneficial effect of p65 on recovery was further evaluated by a spin-rod test performed on D35 (Figure 10B).

2.2) Decreased D5 reactive gliosis

To assess the effect of the peptide, expression of glial acid fibrillary protein (PFGA) expression was quantified as a reactive gliosis index in a subset of animals at D5 after surgery. These animals were blindly selected for quantitation. Three animals were analyzed for p65 (10 slides per animal) and 3 for inverse p65 (10 slides per animal). Double labeling was performed with MenB anti-APS antibody. The quantification was also performed blindly.

A significant difference was observed between p65-treated and reverse p65-treated animals (Figure 11 A and B), demonstrating that p65 treatment reduced reactive gliosis by 40% compared with reverse p65 treatment, possibly avoiding migration within the scar or inhibiting other processes involved, such as the action of inflammatory cytokines. In any case, these results support the fact that functional recovery is better in p65 treated rats.

LIST OF SEQUENCES &lt;110&gt; NATIONAL CENTER OF LA RECHERCHE SCIENTIFIQUE

UNIVERSITE DE LA MEDITERRANEE AIX MARSEILLE II

SCHAFER-N

UNIVERSITAET HAMBURG

ROUGON Geneviève

TORREGROSSA Pascal

SCHACHNER Melitta

SCHAFER NIELSEN Claus &lt;120&gt; Use of poly-alpha-2,8 sialic acid mimetic peptides to regulate MACN functions &lt;130&gt; F644PCT83 &lt;160&gt; 32 &lt;170&gt; Patentln version 3.1 &lt;210&gt; 1 &lt;211&gt; 12 &lt; 212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 1

Asp Ser Pro Leu Goes To Phe Ile Asp Phe His Pro 1 5 10

<td> &lt;210&gt;</td><td> 2</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 2 your Trp Gin Pro Pro Read Ile Pro Gly Ile Asp Phe 15 10

<td> &lt;210&gt;</td><td> 3</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 3

Gin lie Glu Pro Trp Phe Thr Pro Glu Asp Phe Pro 1 5 10

<td> &lt;210&gt;</td><td> 4</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td>

&lt;400&gt; 4

Thr Arg Leo Pro Wing Read Go Phe Pro Read Asp Tyr 1 5 3.0

<td> &lt;210&gt;</td><td> 5</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 5

Ser Trp Read Gin Met Pro Trp Your Wing Go Arg Thr 1 5 10

<td> &lt;210&gt;</td><td> 6</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 6

Glu Ile His Arg Met Ile Lys Gin Ile Thr Ile

<td> 1</td><td> 5 10</td>

<td> &lt;210&gt;</td><td> 7</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

&lt;220&gt;

&lt;223&gt; synthetic peptide

<td> &lt;400&gt;</td><td> 7</td><td></td>

<td colspan="3">Trp His Leu Glu Tyr Met Trp Arg Trp Pro Arg Leu 1 5 10</td>

<td> &lt;210&gt;</td><td> 8</td><td></td>

<td> &lt;211&gt;</td><td> 12</td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td>

<td> &lt;213&gt;</td><td>artificial sequence</td><td></td>

<td> &lt;220&gt;</td><td></td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td><td></td>

<td> &lt;400&gt;</td><td> 8</td><td></td>

<td>Read Ile 1</td><td>Glu Gin Arg Leu Pro Lys His 5th</td><td>Ile Leu Thr 10</td>

<td> &lt;210&gt;</td><td> 9</td><td></td>

<td> &lt;211&gt;</td><td> 12</td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td>

<td> &lt;213&gt;</td><td>artificial sequence</td><td></td>

<td> &lt;220&gt;</td><td></td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td><td></td>

<td> &lt;400&gt;</td><td> 9</td><td></td>

<td>Tyr Glu 1</td><td colspan="2">Thr Ser Ser Ser Arg Arg Leu Wing Tyr Wing 5 10</td>

<td> &lt;210&gt;</td><td> 10</td><td></td>

<td> &lt;211&gt;</td><td> 12</td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td>

&lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 10

Thr Leu Wing Be Gin Leu Be ftsn Thr Be Wing Tyr

<td> 1</td><td> 5 10</td>

<td> &lt;210&gt;</td><td> 11</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 11

Be Asp Gin Gly Go Asn Gly Be Trp Be Asn Pro 15 10

<td> &lt;210&gt;</td><td> 12</td>

<td> &lt;211&gt;</td><td> 13</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td>

<td> &lt;400&gt;</td><td> 12</td>

<td>Trp!</td><td>Aen Trp Asn Read Trp Wing Pro Wing Be Pro Thr</td>

5 3 0 &lt;210&gt; 13 &lt;211&gt; 12

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 13

Trp His Trp Gin Trp Thr Pro Trp Ser lie Gin Pro

<td> 1</td><td> 5 10</td>

<td> &lt;210&gt;</td><td> 14</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td>

<td> &lt;400&gt;</td><td> 14</td>

lie Lvs Be Pro your Thr Trp Leu Go Pro Pro Asp 1 5 10

<td> &lt;210&gt;</td><td> 15</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td>

&lt;400&gt; 15

Be His Leu Asp Leu Be Thr Gly His Arg Thr

<td> 1</td><td> 5 10</td>

<td> &lt;210&gt;</td><td> 16</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 16

Cys Tyr Pro Read Asn Pro Glu Val Tyr His Cys Gly 1 5 10

<td> &lt;210&gt;</td><td> 17</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td>

&lt;400&gt; 17

Cys Trp Pro Reads His Be Val Val Val Cys Gly 15 10 &lt;210&gt; 18 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 18

Cys 3e&gt; r Will Go Thr Thr Trp Thr Thr Gly Cys Gly 1 5 10 &lt;210&gt; 19 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 19

Cys Tyr Met Wing Be Gly Will Phe Cys Gly 1 5 10 &lt;210&gt; 20

211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 20

Cys Trp Pro Read Gly Pro Be Thr Tyr Ile Cys Gly 1 5 10 &lt;210&gt; 21 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 21

Cys Set 'Leu Ile Wing Be Met Glu Thr Gly Cys Gly

<td> 1</td><td> 5 10</td>

<td> &lt;210&gt;</td><td> 22</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td>

<td> &lt;400&gt;</td><td> 22</td>

Cys Be Lys Ile Wing Be Met Glu Thr Gly

<td> 1</td><td> 5 10</td>

<td> &lt;210&gt;</td><td> 23</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

&lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 23

Cys Tyr lie Gly Asp Pro Pro Phe Asn Pro Cys Gly 1 5 10 &lt;210&gt; 24 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 24

Cys Trp Pro Read Gly Asp Ser Thr Val Ile Cys Gly 15 10 &lt;210&gt; 25 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 25

Cys Pro Read Arg Read Le Phe Wing Phe Thr Gly Cys Gly 15 10 &lt;210&gt; 26 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 26

Cys Thr Arg ffefc Being His Gly Tyr Trp Ile Cys Gly

10

<td> &lt;210&gt;</td><td> 27</td>

<td> &lt;211&gt;</td><td> 20</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>initiator</td>

<td> &lt;400&gt;</td><td> 27</td>

tagcgtaacg ccctcatagt 20

<td> &lt;210&gt;</td><td> 28</td>

<td> &lt;211&gt;</td><td> 12</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>synthetic peptide</td>

<td> &lt;400&gt;</td><td> 28</td>

Pro Asp His Ile Phe Val Pro Phe Ser Asp Leu Pro 15 10 &lt;210&gt; 29 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; synthetic peptide &lt;400&gt; 29

Asp Ser Pro Read Val Pro Phe Ile Asp Phe Hxs Pro Cys 1 5 10

<td> &lt;210&gt;</td><td> 30</td>

<td> &lt;211&gt;</td><td> 13</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

&lt;223&gt; synthetic peptide &lt;400&gt; 30

Pro Asp Sis Ile Phe Val Phe Ser Pro Asp Leu Pro C; 1 5 10

<td> &lt;210&gt;</td><td> 31</td>

<td> &lt;211&gt;</td><td> 7</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Artificial Sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>Description of Artificial Sequence: Peptide</td>

APS mimetic &lt;400&gt; 31

Asp Hxs Gin Arg Phe Phe Val

<td> 1</td><td> 5</td>

<td> &lt;210&gt;</td><td> 32</td>

<td> &lt;211&gt;</td><td> 7</td>

&lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Description of artificial sequence: APS mimetic &lt;400&gt; 32

Ala His Gin Ala Ser Phe Vai 1 5 peptide