WO2016149116A1 - Anticorps ciblant la protéine s100b et procédés d'utilisation - Google Patents

Anticorps ciblant la protéine s100b et procédés d'utilisation Download PDF

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WO2016149116A1
WO2016149116A1 PCT/US2016/022104 US2016022104W WO2016149116A1 WO 2016149116 A1 WO2016149116 A1 WO 2016149116A1 US 2016022104 W US2016022104 W US 2016022104W WO 2016149116 A1 WO2016149116 A1 WO 2016149116A1
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antibody
seq
sloob
amino acid
acid sequence
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PCT/US2016/022104
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Danna ZIMMER
Eric W. Mcintush
Henry F. Carwile
John M. CARWILE
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University Of Maryland, Baltimore
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4727—Calcium binding proteins, e.g. calmodulin

Definitions

  • ASCII text file A sequence listing in electronic (ASCII text file) format is filed with this application and incorporated herein by reference.
  • the name of the ASCII text file is
  • Ca 2+ ions are important second messengers in all living cells [1].
  • Ca 2+ -binding proteins including members of the calmodulin/troponin/SlOO superfamily, maintain the integrity of the Ca 2+ signal and transmit it in a temporally and spatially coordinated manner [2].
  • SI 00s were discovered in 1965 [3], and as with other EF-hand containing proteins, SlOOs also transduce changes in [Ca 2+ ]intracellular levels (i.e., [Ca 2+ ]i) into cellular responses by binding Ca 2+ , changing conformation, and then interacting with and modulating the activity of other proteins (target proteins).
  • SI 00 proteins are distinguished from other members of the calmodulin/troponin/SlOO superfamily of EF-hand Ca 2+ sensors by their 3D structure and highly conserved 14 amino acid Ca 2+ binding loop [24]. Upon binding Ca 2+ , SI 00 proteins undergo a conformational change which exposes a hydrophobic patch necessary for interacting with numerous intra- and extracellular protein targets and subsequent exertion of their biological effects [23]. S100 proteins regulate a large number of diverse cellular processes that include energy metabolism, cell proliferation, cytoskeletal organization, Ca 2+ homeostasis, and signal transduction pathways.
  • the amino acid homology between the current family members ranges from approximately 20% to 55% [4]. Because of the extensive amino acid homology between S100B and S100A1, the first two family members identified, early models predicted that individual members were functionally redundant and essentially interchangeable. As the number of family members discovered has increased and differences in their cellular/subcellular localization, physical properties, and target proteins have expanded, additional models have arisen with specific S100 family members having unique biological functions [5-12, 20]. This multigenic family now contains at least twenty-one members (human) whose phylogenetic distribution is restricted to higher chordates. Oligomerization properties, affinities for divalent metal ions (Ca 2+ , Zn , Cu ), and posttranslational modifications also contribute to diversity among SI 00 family members [7, 12].
  • S100B is of particular interest. In addition to intracellular signaling, S100B is released into the extracellular space where it participates in local intercellular communication (autocrine and paracrine). It also enters the systemic circulation where it can coordinate biological events over long distances.
  • the biological effects of extracellular S100B are concentration-dependent; nanomolar S100B levels beneficially promote neuronal survival while micromolar levels detrimentally promote apoptosis [25].
  • S100B lacks a signal peptide for secretion via the conventional Golgi -mediated pathway, and there is debate as to whether it is actively secreted from live cells or passively released.
  • the short half-life (30 minutes) and renal clearance (2 hours) for S100B indicate that persistent elevations reflect active secretion or passive release [26].
  • S100B release/secretion is gender- and age-specific, and can be augmented by forskolin, lysophosphatidic acid, serotonin, glutamate, IL- ⁇ , metabolites, and the neurotoxic ⁇ peptide [27-32].
  • Biochemical and cellular studies have identified a number of receptors for extracellular S100B including the advanced glycation end-products receptor (AGER or RAGE), toll-like receptor-4 (TLR4), FGF-receptor 1 (FGFR1), activated leukocyte adhesion molecule (ALCAM), and formyl peptide receptor like 1 (FPRL1) [33-38].
  • AGER advanced glycation end-products receptor
  • TLR4 toll-like receptor-4
  • FGFR1 FGF-receptor 1
  • ACAM activated leukocyte adhesion molecule
  • FPRL1 formyl peptide receptor like 1
  • novel antibodies that exhibit binding specificity for S100B as well as variants and fragments thereof that retain the binding specificity of the antibodies from which they are derived, collectively termed S100B binding agents. Methods of using these binding agents in the treatment and prevention of SlOOB-associated diseases are also provided. Methods of using these binding agents in the screening and diagnosis of subjects for SlOOB- associated diseases are further provided.
  • the present invention is directed to agents that exhibit binding specificity for the S100B polypeptide.
  • S100B binding agents include antibodies and variants thereof that exhibit binding specificity for S100B, as well as fragments thereof that exhibit binding specificity for S100B.
  • the invention is directed to antibodies having binding specificity for an epitope of S100B, wherein the epitope comprises the amino acid sequence set forth in SEQ ID NO:5.
  • the invention is directed to antibodies having binding specificity for S100B, wherein the antibody was raised against an epitope of S100B comprising the amino acid sequence set forth in SEQ ID NO:5.
  • Antibodies having binding specificity for S100B and S100B epitopes can be defined, in non-limiting aspects of the invention, based on their variable regions, i.e., as antibodies comprising one or more of (a) a V L region comprising the amino acid sequence of SEQ ID NO: 11, (b) a V H region comprising the amino acid sequence of SEQ ID NO:9, (c) a V L region comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO: 11, and (d) a V H region comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:9.
  • the invention is directed to antibodies having binding specificity for S100B comprising (a) a V L region comprising the amino acid sequence of SEQ ID NO: 11 and a V H region comprising the amino acid sequence of SEQ ID NO:9; or (b) a V L region comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO: 11 and a V H region comprising the amino acid sequence of SEQ ID NO:9; or (c) a V L region comprising the amino acid sequence of SEQ ID NO: 11 and a V H region comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO :9; or (d) a V L region comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO: 1 1 and a V H region comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:9.
  • Antibodies having binding specificity for S 100B and S 100B epitopes can also be defined, in non-limiting aspects of the invention, based on their heavy and light chains, i.e., as antibodies comprising one or more of (a) a light chain comprising the amino acid sequence of SEQ ID NO: 4, (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, (c) a light chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:4, and (d) a heavy chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:2.
  • the invention is directed to antibodies having binding specificity for S 100B comprising (a) a light chain comprising the amino acid sequence of SEQ ID NO:4 and a heavy chain comprising the amino acid sequence of SEQ ID NO:2; or (b) a light chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:4 and a heavy chain comprising the amino acid sequence of SEQ ID NO:2; or (c) a light chain comprising the amino acid sequence of SEQ ID NO:4 and a heavy chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:2; or (d) a light chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:4 and a heavy chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO :2.
  • the invention is directed to the 1 C8 antibody, a S 100B binding antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:4 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:2.
  • the antibodies may be polyclonal or monoclonal antibodies, and the antibodies may be in the form of an antiserum comprising the antibodies.
  • the antibodies may be isolated antibodies, purified antibodies, exogenous antibodies, endogenous antibodies, or a combination thereof. Further, the antibodies may be recombinant antibodies.
  • the antibodies may be raised in humans, mice, rats, goats, rabbits, pigs, birds, or other animals. The antibodies may be raised against an epitope of S 100B from human, mouse, rat, goat, rabbit, or pig S 100B, or S 100B from some other mammal.
  • the SIOOB binding agents include variants of each of the antibodies defined herein that retain the ability to bind S100B or an S100B epitope.
  • the variants include, but are not limited to, humanized antibodies, chimeric antibodies, and fully human antibodies.
  • the S100B binding agents include fragments of the antibodies and variants defined herein that retain the ability to bind S100B or an S 100B epitope.
  • the fragments include, but are not limited to, Fab fragments, F(ab') 2 fragments, single chain Fv (scFv) antibodies, and fragments produced by an Fab expression library.
  • the variation in sequence may be limited to the one or more of the framework regions (FRs) or limited to one or more of the complementarity determining regions (CDRs). The variations may also be found in one or more of the FRs and in one or more of the CDRs.
  • FRs framework regions
  • CDRs complementarity determining regions
  • the antibodies, variants, and fragments exhibit at least 75% of the binding affinity for mouse S100B that is exhibited by the 1C8 antibody described herein.
  • the invention includes polynucleotides comprising nucleotide sequences encoding each the S100B binding agents provided in the various embodiments and aspects defined herein, as well as complementary strands thereof.
  • the invention also includes cloning and expression vectors comprising the polynucleotides, and host cells comprising the polynucleotides and/or cloning and expression vectors.
  • the invention further includes methods of producing the S100B binding agents defined herein, comprising culturing the host cells under conditions promoting expression of the binding agents encoded by the expression vectors, and recovering the binding agents from the cell cultures.
  • the invention is directed to pharmaceutical formulations comprising one or more of the S100B binding agents defined herein and a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical formulations include those comprising one or more of the antibodies defined herein and a pharmaceutically acceptable carrier; one or more the humanized variants defined herein and a pharmaceutically acceptable carrier; and one or more the SlOOB-binding fragments defined herein and a pharmaceutically acceptable carrier. Kits, Cells Lines, and Agents
  • kits comprising (a) one or more of the S100B binding agents as defined herein, and (b) a labeled secondary antibody recognizing the S100B binding agent(s) of (a).
  • the S100B binding agents include one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the invention is directed to cell lines which produce one or more of the S100B binding agents defined herein.
  • the cell lines of the invention include those which produce one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the invention is directed to diagnostic reagents for Alzheimer disease comprising one or more of the S100B binding agents defined herein.
  • the diagnostic reagents of the invention include those comprising one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the invention is directed to therapeutic agents for a S100B- associated disease or condition comprising one or more of the S100B binding agents defined herein.
  • the therapeutic agents of the invention include those comprising one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the invention is directed to methods of using S100B binding agents in the treatment or prevention of S lOOB-associated diseases or conditions in a subject.
  • Such methods generally encompass administering one or more of the S100B binding agents defined herein, or a pharmaceutical formulation comprising one or more of the S100B binding agents, to a subject in need thereof.
  • Such methods can be used to inhibit S100B activity and/or inhibit S100B binding, e.g. to a target protein or receptor.
  • Such methods can be used to treat or prevent a symptom of a disease or condition associated with aberrant S100B activity.
  • Such methods can further be used to treat or prevent a disease or condition associated with aberrant S100B activity.
  • the invention is directed to a method of treating or preventing a SlOOB-associated disease or condition in a subject comprising administering a therapeutically-effective amount of one or more S100B binding agents to a subject in need thereof, thereby treating or preventing a SlOOB-associated disease or condition in the subject.
  • the one or more S100B binding agents may be administered as a pharmaceutical formulation as defined herein.
  • SlOOB-associated diseases and conditions include, but are not limited to, aging, neurological disorders, acute injury, psychiatric and affective disorders, and cancer.
  • Neurological disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, Down syndrome, COPD-induced cognitive decline, autoimmune-induced cognitive decline, postoperative cognitive decline, radiation-induced cognitive decline, vertigo and aging.
  • Acute injuries include, but are not limited to traumatic brain injury, stroke/ischemia, and hypoxic ischemia at term.
  • Psychiatric and affective disorders include, but are not limited to, disease- induced depression, mood disorders, suicide, schizophrenia, and delirium.
  • Cancers include, but are not limited to, melanoma, astrocytoma, glioma, and brain cancer.
  • the therapeutically-effective amount of the S100B binding agent is between 10 ug/kg and 100 mg/kg of the agent per body weight of the subject.
  • the S100B binding agent is administered to the subject via intravenous, subcutaneous, or intransal means, or other means.
  • the invention is directed to methods of using S100B binding agents in the screening for and/or quantifying of S100B in a biological sample.
  • Such methods generally encompass using one or more of the S100B binding agents defined herein to detect S 100B in a biological sample, such as a sample obtained from a subject.
  • the invention is directed to a method of screening a biological sample for the presence of S100B comprising contacting a biological sample with one or more of the S100B binding agents defined herein and detecting binding of S100B by the one or more S100B binding agents.
  • the invention is directed to a method of quantifying the amount of S100B in a biological sample comprising contacting a biological sample with one or more of the S100B binding agents defined herein, detecting binding of S100B by the one or more S100B binding agents, and quantifying the amount of S100B detected.
  • the biological sample includes, but is not limited to, blood, serum, cerebrospinal fluid (CSF), tissue sample, tears, sweat, urine, lymph, tumor exudate, ascites fluid, and pleural fluids.
  • CSF cerebrospinal fluid
  • binding of S100B by the one or more S100B binding agents is via an assay that includes, but is not limited to, an ELISA assay, an affinity column, a Western/immunoblot, immunohistochemistry, or a proximity ligation assay.
  • the invention is directed to methods of using S100B binding agents in the diagnosing and/or monitoring of SlOOB-associated diseases or conditions in a subject.
  • Such methods generally encompass using one or more of the S100B binding agents defined herein to quantifying the amount of S100B in a biological sample obtained from a subject and using the quantified amount of S100B to monitoring disease progression or to make a diagnosis of a SlOOB-associated disease or condition in the subject.
  • Such methods include administering one or more of the S100B binding agents defined herein to a subject having a SlOOB-associated disease or condition, or suspected of having a SlOOB-associated disease or condition, and detecting binding of the one or more S100B binding agents to S100B in the subject.
  • the invention is directed to a method of monitoring a SlOOB-associated disease or condition in a subject comprising (a) contacting a biological sample obtained from a subject having a SlOOB-associated disease or condition with one or more of the S100B binding agents defined herein, (b) detecting binding of S100B by the one or more S100B binding agents, (c) quantifying the amount of S100B detected, (d) comparing the amount quantified in (c) with an amount quantified in a biological sample from the same subject at an earlier time point, thereby monitoring a SlOOB-associated disease or condition in the subject.
  • the invention is directed to a method of diagnosing a SlOOB-associated disease or condition in a subject comprising (a) contacting a biological sample obtained from a subject with one or more of the S100B binding agents defined herein, (b) detecting binding of S100B by the one or more S100B binding agents, (c) quantifying the amount of S100B detected, (d) comparing the amount quantified in (c) with one or more control amounts quantified in a biological sample obtained from a subject with a SlOOB- associated disease or condition, thereby diagnosing a SlOOB-associated disease or condition in the subject.
  • the invention is directed to a method of diagnosing a SlOOB-associated disease or condition in a subject comprising administering one or more of the S100B binding agents defined herein to a subject having a SlOOB-associated disease or condition, or suspected of having a SlOOB-associated disease or condition, and detecting binding of the one or more S100B binding agents to S100B in the subject.
  • the biological sample includes, but is not limited to, blood, serum, cerebrospinal fluid (CSF), tissue sample, tears, sweat, urine, lymph, tumor exudate, ascites fluid, and pleural fluids.
  • CSF cerebrospinal fluid
  • binding of S100B by the one or more S100B binding agents is via an assay that includes, but is not limited to, an ELISA assay, an affinity column, a Western/immunoblot, immunohistochemistry, a proximity ligation assay or in vivo imaging.
  • an assay that includes, but is not limited to, an ELISA assay, an affinity column, a Western/immunoblot, immunohistochemistry, a proximity ligation assay or in vivo imaging.
  • FIG. 1 Structure of S100B.
  • the four helices, two loops (LI and L2) and the hinge/linker domain are shown.
  • the pseudo EF-hand comprises helices 1 and 2 and loop 1; the typical EF-hand comprises helices 3 and 4 and loop 2.
  • the upper amino acid sequence is human S100B (SEQ ID NO:6).
  • the lower amino acid sequence is mouse S100B (SEQ ID NO:7).
  • the epitope used in the production of antibodies (SEQ ID NO:5) is double underlined. The single amino acid change between human and mouse sequences is underlined.
  • S100B*RAGE (Fig. 2C), but not S100B*D2R (Fig. 2D), complexes are present in sporadic and familial human AD specimens.
  • Representative micrographs of sporadic (olfactory cortex) and familial (cingulate gyrus) AD human specimens processed for proximity ligation assays using S100B (BL356) + TLR4, FGFR1, AGER or D2R primary antibodies (brown; solid arrows) and counterstained with hematoxylin to visualize nuclei (blue; dashed arrows). Bars 50 ⁇ .
  • S100B*RAGE (Fig. 3C) complexes exhibit age-dependent increases in the PSAPP AD mouse model.
  • FIG. 4A-4C Anti-SlOOB (BL356) antibody crosses the blood brain barrier and neutralizes S100B.
  • Fig. 4B contains ex vivo images of brain tissue from PSAPP mice 48 hours after receiving a single IV injection of IRDye® 800CW-labeled anti-SlOOB antibody (Bethyl Laboratories) and untreated mice
  • FIG. 5A-5B Neutralization of extracellular S 100B blocks TLR4/NF-KB signaling.
  • Representative micrographs of parasagittal cortical sections from 7.5 month old PSAPP mice treated with an anti-SlOOB antibody (BL356) or isotype control -IgG/placebo and processed for proximity ligation assays using S100B + TLR4 primary antibodies (brown; solid arrows) and counterstained with hematoxylin to visualize nuclei (blue; dashed arrows) (Fig. 5A). Bars 50 ⁇ .
  • Fig. 5B contains representative micrographs of adjacent sections processed for
  • FIGs 7A-7B Neutralization of extracellular S100B reduces neuroinflammation.
  • Representative micrographs of parasagittal cortical sections from 7.5 month old PSAPP mice receiving anti-SlOOB (BL356) antibody or an isotype control -IgG/placebo stained with the astrocytic marker GFAP (Fig. 7A) or the microglial marker Ibal (Fig. 7B) and DAPI (blue) to visualize nuclei. Bars 100 ⁇ .
  • Analysis by an independent samples t-test, asterisks denote p ⁇ 0.05 when compared to the placebo group.
  • FIG. 8 Neutralization of extracellular S100B reduces amyloidosis.
  • Representative micrographs of parasagittal cortical sections from 7.5 month old PSAPP mice receiving anti- Si 00B (BL356) antibody or isotype control -IgG/placebo stained with thioflavin S (green) to visualize plaques. Bars 100 ⁇ .
  • Analysis by an independent samples t-test, asterisks denote p ⁇ 0.05 when compared to the placebo group.
  • FIG. 10 Dose-response curves for engineered antibodies. A direct ELISA and serial dilutions ranging from 0.0025 to 83 micrograms were used to generate dose-response curves for engineered antibodies. All five antibodies exhibited greater sensitivity (concentration for 10% biding) than the BL356 prototype. However, only three exhibited a lower ECso/higher K D (concentration for 50% binding) than the BL356 prototype.
  • Figure 13 Detection of muBL356(lC8 S100B complexes. Indirect ELISAs were used to assess the ability of various capture antibodies (2 fold excess) to detect
  • Figure 14A The nucleotide sequence (SEQ ID NO: 1) encoding the mouse 1C8 antibody heavy chain.
  • the various domains encoded by the heavy chain sequence are indicated with underlining, ordered as shown.
  • FR framework region;
  • CDR complementarity determining region.
  • Figure 14B The amino acid sequence (SEQ ID NO:2) of the mouse 1C8 antibody heavy chain.
  • the various domains of the heavy chain are indicated with underlining, ordered as shown.
  • FR framework region 1;
  • CDR complementarity determining region.
  • Figure 14C The nucleotide sequence (SEQ ID NO:8) encoding the mouse 1C8 antibody heavy chain variable region.
  • the various domains encoded by the heavy chain variable region sequence are indicated with underlining, ordered as shown.
  • FR framework region;
  • CDR complementarity determining region.
  • FIG. 15B The amino acid sequence (SEQ ID NO:4) of the mouse 1C8 antibody light chain.
  • the various domains of the light chain are indicated with underlining, ordered as shown.
  • FR framework region;
  • CDR complementarity determining region.
  • Figure 15C The nucleotide sequence (SEQ ID NO: 10) encoding the mouse 1C8 antibody light chain variable region.
  • the various domains encoded by the light chain variable region sequence are indicated with underlining, ordered as shown.
  • FR framework region;
  • CDR complementarity determining region.
  • Figure 15D The amino acid sequence (SEQ ID NO: 11) of the mouse 1C8 antibody light chain variable region.
  • the various domains of the light chain variable region are indicated with underlining, ordered as shown.
  • FR framework region;
  • CDR complementarity determining region.
  • FIG. 16 Neutralizing extracellular SI 008 antibodies halt primary melanoma tumorigenesis. Intralesional administration of an anti-SlOOB (BL356; prototype) antibody and candidate antibody 1C8 (35 ⁇ g/100mm 3 ) twice weekly for 25 days halted primary tumor growth in an immune competent BRAF wild-type mouse melanoma model. * denotes p ⁇ 0.05.
  • Figures 17A-17B Inhibition of extracellular S100B reduces ERK/AKT activation.
  • Immune competent BRAF wild-type animals received intralesional doses of candidate antibody 1C8 (35 ⁇ g/100 mm 3 ) twice weekly. Twenty -five days later tissues were processed for PO 4 -ERK and PO 4 -AKT detection using fluorescence (Fig. 17 A) or brightfield (Fig. 17B) microscopy.
  • FIG. 18A-18B. S100B _/" mice had improved retention memory function after TBI.
  • Fig. 18 A TBI induced sensorimotor impairments at all time points when compared with sham- injured mice in the beam walk test ( *** p ⁇ 0.001, ** p ⁇ 0.01, vs. sham).
  • S100B _/" mice failed to show any improvement in sensorimotor function. Analysis by repeated measures two- way ANOVA followed by Tukey's post-hoc.
  • Fig. 18B. S100B _/" mice showed significant improvement ( + p ⁇ 0.05, vs. S100B +/+ ) in Discrimination Index (D.I.) in the Novel Object Recognition (NOR) task.
  • D.I. Discrimination Index
  • NOR Novel Object Recognition
  • FIGs 20A-20B S100B _/" mice had reduced microglial activation in the injured cortex after TBI. Stereological assessment of microglial cell number and activation phenotype was performed in the cortex at 7 and 28 days after TBI.
  • Fig. 20A Representative images of the Iba-1 stained sections show morphological features of ramified, and hypertrophic and bushy (activated) microglia.
  • Fig. 20B. S100B _/" mice showed statistically significant increases in the numbers of ramified microglia on 28 days after injury ( ** p ⁇ 0.01, vs. sham; + p ⁇ 0.05, vs.
  • Figures 21A-21B Systemic administration of a neutralizing S100B antibody
  • FIGs 22A-22D Systemic administration of a neutralizing S100B antibody (BL356) reduced lesion volume, improved neuronal survival and attenuated microglial activation in the cortex after TBI.
  • Fig. 22A Histological assessment using unbiased stereology showed that vehicle-treated mice developed a large lesion following TBI, whereas anti-SlOOB IgG treatment resulted in significant reduction in lesion size ( *** p ⁇ 0.001, vs. vehicle).
  • Systemic administration of IgG control significantly reduced the lesion volume ( ** p ⁇ 0.01, vs. vehicle, + p ⁇ 0.05, vs. anti- Si 00B IgG treatment). Analysis by one-way ANOVA followed by Tukey's post-hoc.
  • FIG. 22B Representative images of the cresyl violet-stained sections show the reduction in lesion volume observed in anti-SlOOB IgG-treated samples.
  • Fig. 22C Representative images of the cresyl violet-stained sections show the reduction in lesion volume observed in anti-SlOOB IgG-treated samples.
  • “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term “about” generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
  • novel antibodies that exhibit binding specificity for S100B as well as variants and fragments thereof that retain the binding specificity of the antibodies from which they are derived, collectively termed S100B binding agents.
  • Such antibodies form the basis of the S100B binding agents of the present invention, along with variants of these antibodies, and fragments of the antibodies and variants.
  • Particular antibodies disclosed herein include a novel anti-SlOOB monoclonal antibody (termed “1C8"), a polyclonal antibody (termed “BL356”), and other monoclonal antibodies that exhibit binding specificity for the S100B protein both in vitro and in vivo.
  • Methods of using these binding agents in the treatment and prevention of S100B- associated diseases are also provided. Methods of using these binding agents in the screening and diagnosis of subjects for SlOOB-associated diseases are further provided.
  • the antibodies of the present invention exhibit binding specificity for the S100B polypeptide.
  • Such antibodies can be defined as those antibodies having binding specificity for an epitope of S100B, wherein the epitope comprises the amino acid sequence set forth in SEQ ID NO: 5.
  • Such antibodies can also be defined as those antibodies having binding specificity for S100B, wherein the antibody was raised against an epitope of S100B comprising the amino acid sequence set forth in SEQ ID NO: 5.
  • Antibodies having binding specificity for S100B and S100B epitopes can be defined, in non-limiting aspects of the invention, based on their variable regions, i.e., as antibodies comprising one or more of (a) a V L region comprising the amino acid sequence of SEQ ID NO: 11, (b) a V H region comprising the amino acid sequence of SEQ ID NO:9, or (c) both a V L region comprising the amino acid sequence of SEQ ID NO: l 1 and a V H region comprising the amino acid sequence of SEQ ID NO:9.
  • the antibodies are not limited with respect to other characteristics.
  • the antibodies may be of any class, such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD or IgE.
  • the present invention includes antibodies having one or more amino acid insertions, deletions and/or substitutions, that also retain binding specificity for S100B.
  • the invention includes an antibody where the V L region has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 11 and the V H region comprises the amino acid sequence of SEQ ID NO:9.
  • the invention also includes an antibody where the V L region comprises the amino acid sequence of SEQ ID NO: 11 and the V H region has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO:9.
  • the invention further includes an antibody where the V L region has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 11 and the V H region has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO:9.
  • the invention includes an antibody where the V L region has at least about 95% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 11, or the V H region has at least about 95% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 9, or the V L region has at least about 95% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 11 and the V H region has at least about 95% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO:9.
  • the isolated antibodies having binding specificity for S100B and S100B epitopes can also be defined, in non-limiting aspects of the invention, based on their heavy and light chains.
  • the S100B binding antibodies comprise one or more of (a) a light chain comprising the amino acid sequence of SEQ ID NO:4, (b) a heavy chain comprising the amino acid sequence of SEQ ID NO:2, or (c) a light chain comprising the amino acid sequence of SEQ ID NO:4 and a heavy chain comprising the amino acid sequence of SEQ ID NO:2.
  • the antibodies are not limited with respect to other characteristics.
  • the antibodies may be of any class, such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD or IgE.
  • alterations can be made to the amino acid sequence of antibodies without affecting the binding activity, for example, alterations can be made to either or both the light and heavy chains, while maintaining the binding activity and/or binding specificity of the antibody.
  • the present invention includes antibodies having one or more amino acid insertions, deletions and/or substitutions, that also retain binding specificity for S100B.
  • the invention includes an antibody where the light chain has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO:4 and the heavy chain comprises the amino acid sequence of SEQ ID NO:2.
  • the invention also includes an antibody where the light chain comprises the amino acid sequence of SEQ ID NO:4 and the heavy chain has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO:2.
  • the invention further includes an antibody where the light chain has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO:4 and the heavy chain has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO:2.
  • the invention is directed to S100B binding antibodies comprising (a) a light chain comprising the amino acid sequence of SEQ ID NO:4 and a heavy chain comprising the amino acid sequence of SEQ ID NO:2; (b) a light chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:4 and a heavy chain comprising the amino acid sequence of SEQ ID NO:2; (c) a light chain comprising the amino acid sequence of SEQ ID NO:4 and a heavy chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:2; and (d) a light chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:4 and a heavy chain comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:2.
  • the invention is directed to the 1C8 antibody, a S100B binding antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:4 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:2.
  • the antibodies of the invention may be polyclonal or monoclonal antibodies, and the antibodies may be in the form of an antiserum comprising the antibodies.
  • the antibodies may be isolated antibodies, purified antibodies, exogenous antibodies, endogenous antibodies, or a combination thereof. Further, the antibodies may be recombinant antibodies.
  • the S100B binding agents of the invention include variants of each of the antibodies defined herein that retain the ability to bind S100B.
  • the term "variant" is intended to encompass modified versions of the antibodies, such as humanized antibodies and chimeric antibodies. It also encompasses fully human antibodies.
  • the S100B binding agents of the invention also include fragments of the antibodies and variants defined herein that retain the ability to bind S100B.
  • the fragments included, but are not limited to, Fab fragments, F(ab') 2 fragments, single chain Fv (scFv) antibodies, and fragments produced by an Fab expression library, as well as bi-specific antibody and triple- specific antibodies.
  • Each of the variants and fragments of the invention exhibits at least 50% of the binding affinity for mouse S100B that is exhibited by the 1C8 antibody described herein, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.
  • the binding affinity can be determined using assays well known in the art, including ELISAs, Western/immunoblots, immunohistochemistry and proximity ligation assays.
  • Antibodies may be produced in any species of animal, though preferably from a mammal such as a human, simian, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog or cat.
  • the antibodies can be human antibodies or humanized antibodies, or any antibody preparation suitable for administration to a human.
  • the selected species of animal can be immunized by injection with one or more antigens, e.g., full-length S100B and/or one or more fragments thereof, a peptide comprising the linker peptide defined herein (ELSFIFLEEIKEQE; SEQ ID NO:5), and a peptide consisting of the linker peptide defined herein (ELSFIFLEEIKEQE; SEQ ID NO:5).
  • one or more antigens e.g., full-length S100B and/or one or more fragments thereof, a peptide comprising the linker peptide defined herein (ELSFIFLEEIKEQE; SEQ ID NO:5), and a peptide consisting of the linker peptide defined herein (ELSFIFLEEIKEQE; SEQ ID NO:5).
  • the antigen may be an epitope of S100B derived from any mammalian species, including, but not limited to, human S100B, mouse S100B, rat S100B, goat S100B, rabbit S100B, pig S100B, or S100B from some other mammal.
  • the antigens may be administered in conjunction with one or more pharmaceutically acceptable adjuvants to increase the immunological response.
  • Suitable adjuvants include, but are not limited to, Freund's Complete and Incomplete Adjuvant, Titermax, Oil in Water adjuvants, as well as aluminum compounds where antigens, normally peptides, are physically precipitated with hydrated insoluble salts of aluminum hydroxide or aluminum phosphate.
  • Other adjuvants include liposome-type adjuvants comprising spheres having phospholipid bilayers that form an aqueous compartment containing the antigen and protect it from rapid degradation, and that provide a depot effect for sustained release.
  • Surface active agents may also be used as adjuvants and include lipoteichoic acid of gram -positive organisms, lipid A, and TDM.
  • Quil A and QS-21 (saponin-type adjuvants), monophosphoryl lipid A, and lipophilic MDP derivatives are suitable adjuvants that have hydrophilic and hydrophobic domains from which their surface-active properties arise.
  • Compounds normally found in the body such as vitamin A and E, and lysolecithin may also be used as surface-active agents.
  • adjuvants include glycan analog, coenzyme Q, amphotericin B, dimethyldioctadecylammonium bromide (DDA), levamisole, and benzimidazole compounds.
  • the immunostimulation provided by a surface active agent may also be accomplished by either developing a fusion protein with non-active portions of the cholera toxin, exotoxin A, or the heat labile toxin from E. coli.
  • Immunomodulation through the use of anti-IL-17, anti IFN- ⁇ , anti-IL-12, IL-2, IL-10, or IL-4 may also be used to promote a strong Th2 or antibody mediated response to the immunogenic formulation.
  • the antibodies of the invention can be prepared using any known technique that provides for the production of antibody molecules. Suitable techniques include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975)), the human B-cell hybridoma technique (Kosbor et al., Immunol Today 4:72 (1983); Cote et al., Proc Natl. Acad. Sci 80:2026-2030 (1983)), and the EBV-hybridoma technique (Cole et al., Monoclonal
  • antibodies can be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl. Acad. Sci. USA 86: 3833-3837 (1989), and in Winter G. and Milstein C, Nature 349:293-299 (1991), both of which is herein incorporated by reference in its entirety.
  • Humanized antibodies are those antibodies where a human antibody has been engineered to contain non-human complementarity-determining regions (CDRs) derived from an antibody produced in a non-human host against a selected antigen.
  • CDRs complementarity-determining regions
  • Means for producing humanized antibodies are well-known in the art and include Vaswani SK, and Hamilton RG, Ann Allergy Asthma Immunol. 81(2): 105-15 (1998) and Kashmiri SV et al., Methods 36 (l):25-34 (2005), each of which is herein incorporated by reference in its entirety.
  • Chimeric antibodies are those where an antigen binding region (e.g., F(ab') 2 or hypervariable region) of a non-human antibody is transferred into the framework of a human antibody by recombinant DNA techniques.
  • Techniques developed for the production of such antibodies include the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • Such techniques are also well known and include: Morrison et al., Proc Natl. Acad. Sci 81 :6851-6855 (1984); Neuberger et al., Nature 312:604-608(1984); Takeda et al., Nature 314:452-454(1985), each of which is herein incorporated by reference in its entirety.
  • Antibody fragments such as F(ab') 2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W. D. et al., Science 256: 1275-1281 (1989), herein incorporated by reference in its entirety).
  • the invention includes polynucleotides comprising nucleotide sequences encoding each the binding agents provided herein, as well as complementary strands thereof.
  • the invention also includes vectors, such as cloning vectors and expression vectors, comprising the polynucleotides, and host cells comprising the polynucleotides and/or the vectors.
  • vectors such as cloning vectors and expression vectors, comprising the polynucleotides, and host cells comprising the polynucleotides and/or the vectors.
  • Suitable expression vectors include, e.g., pcDNA3.1 and pSec-His.
  • Suitable host cells include, e.g., Chinese hamster ovary cells (CHO cells) and human embryonic kidney cells 293 (F£EK 293 cells).
  • the invention further includes methods of producing the binding agents defined herein, comprising culturing the host cells under conditions promoting expression of the binding agents encoded by the polynucleotides or the expression vectors, and recovering the binding agents from the cell cultures.
  • kits comprising (a) one or more of the S100B binding agents as defined herein, and optionally (b) a labeled secondary antibody recognizing the S100B binding agent(s) of (a), and further optionally instructions for the use of the kit.
  • the S100B binding agents include one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the labeled secondary antibody may be any antibody that can bind the S100B binding agent and that is tagged with a detectable label.
  • the invention includes cell lines which produce one or more of the S100B binding agents defined herein.
  • the cell lines of the invention include those which produce one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the invention includes diagnostic reagents for Alzheimer disease comprising one or more of the S100B binding agents defined herein.
  • the diagnostic reagents of the invention include those comprising one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the invention includes therapeutic agents for a SlOOB-associated disease or condition comprising one or more of the S100B binding agents defined herein.
  • the therapeutic agents of the invention include those comprising one or more of the antibodies defined herein, one or more the humanized variants defined herein, and one or more the SlOOB-binding fragments defined herein.
  • the S100B binding agents defined in the present invention can be used in a variety of applications, including methods of treating, preventing, screening for, or diagnosing SlOOB-associated diseases and conditions.
  • the invention includes pharmaceutical formulations comprising the S100B binding agents and a pharmaceutically acceptable carrier or diluent (also termed antibody formulations herein).
  • Suitable examples of carriers and diluents are well known to those skilled in the art and include water, water-for-injection, saline, buffered saline, dextrose, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80TM), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL), poloxamer 407 and 188, hydrophilic and hydrophobic carriers, and combinations thereof.
  • Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes.
  • the terms specifically exclude cell culture medium.
  • the formulations may further comprise stabilizing agents, buffers, antioxidants and preservatives, tonicity agents, bulking agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, and combinations thereof.
  • compositions for intramuscular preparations can be prepared where the carrier is water-for-injection, 0.9% saline, or 5% glucose solution.
  • Pharmaceutical formulations may also be prepared as liquid or powdered atomized dispersions for delivery by inhalation. Such dispersion typically contain carriers common for atomized or aerosolized dispersions, such as buffered saline and/or other compounds well known to those of skill in the art.
  • the delivery of the pharmaceutical formulations via inhalation has the effect of rapidly dispersing the immunogenic formulation to a large area of mucosal tissues as well as quick absorption by the blood for circulation.
  • One example of a method of preparing an atomized dispersion is described in U.S. Patent No.
  • the pharmaceutical formulations may be administered in a liquid form.
  • the liquid can be for oral dosage, for ophthalmic or nasal dosage as drops, or for use as an enema or douche.
  • the pharmaceutical formulation is formulated as a liquid, the liquid can be either a solution or a suspension of the pharmaceutical formulation.
  • suitable formulations for the solution or suspension of the pharmaceutical formulations that are well known to those of skill in the art, depending on the intended use thereof.
  • formulations for oral administration prepared in water or other aqueous vehicles may contain various suspending agents such as methylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol.
  • the liquid formulations may also include solutions, emulsions, syrups and elixirs containing, together with the active compound(s), wetting agents, sweeteners, and coloring and flavoring agents.
  • the S100B binding agents and pharmaceutical formulations defined herein can be used in the treatment or prevention of SlOOB-associated diseases or conditions in a subject. Such methods generally encompass administering one or more of the S100B binding agents defined herein, or a pharmaceutical formulation comprising one or more of the S100B binding agents, to a subject in need thereof. Such methods can be used to inhibit S100B activity, inhibit S100B binding, neutralize S100B activity, and/or neutralize S100B binding, e.g. to a binding partner, target protein and/or receptor. Such methods can be used to treat or prevent a symptom of a disease or condition associated with aberrant S100B activity.
  • Such methods can further be used to treat or prevent a disease or condition associated with aberrant S100B activity (i.e., a S100B- associated disease or condition).
  • the S100B binding agents of the invention can have neutralizing activity.
  • S100B binding agents having neutralizing activity such as S100B neutralizing antibodies, can be used in the pharmaceutical formulations and methods of treatment and prevention described herein.
  • Methods of inhibiting S100B activity and/or inhibiting S100B binding can be practiced in vitro, in vivo or ex vivo.
  • Such methods include a method of inhibiting S100B activity in a subject comprising administering a therapeutically-effective amount of one or more S 100B binding agents to a subject in need thereof, thereby inhibiting S100B activity in the subject.
  • the one or more S100B binding agents may be administered as a pharmaceutical formulation as defined herein.
  • Such methods also include a method of inhibiting S100B binding in a subject comprising administering a therapeutically-effective amount of one or more S100B binding agents to a subject in need thereof, thereby inhibiting S100B binding in the subject.
  • the one or more S100B binding agents may be administered as a pharmaceutical formulation as defined herein.
  • the method includes inhibiting S100B binding to a target protein, inhibiting S100B binding to a receptor, or both.
  • the methods of treatment and/or prevention include methods of treating or preventing a SlOOB-associated disease or condition in a subject comprising administering a therapeutically- effective amount of one or more S100B binding agents to a subject in need thereof, thereby treating or preventing a SlOOB-associated disease or condition in the subject.
  • the one or more S100B binding agents may be administered as a pharmaceutical formulation as defined herein.
  • the terms “treat”, “treating” and “treatment” have their ordinary and customary meanings, and include one or more of, ameliorating a symptom of a SlOOB-associated disease or condition; blocking or ameliorating a recurrence of a symptom of a SlOOB-associated disease or condition; decreasing in severity and/or frequency a symptom of a SlOOB-associated disease or condition; blocking progression of a SlOOB-associated disease or condition; and curing or resolving a SlOOB-associated disease or condition.
  • Treatment means ameliorating, etc. by about 1% to about 100% versus a subject to which the treatment has not been administered.
  • the treatment may begin prior to, concurrent with, or after the onset of clinical symptoms of the disease or condition.
  • the subject may be showing symptoms of a SlOOB-associated disease or condition, or merely diagnosed as having a SlOOB-associated disease or condition but so far free of symptoms of the disease or condition.
  • the results of the treatment may be permanent or may continue for a period of days (such as 1, 2, 3, 4, 5, 6 or 7 days), weeks (such as 1, 2, 3 or 4 weeks) or months (such as 1, 2, 3, 4, 5, 6 or more months).
  • the terms "prevent”, “preventing” and “prevention” have their ordinary and customary meanings, and include one or more of, stopping, averting, avoiding, or blocking the occurrence of a symptom of a SlOOB-associated disease or condition, the recurrence of a symptom of a SlOOB-associated disease or condition, or the development of a SlOOB- associated disease or condition.
  • Prevention means stopping, etc. by at least about 95% versus a subject to which the prevention has not been administered.
  • the stopping is about 100%, about 99%, about 98%, about 97%, about 96% or about 95%.
  • the course of therapy may begin prior to, concurrent with, or after the onset of clinical symptoms of the SlOOB-associated disease or condition.
  • the subject may have a SlOOB-associated disease or condition or merely be susceptible to SlOOB-associated disease or condition.
  • the results of the prevention may be permanent or may continue for a period of days (such as 1, 2, 3, 4, 5, 6 or 7 days), weeks (such as 1, 2, 3 or 4 weeks) or months (such as 1, 2, 3, 4, 5, 6 or more months).
  • the one or more S100B binding agents or pharmaceutical formulations are administered in a
  • agents and pharmaceutical formulations may be administered to a subject using different schedules, depending on the particular disease or condition being treated or prevented, and the severity thereof; the age and size of the subject; and the general health of the subject, to name only a few factors to be considered.
  • the agents and pharmaceutical formulations may be administered once, or twice, three times, four times, five times, six times or more, over a course of treatment or prevention.
  • the timing between each dose in a dosing schedule may range between days, weeks, months, or years, an includes administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more weeks.
  • the same quantity of protein in the formulation may be administered in each dose of the dosing schedule, or the amounts in each dose may vary.
  • the identity of the particular binding agents in the formulation may also vary or remain the same in each dose in a dosing schedule.
  • the amount of the S100B binding agent administered to a subject in a dose when the methods of the present invention are practiced will again vary. However, the amount
  • pharmaceutical formulation of the present invention is typically between about 0.1 ug to about 200 mg per kg of body weight of the subject to which the dose of the pharmaceutical formulation is being administered.
  • Additional ranges of therapeutically-effective amounts of the S100B binding agents include, but are not limited to, about 10 ug/kg to 200 mg/kg; about 1 ug/kg to 200 mg/kg; about 100 ug/kg to 200 mg/kg; about 1 mg/kg to 200 mg/kg; about 10 mg/kg to 200 mg/kg; about 100 ug/kg to 100 mg/kg; about 1 mg/kg to 100 mg/kg; about 10 mg/kg to 100 mg/kg; about 1 mg/kg to 50 mg/kg; about 0.1 ug/kg to 50 mg/kg; about 1 ug/kg to 50 mg/kg; about 10 ug/kg to 50 mg/kg and about 100 ug/kg to 50 mg/kg.
  • Administration of the pharmaceutical formulations may be via any of the means commonly known in the art of vaccine delivery.
  • routes include intravenous, intraperitoneal, intramuscular, subcutaneous and intradermal routes of administration, as well as nasal application, by inhalation, ophthalmically, orally, rectally, vaginally, or by any other mode that results in the formulation contacting mucosal tissues.
  • SlOOB-associated diseases and conditions include, but are not limited to, aging, neurological disorders, acute injury, psychiatric and affective disorders, and cancer.
  • Neurological disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, Down syndrome, COPD-induced cognitive decline, autoimmune-induced cognitive decline, postoperative cognitive decline, radiation-induced cognitive decline, vertigo, and aging.
  • Acute injuries include, but are not limited to traumatic brain injury, stroke/ischemia, and hypoxic ischemia at term.
  • Psychiatric and affective disorders include, but are not limited to, disease- induced depression, mood disorders, suicide, schizophrenia, and delirium.
  • Cancers include, but are not limited to, melanoma, astrocytoma, glioma, and brain cancer.
  • S100B overexpression has been used as a diagnostic marker for primary and metastatic melanoma since the early 1980s [75-77]. It has been demonstrated that intracellular S100B enhances tumor cell proliferation by binding to/promoting the degradation of the tumor suppressor p53, and subsequent MAPK activation [73, 74, 78, 79]. However, it is unlikely that the dramatic effects of S100B inhibition on tumor cell growth are limited to intracellular S100B [72, 80-82], as S100B is present in tumor exudates, ascites and pleural fluids from patients [83]. Serum S100B levels have been used as a prognostic indicator and to monitor response to treatment, including targeted therapies, in melanoma [84-85].
  • Extracellular S100B is a damage- associated molecular pattern molecule (DAMP) and regulates inflammatory responses in cancers [86-88] as well as fat [89], brain [46, 90, 91], gut [92] lung [93], and the immune system [94].
  • the metastatic switch is associated with an 8 fold increase in extracellular S100B [102] and extracellular S100B promotes invasion and metastasis in in vitro assays [103].
  • Each of the methods of treatment and prevention provided herein may be performed in conjunction with other types of therapy.
  • methods of treating cancer via administration of one or more of the S100B binding agents of the invention concurrent administration of traditional chemotherapeutics may occur.
  • subject includes, but is not limited, to a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
  • the S100B binding agents of the invention can be utilized in different capacities, including screening for the presence of S100B in a sample, determining the amount of S100B in a sample, monitoring progression of SlOOB-associated diseases or conditions in a subject, and diagnosing SlOOB-associated diseases or conditions in a subject.
  • Each of these methods will generally utilize (i) a sample, such as a biological sample obtained from a subject, (ii) one or more of the S100B binding agents of the invention, such as the 1C8 antibody, where the S100B binding agent may be conjugated to a detectable label, and (iii) an assay or means for detecting binding between the S100B binding agent and S100B in the sample.
  • the assay or means may further quantify the amount of S100B in the sample.
  • the ability to quantify the amount of S100B will be important in determining whether there has been an increase or decrease in the amount of S100B produced in a subject, relative to an earlier time point.
  • Samples can be obtained from the same subject at different time points, such as before and after therapeutic intervention, and the disease or condition can be monitored in the subject.
  • the amount of S100B in a sample obtained from a subject can be measured, and the amount thus quantified can be compared to the amount present in a sample from the same or similar source from a subject known to have a particular SlOOB-associated disease or condition, thereby permitting diagnosis of a SlOOB-associated disease or condition.
  • the binding of S100B by the one or more S100B binding agents may also be detected and/or quantified by direct in vivo imaging of a live subject. Whether based on the information regarding S100B levels alone, or in conjunction with other signs and symptoms of a SlOOB-associated disease or condition, a diagnosis regarding a particular SlOOB-associated disease or condition can be made.
  • the present invention is directed to methods of using S100B binding agents in the screening for and/or quantifying of S100B in a biological sample. Such methods generally encompass using one or more of the S100B binding agents defined herein to detect S100B in a biological sample, such as a sample obtained from a subject.
  • the invention is directed to a method of screening a biological sample for the presence of S100B comprising contacting a biological sample with one or more of the S100B binding agents defined herein and detecting binding of S100B by the one or more S100B binding agents.
  • the invention is directed to a method of quantifying the amount of S100B in a biological sample comprising contacting a biological sample with one or more of the S100B binding agents defined herein, detecting binding of S100B by the one or more S100B binding agents, and quantifying the amount of S100B detected.
  • the S100B binding agent may be conjugated to a detectable label.
  • Methods of using the S100B binding agents in the diagnosing and/or monitoring of SlOOB-associated diseases or conditions in a subject generally encompass using one or more of the S100B binding agents defined herein to quantifying the amount of S100B in a biological sample obtained from a subject and using the quantified amount of S100B to monitoring disease progression or to make a diagnosis of a SlOOB-associated disease or condition in the subject.
  • binding of S100B by the S100B binding agents can be directly imaged in the living subject.
  • the invention is directed to a method of monitoring a SlOOB-associated disease or condition in a subject comprising (a) contacting a biological sample obtained from a subject having a SlOOB-associated disease or condition with one or more of the S100B binding agents defined herein, (b) detecting binding of S100B by the one or more S 100B binding agents, (c) quantifying the amount of S100B detected, (d) comparing the amount quantified in (c) with an amount quantified in a biological sample from the same subject at an earlier time point, thereby monitoring a SlOOB-associated disease or condition in the subject.
  • the S100B binding agent may be conjugated to a detectable label.
  • the invention is directed to a method of diagnosing a SlOOB- associated disease or condition in a subject comprising (a) contacting a biological sample obtained from a subject with one or more of the S100B binding agents defined herein, (b) detecting binding of S100B by the one or more S100B binding agents, (c) quantifying the amount of S100B detected, (d) comparing the amount quantified in (c) with one or more control amounts quantified in a biological sample obtained from a subject with a S lOOB-associated disease or condition, thereby diagnosing a SlOOB-associated disease or condition in the subject.
  • the S100B binding agent may be tagged with a detectable label.
  • the invention is directed to a method of diagnosing a SlOOB- associated disease or condition in a subject comprising administering one or more of the S100B binding agents defined herein to a subject having a SlOOB-associated disease or condition, or suspected of having a SlOOB-associated disease or condition, and detecting binding of the one or more S100B binding agents to S100B in the subject.
  • the S100B binding agent may be conjugated to a detectable label.
  • biological sample includes, but is not limited to blood, serum, cerebrospinal fluid (CSF), tissue sample, tears, sweat, urine, lymph, tumor exudate, ascites fluid, or pleural fluids.
  • CSF cerebrospinal fluid
  • the S100B binding agents can be conjugated to detectable labels such as an enzyme (e.g., peroxidase, alkaline phosphatase, glucose oxidase), a metal (e.g., gold for electron microscopy applications), a fluorescent marker (e.g., for immunofluorescence and flow cytometry applications, including CYE dyes, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine), a fluorescence-emitting metals (e.g., 152 Eu), a radioactive marker (e.g., radioisotopes for diagnostic
  • an enzyme e.g., peroxidase, alkaline phosphatase, glucose oxidase
  • a metal e.g., gold for electron microscopy applications
  • a fluorescent marker e.g., for immunofluorescence and flow cyto
  • chemiluminescent marker e.g., luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester
  • protein tag e.g., biotin, phycobiliprotein, c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS.
  • Suitable assays and means for detecting binding of S 100B by the one or more S 100B binding agents include, but are not limited to, ELISA assays, affinity columns,
  • Suitable assays and means for quantifying S 100B bound by the one or more S 100B binding agents include, but are not limited to, ELISA assays, Western/immunoblots,
  • S 100B expression is elevated in familial as well as sporadic Alzheimer's disease (AD), and the highest levels are observed in the most severely affected regions [13-15, 39]. S 100B levels also increase in the precuneu and posterior cingulate gyrus during aging, two areas of the brain that exhibit early alterations during the course of AD [40].
  • CSF serum/cerebrospinal fluid
  • PSAPP mice The mouse PSAPP double transgenic line was generated by crossing the mouse Tg2576 line ("Swedish" APPK670N/M671L mutation) with the mouse 6.2 line (PS- 1M146L) [49-51]. This mouse model mimics many facets of the human disease including ⁇ plaque deposition, dystrophic neurites, glial activation, and memory deficits.
  • the PSAPP line was maintained as previously described and all experimental animals were generated from PSAPP male X wildtype female (B6SJLF 1 /J, strain 100012, Jackson Laboratories, Bar Harbor, ME) crosses [46,52].
  • BL356 mouse anti-SlOOB polyclonal antibody was used to neutralize extracellular S100B (generous gift of Bethyl Laboratory Inc., Montgomery, TX).
  • PSAPP mice received IP injections (10 mg/kg) of BL356 anti-SlOOB or isotype IgG control (Bethyl Laboratory Inc.) on day 0, 14 and 28.
  • animals were euthanized, specimens harvested and processed as described below.
  • BL356 was labeled with the infrared dye IRDye® 800CW as described by the manufacturer (#928-38040, LI-COR Biosciences, Lincoln, E).
  • GFAP astrocytosis
  • Ibal microgliosis
  • TLR4 and FGFR are primary extracellular S100B receptors in AD. Extracellular S100B (CSF/serum) is present in AD patients and biochemical/cellular studies have identified AGER/RAGE, TLR4, FGFRl and DRD2 (dopamine receptor 2; D2R) as S100B receptors/target proteins. However, none of these protein-protein interactions have been verified in vivo.
  • Proximity ligation assays and antibodies that recognize extracellular domains on putative SI 00 receptors were used to identify extracellular S100B receptors in sporadic and familial AD autopsy specimens.
  • S100B*TLR4 and S100B*FGFR complexes were readily detectable in sporadic (olfactory cortex) and familial (cingulate gyrus) human AD specimens ( Figure 2).
  • Significantly fewer S100B*AGER complexes were present, and S100B*DRD2 (D2R) complexes were undetectable ( Figures 2A-2D).
  • the inability to detect some complexes was not due to technical issues since S100B*receptor complexes were readily discernible in adjacent sections and the suitability of all pairs of primary antibodies was verified by
  • S100B*receptor complexes in sporadic versus familial AD specimens may reflect differences in receptor levels (TLR4, FGFR and AGER), ligand levels (S100B, ⁇ , HMGB, etc) or as of yet unidentified S100B receptors. This is the first direct demonstration of in vivo S100B
  • TLR4, FGFR and AGER intensity/number of complexes may reflect differences in the levels of TLR4, FGFR and AGER, or their respective ligands (S100B, ⁇ , HMGA, FGF, etc).
  • Extracellular S100B activates TLR4 NF-KB and FGFR/Akt signaling.
  • BL356 antibody which is also an S100B neutralizing antibody, was used to ascertain the contribution of extracellular S100B to AD pathobiology in the PSAPP mouse model. This antibody was specific for S100B, entered the CNS and bound SlOOB/blocked S100B staining ( Figures 4A-4C).
  • extracellular S100B signaling contributes to the phenotypic changes observed in AD mouse models in response to modulation of S100B expression.
  • Extracellular S100B signaling modulates neuroinflammation and amyloidosis.
  • microgliosis in the AD brain and are consistent with cellular studies demonstrating toxic effects of exogenous S100B on astrocytes and microglia.
  • plaque load was quantified in PSAPP mice treated with BL356 anti-SlOOB or isotype IgG antibodies.
  • Acute neutralization of extracellular S100B resulted in a 5-fold decrease in hippocampal (0.07 ⁇ 0.03 and 0.32 ⁇ 0.11 percent area) and an 8-fold decrease in cortical (0.06 ⁇ 0.01 and 0.41 ⁇ 0.10 percent area) plaque load when compared to isotype-IgG treated animals ( Figure 8).
  • These fold changes are larger than the 3-fold change in cortical and no change in hippocampal plaque load observed in response to genetic ablation [46].
  • Extracellular S100B signaling modulates neuronal integrity. Structural degeneration in AD patients and mouse models is linked to cellular hyperexcitability, and S100B overexpressing mice exhibit reductions in dendritic density [56,57].
  • PSD95 The post-synaptic marker PSD95 was used to determine if acute inhibition of extracellular S100B altered neuronal integrity [58].
  • PSAPP mice exhibited reduced dendritic and somatic PSD95 staining when compared to age-matched non-transgenic animals (Figure 9). Treatment of PSAPP mice with the BL356 anti-SlOOB antibody normalized the dendritic/somatic PSD95 staining pattern, while isotype IgG treatment had no effect.
  • S100B is expressed in a variety of extracerebral tissues (adipose, bone/cartilage and skin), the limited information that is available indicates that changes in serum S100B levels in neurological diseases are primarily due to release from the brain [65]. In traumatic brain injury (TBI), release appears to occur via the glymphatic system [66]. Microglial activation is associated with increased blood brain barrier permeability and could also contribute to S100B release in neurological disorders [67,68].
  • affinity purified antibodies were isolated from conditioned media (1 liter) of the hybridoma cell lines using Protein-G column chromatography (Paragon Bioservices, Baltimore, MD). Antibody-containing fractions were identified by gel
  • Body weight (semi -weekly) was used to assess systemic toxicity. Seven days later, animals were euthanized and visual inspection was used to evaluate gross tissue toxicity. The brain was removed, fixed and embedded in paraffin for further histological evaluation of necrosis, hemorrhage and edema.
  • Proximity ligation assays PHAs
  • TLR4 toll-like receptor 4
  • ELISAs are the device/method of choice for companion diagnostics for complex biologies/ antibodies. ELISAs were developed that measure 1C8 levels, 1C8 activity (e.g., 1C8 » S100B complexes), and anti-lC8 antibodies. Direct/indirect ELISAs used recombinant S100B protein [70] and a variety of capture and detection antibodies that included the BL356 prototype as well as clones from the S100B monoclonal antibody panel described above (e.g., 8D11 and 1C8).
  • HRP-conjugated detection antibodies, detection reagents, and blocking reagents were obtained from Bethyl Laboratories (Montgomery, TX) and used in accordance with the manufacturer's recommendations.
  • the dose-response curves for 1C8 also termed "muBL356" in presence of serum were shifted to the right when compared to curves for samples containing PBS only, indicating increased sensitivity with no change in dynamic range.
  • 1C8 » S100B complexes were undetectable with an indirect ELISA (capture antibody #1, Figure 13). To determine if these results were due to masking of the capture antibody binding site on S100B by 1C8, other S100B antibodies were screened for use as capture antibodies (Figure 13).
  • Example 4 1C8 anti-SlOOB Monoclonal Antibody
  • 1C8 mouse anti-SlOOB monoclonal antibody total RNA was extracted from frozen 1C8 hybridoma cells and cDNA was synthesized from the RNA. PCR was then performed to amplify the variable regions (heavy and light chains) and constant regions of the antibody, which were then cloned into a standard cloning vector separately and sequenced.
  • RNA was isolated from the hybridoma cells following the technical manual of TRIzol® Reagent (Ambion, Cat. No. 15596-026). The total RNA was analyzed by agarose gel electrophoresis. Total RNA was reverse transcribed into cDNA using isotype-specific anti-sense primers or universal primers following the technical manual of PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Cat. No. : 6110A). The antibody fragments of VH, VL, CH and CL were amplified according to the standard operating procedure of RACE of GenScript. Amplified antibody fragments were separately cloned into a standard cloning vector using standard molecular cloning procedures. Colony PCR screening was performed to identify clones with inserts of correct sizes. More than five single colonies with inserts of correct sizes were sequenced for each antibody fragment.
  • 14A-14D provide the nucleic acid sequence encoding the 1C8 heavy chain (SEQ ID NO: l), the amino acid sequence of the 1C8 heavy chain (SEQ ID NO:2), the nucleic acid sequence encoding the 1C8 heavy chain variable region (SEQ ID NO:8), and the amino acid sequence of the 1C8 heavy chain variable region (SEQ ID NO:9), respectively.
  • 15A-15D provide the nucleic acid sequence encoding the 1C8 light chain (SEQ ID NO:3), the amino acid sequence of the 1C8 light chain (SEQ ID NO:4), the nucleic acid sequence encoding the 1C8 light chain variable region (SEQ ID NO: 10), and the amino acid sequence of the 1C8 light chain variable region (SEQ ID NO: 11), respectively.
  • genotypes/subclasses express high levels of S100B [23].
  • the presence of extracellular S100B in clinical melanoma specimens and correlations between S100B levels and prognosis/treatment response in some melanoma patients suggest that antibodies having binding specificity for S100B may be a therapeutic agent in the treatment of cancer, such as melanoma and cancers associated with aberrant S100B expression.
  • drugs can be delivered directly to the tumor (intratumoral) via anti-SlOOB antibodies without optimization for systemic delivery and/or minimization of toxic effects on normal cells.
  • intratumoral delivery can achieve significantly higher drug concentrations at the site of action than systemic delivery.
  • the Tyr: :RASoi 2 v/I K4a/ARF -/- mouse line is bigenic and contains two genomic mutations on an FVB background: a mutated H-ras GOV transgene on the Y chromosome and inactivated
  • This model is not commercially available and a breeding colony is maintained that generates experimental animals as well as breeders for the individual Tyr: :RASoi 2 v and F K4a/ARF -/- lines. Founders for both lines were obtained from the National Cancer Institute Mutant Mouse Resource (Frederick, MD).
  • experimental Tyr: :RAS G i 2 v/INK4a/ARF -/- males develop spontaneous cutaneous melanomas in the pinna of the ears (30%), torso (23%), and tail (20%) without distant metastasis.
  • the intratumoral in vivo screening protocol for BL356 and muBL356 (1C8) used a longitudinal design with a study period of 3-8 weeks and relative tumor proliferation rate (the tumor volume at a particular treatment interval/tumor volume at the time of treatment initiation) as the primary endpoint [72].
  • S100B Neuroinflammation following traumatic brain injury (TBI) is increasingly recognized as contributing to chronic tissue loss and neurological dysfunction. Circulating levels of S100B increase after TBI, and have been used as a biomarker. S100B is produced by activated astrocytes and can promote microglial activation; signaling by S100B through interaction with the multiligand Advanced Glycation End Product-specific Receptor (AGER) has been implicated in brain injury and microglial activation during chronic neurodegeneration. The effects of S100B inhibition were examined in a controlled cortical impact (CCI) model, using S100B knockout mice or administration of neutralizing S100B antibody (BL356).
  • CCI controlled cortical impact
  • BL356 neutralizing S100B antibody
  • neutralizing S100B antibody significantly reduced TBI-induced lesion volume, improved retention memory function and attenuated microglial activation.
  • the neutralizing antibody also significantly reduced sensorimotor deficits and improved neuronal survival in the cortex. The results strongly implicate S100B in TBI-induced neuroinflammation, cell loss and neurological dysfunction, thereby indicating that it is a potential therapeutic target for TBI.
  • Controlled cortical impact (CCI).
  • a CCI-injury device [104, 105] was designed that consists of a microprocessor-controlled pneumatic impactor with a 3.5 mm diameter tip.
  • Male mice were anesthetized with isoflurane (4% induction, 2% maintenance) evaporated in a gas mixture containing 70% N 2 0 and 30% 0 2 and administered through a nose mask.
  • the mouse was placed on a heated pad and a core body temperature was maintained at 37°C.
  • a 10-mm midline incision was made over the skull, the skin and fascia were reflected, and a 4-mm craniotomy was made on the central aspect of the left parietal bone.
  • Moderate injury was induced using an impactor velocity of 6 m/s and deformation depth of 2 mm, as previously detailed [104, 105]. Sham animals underwent the same procedure as injured mice except for the impact. All experiments involving animals were approved by the Institutional Animal Care and Use
  • Novel object recognition Retention or intact memory was assessed by the novel object recognition (NOR) test on post-injury day 21.
  • the apparatus consists of an open field (22.5 cm x 22.5 cm) with two adjacently-located imaginary circular zones, as previously designed [105]. The zones were equally spaced from the sides in the center of the square and designated as "old object” and "novel object” zones using the AnyMaze video tracking system.
  • On post-injury day 20 all animals were placed in the open field for 5 minutes each without any objects present for habituation.
  • Two 5 minute trials were performed on post-injury day 21 : the first (training) trial with 2 old objects in both zones and the second (testing) phase with one old object and one novel object present in the respective zones of the open field. There was an inter- trial interval of 60 minutes.
  • Lesion volume was quantified based on the Cavalieri method of unbiased stereology using Stereoinvestigator software (MBF Biosciences, Williston, VT) [105]. The lesion volume was quantified by outlining the missing tissue on the injured hemisphere using the Cavalieri estimator with a grid spacing of 0.1 mm. Out of the total ninety-six 60 ⁇ sections, every eighth section was analyzed beginning from a random start point.
  • the total number of surviving neurons was quantified in the cortex and thalamus using the optical fractionator method of unbiased stereology at 28 days post-TBI, as described previously [105].
  • the optical dissector had a size of 50 ⁇ by 50 ⁇ in the x and y-axis, respectively with a height of 10 ⁇ and guard-zone of 4 ⁇ from the top of the section.
  • a grid spacing of 400 ⁇ in the x-axis and 400 ⁇ in the y-axis was used, resulting in an area fraction of one-sixty-fourth.
  • CA2 Cornu Ammonis
  • DG dentate gyrus sub-regions of the hippocampus every fourth 60 ⁇ section between -1.22 mm and -2.54 mm from bregma was analyzed beginning from a random start point.
  • the optical dissector had a size of 50 ⁇ by 50 ⁇ in the x and y- axis, respectively with a height of 10 ⁇ and guardzone of 4 ⁇ from the top of the section.
  • CA2 and CA3 sub-regions a grid spacing of 75 ⁇ in the x-axis and 100 ⁇ in the y- axis was used, resulting in an area fraction of one-twelfth.
  • a grid spacing of 175 ⁇ in the x-axis and 100 ⁇ in the y-axis was used, resulting in an area fraction of one- twenty-eighth.
  • the volume of the hippocampal subfield was measured using the Cavalieri estimator method with a grid spacing of 50 ⁇ .
  • the estimated number of surviving neurons in each field was divided by the volume of the region of interest to obtain the neuronal cellular density, expressed as counts/mm 3 .
  • Every fourth 60 ⁇ section was analyzed beginning from a random start point.
  • the optical dissector had a size of 50 ⁇ by 50 ⁇ in the x and y-axis, respectively with a height of 10 ⁇ and guard zone of 4 ⁇ from the top of the section.
  • a grid spacing of 150 ⁇ in the x-axis and 150 ⁇ in the y-axis was used, resulting in an area fraction of one-ninth.
  • the volume of the region of interest was measured using the Cavalieri estimator method with a grid spacing of 100 ⁇ for the cortex.
  • the estimated number of microglia in each morphological class was divided by the volume of the region of interest to obtain the cellular density expressed in counts/mm 3 .
  • Proximity Ligation Assays Formalin fixed, paraffin embedded sections on glass slides collected from 7 day samples from TBI and sham groups were deparaffinized, rehydrated and processed for PLA using a DUOLink II brightfield kit (#92012, OLINK, Uppsala, Sweden) as previously described [52].
  • Primary antibodies included mouse anti-SlOOB (1-50 dilution of #612376, BD Transduction Laboratories) and rabbit anti-AGER (1- 20 dilution of #AP4861a, Abgent).
  • the lesion volume data were analyzed by unpaired Student's t test (genetic knockout study) or one-way ANOVA (anti-SlOOB IgG study).
  • the stereological assessments of neuronal cell loss and microglial activation were analyzed by one-way ANOVA followed by Tukey's post hoc test.
  • In vitro studies were analyzed by one-way ANOVA followed by Tukey's post hoc test.
  • Data were analyzed using SigmaPlot 12 (Systat Software, San Jose, CA) or GraphPad Prism Version 4.0 for Windows (GraphPad Software, San Diego, CA). A p ⁇ 0.05 was considered statistically significant. [00170] Results
  • S100B +/+ mice showed significant impairments in retention memory function (Fig. 18B; p ⁇ 0.05, vs. sham). In contrast, S100B _/" mice showed a significant improvement in retention memory function, when compared to S100B mice (Fig 18B; p ⁇ 0.05, vs. S100B +/+ ).
  • TBI resulted in significant neuronal cell loss in the cortex (Fig. 19B, p ⁇ 0.05, vs. sham), thalamus (Fig. 19C, p ⁇ 0.01 or 0.05, vs. sham), and CA1 hippocampal sub-region (Fig. 19D, p ⁇ 0.01, vs. sham).
  • S100B _/" mice did not show any improvement in neuronal survival in all the assessed brain regions.
  • S100B _/ mice showed significant attenuation in numbers of activated microglia at 28 days (p ⁇ 0.05, vs.S100B +/+ , 34,325.9 ⁇ 5,965.02 counts/mm 3 ), when compared to S100B +/+ mice.
  • TBI resulted in significant neuronal cell loss in the cortex (Fig. 22C, p ⁇ 0.01, vs. sham), thalamus (data not shown), and all hippocampal sub-regions (data not shown).
  • Systemic administration of anti-SlOOB IgG significantly improved neuronal survival in the cortex (Fig. 22C; p ⁇ 0.05, vs.
  • anti-SlOOB IgG significantly attenuated microglial activation at 7 days after injury (p ⁇ 0.001, vs. vehicle and IgG control, 28,571.64 ⁇ 5,702.16 counts/mm 3 ), when compared with vehicle- (74,305.19 ⁇ 7,811.68 counts/mm 3 ), and IgG control -treated (69,807.02 ⁇ 8,947.65 counts/mm 3 ) samples.
  • anti-SlOOB IgG continued to show significant reduction in numbers of activated microglia at 28 days post-injury (p ⁇ 0.05, vs. vehicle, 28,727.6 ⁇ 3901.79 counts/mm 3 ) when compared with vehicle-treated (53,348.67 ⁇ 4,536.09 counts/mm 3 ) samples.
  • Van Eldik LJ, Wainwright MS The Janus face of glial-derived S100B: beneficial and detrimental functions in the brain. Restor Neurol Neurosci 2003, 21 :97-108.
  • Leclerc E, Sturchler E, Vetter SW The S100B/RAGE Axis in Alzheimer's Disease. Cardiovasc Psychiatry Neurol 2010, 2010:539581.
  • Hu J, Castets F, Guevara JL, Van Eldik LJ: SI 00 beta stimulates inducible nitric oxide synthase activity and mRNA levels in rat cortical astrocytes. J Biol Chem 1996, 271 :2543-2547.
  • Goncalves C Induction of S100B secretion in C6 astroglial cells by the major metabolites accumulating in glutaric acidemia type I. Metabolic Brain Disease 2010, 25(2): 191-8. Nogueira MI, Abbas SY, Campos LG, Allemandi W, Lawson P, Takada SH, Azmitia EC: SlOObeta protein expression: gender- and age-related daily changes. Neurochem Res 2009, 34: 1355-1362.
  • POCD postoperative cognitive dysfunction
  • Kitazawa M Cheng D, Tsukamoto MR, Koike MA, Wes PD,ierivko V, Cribbs DH, Laferla FM: Blocking IL-1 Signaling Rescues Cognition, Attenuates Tau Pathology, and Restores Neuronal beta-Catenin Pathway Function in an Alzheimer's Disease Model. J Immunol 2011, 187(12):6539-49.
  • Transgenic mice overexpressing the neurotrophic factor S-100 beta show neuronal cytoskeletal and behavioral signs of altered aging processes: implications for Alzheimer's disease and Down's syndrome. Brain Res 1997, 776:51-60.
  • Motin VG, Nikitin VP, Sherstnev VV Effects of antibodies against protein SI 00b on synaptic transmission and long-term potentiation in CA-1 hippocampal neurons in rats. Bull Exp Biol Med 2002, 133 : 110-113.
  • Minogue AM Age- associated dysregulation of microglial activation is coupled with enhanced blood-brain barrier permeability and pathology in APP/PSl mice. Neurobiol Aging 2014, 35(6): 1442- 52.
  • Tyrosinase-related protein 1 mRNA expression in lymph node metastases predicts overall survival in high-risk melanoma patients, Br J Cancer 108, 1641-1647.
  • Enteroglial-derived S100B protein integrates bacteria-induced Toll-like receptor signalling in human enteric glial cells, Gut 63, 105-115.
  • metabotropic glutamate receptor 5 modulates microglial reactivity and neurotoxicity by inhibiting NADPH oxidase. J. Biol. Chem. 2009; 284(23): 15629-39.

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Abstract

L'invention concerne de nouveaux anticorps qui présentent une spécificité de liaison pour la protéine S100B, ainsi que des variantes et des fragments de ceux-ci qui conservent la spécificité de liaison des anticorps à partir desquels ils sont dérivés, collectivement appelés agents de liaison S100B. L'invention concerne également des procédés d'utilisation desdits anticorps dans le traitement et la prévention de maladies associées à la S100B. L'invention concerne également des procédés d'utilisation desdits anticorps dans le dépistage et le diagnostic de maladies associées à la S100B chez des sujets.
PCT/US2016/022104 2015-03-13 2016-03-11 Anticorps ciblant la protéine s100b et procédés d'utilisation WO2016149116A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181908A (zh) * 2021-07-23 2022-03-15 无锡傲锐东源生物科技有限公司 抗人s100b蛋白鼠单克隆抗体及其应用
CN116903739A (zh) * 2023-03-28 2023-10-20 中国人民解放军军事科学院军事医学研究院 一种抗s100b蛋白的抗体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007090807A1 (fr) * 2006-02-06 2007-08-16 Metheresis Translational Research S.A. Anticorps monoclonal anti-met, fragments et vecteurs de cet anticorps destinés à traiter des tumeurs, et produits associés
EP2127683A1 (fr) * 2008-05-29 2009-12-02 Metheresis Translational Research SA Anticorps monoclonal anti-met, fragments et dérivés correspondants pour une utilisation dans l'imagerie de tumeurs, compositions et kits correspondants
WO2010119704A1 (fr) * 2009-04-17 2010-10-21 Immunas Pharma, Inc. Anticorps qui se lient spécifiquement aux oligomères a bêta et leur utilisation
US20110305699A1 (en) * 2010-06-15 2011-12-15 National Cheng Kung University Treating Oral Cancer with Anti-IL-20 Antibody
US8858949B2 (en) * 2009-08-06 2014-10-14 Immunas Pharma, Inc. Antibodies that specifically bind to a beta oligomers and use thereof
WO2014194329A1 (fr) * 2013-05-31 2014-12-04 Banyan Biomarkers, Inc. Marqueur neuronal spécifique s100β pour dosages de biomarqueurs et dispositifs utilisés dans la détection d'une affection neurologique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007090807A1 (fr) * 2006-02-06 2007-08-16 Metheresis Translational Research S.A. Anticorps monoclonal anti-met, fragments et vecteurs de cet anticorps destinés à traiter des tumeurs, et produits associés
EP2127683A1 (fr) * 2008-05-29 2009-12-02 Metheresis Translational Research SA Anticorps monoclonal anti-met, fragments et dérivés correspondants pour une utilisation dans l'imagerie de tumeurs, compositions et kits correspondants
WO2010119704A1 (fr) * 2009-04-17 2010-10-21 Immunas Pharma, Inc. Anticorps qui se lient spécifiquement aux oligomères a bêta et leur utilisation
US8858949B2 (en) * 2009-08-06 2014-10-14 Immunas Pharma, Inc. Antibodies that specifically bind to a beta oligomers and use thereof
US20110305699A1 (en) * 2010-06-15 2011-12-15 National Cheng Kung University Treating Oral Cancer with Anti-IL-20 Antibody
WO2014194329A1 (fr) * 2013-05-31 2014-12-04 Banyan Biomarkers, Inc. Marqueur neuronal spécifique s100β pour dosages de biomarqueurs et dispositifs utilisés dans la détection d'une affection neurologique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHLOMCHIK, M ET AL.: "Anti-DNA Antibodies from Autoimmune Mice Arise by Clonal Expansion and Somatic Mutation.", JOUMAL OF EXPERIMENTAL MEDICINE., vol. 171, 1 January 1990 (1990-01-01), pages 265 - 267, XP001199896 *

Cited By (4)

* Cited by examiner, † Cited by third party
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CN114181908A (zh) * 2021-07-23 2022-03-15 无锡傲锐东源生物科技有限公司 抗人s100b蛋白鼠单克隆抗体及其应用
CN114181908B (zh) * 2021-07-23 2023-11-17 无锡傲锐东源生物科技有限公司 抗人s100b蛋白鼠单克隆抗体及其应用
CN116903739A (zh) * 2023-03-28 2023-10-20 中国人民解放军军事科学院军事医学研究院 一种抗s100b蛋白的抗体及其应用
CN116903739B (zh) * 2023-03-28 2023-12-15 中国人民解放军军事科学院军事医学研究院 一种抗s100b蛋白的抗体及其应用

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