Classifications

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  • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
  • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
• • 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
• • 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
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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  • 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
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/567—Framework region [FR]
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  • 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
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  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present invention relates to Toll-Like Receptor 3 (TLR3) antibody antagonists, polynucleotides encoding TLR3 antibody antagonists or fragments thereof, and methods of making and using the foregoing.
• TLR3 Toll-Like Receptor 3
• TLRs Toll-like receptors
• the plasma membrane localized TLRs, TLR1, TLR2, TLR4 and TLR6 recognize ligands including protein or lipid components of bacteria and fungi.
• the predominantly intracellular TLRs, TLR3, TLR7 and TLR9 respond to dsRNA, ssRNA and unmethylated CpG DNA, respectively.
• Dysregulation of TLR signaling is believed to cause a multitude of problems, and therapeutic strategies are in development towards this axis (Hoffman et al . , Nat. Rev. Drug Discov. 4:879-880, 2005; Rezaei, Int. Immunopharmacol . 6:863-869,
• TLR4 and TLRs 7 and 9 are in clinical development for severe sepsis and lupus, respectively
• TLR3 signaling is activated by dsRNA, mRNA or RNA released from necrotic cells during inflammation or virus infection. TLR3 activation induces secretion of interferons and pro-inflammatory cytokines and triggers immune cell activation and recruitement that are protective during certain microbial infections.
• TLR3 deficiency is associated with decreased survival upon coxsackie virus challenge
• TLR3 signaling has been shown to contribute to morbidity and mortality in certain viral infection models including West Nile, phlebovirus, vaccinia, and influenza A (Wang et al . , Nat. Med. 10:1366-1373, 2004; Gowen et al., J. Immunol. 177:6301-6307, 2006; Hutchens et al., J. Immunol. 180:483-491, 2008; Le Goffic et al . , PloS Pathog. 2 :E53, 2006) .
• TLR3 adopts the overall shape of a solenoid horseshoe decorated by glycans and has 23 tandem units of leucine-rich repeat (LRR) motifs.
• LRR leucine-rich repeat
• the singaling assembly has been proposed to consist of 1 dsRNA and two TLR3 extracellular domains (Leonard et al . , Proc. Natl. Acad. Sci. (USA) 105: 258-263, 2008) .
• TLR3 has been shown to drive pathogenic mechanisms in a spectrum of inflammatory, immune-mediated and autoimmune diseases including, for example, septic shock (Cavassani et al., J. Exp. Med. 205:2609-2621, 2008), acute lung injury (Murray et al . , Am. J. Respir. Crit. Care Med. 178:1227-1237, 2008), rheumatoid arthritis (Kim et al., Immunol. Lett.
• necrotic conditions the release of intracellular content including endogenous mRNA triggers secretion of cytokines, chemokines and other factors that induce local inflammation, facilitate clearance of dead cell remnants and repair the damage. Necrosis often perpetuates inflammatory processes, contributing to chronic or exaggerated
• TLR3 antagonism may be beneficial in a variety of disorders involving chronic or exaggerated inflammation and/or necrosis.
• Down-modulation of TLR3 activation may also represent a novel treatment strategy for oncologic indications including renal cell carcinomas and head and neck squamous cell carcinomas (Morikawa et al., Clin. Cancer Res. 13:5703-5709, 2007; Pries et al., Int. J. Mol. Med. 21:209-215, 2008).
• TLR3 L423F allele encoding a protein with reduced activity has been associated with protection against advanced "dry" age-related macular degeneration (Yang et al . , N. Engl. J. Med. 359:1456-1463, 2008), indicating that TLR3 antagonists may be beneficial in this disease.
• Fig. 1 shows the effect of anti-human TLR3 (huTLR3) mAbs in an NF- ⁇ reporter gene assay.
• Figs. 2A and 2B show the effect (% inhibition) or anti- huTLR3 mAbs in a BEAS-2B assay.
• Figs. 3A and 3B show the effect of anti-huTLR3 mAbs in a NHBE assay.
• Fig. 4 shows the effect of anti-huTLR3 mAbs in a PBMC assay .
• Figs. 5A and 5B show the effect of anti-huTLR3 mAbs in a HASM assay.
• Figs. 6A, 6B and 6C show the binding of anti-huTLR3 mAbs to TLR3 mutants .
• Fig. 7A shows epitopes for mAb 15EVQ (black) and C1068 mAb
• Fig. 7B shows localized H/D exchange perturbation map of TLR3 ECD protein complexed with mAb 15EVQ.
• Figs. 8A and 8B show the effect of rat/mouse anti-mouse
• Fig. 9 shows the effect of the surrogate mAbs (mAb 5429, mAb cl811) in the MEF CXCLlO/IP-10 assay.
• Fig. 10 shows specificity of binding of the surrogate mAb to TLR3.
• Top panel isotype control; bottom panel: mAb cl811.
• Fig. 11 shows effect of the surrogate mAbs on penH level in an AHR model.
• Fig. 12 shows effect of the surrogate mAbs on total neutrophil numbers in BAL fluid in an AHR model.
• Fig. 13 shows effect of the surrogate mAbs on CXCL10/IP- 10 levels in BAL fluid in an AHR model.
• Fig. 14 shows effect of the surrogate mAb on
• Fig. 15 shows effect of the surrogate mAb on A) histopathology scores and B) neutrophil influx in a T-cell transfer model.
• Fig. 16 shows effect of the surrogate mAb on clinical scores in a CIA model.
• Fig. 17 shows effect of the surrogate mAb on the clinical AUC scores in a CIA model.
• Fig. 18 shows effect of the surrogate mAb on the survival of C57BL/6 mice following intranasal administration of influenza A/PR/8/34. mAb dosing began at day -1.
• Fig. 19 shows effect of the surrogate mAb on clinical scores following influenza A/PR/8/34 administration. mAb dosing began at day -1.
• Fig. 20 shows effect of the surrogate mAb on body weight over 14 days after administration of influenza A/PR/8/34. mAb dosing began at day -1.
• Fig. 21 shows effect of the surrogate mAbs on blood glucose levels in (A) WT DIO and (B) TLR3KO DIO animals after glucose challenge.
• Fig. 22 shows effect of the surrogate mAb on insulin levels in WT DIO animals.
• Fig. 23 shows effect of mAb 15EVQ on (A) NTHi and (B) rhinovirus induced CXCLlO/IP-10 and CCL5/RANTES levels in NHBE cells.
• Fig. 24 shows effect of mAb 15EVQ on (A) sICAM-1 levels and (B) viability in HUVEC cells.
• Fig. 25 shows survival of animals following
• Fig. 26 shows clinical scores following administration of the surrogate mAb 3 days post infection with influenza A.
• Fig. 27 shows body weight change of animals following administration of the surrogate mAb 3 days post infection with influenza A.
• Fig. 28 shows the molecular structure of the quaternary complex of huTLR3 ECD with Fab 12QVQ/QSV, Fab 15EVQ and Fab cl068 in A. in ribbon and surface representations.
• the TLR3 ECD is in light gray with the N-terminus labeled N; all Fab molecules are shown in dark gray in ribbons representation.
• B. The epitopes are colored light gray and labeled on the TLR3 ECD as for the Fabs in A.
• the Fab 12QVQ/QSV, Fab cl068 and Fab 15EVQ are abbreviated to Fabl2, Fabl068 and Fabl5, respectively in the labels for clarity.
• Fig 29 Shows a mechanism of neutralization by Fab 15EVQ .
• A. dsRNA TLR3 signaling unit (SU) is shown with the Fab 15EVQ epitope highlighted (light gray) in one of the two TLR3 ECD (light and dark gray, and labeled TLR3) .
• the dsRNA ligand is shown as a double helix in light gray.
• Fig. 30 shows a mechanism of Fab 12QVQ/QSV and Fab cl068 and clustering of TLR3 signaling units (SU) .
• 12QVQ/QSV and Fab cl068 can bind (or co-bind) a single SU.
• Fab 12QVQ/QSV and Fab cl068 prevents SU clustering due to steric clashes between the antibodies and neighboring SUs.
• the two left-pointing arrows qualitatively represent different degrees of separation of SUs due to the antibodies (bottom arrow for Fab 12QVQ/QSV and top arrow for Fab cl068) .
• Fig. 31 shows the correspondence between sequential, Rabat, and Chothia numbering for exemplary antibodies. The CDRs and HVs are highlighted in gray.
• Fig. 32 shows alignment of VL of mAb 15EVQ with human Vkl frameworks. Chothia hypervariable loops are underlined, paratope residues double underlined and the framework differences highlighted in gray. The VKI genes are *01 alleles unless otherwise indicated. Residue numbering is sequential .
• Fig. 33 shows alignment of VH of mAb 15EVQ with human
• Fig. 34 shows alignment of VL of mAb 12QVQ/QSV with human Vk3 frameworks. Sequence features indicated as in Fig. 32.
• Fig. 35 shows alignment of VL and VH of mAb 15EVQ or mAb
• One aspect of the invention is an isolated antibody or fragment thereof, wherein the antibody binds toll-like receptor 3 (TLR3) amino acid residues K416, K418, L440, N441, E442, Y465, N466, K467, Y468, R488, R489, A491, K493, N515, N516, N517, H539, N541, S571, L595, and K619 of SEQ ID NO: 2.
• TLR3 toll-like receptor 3
• Another aspect of the invention is an isolated antibody or fragment thereof, wherein the antibody binds toll-like receptor 3 (TLR3) amino acid residues S115, D116, K117, A120, K139, N140, N141, V144, K145, T166, Q167, V168, S188, E189, D192, A195, and A219 of SEQ ID NO: 2.
• TLR3 toll-like receptor 3
• Another aspect of the invention is an isolated antibody having a heavy chain variable region and a light chain variable region or fragment thereof, wherein the antibody binds TLR3 having an amino acid sequence shown in SEQ ID NO: 2 with the heavy chain variable region Chothia residues W33, F50, D52, D54, Y56, N58, P61, E95, Y97, Y100, and DIOOb and the light chain variable region Chothia residues Q27, Y32, N92, T93, L94, and S95.
• Another aspect of the invention is an isolated antibody having a heavy chain variable region and a light chain variable region or fragment thereof, wherein the antibody binds TLR3 having an amino acid sequence shown in SEQ ID NO: 2 with the heavy chain variable region Chothia residues N31a, Q52, R52b, S53, K54, Y56, Y97, P98, F99, and Y100, and the light chain variable region Chothia residues G29, S30, Y31, Y32, E50, D51, Y91, D92, and D93.
• Another aspect of the invention is an isolated antibody reactive with TLR3, wherein the antibody has at least one of the following properties:
• a. binds to human TLR3 with a Kd fo ⁇ 10 nM; b. reduces human TLR3 biological activity in an in vitro poly(I:C) NF-kB reporter gene assay >50% at 1 ⁇ g/ml;
• c. inhibits >60% of IL-6 or CXCLlO/IP-10 production from BEAS-2B cells stimulated with ⁇ 100 ng/ml poly(I:C) at 10 ⁇ g/ml;
• Another aspect of the invention is an isolated antibody reactive with TLR3 that competes for TLR3 binding with a monoclonal antibody, wherein the monoclonal antibody comprises the amino acid sequences of certain heavy chain complementarity determining regions (CDRs) 1, 2 and 3, the amino acid sequences of certain light chain CDRs 1, 2 and 3, the amino acid sequences of certain heavy chain variable regions (VH) or the amino acid sequence of certain light chain variable regions (VL) .
• CDRs heavy chain complementarity determining regions
• Another aspect of the invention is an isolated antibody reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises the amino acid sequences of certain heavy chain complementarity determining regions (CDRs) 1, 2 and 3 and the amino acid sequences of certain light chain CDRs 1, 2 and 3.
• CDRs heavy chain complementarity determining regions
• Another aspect of the invention is an isolated antibody reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises the amino acid sequences of certain heavy chain variable regions (VH) and the amino acid sequences of certain light chain variable regions (VL) .
• Another aspect of the invention is an isolated antibody reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises the amino acid sequence of certain heavy chains and the amino acid sequence of certain light chains.
• Another aspect of the invention is an isolated antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 124, 125, 126, 127, 128, 129, 159, 198, 200, 202, 164, 212, 213, 214, 215 or 216.
• Another aspect of the invention is an isolated antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 102, 130, 131, 132, 133, 134, 135, 160, 204, 206, 208, 220, 166 or 168.
• Another aspect of the invention is an isolated antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 155, 156, 157, 158, 203, 205, 207, 165, 167, or 227.
• Another aspect of the invention is an isolated
• polynucleotide encoding an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 124, 125, 126, 127, 128, 129, 159, 198, 200, 202, 164, 212, 213, 214, 215 or 216.
• Another aspect of the invention is an isolated
• polynucleotide encoding an antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 122, 123, 197, 199, 201, 163, 209, 210, 211, or 225.
• Another aspect of the invention is an isolated
• polynucleotide encoding an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 102, 130, 131, 132, 133, 134, 135, 160, 204, 206, 208, 220, 166 or 168.
• Another aspect of the invention is an isolated
• polynucleotide encoding an antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 155, 156, 157, 158, 203, 205, 207, 165, 167, or 227.
• Another aspect of the invention is a pharmaceutical composition
• a pharmaceutical composition comprising the isolated antibody of the invention and a pharmaceutically acceptable carrier.
• Another aspect of the invention is a vector comprising at least one polynucleotide of the invention.
• Another aspect of the invention is a host cell
• Another aspect of the invention is a method of making an antibody reactive with TLR3 comprising culturing the host cell of the invention and recovering the antibody produced by the host cell.
• Another aspect of the invention is a method of treating or preventing an inflammatory condition comprising
• Another aspect of the invention is a method of treating or preventing a systemic inflammatory condition comprising administering a therapeutically effective amount of the isolated antibody of the invention to a patient in need thereof for a time sufficient to treat or prevent the systemic inflammatory condition.
• Another aspect of the invention is a method of treating type II diabetes comprising administering a therapeutically effective amount of the isolated antibody of the invention to a patient in need thereof for a time sufficient to treat type II diabetes.
• Another aspect of the invention is a method of treating hyperglycemia comprising administering a therapeutically effective amount of the isolated antibody of the invention to a patient in need thereof for a time sufficient to treat the hyperglycemia .
• Another aspect of the invention is a method of treating hyperinsulinemia comprising administering a therapeutically effective amount of the isolated antibody of the invention to a patient in need thereof for a time sufficient to treat the insulin resistance.
• Another aspect of the invention is a method of treating or preventing viral infections comprising administering a therapeutically effective amount of the isolated antibody of the invention to a patient in need thereof for a time sufficient to treat or prevent viral infections.
• antagonist means a molecule that partially or completely inhibits, by any mechanism, an effect of another molecule such as a receptor or
• TRL3 antibody antagonist or an antibody “reactive with TLR3” describes an antibody that is capable of, directly or indirectly, substantially
• TLR3 biological activity or TLR3 receptor activation can be counteracting, reducing or inhibiting TLR3 biological activity or TLR3 receptor activation.
• an antibody reactive with TLR3 can bind directly to TLR3 and neutralize TLR3 activity, i.e, block TLR3 signaling to reduce cytokine and chemokine release or NF- ⁇ activation.
• antibodies as used herein is meant in a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies and antibody fragments.
• antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen.
• Intact antibodies are heterotetrameric glycoproteins, composed of two identical light chains and two identical heavy chains.
• each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
• Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
• Each heavy chain has at one end a variable domain (variable region) (VH) followed by a number of constant domains
• Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain.
• Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa ( ⁇ ) and lambda ( ⁇ ) , based on the amino acid sequences of their constant domains.
• Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
• IgA and IgG are further sub-classified as the isotypes IgAi, IgA 2 , IgGi, IgG 2 , IgG 3 and IgG 4 .
• antibody fragments means a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody.
• antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments, diabodies, single chain antibody molecules and multispecific antibodies formed from at least two intact antibodies.
• An immunoglobulin light chain variable region or heavy chain variable region consists of a "framework" region interrupted by three "antigen-binding sites".
• the antigen- binding sites are defined using various terms as follows: (i) the term Complementarity Determining Regions (CDRs) is based on sequence variability (Wu and Rabat, J. Exp. Med. 132:211- 250, 1970) . Generally, the antigen-binding site has six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) , and three in the VL (LCDR1, LCDR2, LCDR3) (Rabat et al . , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) The term “hypervariable region", "HVR", or “HV” refers to the regions of an antibody variable domain which are
• the "IMGT-CDRs" as proposed by Lefranc (Lefranc et al . , Dev. Comparat. Immunol. 27:55-77, 2003) are based on the comparison of V domains from immunoglobulins and T-cell receptors.
• the International ImMunoGeneTics (IMGT) database (http : //www_imgt_org) provides a standardized numbering and definition of these regions.
• the antigen-binding site can also be delineated based on Specificity Determining Residue Usage (SDRU) (Almagro, Mol. Recognit. 17:132-143, 2004), where
• SDR Specificity Determining Residues
• SDRU is a precise measure of a number and distribution of SDR for different types of antigens as defined by analyses of crystal structures of antigen-antibody complexes,
• the antigen-binding site can also be defined as the antibody paratope residues identified from crystal structure of the antigen-antibody complex.
• composite sequences as used herein means an antigen-binding site defined to include all amino acid residues delineated individually by Rabat, Chothia or IMGT, or any other suitable antigen-binding site delineation.
• Chothia residues as used herein are the antibody VL and VH residues numbered according to Al-Lazikani (Al- Lazikani et al., J. Mol. Biol. 273:927-48, 1997) .
• Framework or “framework sequences” are the remaining sequences of a variable region other than those defined to be antigen-binding site.
• the framework is typically divided into four regions, FR1, FR2, FR3, and FR3, which form a scaffold for the three antigen-binding sites in each variable reigon. Because the antigen-binding site can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined.
• a light chain variable region kappa 1 (VKI) framework or “VKI” as used herein refers to a framework having an amino acid sequence encoded by any of the human VKI functional genes or alleles thereof.
• exemplary functional human Vkl genes are IGKV1-5*01, IGKV1-6*01, IGKV1-8*01, IGKV1-9*01, IGKV1-12*01, IGKV1-13*02, IGKV1-16*01, IGKV1-17*01, IGKV1- 27*01, IGKV1-33*01, IGKV1-37*01, IGKV1-39*01, IGKV1D-8*01, IGKV1D-12*01, IGKV1D-13*01, IGKV1D-16*01, IGKV1D-17*01, IGKV1D-33*01, IGKV1D-37 * 01 , IGKV1D-39*01
• a light chain variable region lambda 3 ( ⁇ 3) framework or “ ⁇ 3” as used herein refers to a framework having an amino acid sequence encoded by any of the human ⁇ 3 functional genes or alleles thereof.
• exemplary functional human ⁇ 3 genes are IGLV3-1*01, IGLV3-9*01, IGLV3-10*01, IGLV3-12*01, IGLV3-
• a heavy chain variable region Vh5 framework or "Vh5" as used herein refers to a framework having an amino acid sequence encoded by any of the human Vh5 functional genes or alleles thereof.
• Exemplary functional human Vh5 genes are IGHV5-51*01 and IGHV5-1*01.
• a heavy chain variable region Vh6 framework or “Vh6” as used herein refers to a framework having an amino acid sequence encoded by any of the human Vh6 functional genes or alleles thereof.
• An exemplary functional human Vh6 gene is IGHV6-1*01.
• a light chain kappa J-region (JK) framework or “JK” as used herein refers to a framework having an amino acid sequence encoded by any of the human JK functional genes or alleles thereof.
• Exemplary functional human VK genes are
• a light chain lambda J-region (J ) framework or " ⁇ ” as used herein refers to a framework having an amino acid sequence encoded by any of the human ⁇ functional genes or alleles thereof.
• exemplary functional human ⁇ genes are
• a heavy chain J-region (Jh) framework or “Jh” as used herein refers to a framework having an amino acid sequence encoded by any of the human Jh functional genes or alleles thereof.
• exemplary functional human Jh genes are IGHJ1,
• IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6 are examples of IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6.
• “Germline genes” or “antibody germline genes” as used herein are immunoglobulin sequences encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin.
• “Scaffold” as used herein refers to amino acid sequences of light or heavy chain variable regions encoded by human germline genes. Thus, the scaffold encompasses both the framework and the antigen-binding site.
• antigen means any molecule that has the ability to generate antibodies either directly or indirectly. Included within the definition of "antigen” is a protein-encoding nucleic acid.
• homolog means protein sequences having between 40% and 100% sequence identity to a reference sequence.
• homologs of human TLR3 include polypeptides from other species that have between 40% and 100% sequence identity to a known human TLR3 sequence. Percent identity between two peptide chains can be determined by pairwise alignment using the default settings of the AlignX module of
• TLR3 human TLR3 (huTLR3) and its homologs.
• the nucleotide and amino acid sequences of the full length huTLR3 are shown in SEQ ID NOs : 1 and 2, respectively.
• the nucleotide and amino acid sequences of the huTLR3 extracellular domain (ECD) are shown in SEQ ID NOs : 3 and 4, respectively.
• substantially identical means that the two antibody or antibody fragment amino acid sequences being compared are identical or have "insubstantial differences". Insubstantial differences are substitutions of 1, 2, 3, 4, 5 or 6 amino acids in an antibody or antibody fragment amino acid sequence . Amino acid sequences
• sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Percent identity can be determined as described above. Exemplary peptide chains being compared are heavy or light chain variable regions.
• inflammatory condition means a localized response to cellular injury that is mediated in part by the activity of cytokines, chemokines, or
• inflammatory cells e.g., neutrophils, monocytes,
• inflammatory pulmonary condition means an inflammatory condition affecting or associated with the lungs.
• mAb monoclonal antibody
• Monoclonal antibodies are highly specific, typically being directed against a single antigenic determinant.
• the modifier "monoclonal” indicates the substantially homogeneous character of the antibody and does not require production of the antibody by any particular method.
• murine mAbs can be made by the hybridoma method of Kohler et al . , Nature 256:495-497, 1975.
• Chimeric mAbs containing a light chain and heavy chain variable region derived from a donor antibody (typically murine) in association with light and heavy chain constant regions derived from an acceptor antibody (typically another mammalian species such as human) can be prepared by the method disclosed in U.S. Pat. No.
• Human-adapted mAbs having CDRs derived from a non-human donor immunoglobulin (typically murine) and the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins can be prepared by techniques known to those skilled in the art such as that disclosed in U.S. Pat. No. 5,225,539. Human
• framework sequences useful for human-adaptation can be selected from relevant databases by those skilled in the art.
• human-adapted mAbs can be further modified by incorporating altered framework support residues to preserve binding affinity by techniques such as those disclosed in Queen et al., Proc. Natl. Acad. Sci . (USA), 86:10029-10032, 1989 and Hodgson et al., Bio/Technology, 9:421, 1991.
• Fully human mAbs lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice by techniques referenced in, e.g., Lonberg et al . , Nature
• Human mAbs can also be prepared and optimized from phage display libraries by techniques referenced in, e.g., Knappik et al . , J. Mol . Biol. 296:57-86, 2000; and Krebs et al., J. Immunol. Meth. 254:67-84 2001.
• Fragments of antibodies e.g., Fab, F(ab')2, Fd, and dAb fragments may be produced by cleavage of the antibodies or by recombinant engineering. For example, Fab and F(ab')2 fragments may be generated by treating the antibodies with an enzyme such as pepsin .
• epitope means a portion of an antigen to which an antibody specifically binds.
• Epitopes usually consist of chemically active (such as polar, non- polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
• An epitope can be linear in nature or can be a discontinous epitope, e.g., a conformational epitope, which is formed by a spatial
• a conformational epitope includes epitopes resulting from folding of an antigen, where amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3- dimensional space.
• paratope refers to a portion of an antibody to which an antigen specifically binds.
• a paratope can be linear in nature or can be discontinuous, formed by a spatial relationship between non-contiguous amino acids of an antibody rather than a linear series of amino acids.
• a "light chain paratope” and a “heavy chain paratope” or “light chain paratope amino acid residues” and “heavy chain paratope amino acid residues” refer to antibody light chain and heavy chain residues in contact with an antigen, respectively .
• the term "specific binding” as used herein refers to antibody binding to a predetermined antigen with greater affinity than for other antigens or proteins.
• the antibody binds with a dissociation constant (K D ) of 10 ⁇ 7 M or less, and binds to the predetermined antigen with a K D that is at least twofold less than its K D for binding to a nonspecific antigen (e.g., BSA, casein, or any other specified polypeptide) other than the predetermined antigen.
• K D dissociation constant
• an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen” or "an antigen specific antibody” e.g. a TLR3 specific antibody.
• TLR3 biological activity or "TLR3 activation'' as used herein refers to any activity occurring as a result of ligand binding to TLR3.
• TLR3 ligands include dsRNA, poly(I:C), and endogenous mRNA, e.g., engodenous mRNA released from necrotic cells.
• An exemplary TLR3 activation results in activation of NF- ⁇ in response to the TLR3 ligand.
• NF- ⁇ activation can be assayed using a reporter- gene assay upon induction of the receptor with poly(I:C) (Alexopoulou et al., Nature 413:732-738, 2001; hacker et al . , EMBO J. 18:6973-6982, 1999).
• poly(I:C) Alexopoulou et al., Nature 413:732-738, 2001;hacker et al . , EMBO J. 18:6973-6982, 1999.
• TLR3- mediated IRF activation results in activation of interferon response factors (IRF-3, IRF-7) in response to the TLR3 ligand.
• IRF-3, IRF-7 interferon response factors
• TLR3- mediated IRF activation can be assayed using a reporter gene driven by an interferon-stimulated response element (ISRE).
• ISRE interferon-stimulated response element
• Another exemplary TLR3 activation results in secretion of pro-inflammatory cytokines and chemokines, for example TNF-a, IL-6, IL-8, IL-12, CXCL5/IP-10 and RANTES .
• circulation can be measured using well-known immunoassays, such as an ELISA immunoassay.
• the present invention provides antibody antagonists capable of inhibiting TLR3 biological activity and uses of such antibodies.
• TLR3 antagonists may have the properties of binding TLR3 and inhibiting TLR3 activation.
• Exemplary mechanisms by which TLR3 activation may be inhibited by such antibodies include in vitro, in vivo or in situ inhibition of ligand binding to TLR3, inhibition of receptor dimerization, inhibition of TLR3 localization to the endosomal compartment, inhibition of kinase activity of downstream signaling pathways, or inhibition of TLR3 mRNA transcription.
• Other antibody antagonists capable of inhibiting TLR3 activation by other mechanisms are also within the scope of the various aspects and embodiments of the invention. These antagonists are useful as research reagents, diagnostic reagents and therapeutic agents.
• Antibody diversity in a natural system, is created by the use of multiple germline genes encoding variable regions and a variety of somatic events.
• the somatic events include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete VH region, and the recombination of variable and joining gene segments to make a complete VL region.
• the recombination process itself can be imprecise, resulting in the loss or addition of amino acids at the V(D)J junctions.
• the invention provides novel antigen-binding sites and immunoglobulin chains derived from human immunoglobulin gene libraries.
• the structure for carrying an antigen-binding site is generally an antibody heavy or light chain or portion thereof, where the antigen-binding site is located to a naturally occurring antigen-binding site as determined as described above.
• the invention provides an isolated antibody or fragment thereof reactive with TLR3 comprising both a heavy chain and a light chain variable region and wherein the antibody comprises the heavy chain complementarity determining region (CDR) amino acid sequences 1, 2 and 3 (HCDR1, HCDR2 and HCDR3) and the light chain complementarity determining region (CDR) amino acid sequences 1, 2 and 3 (LCDR1, LCDR2 and LCDR3) as shown in Table la.
• CDR heavy chain complementarity determining region
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises a HCDR2 amino acid sequence as shown in SEQ ID NO: 192, wherein the HCDR2 of SEQ ID NO: 192 is defined as shown in Formula (I) :
• Xaa 6 may be Arg or Lys
• Xaa 7 may be Tyr, His or Ser
• Xaa 8 may be Met, Arg or Tyr
• Xaa 9 may be Lys or Arg.
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises a HCDR2 amino acid sequence as shown in SEQ ID NO: 194, wherein the HCDR2 of SEQ ID NO: 194 is defined as shown in Formula (III) :
• Xaais may be Lys, Thr or lie;
• Xaai 6 may be Asn or Asp
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises a HCDR2 amino acid sequence as shown in SEQ ID NO: 196, wherein the HCDR2 of SEQ ID NO: 196 is defined as shown in Formula (V) :
• Xaa 2 4 may be Phe or Arg
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises a LCDR3 amino acid sequence as shown in SEQ ID NO: 191, wherein the LCDR3 of SEQ ID NO: 191 is defined as shown in Formula (II) : Xaai-S-Y-D-Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 -T-V,
• Xaai may be Ala, Gin, Gly or Ser;
• Xaa 2 may be Gly, Glu or Ser
• Xaa 3 may be Asp or Asn
• Xaa 4 may be Glu or Ser
• Xaa 5 may be Phe, Ala or Leu.
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises a LCDR3 amino acid sequence as shown in SEQ ID NO: 193, wherein the LCDR3 of SEQ ID NO: 193 is defined as shown in Formula (IV) :
• Xaaio may be Gin or Ser
• Xaan may be Thr, Glu or Asp
• Xaai 3 may be Tyr or Phe
• Xaai 4 may be Ser, Asn or Gin.
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises a LCDR3 amino acid sequence as shown in SEQ ID NO: 195, wherein the LCDR3 of SEQ ID NO: 195 is defined as shown in Formula (VI) :
• Xaais may be Tyr, Gly or Ala
• Xaaig may be Gly, Glu or Asn
• Xaa 2 2 may be Ser or Leu
• Xaa 2 3 may be lie, Ser, Pro or Tyr .
• the invention also provides an isolated antibody or fragment reactive with TLR3 having the heavy chain
• CDR complementarity determining region amino acid sequences 1,2 and 3 (HCDR1, HCDR2 and HCDR3) and light chain
• CDR complementarity determining region
• Antibodies whose antigen-binding site amino acid sequences differ insubstantially from those shown in Table la are encompassed within the scope of the invention. Typically, this involves one or more amino acid substitutions with an amino acid having similar charge, hydrophobic, or stereochemical characteristics.
• substitutions in the framework regions in contrast to antigen- binding sites may also be made as long as they do not adversely affect the properties of the antibody. Substitutions may be made to improve antibody properties, for example stability or affinity. One, two, three, four, five or six substitutions can be made to the antigen binding site. 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of the framework residues can be substituted, as long as the resulting antibody retains desired properties.
• Substantial modifications in the functional and/or chemical characteristics of the molecules may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (1) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical
• a "conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
• any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al . , Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasaki et al . , Adv. Biophys . 35:1-24, 1998) . Desired amino acid
• amino acid amino acid
• substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein. Exemplary amino acid substitutions are shown in Table lb.
• conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems.
• Amino acid substitutions can be done for example by PCR mutagenesis (US Pat. No. 4,683,195).
• Libraries of variants can be generated using well known methods, for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (ACDEGKNRSYW) , and screening the libararies for variants with desired
• the antigen-binding site residues of the antibodies of the invention and subsequently the framework residues may vary slightly for each heavy and light chain.
• Table 2a and 2b shows the antigen-binding site residues of exemplary antibodies of the invention delineated according to Rabat, Chothia and IMGT, and their composite sequences.
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region and wherein the antibody comprises the amino acid sequences of the heavy chain variable (VH) and the light chain variable (VL) regions and also provides for each isolated heavy chain variable and light chain variable region as shown in Table 3a.
• F17, F18 and F19 represent antibody variants comprising consensus amino acid sequences for families 17, 18 and 19, respectively (see Example 1) .
• variable regions one from a heavy and one from a light chain
• alternative embodiments may comprise single heavy or light chain variable regions.
• the single variable region can be used to screen for a second variable region capable of forming a two-domain specific antigen-binding fragment capable of, for example, binding to TLR3.
• the screening may be accomplished by phage display screening methods using for example hierarchical dual combinatorial approach disclosed in PCT Publ. No. WO92/01047.
• the invention provides an isolated antibody or fragment reactive with TLR3 comprising both a heavy chain variable region and a light chain variable region having amino acid sequences at least 95% identical to the variable region amino acid sequences shown in Table 3a.
• the invention provides an isolated antibody having certain heavy chain and light chain amino acid sequences as shown in Table 3b. Another aspect of the invention is isolated
• polynucleotides encoding any of the antibodies of the invention or their complement. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the antibody antagonists of the invention are also within the scope of the invention. Table 2b.
• Exemplary antibody antagonists may be antibodies of the
• antibody antagonists can be post-translationally modified by processes such as glycosylation, isomerization,
• PEG polyethylene glycol
• the antibodies of the invention can be conjugated to polyethylene glycol (PEGylated) to improve their
• Conjugation can be carried out by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not interfering with function.
• IgG4 isotype heavy chains contain a Cys-Pro-Ser-Cys (CPSC) motif in the hinge region capable of forming either inter- or intra-heavy chain disulfide bonds, i.e., the two Cys residues in the CPSC motif may disulfide bond with the corresponding Cys residues in the other heavy chain (inter) or the two Cys residues within a given CPSC motif may disulfide bond with each other (intra) .
• CPSC Cys-Pro-Ser-Cys
• the heavy:light chain (H:L) pairs in those IgG4 molecules with intra-heavy chain bonds in the hinge region are not covalently associated with each other, they may dissociate into H:L monomers that then reassociate with H:L monomers derived from other IgG4 molecules forming bispecific, heterodimeric IgG4 molecules.
• H:L heavy:light chain
• the two Fabs of the antibody molecule differ in the epitopes that they bind.
• the antibodies of the invention will comprise an IgG4 Fc domain with a S to P mutation in the CPSC motif.
• the location of the CPSC motif is typically found at residue 228 of a mature heavy chain but can change depending on CDR lengths .
• sites can be removed that affect binding to Fc receptors other than an FcRn salvage receptor in the
• the Fc receptor binding regions involved in ADCC activity can be removed in the antibodies of the invention.
• mutation of Leu234/Leu235 in the hinge region of IgGl to L234A/L235A or Phe235/Leu236 in the hinge region of IgG4 to P235A/L236A minimizes FcR binding and reduces the ability of the
• the antibodies of the invention will comprise an IgG4 Fc domain with P235A/L236A mutations.
• antibodies having P235A/L236A mutations are antibodies having heavy chain amino acid sequences shown in SEQ ID NOs: 218, 219 or 220.
• Fully human, human-adapted, humanized and affinity- matured antibody molecules or antibody fragments are within the scope of the invention as are fusion proteins and chimeric proteins.
• Antibody affinity towards an antigen may be improved by rational design or random affinity maturation using well-known methods such as random or directed
• mutagenesis or employing phage display libraries.
• substitutions can be made to the Vernier Zone residues that mostly reside in the framework region or to the ""Affinity Determining Residues", ADRs, to modulate affinity of an antibody (US Pat. No. 6,639,055; PCT Publ . No.
• Fully human, human-adapted, humanized, affinity-matured antibody molecules or antibody fragments modified to improve stability, selectivity, cross-reactivity, affinity,
• Stability of an antibody is influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protein/protein interface
• Tm thermal transition midpoint
• DSC differential scanning calorimetry
• the antibody antagonists of the invention may bind TLR3 with a K d less than or equal to about 10 "7 , 10 "8 , 10 “9 , 10 "10 , 10 "11 or 10 "12 M.
• the affinity of a given molecule for TLR3, such as an antibody can be determined experimentally using any suitable method. Such methods may utilize Biacore or
• Antibody antagonists binding a given TLR3 homolog with a desired affinity can be selected from libraries of variants or fragments by techniques including antibody affinity maturation. Antibody antagonists can be identified based on their inhibition of TLR3 biological activity using any suitable method. Such methods may utilize reporter-gene assays or assays measuring cytokine production using well known methods and as described in the application.
• Another embodiment of the invention is a vector comprising at least one polynucleotide of the invention.
• Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides of the invention into a given organism or genetic background by any means .
• Another embodiment of the invention is a host cell comprising any of the polynucleotides of the invention such as a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 124, 125, 126, 127, 128, 129, 159, 198, 200, 202, 164, 212, 213, 214, 215 or 216 or an immunoglobulin light chain variable region having the amino acid sequence shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 122, 123, 197, 199, 201, 163, 209, 210, 211, or 225.
• Another embodiment of the invention is a host cell comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain having the amino acid sequence shown in SEQ ID NO: 102, 130, 131, 132, 133, 134, 135, 160, 204, 206, 208, 220, 166 or 168, or an immunoglobulin light chain having the amino acid sequence shown in SEQ ID NO: 155, 156, 157, 158, 203, 205, 207, 165, 167, or 227.
• Such host cells may be eukaryotic cells, bacterial cells, plant cells or archeal cells. Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins.
• Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC) , Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC) , Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines.
• An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196) .
• Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologies, Walkersville, MD) , CHO-K1 (ATCC CRL-61) or DG44.
• Another embodiment of the invention is a method of making an antibody reactive with TLR3 comprising culturing a host cell of the invention and recovering the antibody produced by the host cell. Methods of making antibodies and purifying them are well known in the art.
• Another embodiment of the invention is a hybridoma cell line that produces an antibody of the invention.
• Another embodiment of the invention is an isolated antibody or fragment thereof, wherein the antibody binds toll-like receptor 3 (TLR3) amino acid residues K416, K418, L440, N441, E442, Y465, N466, K467, Y468, R488, R489, A491, K493, N515, N516, N517, H539, N541, S571, L595, and K619 of SEQ ID NO: 2.
• TLR3 toll-like receptor 3
• Another embodiment is an isolated antibody or fragment thereof, wherein the antibody binds toll-like receptor 3 (TLR3) amino acid residues S115, D116, K117, A120, K139, N140, N141, V144, K145, T166, Q167, V168, S188, E189, D192, A195, and A219 of SEQ ID NO: 2.
• TLR3 toll-like receptor 3
• binding site is first localized to a region on the protein, such as by docking, segment
• mutagenesis or H/D exchange When the structures of both individual components are known, in silico protein-protein docking can be carried out to identify compatible sites of interaction. Co-crystal structure of antibody-antigen complex can be used to identify residues contributing to the epitope and paratope .
• Another embodiment of the invention is an isolated antibody or fragment thereof, wherein the antibody binds TLR3 having an amino acid sequence shown in SEQ ID NO: 2 with the heavy chain variable region Chothia residues W33, F50, D52, D54, Y56, N58, P61, E95, Y97, Y100, and DIOOb, and with the light chain variable region Chothia residues Q27, Y32, N92, T93, L94, and S95.
• the heavy chain paratope and the light chain paratope Chothia residues correspond to heavy chain residues W33, F50, D52, D55, Y57, N59, P62, E99, Y101, Y104, and D106 of SEQ ID NO: 216 and light chain residues Q27, Y32, N92, T93, L94, and S95 of SEQ ID NO: 41.
• Another embodiment of the invention is an isolated antibody or fragment thereof, wherein the antibody binds TLR3 having an amino acid sequence shown in SEQ ID NO: 2 with the heavy chain variable region Chothia residues N31a, Q52, R52b, S53, K54, Y56, Y97, P98, F99, and Y100, and with the light chain variable region Chothia residues G29, S30, Y31, Y32, E50, D51, Y91, D92, and D93.
• the heavy chain paratope and the light chain paratope Chothia residues correspond to heavy chain residues N32, Q54, R56, S57, K58, Y60, Y104, P105, F106, and Y107 of SEQ ID NO: 214 and light chain residues G28, S29, Y30, Y31, E49, D50, Y90, D91, and D92 of SEQ ID NO: 211.
• Isolated antibodies having certain paratope residues that bind TLR3 can be made by for example grafting the paratope residues into a suitable scaffold, assembling the engineered scaffolds into full antibodies, expressing the resulting antibodies, and testing the antibodies for binding to TLR3 or for an effect on TLR3 biological activity.
• Exemplary scaffolds are amino acid sequences of human antibody variable regions encoded by human germline genes.
• the scaffolds can be selected based on for example overall sequence homology, % identity between the paratope residues, or canonical structure class identity between the scaffold and an exemplary antibody, such as mAb 15EVQ or mAb
• Exemplary human germline genes that can be used as scaffolds onto which the paratope residues are grafted are the genes encoded by the VKI, ⁇ 3, Vh5, Vh6, JK , J ⁇ , and the Jh frameworks.
• the germline J-regions are used in their entirety or in part to select FR4 sequences.
• the mAb 15EVQ light chain paratope residues can be grafted to a VKI framework encoded by IGKV1-39*01 that is joined directly to the J region sequence encoded by IGKJ1.
• VKI genes can also be used, and the FR4 sequences of other JK genes can be substituted in place of IGKJ1.
• the mAb 15EVQ heavy chain paratope residues can be grafted to a Vh5 framework encoded by IGHV5-51*01, followed by about 11-13 residues, for example 12 residues,
• HCDR3 and the FR4 sequence encoded by IGHJ1 constituting HCDR3 and the FR4 sequence encoded by IGHJ1.
• the 11-13 residues span between the end of the FR3 region ("CAR") and the start of the FR4 region (WGQ for most JH regions) and include 4 defined paratope residues from mAb 15EVQ Vh .
• Sequences from other Vh5 genes can also be used, and the FR4 sequences of other Jh genes can be substituted in place of IGJH1.
• the mAb 12QVQ/QSV light chain paratope residues can be grafted to a ⁇ 3 framework encoded by IGLV3-1*01 that is joined directly to the J region sequence encoded by IGJL2. Sequences of other ⁇ 3 and J ⁇ genes can also be used.
• the length of LCDR3 is maintained at about 9-11 residues, for example 10 residues. These about 9- 11 residues span between the end of the FR3 region ("YYC” for most V lambda scaffolds) and the start of the FR4 region ("FGG” for most JL regions) and include 3 defined paratope residues from mAb 12QVQ/QSV .
• the mAb 12QVQ/QSV heavy chain paratope residues can be grafted to a Vh6 framework encoded by IGHV6-1*01, followed by about 9-11 residues, for example 10 residues, constituting HCDR3, and the FR4 sequence encoded by IGJH1.
• the about 9-11 residues span between the end of the FR3 region ("CAR") and the start of the FR4 region (WGQ for most JH regions) and include 4 defined paratope residues from mAb 12QVQ/QSV Vh .
• the FR4 sequences of other Jh genes can be substituted in place of IGHJ1.
• the binding to TLR3 and biological activity of the resulting antibody can be evaluated using standard methods. Alignments of the mAb 15EVQ and the mAb 12QVQ/QSV light chain variable regions and heavy chain variable regions with the exemplary VKI, Vh5, ⁇ 3, Vh6, JK, ⁇ or Jh genes are shown in Figures 32 - 35.
• the paratope-grafted engineered antibodies can further be modified by substitutions of the Vernier Zone residues or the Affinity Determining Residues to improve antibody properties, for example affinity, as described above.
• the framework amino acid sequence in the paratope-grafted antibody may be 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the the mAb 15EVQ or 12QVQ/QSV framework sequences .
• Sequences from the antigen-binding sites can be grafted in addition to the paratope residues using standard methods. For example, a complete HCDR3 or LCDR3 may be grafted.
• Another aspect of the invention is an isolated antibody or fragment thereof reactive with TLR3 that competes for TLR3 binding with a monoclonal antibody, wherein the monoclonal antibody comprises the amino acid sequences of certain heavy chain complementarity determining regions (CDRs) 1, 2 and 3, the amino acid sequences of certain light chain CDRs 1, 2 and 3, the amino acid sequences of certain heavy chain variable regions (VH) or the amino acid sequence of certain light chain variable regions (VL) .
• CDRs heavy chain complementarity determining regions
• Examplary monoclonal antibodies of the invention are an isolated antibody comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 216 and a light chain variable region amino acid sequence shown in SEQ ID NO: 41, and an antibody comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 214 and a light chain variable region amino acid sequence shown in SEQ ID NO: 211.
• TLR3 Competition between binding to TLR3 can be assayed in vitro using well known methods. For example, binding of MSD Sulfo-TagTM NHS-ester -labeled antibody to TLR3 in the presence of an unlableled antibody can be assessed by ELISA.
• Exemplary antibodies of the invention are mAb 12, mAb 15 and mAb cl811 (see Table 3a) .
• Previously described anti-TLR3 antibodies cl068 and its derivatives described in PCT Publ . No.
• TLR3.7 eBiosciences, cat no 14-9039
• Imgenex IMG-315A Imgenex IMG-315A; generated against human TLR3 amino acids amino acids 55-70, VLNLTHNQLRRLPAAN
• Another aspect of the invention is an isolated antibody reactive with TLR3, wherein the antibody has at least one of the following properties:
• a. binds to human TLR3 with a Kd of ⁇ 10 nM
• b. reduces human TLR3 biological activity in an in vitro poly(I:C) NF-kB reporter gene assay >50% at 1 ⁇ g/ml; c. inhibits >60% of IL-6 or CXCL5/IP-10 production from BEAS-2B cells stimulated with ⁇ 100 ng/ml poly(I:C) at 10 ⁇ g/ml ;
• g. inhibits >20% of poly ( I : C) -induced IFN- ⁇ , IL-6 or IL-12 production by PBMC cells at 1 ⁇ g/ml .
• h. inhibits cynomologus TLR3 biological activity in an in vitro NF-kB reporter gene assay with IC50 ⁇ 10 ⁇ g/ml; or i. inhibits cynomologus TLR3 biological activity in an in vitro ISRE reporter gene assay with IC50 ⁇ 5 ⁇ g/ml.
• TLR3 antagonists of the invention for example TLR3 antibody antagonists, can be used to modulate the immune system. While not wishing to be bound by any particular theory, the antagonists of the invention may modulate the immune system by preventing or reducing ligand binding to TLR3, dimerization of TLR3, TLR3 internalization or TLR3 trafficking. The methods of the invention may be used to treat an animal patient belonging to any classification.
• the antibodies of the invention are useful in antagonizing TLR3 activity, in the treatment of inflammation, inflammatory and metabolic diseases and are also useful in the preparation of a medicament for such treatment wherein the medicament is prepared for administration in dosages defined herein.
• inflammatory conditions, infection-associated conditions or immune-mediated inflammatory disorders that may be prevented or treated by administration of the TLR3 antibody antagonists of the invention include those mediated by cytokines or chemokines and those conditions which result wholly or partially from activation of TLR3 or signaling through the TLR3 pathway.
• inflammatory conditions include sepsis-associated conditions, inflammatory bowel diseases, autoimmune disorders, inflammatory disorders and infection-associated conditions. It is also thought that cancers, cardiovascular and metabolic conditions, neurologic and fibrotic conditions can be prevented or treated by administration of the TLR3 antibody antagonists of the invention. Inflammation may affect a tissue or be systemic.
• Exemplary affected tissues are the respiratory tract, lung, the gastrointestinal tract, small intestine, large intestine, colon, rectum, the cardiovascular system, cardiac tissue, blood vessels, joint, bone and synovial tissue, cartilage, epithelium, endothelium, hepatic or adipose tissue.
• Exemplary systemic inflammatory conditions are cytokine storm or hypercytokinemia, systemic inflammatory response syndrome (SIRS) , graft versus host disease (GVHD) , acute respiratory distress syndrome (ARDS) , severe acute respiratory distress syndrome (SARS) , catastrophic anti-phospholipid syndrome, severe viral infections, influenza, pneumonia, shock, or sepsis .
• SIRS systemic inflammatory response syndrome
• GVHD graft versus host disease
• ARDS acute respiratory distress syndrome
• SARS severe acute respiratory distress syndrome
• catastrophic anti-phospholipid syndrome severe viral infections, influenza, pneumonia, shock, or sepsis .
• Inflammation is a protective response by an organism to fend off an invading agent. Inflammation is a cascading event that involves many cellular and humoral mediators. On one hand, suppression of inflammatory responses can leave a host immunocompromised; hovewer, if left unchecked,
• inflammation can lead to serious complications including chronic inflammatory diseases (e.g. asthma, psoriasis, arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and the like) , septic shock and multiple organ failure.
• chronic inflammatory diseases e.g. asthma, psoriasis, arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and the like
• septic shock e.g. asthma, psoriasis, arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and the like
• these diverse disease states share common inflammatory mediators, such as
• cytokines cytokines, chemokines, inflammatory cells and other mediators secreted by these cells.
• TLR3 activation by its ligands poly(I:C), dsRNA or endogenous mRNA leads to activation of signaling pathways resulting in synthesis and secretion of pro-inflammatory cytokines, activation and recruitment of inflammatory cells, such as macrophages, granulocytes, neutrophils and
• TLR3 induces secretion of IL-6, IL-8, IL-12, TNF-a, MIP-1,
• TLR3 ligand endogenous mRNA is released from necrotic cells during inflammation, and may result in a positive feedback loop to activate TLR3 and perpetuate inflammation and further tissue damage.
• TLR3 antagonists such as TLR3 antibody antagonists, may normalize cytokine secretion, reduce recruitment of inflammatory cells, and reduce tissue damage and cell death. Therefore, TLR3 antagonists have therapeutic potential to treat inflammation and a spectrum of inflammatory conditions.
• inflammatory condition is sepsis-associated condition that may include systemic inflammatory response syndrome (SIRS) , septic shock or multiple organ dysfunction syndrome (MODS) .
• SIRS systemic inflammatory response syndrome
• MODS multiple organ dysfunction syndrome
• dsRNA released by viral, bacterial, fungal, or parasitic infection and by necrotic cells can contribute to the onset of sepsis. While not wishing to be bound by an particular theory, it is believed that treatment with TLR3 antagonists can provide a
• therapeutic benefit by extending survival times in patients suffering from sepsis-associated inflammatory conditions or prevent a local inflammatory event (e.g., in the lung) from spreading to become a systemic condition, by potentiating innate antimicrobial activity, by demonstrating synergistic activity when combined with antimicrobial agents, by
• Such intervention may be sufficient to permit additional treatment (e.g., treatment of underlying infection or reduction of cytokine levels) necessary to ensure patient survival.
• Sepsis can be modeled in animals, such as mice, by the adminstration of D-galactosamine and poly(I:C) .
• D-galactosamine is a hepatotoxin which functions as a sepsis sensitizer and poly(I:C) is a sepsis-inducing molecule that mimics dsRNA and activates TLR3.
• TLR3 antagonist treatment may increase animal survival rates in a murine model of sepsis, and thus TLR3 antagonists may be useful in the treatment of sepsis.
• Gastrointestinal inflammation is inflammation of a mucosal layer of the gastrointestinal tract, and encompasses acute and chronic inflammatory conditions. Acute
• Mucosal layer may be mucosa of the bowel (including the small intestine and large intestine) , rectum, stomach (gastric) lining, or oral cavity.
• colitis induced by environmental insults e.g., gastrointestinal inflammation (e.g., colitis) caused by or associated with (e.g., as a side effect) a therapeutic regimen, such as administration of chemotherapy, radiation therapy, and the like
• infections colitis ischemic colitis
• collagenous or lymphocytic colitis necrotizing
• enterocolitis colitis in conditions such as chronic granulomatous disease or celiac disease, food allergies, gastritis, infectious gastritis or enterocolitis (e.g., Helicobacter pylori-infected chronic active gastritis) and other forms of gastrointestinal inflammation caused by an infectious agent.
• enterocolitis colitis in conditions such as chronic granulomatous disease or celiac disease, food allergies, gastritis, infectious gastritis or enterocolitis (e.g., Helicobacter pylori-infected chronic active gastritis) and other forms of gastrointestinal inflammation caused by an infectious agent.
• IBD Inflammatory bowel disease
• UC ulcerative colitis
• CD Crohn's disease
• IBD IBD pathogenesis
• inflammatory bowel diesase the tissue damage results from an inappropriate or exaggerated immune response to antigens of the gut microflora.
• TNBS 2, 4, 6-trinitrobenesulfonic acid/ethanol
• oxazalone the oxazalone model, which induce chronic inflammation and ulceration in the colon (Neurath et al . , Intern. Rev. Immunol 19:51-62, 2000) .
• DSS dextran sulfate sodium
• antagonists of the present invention in any of these models can be used to evaluate the potential efficacy of those antagonists to ameliorate symptoms and alter the course of diseases associated with inflammation in the gut, such as inflammatory bowel disease.
• IBD inflammatory bowel disease
• anti-TNF-a antibody therapies have been used for a decade to treat Crohn's disease (Van Assche et al . , Eur. J. Pharmacol. Epub Oct 2009).
• a significant percentage of patients are refractory to the current treatments (Hanauer et al . , Lancet 359:1541-1549, 2002; Hanauer et al., Gastroenterology 130:323-333, 2006), and thus new therapies targeting refractory patient
• an inflammatory condition is an inflammatory pulmonary condition.
• exemplary inflammatory pulmonary conditions include infection-induced pulmonary conditions including those associated with viral, bacterial, fungal, parasite or prion infections; allergen-induced pulmonary conditions; pollutant-induced pulmonary conditions such as asbestosis, silicosis, or berylliosis; gastric aspiration-induced pulmonary conditions, immune
• predisposition such as as cystic fibrosis, and physical trauma-induced pulmonary conditions, such as ventilator injury.
• These inflammatory conditions also include asthma, emphysema, bronchitis, chronic obstructive pulmonary disease (COPD) , sarcoidosis, histiocytosis, lymphangiomyomatosis , acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, community- acquired pneumonia, nosocomial pneumonia, ventilator - associated pneumonia, sepsis, viral pneumonia, influenza infection, parainfluenza infection, rotavirus infection, human metapneumovirus infection, respiratory syncitial virus infection and aspergillus or other fungal infections.
• COPD chronic obstructive pulmonary disease
• Exemplary infection-associated inflammatory diseases may include viral or bacterial pneumonia, including severe pneumonia, cystic fibrosis, bronchitis, airway exacerbations and acute respiratory distress syndrome (ARDS) .
• Such infection-associated conditions may involve multiple infections such as a primary viral infection and a secondary bacterial infection.
• Asthma is an inflammatory disease of the lung that is characterized by airway hyperresponsiveness ("AHR") , bronchoconstriction, wheezing, eosinophilic or neutrophilic inflammation, mucus hypersecretion, subepithelial fibrosis, and elevated IgE levels.
• AHR airway hyperresponsiveness
• wheezing wheezing
• eosinophilic or neutrophilic inflammation wheosinophilic or neutrophilic inflammation
• mucus hypersecretion e.g., mucus hypersecretion
• subepithelial fibrosis e.g.
• Asthmatic attacks can be triggered by environmental factors (e.g. ascarids, insects, animals (e.g., cats, dogs, rabbits, mice, rats, hamsters, guinea pigs and birds) , fungi, air pollutants (e.g., tobacco smoke), irritant gases, fumes, vapors, aerosols, chemicals, pollen, exercise, or cold air.
• environmental factors e.g. ascarids, insects, animals (e.g., cats, dogs, rabbits, mice, rats, hamsters, guinea pigs and birds)
• fungi e.g., air pollutants (e.g., tobacco smoke), irritant gases, fumes, vapors, aerosols, chemicals, pollen, exercise, or cold air.
• air pollutants e.g., tobacco smoke
• irritant gases irritant gases
• fumes e.g., irritant gases
• aerosols e.g., smoke
• chemicals e
• COPD chronic obstructive pulmonary disease
• bacterial pneumonia and cystic fibrosis bacterial pneumonia and cystic fibrosis
• diseases such as COPD, allergic rhinitis, and cystic fibrosis are characterized by airway hyperresponsiveness (Fahy and O'Byrne, Am. J. Respir. Crit. Care Med. 163:822-823, 2001).
• airway hyperresponsiveness Fahy and O'Byrne, Am. J. Respir. Crit. Care Med. 163:822-823, 2001.
• inflammation include the ovalbumin challenge model and methacholine sensitization models (Hessel et al . , Eur. J. Pharmacol. 293:401-412, 1995). Inhibition of cytokine and chemokine production from cultured human bronchial epithelial cells, bronchial fibroblasts or airway smooth muscle cells can also be used as in vitro models.
• the administration of antagonists of the present invention to any of these models can be used to evaluate the use of those antagonists to ameliorate symptoms and alter the course of asthma, airway inflammation, COPD and the like.
• inflammatory conditions and neuropathies which may be prevented or treated by the methods of the invention are those caused by autoimmune diseases.
• These conditions and neuropathies include multiple sclerosis, systemic lupus erythematous, and neurodegenerative and central nervous system (CNS) disorders including Alzheimer's disease,
• Parkinson's disease Huntington's disease, bipolar disorder and Amyotrophic Lateral Sclerosis (ALS) , liver diseases including primary biliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fatty liver
• autoimmune hepatitis fibrosis, hepatitis C virus (HCV) and hepatitis B virus (HBV)
• diabetes and insulin resistance cardiovascular disorders including atherosclerosis, cerebral hemorrhage, stroke and myocardial infarction, arthritis, rheumatoid arthritis, psoriatic arthritis and juvenile rheumatoid arthritis (JRA) , osteoporosis, osteoarthritis, pancreatitis, fibrosis, encephalitis, psoriasis, Giant cell arteritis, ankylosing spondolytis, autoimmune hepatitis, human immunodeficiency virus (HIV) , inflammatory skin conditions, transplant, cancer, allergies, endocrine diseases, wound repair, other autoimmune disorders, airway hyperresponsiveness and cell, virus, or prion-mediated infections or disorders.
• HCV hepatitis C virus
• HBV hepatitis B virus
• HCV hepatitis C
• rheumatoid arthritis is a systemic disease that affects the entire body and is one of the most common forms of arthritis. Since rheumatoid arthritis results in tissue damage, TLR3 ligands could be present at the site of the inflammation. Activation of TLR3 signaling may perpetuate inflammation and further tissue damage in the inflamed joint.
• RA rheumatoid arthritis
• CIA chronic inflammatory arthritis
• TLR3 antagonists of the present invention administered to the CIA model mice can be used to evaluate the use of these
• Diabetes mellitus refers to a disease process derived from multiple causative factors and characterized by hyperglycemia (LeRoith et al . , (eds.), Diabetes Mellitus, Lippincott-Raven Publishers, Philadelphia, Pa. U.S.A. 1996), and all references cited therein. Uncontrolled hyperglycemia is associated with increased and premature mortality due to an increased risk for microvascular and macrovascular diseases, including nephropathy, neuropathy, retinopathy, hypertension, cerebrovascular disease and coronary heart disease. Therefore, control of glucose homeostasis is a critically important approach for the treatment of diabetes.
• Type I diabetes insulin dependent diabetes mellitus, IDDM
• type 2 diabetes non-insulin dependent diabetes mellitus, NIDDM
• Type 2 diabetes is characterized by insulin resistance accompanied by relative, rather than absolute, insulin deficiency.
• the body secretes abnormally high amounts of insulin to compensate for this defect.
• inadequate amounts of insulin are present to compensate for insulin resistance and adequately control glucose, a state of impaired glucose tolerance develops.
• insulin secretion declines further and the plasma glucose level rises, resulting in the clinical state of diabetes.
• Adipocity- associated inflammation has been stronly implicated in the development of insulin resistance, type 2 diabetes,
• TLR3 activation on macrophages may contribute to the pro-inflammatory status of the adipose.
• Several animal modes of insulin resistance are known. For example, in a diet-induced obesity model (DIO) animals develop hyperglycemia and insulin resistance accompanied by weight gain.
• DIO diet-induced obesity model
• antagonists of the present invention to the DIO model can be used to evaluate the use of the antagonists to ameliorate complications associated with type 2 diabetes and alter the course of the disease.
• Fatty liver disease encompasses a spectrum of liver conditions and is typically classified as either alcoholic or nonalcoholic. In either case, fatty liver disease ranges from simple hepatic steatosis (lipid accumulation and deposition) to alcoholic and non-alcoholic steatohepatitis (ASH or NASH) , which often progresses to hepatic fibrosis, cirrhosis, and probably hepatocellular carcinoma.
• Alcoholic (AFLD) and nonalcoholic fatty liver disease (NAFLD) are histologically indistinguishable; however, by definition NAFLD develops in patients who consume little or no alcohol.
• NAFLD is frequently found in individuals with obesity, metabolic syndrome, and type 2 diabetes and is closely linked to insulin resistance (Utzschneider et al . , J Clin Endocrinol Metab 91:4753-4761, 2006). With the dramatic recent increase in the prevalence of obesity and insulin resistance, NAFLD has surpassed AFLD and viral hepatitis- induced liver disease as the most common chronic liver disease. It has been estimated that approximately 75% of those with obesity have NAFLD and as many as 20% may have NASH (Clark, J Clin Gastroenterol 40 (Suppl 1):S5-S10, 2006; Lazo et al., Semin Liver Dis 28:339-350, 2008).
• Atherosclerotic coronary heart disease represents the major cause for death and cardiovascular morbidity in the western world. Risk factors for atherosclerotic coronary heart disease include hypertension, diabetes mellitus, family history, male gender, cigarette smoke, high serum
• LDL high low density lipoprotein
• HDL low high density lipoprotein
• Exemplary cancers may include at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to leukemia, acute leukemia, acute lymphoblastic leukemia (ALL) , B-cell or T-cell ALL, acute myeloid leukemia (AML) , chronic myelocytic leukemia (CML) , chronic lymphocytic leukemia (CLL) , hairy cell leukemia, myelodyplastic syndrome (MDS) , a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin' s lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, renal cell carcinoma, breast cancer, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, adenocar
• Exemplary cardiovascular diseases may include
• cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic atherosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, syphilis of the cardiovascular system, heart failure, cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or
• arrhythmias arrhythmias, atrioventricular block, bundle branch block, myocardial ischemic disorders, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy, restrictive
• cardiomyopathy valvular heart diseases, endocarditis, pericardial disease, cardiac tumors, aordic and peripheral aneurysms, aortic dissection, inflammation of the aorta, occulsion of the abdominal aorta and its branches, peripheral vascular disorders, occulsive arterial disorders, peripheral atherosclerotic disease, thromboangitis obliterans,
• Exemplary neurological diseases may include neurologic disease in a cell, tissue, organ, animal or patient,
• neurodegenerative diseases including, but not limited to neurodegenerative diseases, multiple sclerosis, migraine headache, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea;
• drug-induced movement disorders such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive supranucleo Palsy; structural lesions of the cerebellum;
• spinocerebellar degenerations such as spinal ataxia
• Friedreich's ataxia cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dej erine-Thomas, Shi- Drager, and Machado-Joseph) ; systemic disorders (Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multisystem disorder) ; demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy body type; Wernicke- Korsakoff syndrome; chronic alcoholism; Creutz feldt-Jakob disease; Subacute sclerosing panencephalitis, Hallerrorden- Spatz disease and
• Exemplary fibrotic conditions may include liver fibrosis (including but not limited to alcohol-induced cirrhosis, viral-induced cirrhosis, autoimmune-induced hepatitis) ; lung fibrosis (including but not limited to scleroderma, idiopathic pulmonary fibrosis) ; kidney fibrosis (including but not limited to scleroderma, diabetic nephritis,
• the fibrosis can be organ specific fibrosis or systemic fibrosis.
• the organ specific fibrosis can be associated with at least one of lung fibrosis, liver fibrosis, kidney fibrosis, heart fibrosis, vascular fibrosis, skin fibrosis, eye fibrosis, bone marrow fibrosis or other fibrosis.
• the lung fibrosis can be associated with at least one of idiopathic pulmonary fibrosis, drug induced pulmonary fibrosis, asthma,
• the liver fibrosis can be associated with at least one of cirrhosis, schistomasomiasis or cholangitis.
• the cirrhosis can be selected from alcoholic cirrhosis, post-hepatitis C cirrhosis, primary biliary cirrhosis.
• the cholangitis is sclerosing cholangitis.
• the kidney fibrosis can be
• the heart fibrosis can be associated with myocardial infarction.
• the vascular fibrosis can be associated with postangioplasty arterial restenosis or atherosclerosis.
• the skin fibrosis can be associated with burn scarring, hypertrophic scarring, keloid, or nephrogenic fibrosing dermatopathy .
• the eye fibrosis can be associated with retro-orbital fibrosis, postcataract surgery or proliferative vitreoretinopathy .
• the bone marrow fibrosis can be associated with idiopathic myelofibrosis or drug induced myelofibrosis.
• the other fibrosis can be selected from Peyronie's disease, Dupuytren' s contracture or
• the systemic fibrosis can be systemic sclerosis or graft versus host disease.
• Administration/Pharmaceutical Compositions
• the "therapeutically effective amount” of the agent effective in the treatment or prevention of conditions where suppression of TLR3 activity is desirable can be determined by standard research techniques. For example, the dosage of the agent that will be effective in the treatment or
• prevention of inflammatory condition such as asthma, Crohn's Disease, ulcerative colitis or rheumatoid arthritis
• relevant animal models such as the models described herein.
• in vitro assays can optionally be employed to help identify optimal dosage ranges. Selection of a particular effective dose can be determined (e.g., via clinical trials) by those skilled in the art based upon the consideration of several factors. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan. The precise dose to be employed in the formulation will also depend on the route of administration, and the severity of disease, and should be decided according to the judgment of the
• Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the TLR3 antagonist may be administered singly or in combination with at least one other molecule.
• additional molecules may be other TLR3 antagonist molecules or molecules with a therapeutic benefit not mediated by TLR3 receptor signaling.
• Antibiotics, antivirals, palliatives and other compounds that reduce cytokine levels or activity are examples of such additional molecules .
• the mode of administration for therapeutic use of the agent of the invention may be any suitable route that delivers the agent to the host.
• Pharmaceutical compositions of these agents are particularly useful for parenteral administration, e.g., intradermal, intramuscular,
• intraperitoneal intravenous, subcutaneous or intranasal.
• the agent of the invention may be prepared as
• compositions containing an effective amount of the agent as an active ingredient in a pharmaceutically acceptable carrier.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered.
• Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration) .
• compositions may contain pharmaceutically acceptable
• auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc.
• concentration of the agent of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected.
• a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 ml sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of a TLR3 antibody antagonist of the invention.
• a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 mg to about 30 mg and preferably 5 mg to about 25 mg of an antagonist of the invention.
• Actual methods for preparing parenterally administrable compositions are well known and are described in more detail in, for example, "Remington's Pharmaceutical Science", 15th ed., Mack Publishing Company, Easton, PA.
• the antibody antagonists of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and protein preparations and art-known lyophilization and reconstitution techniques can be employed.
• TLR3 antigen generated from the expression of amino acids 1-703 of human TLR3 (huTLR3) (SEQ ID NO: 4) with a C-terminal poly-histidine tag and purified by immobilized metal affinity
• Amino acids 1-703 correspond to the predicted extracellular domain (ECD) of huTLR3.
• Fab fragments (Fabs) that bound specifically to huTRL3 ECD were selected by presenting the TLR3 protein in a variety of ways so that a diverse set of antibody fragments could be identified, sequenced and confirmed as unique. From different panning strategies, 62 candidates (different V- region sequences) were identified as unique hTLR3 ECD binders.
• the 62 candidates identified as huTLR3 ECD binders were screened for neutralizing activity in a range of cell-based assays relevant to identifying anti-inflammatory activity.
• four candidates (Fabs 16-19) defining families 16-19 were selected from the 62 as parents for CDR maturation of heavy chain CDR2 (HCDR2 ) and light chain CDR3 (LCDR3) .
• One of the parental candidates (candidate 19) exhibited an N-linked glycosylation site in HCDR2; a Ser to Ala (S to A) mutation was made in this candidate to delete the site.
• progeny candidates were identified for further characterization as described in Example 2 below.
• a listing of the light and heavy chain variable regions present in each of the 19 candidates is shown in Table 3 above.
• the candidates are herein referred to as mAbs 1-19 or Fabs 1-19, depending whether they were Fabs or cloned as full length antibody chains (Example 3) .
• Due to expression vector design, the mature amino termini of the variable regions for all candidates were QVE for heavy chain and DI for the light chain.
• the preferred sequences at these termini are those in the respective germline genes with high identity to the candidate sequences.
• the germline sequences are QVQ for VH and SY for VL .
• the sequences are EVQ for VH and DI for VL .
• the SY sequence is unique to the lambda subgroup 3 and there are reports of heterogeneity with either S or Y as the amino terminal residue.
• the QSV consensus terminus from the prominent lambda subgroup 1 was considered a more suitable replacement for DIE for VL of families 17 and 18.
• the amino acid sequences of the light chain variable region N-terminal germline variants of candidates 9, 10 and 11 are shown in SEQ ID NO : s 209-211, and the amino acid sequences of the heavy chain variable region N-terminal germline variants for candidates 9, 10, 12, 14, and 15 are shown in SEQ ID NO : s 212-216, respectively.
• the N-terminal variants of the candidates are herein referred to as candidate/mAb/Fab 9QVQ/QSV, 10QVQ/QSV, 12QVQ/QSV, 14EVQ or 15EVQ.
• the N-terminal germline variants were expressed as mAbs and showed no effect on binding to TLR3 or in their ability to inhibit TLR3 biological activity when compared to their parent counterparts (data not shown) .
• the 15 CDR-matured candidates described above were selected as potential human therapeutics and a range of binding and neutralizing activities were determined.
• the activity assays and results for the four parental Fabs, Fabs 16-19 and 15 CDR-matured Fabs, Fabs 1-15 or their non- germline V-region variants are described below.
• 293T cells were grown in DMEM and GlutaMax media
• the phRL-TK plasmid contains the Renilla luciferase gene driven by the HSV-1 thymidine kinase promoter (Promega, Madion, WI) . TLR3 antibodies were incubated 30-60 min .
• Fig. 1 The results for the NF- ⁇ assays are shown in Fig. 1 and are expressed as % inhibition of the Firefly/Renilla ratio with 5465 as the positive control (neutralizing anti-human TLR3 Mab) and an anti-human tissue factor mAb (859) as the human IgG4 isotype control. >50% inhibition was achieved with mAb concentrations 0.4-10 ⁇ g/ml. cl068 and TLR3.7 inhibited about 38% and 8% of TLR3 biological activity at 10 ⁇ g/ml . Similar results were obtained with the ISRE reporter gene assay (data not shown) .
• BEAS-2B cells (SV-40 transformed normal human bronchial epithelial cell line) were seeded in a collagen type I coated dishes and incubated with or without anti-human TLR3
• Cytokine release was also assayed in normal human bronchial epithelial (NHBE) cells (Lonza, Walkersville , MD) .
• NHBE cells were expanded and transferred to collagen-coated dishes and incubated for 48 hours after which the media was removed and replenished with 0.2 ml of fresh media. The cells were then incubated with or without anti-human TLR3 mAbs 60 minutes prior to the addition of poly (I:C) .
• Cytokine release was also assayed in human peripheral blood mononuclear cells (PBMC) .
• PBMC peripheral blood mononuclear cells
• PBMCs that formed a white layer just above the Ficoll, were recovered and plated.
• the PBMCs were then incubated with or without anti-human TLR3 mAbs prior to the addition of 25 ⁇ g/ml poly(I:C) . After 24 hrs, supernatants were collected and cytokine levels were determined using Luminex technology. Results are graphed in Fig. 4 as cumulative percentage inhibition of IFN- ⁇ , IL-12 and IL-6 using a single dose of mAb (0.4 g/ml) with 5465 is a positive control; hIgG4 is an isotype control.
• HASM human airway smooth muscle
• results from the in vitro assays in human cells confirm the ability of the antibodies of the invention to reduce cytokine and chemokines release as a result of binding to huTLR3.
• the four parental Fabs (candidate nos . 16-19) and 15 progeny Fabs (candidate nos. 1-15) heavy chains were cloned onto a human IgG4 background with a S229P Fc mutation.
• Candidates 9QVQ/QSV, 10QVQ/QSV, 12QVQ/QSV, 14EVQ or 15EVQ were cloned onto a human IgG4 background with F235A/L236A and S229P Fc mutations.
• these heavy chain sequences can include an N-terminal leader sequence such as MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 103) .
• N-terminal leader sequence such as MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 103) .
• Exemplary nucleotide sequences encoding the heavy chain of candidates 14EVQ and 15EVQ with a leader sequence and the mature form (without a leader sequence) are shown in SEQ ID NOs: 104 and 105, respectively.
• the light chain sequences of the antibodies of the invention can include an N-terminal leader sequence such as MGVPTQVLGLLLLWLTDARC (SEQ ID NO: 106) .
• Exemplary nucleotide sequences encoding the light chain of codon optimized candidate 15 with a leader sequence and the mature form (without a leader sequence) are shown in SEQ ID NOs : 107 and 108, respectively.
• EC50 values for the binding of the mAbs to human TLR3 extracellular domain (ECD) were determined by ELISA.
• Human TLR3 ECD protein was diluted to 2 ⁇ g/ml in PBS and 100 ⁇ aliquots were dispensed to each well of a 96-well plate (Corning Inc., Acton, MA) . After overnight incubation at 4°C, the plate was washed 3 times in wash buffer consisting of 0.05% Tween-20 ( Sigma-Aldrich) in PBS. The wells were blocked with 200 ⁇ blocking solution consisting of 2% I- Block (Applied Biosystems, Foster City, CA) and 0.05% Tween- 20 in PBS. After blocking for 2 hours at room temperature the plate was washed 3 times followed by addition of serial
• Binding affinity for huTLR3 ECD was also determined by
• Epitope binding experiments were performed to determine the anti-TLR3 antibody competition groups or "epitope bins”.
• MSD Blocker A buffer (Meso Scale Discovery) was added to each well and incubated for 2 hr at room
• MSD Read Buffer T was diluted with distilled water (4-fold) and dispensed at a volume of 150 ⁇ /well and analyzed with a SECTOR Imager 6000.
• Antibodies were labeled with MSD Sulfo-TagTM NHS-ester according to manufacturer's instructions (Meso Scale).
• anti-TLR3 antibodies were evaluated: mAbs 1-19 obtained from a MorphoSys Human Combinatorial Antibody Library (shown in Table 3a); cl068 (described in
• cl811 rat anti-mouse TLR3 mAb produced by a hybridoma generated from rats immunized with mouse TLR3 protein
• TLR3.7 eBiosciences , San Diego, CA, cat no 14- 9039
• IMG-315A generated against human TLR3 amino acids amino acids 55-70 (VLNLTHNQLRRLPAAN) from Imgenex, San Diego, CA) .
• mAbs 9, 10, 12, 14 and 15, variants 9QVQ/QSV, 10QVQ/QSV, 12QVQ/QSV, 14EVQ or 15EVQ were used in this study.
• anti-TLR3 antibodies were assigned to five distinct bins.
• Bin A mAbs 1, 2, 13, 14EVQ, 15EVQ, 16, 19;
• Bin B mAbs 3, 4, 5, 6, 7, 8, 9QVQ/QSV,
• Bin C antibody Imgenex IMG-315A
• Bin D antibodies TLR3.7, cl068
• Bin E antibodies
• Example 5 Representative antibodies from distinct epitope bins as described in Example 5 were selected for further epitope mapping.
• Epitope mapping was performed using various approaches, including TLR3 segment swapping experiments, mutagenesis, H/D exchange and in silico protein-protein docking (The Epitope Mapping Protocols, Methods in Molecular Biology, Volume 6, Glen E. Morris ed., 1996) .
• TLR.3 segment swapping TLR3 human-mouse chimeric proteins were used to locate gross antibody binding domains on TLR3.
• the human TLR3 protein extracellular domain was divided into three segments (aa 1-209, aa 210-436, aa 437-708 according to amino acid numbering based on human TLR3 amino acid sequence, GenBank Acc. No. NP_003256) .
• MT5420 chimeric protein was generated by replacing human TLR3 amino acids
• mice TLR3, GenBank Acc. No. NP_569054, amino acids 211-437 and 438-709 mouse amino acids
• the MT6251 chimera was generated by replacing human amino acids at positions 437-708 by mouse TLR3 amino acids (mouse TLR3, GenBank Acc. No. NP_569054, amino acids
• mAb cl068 bound human TLR3 ECD with high affinity but did not bind well to murine TLR3.
• cl068 lost its ability to bind to both MT5420 and MT6251, demonstrating that the binding site was located within the amino acids 437- 708 of the WT human TLR3 protein.
• mAb 12QVQ/QSV bound both chimeras, indicating that the binding site for mAb 12QVQ/QSV was located within the amino acids 1-209 of the human TLR3 protein having a sequence shown in SEQ ID NO : 2.
• ZDOCKpro 1.0 (Accelrys, San Diego, CA) , which is equivalent to ZDOCK 2.1 (Chen and Weng, Proteins 51: 397-408, 2003) with an angular grid of 6 degrees.
• Known N-linked glycosylation site Asn residues in human TLR3 (Asn 52, 70, 196, 252, 265, 275, 291, 398, 413, 507 and 636) (Sun et al . , J. Biol. Chem. 281:11144-11151, 2006) were blocked from participating in the antibody-antigen complex interface by an energy term in the ZDOCK algorithm.
• Crystallization of Fab 15EVQ was carried out by the vapor-diffusion method at 20°C (Benvenuti and Mangani, Nature Protocols 2:1633-51, 2007).
• the initial screening was set up using a Hydra robot in 96-well plates.
• the experiments were composed of droplets of 0.5 ⁇ of protein solution mixed with 0.5 ⁇ of reservoir solution. The droplets were equilibrated against 90 ⁇ of reservoir solution.
• the Fab solution in 20 mM Tris buffer, pH 7.4, containing 50 mM NaCl was
• the screening was performed with the Wizard I & II (Emerald BioSystems, Bainbridge Island, WA) and in-house
• X-ray diffraction data were collected and processed using the Rigaku MicroMaxTM-007HF microfocus X-ray generator equipped with an OsmicTM VariMaxTM confocal optics, Saturn 944 CCD detector, and an X-streamTM 2000 cryocooling system (Rigaku, Woodlands, TX) . Diffraction intensities were detected over a 270° crystal rotation with the exposure time of 120 s per half-degree image.
• the X-ray data were processed with the program D*TREK (Rigaku) .
• the structure was determined by the molecular replacement method using the program Phaser or CNX (Accelrys, San Diego, CA) .
• Atomic positions and temperature factors were refined with REFMAC using all data in the resolution range 15-2.2 A for Fab 15EVQ and 50-1.9 A for Fab 12QVQ/QSV. Water molecules were added at the (F 0 -F c ) electron density peaks using the cut-off level of 3o. All crystallographic calculations were performed with the CCP4 suite of programs (Collaborative Computational Project, Number 4. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D50 : 760-763) .
• the resolved crystal structure of mAb 15EVQ showed that the antibody combining site was characterized by a number of negatively charged residues in the heavy chain (D52, D55, E99, D106 and D109) . Thus, recognition between mAb 15EVQ and TLR3 most likely involved positively charged residues.
• the protein-protein docking simulations performed suggested that two large patches on TLR3 involving multiple positive charge residues showed good complementarity to the antibody.
• the residues on TLR3 in the interface of the TLR3 - anti-TLR3 antibody simulated complexes were R64, K182, K416, K467, Y468, R488, R489 and K493.
• oligonucleotides shown in Table 5a Mutations were verified by DNA sequencing.
• the proteins were expressed under a CMV promoter as C-terminal His-tag fusions in HEK293 cells, and purified as described in Example 1.
• Binding assays The binding activity of mAb 12QVQ/QSV and mAb 15EVQ to human TLR3 and generated variants was evaluated by ELISA. To expedite the process, mutants in the predicted mAb 15EVQ binding site were co-expressed in HEK cells by co-transfection of TLR3 ECD mutant containing a C- terminal His tag with mAb 12QVQ/QSV, followed by purification by metal affinity chromatography. The recovered sample was a complex of the TLR3 mutant with mAb 12. This approach was feasible because the mAb 12QVQ/QSV and mAb 15EVQ binding sites are distant from one another; and thus, point mutations at one site are unlikely to affect the epitope at the other site.
• Table 5a Sequences of the sense oligonucleiotides are shown.
• the anti-sense oligonucleotides with complementary sequences were used in the mutagenesis reaction.
• MSD Blocker A buffer (Meso Scale Discovery, Gaithersburg, MD) at 4°C. The following day the plates were washed and the MSDSulfo-tag labeled mAb 15EVQ added at concentrations from 500 pM to 1 pM for 1.5 hours. After washes the labeled antibody was detected using MSD Read Buffer T and the plates were read using a SECTOR Imager 6000. To evaluate the binding activity of mAb 12QVQ/QSV to human TLR3 and variants, co-expression was carried out with mAb 15EVQ and binding ELISAs were performed as described for mAb 15EVQ, except that the detecting antibody was labeled mAb 12QVQ/QSV.
• mAb 12QVQ/QSV The binding site for mAb 12QVQ/QSV was located within the amino acids 1-209 of the human TLR3 protein as determined in the segment swap studies. The following TLR3 mutants were evaluated: D116R, N196A, N140A, V144A, K145E, K147E, K163E, and Q167A. The wild type TLR3 and V144A mutant showed comparable binding to mAb 12QVQ/QSV ( Figure 6A) . The antibody did not bind to TLR3 D116R mutant and had significantly reduced binding affinity to the K145E mutant. Thus, residues D116 and K145 which are closely apposed on the surface of TLR3 were identified as key epitope sites for mAb 12QVQ/QSV ( Figure 7A) .
• the two critical residues of the mAb 12QVQ/QSV binding epitope were located near the face of the dsRNA binding site at the N-terminal segment of the TLR3 ectodomain (Pirher, et al., Nature Struct. & Mol . Biol., 15:761-763, 2008). The complete epitope will contain other residues in the
• 12QVQ/QSV on its TLR3 epitope may directly or indirectly interfere with dsRNA binding on TLR3 ectodomain, thereby disrupting receptor dimerization and activation of downstream signaling pathways.
• TLR3 mutants The following TLR3 mutants were evaluated: R64E, K182E, K416E, Y465A, K467E, R488E, R489E, N517A, D536A, D536K, Q538A, H539A, H539E, N541A, E570R, K619A, K619E, a double mutant K467E/Y468A, a triple mutant T472S/R473T/N474S, and a triple mutant R488E/R489E/K493E .
• T472S/R473T/N474S showed comparable binding to mAb 15EVQ
• Figure 7A shows binding epitope sites for mAbs 12QVQ/QSV and 15EVQ (black) and C1068 mAb (grey) superimposed on the structure of human TLR3.
• the epitope for mAb 15EVQ covers residues Y465, K467, Y468, R488, R489, N517, D536, Q538, H539, N541, E570, and K619.
• TLR3 ECD Recombinant TLR3 ECD (expressed from Sf9 cells with C-terminal His-tag and purified) was incubated in a deuterated water solution for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms .
• the deuterated TLR3 ECD was captured on a column containing immobilized mAb 15EVQ and then washed with aqueous buffer.
• the back-exchanged TLR3 ECD protein was eluted from the column and localization of deuterium containing fragments was determined by protease digestion and mass spec analysis.
• TLR3 ECD sample was processed similarly except it was exposed to deuterated water only after capture on the antibody column and then washed and eluted in the same manner as the
• TLR3 ECD perturbation of TLR3 ECD by mAb 15EVQ are shown in Figure 7B. Only the segment of TLR3 around the portion affected by mAb 15EVQ is shown for clarity. The remainder of the protein extending to the amino and carboxyl termini of TLR3 ECD was not affected appreciably.
• H/D exchange studies identified peptide segments 465 YNKYLQL 47 i, 514 SNNNIANINDDML 526 and 529 LEKL 532 of SEQ ID NO: 2 as regions where exchange on TLR3 was particularly altered by binding to mAb 15EVQ.
• H/D exchange is a linear mapping method and usually cannot define which residues within the peptide segment are most affected by antibody binding.
• the extensive overlap between the H/D exchange and mutational results gives added confidence that the surface shown in Figure 7A is the binding site for mAb 15EVQ. This binding site was in same linear amino acid sequence region as previously described for mAb cl068 (PCT Publ . no. WO06/060513A2) but it was found to be located on a completely non-overlapping surface ( Figure 7A) in agreement with the lack of cross-competition between these antibodies.
• the mAb 15EVQ binding epitope was spatially proximal to the dsRNA binding site at the C-terminal segment on TLR3 (Bell et al., Proc. Natl. Acad. Sci . (USA) 103: 8792-8797, 2006; Ranj ith-Kumar et al., J Biol Chem, 282: 7668-7678, 2007; Liu et al., Science, 320: 379-381, 2008).
• binding of mAb 15EVQ on its TLR3 epitope causes steric clashes with a ligand dsRNA molecule and/or the dimer partner, preventing ligand binding and ligand-induced receptor dimerization .
• Structure-based engineering was conducted to generate antibody variants with increased thermal stability, with simultaneous efforts to maintain the biological activity and minimize immunogenicity .
• mAb 15EVQ was selected for engineering. To minimize immunogenicity, only germline mutations predicted to be beneficial based upon structural considerations were pursued.
• the VL and VH sequences of mAb 15EVQ (SEQ ID NO: 41 and SEQ ID NO: 216, respectively) were aligned with the human germline genes using BLAST searches. The closest germline sequences identified were GenBank Acc . No. AAC09093 and X59318 for VH and VL, respectively.
• VH germline VH, VL and those of the mAb 15EVQ VH and VL sequences: (VH) V34I, G35S, F50R, A61S, and Q67H; (VL) G30S, L31S, and A34N.
• the identified sequence differences were mapped onto the crystal structure of the mAb 15EVQ, and residues predicted to alter packing and interface interactions were selected for engineering. Based upon the crystal structure of the antibody (see Example 6) , potential structure destabilizing residues were identified. (1) A small enclosed cavity was identified in the core of VH near V34. This cavity was large enough to accommodate a slightly larger sidechain such as lie.
• E99 of VH CDR3 was buried at the VH/VL interface without a H-bonding network.
• the negatively charged carboxylate group of E99 was in a generally hydrophobic environment with mostly van der Waals (vdw) contacts to neighboring residues. Burying a charge group is usually energetically unfavorable and thus has destabilizing effect.
• F50 of VH is a VH/VL interface residue. Its aromatic sidechain is bulky and thus may have negative impact upon the pairing. H-bonding and vdw packing networks for the Fv were calculated and visually inspected in Pymol
• Binding of mAbs 15-1 - 15-9 to TLR3 was evaluated by ELISA immunoassay.
• Human TLR3 ECD 100 ⁇ of 2 g/ml TLR3- ECD was bound to a black Maxisorb plate (eBioscience) overnight at 4°C. The plates were washed and blocked, and diluted antibodies were aliquoted at 50 ⁇ per well in duplicate onto the wells. The plate was incubated at RT for 2 hours shaking gently. Binding was detected using
• DSC experiments were performed on a MicroCal' s Auto VP- capillary DSC system (MicroCal, LLC, Northampton, MA) in which temperature differences between the reference and sample cells were continuously measured, and calibrated to power units. Samples were heated from 10 °C to 95 °C at a heating rate of 60 °C/hour. The pre-scan time was 15 minutes and the filtering period was 10 seconds. The concentration used in the DSC experiments was about 0.5 mg/ml . Analysis of the resulting thermograms was performed using MicroCal Origin 7 software (MicroCal, LLC) .
• Tm The thermal stability (Tm) of the generated variants was measured by DSC (Table 8) . Binding of the antibody variants to TLR3 was comparable to that of the parental antibody. Table 8. Summary of melting temperatures (T M ) of the variants and rationale for making them.
• a chimeric antagonistic rat/mouse anti-mouse TLR3 antibody, herein named mAb 5429 was generated to evaluate effects of inhibiting TLR3 signaling in various in vivo models, as the humanized antibodies generated in Example 1 did not have sufficient specificity or antagonist activity for mouse TLR3.
• the surrogate chimeric mAb 5429 as well as its parent rat anti-mouse TLR3 antibody cl811 inhibited mouse TLR3 signaling in vitro, and in vivo, and ameliorated pathogenic mechanisms in several disease models in the mouse.
• Data discussed below suggests a role for TLR3 in the induction and perpetuation of detrimental inflammation, and contribute to the rationale for the therapeutic use of TLR3 antagonists and TLR3 antibody antagonists, for example acute and chronic inflammatory conditions including
• CD rats were immunized with recombinant murine TLR3 ectodomain (amino acids 1-703 of seq ID NO: 162, GenBank Acc . No. NP_569054) generated using routine methods. Lymphocytes from two rats demonstrating antibody titers specific to murine TLR3 were fused to FO myeloma cells. A panel of monoclonal antibodies reactive to murine TLR3 were identified and tested for in vitro antagonist activity in the murine luciferase reporter and murine embryonic fibroblast assays. The hybridoma line C1811A was selected for further work.
• Functional variable region genes were sequenced from mAb cl811 secreted by the hybridoma. Cloned heavy chain and light chain variable region genes were then respectively inserted into plasmid expression vectors that provided coding sequences for generating a chimeric Rat/Balb C muIgGl/ ⁇ mAb designated as mAb 5429 using routine methods. The antibodies were expressed as described in Example 3. T he amino acid sequences of the mAb 5429 heavy and light chain variable regions are shown in SEQ ID NO: 164 and SEQ ID NO: 163, respectively, and the heavy and light chain full length sequences are shown in SEQ ID NO: 166 and SEQ ID NO: 165, respectively. The heavy and light chain full length
• mAb 5429 was characterized in a panel of in vitro assays for its neutralizing ability on TLR3 signaling. The activity assays and results are described below.
• the murine TLR3 cDNA (SEQ ID NO: 161, GenBank Acc . No: NM_126166) was amplified by PCR from murine spleen cDNA (BD Biosciences, Bedford, MA) , and cloned into the pCEP4 vector (Life Technologies, Carslbad, CA) using standard methods.
• HEK293T cells 200 ⁇ HEK293T cells were plated in 96 well white clear- bottom plates at a concentration of 4 x 10 4 cells/well in complete DMEM, and used the following day for transfections using Lipofectamine 2000 (Invitrogen Corp., Carslbad, CA) using 30 ng pNF- ⁇ firefly luciferase (Stratagene, San Diego, CA) or 30 ng pISRE firefly luciferase (BD Biosciences,
• the relative light units were measured using a FLUOstar OPTIMA multi-detection reader with OPTIMA software (BMG Labtech GmbH, Germany) . Normalized values (luciferase ratios) were obtained by dividing the firefly relative light units (RLUs) by the Renilla RLUs .
• mAb 5429 as well as its parent mAb cl811 and mAb 15 (Table 3a) reduced poly(I:C) - induced NF-kB and ISRE activation in a dose-dependent fashion ( Figure 8A and 8B) , demonstrating their abilities to
• IC50s measured in the ISRE assay were 0.5, 22, and 0.7 ⁇ g/ml for mAb 5249, mAB 15 and mAb cl811, respectively.
• C57BL/6 MEF cells were obtained from Artis Optimus (Opti-MEFTM C57BL/6 - 0001) .
• the cells were plated in 96-well flat bottom plates (BD Falcon) at 20,000 cells/well in 200 ⁇ MEF media (DMEM with glutamax, 10% heat inactivated-FBS , lx NEAA, and 10 g/ml gentamycin) . All incubations were done at 37°C/5%C0 2 . 24 hours after plating, mAb 5429 or mAb cl811 were added into wells. The plates were incubated with the mAbs for lhr, after which Poly(I:C) was added at 1 g/ml in each well.
• TLR3KO C57BL/6 and TLR3 knockout (C57BL/6 background; female, 8-12 weeks of age, Ace Animals, Inc.), 10 per group, were dosed intraperitoneally with 1 ml of 3% Thioglycollate medium (Sigma) and 96 hrs later, the mice were euthanized and the peritoneum from each mouse was lavaged with 10 ml sterile PBS. Thioglycollate-elicited peritoneal macrophages were resuspended in PBS and cell viability was assessed using Trypan Blue staining.
• the cells were distributed at 10 6 cells in 100 ⁇ /well for surface staining.
• Alexa-Fluor 647 (Molecular Probes ) -conj ugated mAb cl811 and mAb 1679 (rat anti-mouse TLR3 antibody that had no TLR3 specificity, and thus used as an isotype control) were added at 0.25 g/10 6 cells and incubated on ice in the dark for 30 minutes.
• the cells were washed and resuspended in 250 ⁇ of FACS Buffer.
• the viability stain, 7-AAD (BD Biosciences, Bedford, MA) , was added at 5 ⁇ /well no more than 30 minutes before acquisition of samples on FACS Calibur to detect a dead cell population. Samples were collected by the FACS Calibur using Cell Quest Pro Software. FCS Express was used to analyze the collected data by forming histograms.
• mAb 5429 was not used in this assay since the mouse Fc region of this chimeric antibody was expected to contribute to non-specific binding.
• mAb cl811 exhibited no binding to TLR3KO macrophages, and increased binding to the cell surfaces of C57BL/6 peritoneal macrophages, suggesting a specificity of the mAb for TLR3 ( Figure 10) .
• mAb 5429 having the same binding regions as mAb cl811, is assumed to have the same binding specificity as mAb cl811.
• TLR3 antibody antagonists protect from TLR3-mediated systemic inflammation
• the Poly ( I : C) -induced systemic cytokine/chemokine model was used as a model of TLR3-mediated systemic inflammation.
• poly(I:C) PIC
• mice Female C57BL/6 mice (8-10 weeks old) or female TLR3KO mice (C57BL/6 background; 8-10 weeks old, Ace Animals, Inc.) were given mAb 5429 at 10, 20 or 50 mg/kg in 0.5 ml PBS, mAb cl811 at 2, 10 or 20 mg/kg in 0.5 ml PBS or 0.5 ml PBS alone (vehicle control) subcutaneously . 24 hours after antibody dosing, mice were given 50 ⁇ g poly(I:C) (Amersham Cat. No. 26-4732 Lot no. IH0156) in 0.1 ml PBS intraperitoneally .
• poly(I:C) Amersham Cat. No. 26-4732 Lot no. IH0156
• Retro-orbital blood was collected 1 and 4 hours after the poly(I:C) challenge. Serum was prepared from whole blood and analyzed for cytokine and chemokine concentrations by
• TLR3-dependent poly(I:C)- induced mediators were IL-6, KC, CCL2/MCP-1 and TNF-a at 1 hr post-poly (I : C) challenge, and IL-la, CCL5/RANTES and TNF-a at 4 hr post-poly (I : C) challenge.
• Both mAb cl811 and mAb 5429 significantly reduced levels of these TLR3-dependent
• TLR3 antagonism can be beneficial in reducing excess TLR3- mediated cytokine and chemokine levels in conditions such as cytokine storm or lethal shock.
• TLR3 antibody antagonists reduce airway hyperresponsiveness
• Airway hyperresponsiveness was induced by Poly(I:C).
• Female C57BL/6 mice (12 weeks old) or female TLR3KO mice (C57BL/6 background; 12 weeks old, Ace Animals, Inc.) were anesthetized with isoflurane and several doses (10-100 g) of poly(I:C) in 50 ⁇ sterile PBS were administered
• mice received three administrations of poly(I:C) (or PBS) with a 24 hour rest period between each administration. 24 hours following the last poly(I:C) (or PBS) administration, lung function and airway
• mice were placed into the whole body plethysmograph chamber and allowed to acclimate for at least 5 minutes. Following baseline readings, mice were exposed to increasing doses of nebulized methacholine (Sigma, St. Louis, MO) . The nebulized methacholine (Sigma, St. Louis, MO) . The nebulized methacholine (Sigma, St. Louis, MO) .
• methacholine was administered for 2 minutes, followed by a 5- minute data collection period, followed by a 10-minute rest period before subsequent increasing-dose methacholine challenges.
• the increased airflow resistance was measured as Enhanced Pause (Penh) and is represented as the average Penh value over the 5-minute recording period (BUXCO system) .
• mice were euthanized and the lungs were cannulated.
• Bronchoalveolar lavages were performed by injecting 1 ml of PBS into the lungs and retrieving the effluent. The lung tissues were removed and frozen. BAL fluids were centrifuged (1200 rpm, 10 min . ) and the cell-free supernatants were collected and stored at -80°C until analysis. Cell pellets were resuspended in 200 ⁇ PBS for total and differential cell counts. The multiplex assay was performed following the manufacturer's protocol and the Multiplex Immunoassay Kit (Millipore, Billercia, MA) .
• TLR3- mediated increases in baseline PenH and airway sensitivity to methacholine were prevented in the anti-TLR3 antibody-treated animals ( Figure 11) .
• TLR3-mediated recruitment of neutrophils into the mouse lung and generation of chemokines in the airways were reduced in the anti-TLR3 antibody - treated animals.
• the neutrophil numbers ( Figure 12) and the CXCLlO/IP-10 levels ( Figure 13) were measured from the collected bronchoalveolar lavage fluid (BALF) . The studies were repeated at least three times with similar results.
• TLR3 antagonists may be beneficial in the treatment or prevention of respiratory diseases characterized by airway hyperresponsiveness, such as asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD) , and cystic fibrosis.
• COPD chronic obstructive pulmonary disease
• TLR3 antibody antagonists protect from inflammatory bowel disease
• the DSS colitis Model was used as a model of
• mice Female C57BL/6 mice ( ⁇ 8 weeks old) or female TLR3KO mice (C57BL/6 background; ⁇ 8 weeks old weighing between
• Biomedicals, Aurora, OH, Catalog no: 160110; 35-50kDa; 18- 20% Sulfur, Lot no. 8247J) was diluted in autoclaved acidified drinking water to a final concentration of 5%.
• mice were allowed to drink water ad libitum throughout the study. All water bottles were weighed every day to record water consumption. On days 0, 2, and 4 mice were dosed intraperitoneally with 5 mg/kg (0.1 mg in 0.1 ml PBS) mAb 5429, mouse anti-TNF-a antibody, or PBS as a control. Mice were monitored daily throughout the study and were weighed on days 0 through 4 and day 7. Mice were euthanized on days 2 and 7 of the study.
• the DSS model may be useful in evaluating therapeutics that may target the human patient population that is non-resposive to anti-TNF-a therapies, and
• neutralizing anti-TLR3 antibodies may have the potential to provide benefit to patients with inflammatory bowel disease who do not respond to anti-TNF- ⁇ therapies.
• the T cell Transfer Model was used as a model of inflammatory bowel disease.
• gut inflammation was induced in SCID mice by the transfer of a population of regulatory T cell-devoid naive T cells from immune-competent mice, which attack antigen-presenting cells in the gut mucosa .
• Naive T-cells (CD4+CD45RB T cells) were injected intraperitoneally into SCID recipients to induce chronic colitis. Mice were given either PBS (500 ⁇ /mouse
• mAb 5429 0.1 mg/mouse intraperitoneally
• anti-TNF- ⁇ antibody 0.05 mg/mouse intraperitoneally; positive control
• mice lost >15% of their original body weight animals were euthanized and colons removed. Colons were fixed, paraffin-embedded and H&E stained.
• Histopathology cell infiltration, crypt abscesses
• TLR3 antagonists may be beneficial for the treatment of inflammatory bowel diseases, including anti-TNF-a-refractory cases, and other immune-mediated pathologies in the gastrointestinal tract.
• TLR3 antibody antagonists protect from collagen-induced arthritis
• the collagen-induced arthritis (CIA) model was used as a model of rheumatoid arthritis.
• mice Male B10RIII mice (6-8 weeks old, Jackson Labs) were divided into groups of 15 per group (arthritis groups) or 4 per group (control mice) . Arthritis groups were anesthetized with Isoflurane and given injections of Type II collagen (Elastin Products) and Freund' s complete adjuvant
• mice with developing type II collagen arthritis were randomized by body weight into treatment groups and were dosed subcutaneously (SC) on days 12, 17, and 22 (dl2, dl7, 2d2) with mAb 5429 (25 mg/kg) , the negative control antibody CVAM (a recombinant mAb of no known specificity in the mouse)
• mice were treated with vehicle (0.1 mg/kg) or anti-TNF-a antibody (5 mg/kg, positive control) .
• control groups of mice were treated with vehicle
• PBS dexamethasone
• SC subcutaneously daily
• Efficacy evaluation was based on animal body weights, and clinical arthritis scores. All animals survived to study termination.
• Clinical data for paw scores were analyzed using AUC for days 1-15, and % inhibition from controls were calculated.
• Dexamethasone (Dex) and anti-mouse TNF-a antibody was used as a positive control, PBS was used as vehicle control, and CVAM was used as a negative control antibody. All treatments were initiated on day 12 of the study, during the development of joint disease. Disease incidence for vehicle- treated disease control animals was 100% by study day 22. Negative control groups treated with vehicle or CVAM antibody had the highest clinical scores. Significantly reduced clinical scores were observed for the groups treated with Dex (p ⁇ 0.05 for dl8-d26), 5 mg/kg anti-TNF-a antibody (p ⁇ 0.05 for dl8-26), or 25 mg/kg mAb 5429 (p ⁇ 0.05 for dl8-d23 and d25- d26) ( Figure 16) .
• Clinical arthritis scores expressed as area under the curve (AUC) were significantly reduced by treatment with 25 mg/kg mAb 5429 (43% reduction), 5 mg/kg anti-TNF-a antibody (52%), or Dex (69%) as compared to vehicle controls.
• Figure 17 shows means and standard deviations for AUC for each group.
• Paw bone resorption was significantly decreased by treatment with 25 mg/kg mAb 5429 (47% decrease) as compared to vehicle controls.
• Positive control mice treated with 5 mg/kg anti-TNF- ⁇ antibody had significantly decreased paw inflammation (33%) , cartilage damage (38%) , and summed paw scores (37%) .
• Treatment with Dex significantly reduced all paw histopathology parameters (73% reduction of summed scores) .
• TLR3 antibody antagonists improve clinical and histopathological disease symptoms in the CIA model, and suggest the use of TLR3 antagonists for treatment of rheumatoid arthritis.
• TLR3 antibody antagonists protect from acute lethal viral infections
• influenza A virus challenge model was used as a model of acute lethal viral infection.
• mice On Day -1, 4, 8, and 12, female C57BL/6 mice (12 weeks old) or female TLR3K0 mice (C57BL/6 background; 12 weeks old, ACE Animals, inc., 15 mice per group) were dosed
• mice subcutaneously 20 mg/kg mAb 5429, or PBS alone. On day 0, the mice were anesthetized by isoflurane and were
• Influenza A/PR/8/34 virus ATCC, Rockland, MD, Lot no. 218171
• 25 ⁇ PBS Equivalent to 10 5 ' 55 CEID50
• Animals were observed two times a day for changes in body weight and survival over the period of 14 days .
• a clinical scoring system was used to evaluate the level of disease progression and subtle improvements in response to Influenza A virus treatment.
• mice significantly reduced in the group receiving 20 mg/kg mAb 5429, as well as in the TLR3KO group (Figure 19) .
• the body weight of the mice was observed over a period of 14 days after influenza virus administration. Body weight decreased steadily in C57BL/6 mice dosed with Influenza A virus.
• TL 3 antibody antagonists improve hyperglycemia and reduce plasma insulin
• the Diet-induced obesity (DIO) model was used as a model of hyperglycemia and insulin resistance, and obesity.
• TLR3KO mice C57BL/6 WT animals (about 3 weeks old, Jackson Labs) and TLR3KO animals (C57BL/6 background; about 3 weeks old, Ace Animals, Inc.) were maintained on a high fat diet for 12 to 16 weeks. Both TLR3KO and WT C57BL/6 mice were fed either with normal chow or high-fat diet (Purina TestDiet cat. no. 58126) consisting of 60.9% kcal fat and 20.8% kcal
• mice were maintained on a 12:12-h light-dark cycle, with water and food ad libitum. The weight of each mouse within each group was measured weekly.
• mAb 5429 was given intraperitoneally twice a week for the first week followed by once a week dosing for total of 7 weeks.
• Fasting retro-orbital blood serum samples were used for insulin measurements at the time points indicated.
• Glucose tolerance tests were performed by i.p administration of glucose at 1.0 mg/g body weight after overnight fast at week 7. In addition, fasting insulin and glucose levels were measured.
• Fasting blood glucose (BG) was determined using glucose oxidase assay.
• Fasting insulin levels were determined using the insulin rat/mouse ELISA kit (Crystal Chem, cat. No. 90060) . Results
• the WT DIO aimals were hyperglycemic and hyperinsulinemic .
• Glucose tolerance was improved in the WT DIO animals but not in the TLR3KO DIO animals upon treatment with mAb 5429.
• Significantly reduced blood glucose levels were observed in mAb 5429 treated animals post glucose challenge at 60, 90, 120, and 180 min when compared to control (PBS only) ( Figure 21A) .
• About 21% reduction in AUC was observed in the mAb 5429 treated WT DIO animals when compared to the WT DIO mice not receiveing the mAb.
• Fasting insulin levels were also reduced in the WT DIO animals treated with mAb 5429 ( Figure 22) .
• TLR3KO DIO animals showed no improvement in fasting insulin upon mAb 5429 treatment.
• Homeostatic model assessment (HOMA) analysis indicated improved insulin sensitivity in the WT DIO animals treated with mAb 5429, but not in the TLR3KO DIO animals.
• the HOMA-IR values were 14.0+9.8, 8.7+4.9, 9.0+3.0 for WT DIO, 5 mg/kg of WT DIO mAb 5429, and 20 mg/kg of WT DIO mAb 5429 animals, respectively. No effect was observed in TLR3KO DIO animals.
• TLR3 antibody antagonists improved insulin resistance and reduced fasting glucose in the DIO model without weight loss, suggesting that TLR3 antagonists may be beneficial for the treatment of
• hyperglycemia insulin resistance
• type II diabetes type II diabetes
• TLR3 antibody antagonists protect from bacteria and virus-induced inflammatory responses
• NHi Nontypeable Haemophilus influenza
• NHBE cells (Lonza, Wakersville , MD) were seeded in Microtest 96-well tissue culture plates (BD Biosciences, Bedford, MA) at 1 x 107well. NTHi grown on agar plates for 16-20 hr were resuspended in growth medium at ⁇ 2 x 10 s cfu/ml, treated with 100 g/ml gentamycin for 30 min . and added at ⁇ 2 x 107well to 96-well plates containing NHBEs . After 3 hours, supernatants were removed and replaced with fresh growth medium with or without antibodies (0.08 to 50 ⁇ g/ml final concentration) . After additional 24 hr incubation, presence of cytokines and chemokines in cell supernatants was assayed in triplicate with a Cytokine 25-plex AB bead kit, Human
• NHBE cells were seeded in Microtest 96-well tissue culture plates (BD Biosciences, Bedford, MA) at 1 x 10 5 cells/well. The next day, antibodies (0.08 to 50 ⁇ g/ml final concentration) were added to NHBE or BEAS-2B cells and incubated for 1 hr, followed by addition of 10 ⁇ /well of rhinovirus. After additional 24 hr incubation, presence of cytokines and chemokines in cell supernatants was assayed by luminex assays as described above.
• TLR3 antibody antagonists suppress inflammatory responses in astroctyes
• astrocytes Normal human astrocytes from 2 donors (Lonza, Walkersville , MD) were plated in a 24 well plate at 75,000 cells/well and allowed to attach overnight. The next day, the astrocytes were treated with 200 ng/ml poly(I:C) and/or 10 ⁇ g/ml mAb 18 for 24 hours. Cytokines were measured by Luminex.
• TLR3 antibody antagonists suppress inflammatory responses in
• HUVEC cells (Lonza, Walkersville , MD) were cultured in serum-containing growth medium recommended by Lonza. Cells were resuspended in serum-free media (Lonza, Walkersville, MD) , plated in 96-well plates at 3xl0 5 cells/ml, and incubated at 37°C, 5%C02 for 24 hrs . Poly(I:C) (GE Healthcare,
• mAb 15EVQ was added to the cells at various concentrations (0 - 50 ⁇ g/ml) and incubated for 30 min, after which 20 ⁇ g/ml poly(I:C) was added for 24 hours.
• Cell supernatants were collected and cytokine levels were measured using the human cytokine 30-plex kit and Luminex MAP technology (Invitrogen Corp., Carslbad, CA) .
• sICAM-1 sICAM-1 expession
• the HUVEC cells were treated with 20 ⁇ g/ml poly(I:C) and various concentrations of mAb 15EVQ (0.8 - 50 ⁇ g/ml) .
• the cell supernatants were analyzed for sICAM-1 expression by ELISA (R&D systems) .
• Cell viability was measured using the CellTiterGlo kit (Promega, Madison, WI) .
• HUVEC cells produced the following cytokines in response to poly(I:C): IL-1RA, IL-2, IL-2R, IL-6, IL-7, CXCL8/IL-8, IL-12 (p40/p70), IL-15, IL-17, TNF-a, IFN-a, IFN- ⁇ , GM-CSF, CCL3/MIP-la, CCL4/MIP-i , CXCLlO/IP-10, CCL5/RANTES,
• mAb 15EVQ dose-dependently reduced levels of all cytokines induced by poly(I:C) (Table 12). The ability of mAb 15EVQ to reduce poly ( I : C) -induced production of TNF-a, CCL2/MCP-1, CCL5/RANTES, and CXCLlO/IP-10 suggested that inhibiton of
• TLR3-mediated activities may protect against leukocyte and T cell infiltration that can lead to atherosclerosis.
• inhibition of VEGF by mAb 15EVQ suggested a potential benefit of TLR3 blockade in pathologies mediated by VEGF including angiogenesis in a variety of cancers and ocular diseases such as age-related macular degeneration.
• TNF-a and IFN- ⁇ function in leukocyte recruitment and increase the expression of adhesion molecules on the
• Soluble Intercellular Adhesion Molecule 1 (sICAM-1) is generated by proteolytic cleavage and is a marker for endothelial cell activation. ICAM-1 plays a key role in leukocyte migration and activation and is upregulated on endothelial cells and epithelial cells during inflammation where it mediates adhesion to leukocytes via integrin molecules LFA-1 and Mac-1. Poly(I:C)
• TLR3 antibody antagonists can inhibit leukocyte trafficking and thus tissue damage caused by the influx of inflammatory cells.
• HUVECs were cultured, plated and stimulated with poly(I:C) as described above.
• KS Kaposi's sarcoma
• KSHV Kaposi's sarcoma herpes virus
• VEGF and cytokine production contribute to the survival of KS cells (Livengood et al . , Cell Immunol. 249:55- 62, 2007) .
• TLR3 antagonists could be beneficial at reducing angiogenic risks associated with KS and other tumors and at preventing cell viability loss and protecting endothelial barrier integrity to prevent vascular leakage, a potentially serious condition associated with organ failure and life- threatening inflammatory conditions such as sepsis.
• TLR3 antagonism may also be beneficial in viral infections involving endothelial cell pathology such as the viral hemorrhagic fevers caused by members of the families
• flaviviridae e.g. Dengue, yellow fever
• filoviridae e.g., Marburg
• bunyaviridae e.g. Hantavirus, Nairovirus
• Phlebovirus Phlebovirus
• arenaviridae e.g. Lujo, Lassa, Argentine, Venezuelan hemorrhagic fevers (Sihibamiya et al . , Blood 113:714-722, 2009).
• cynomolgus or murine TLR3 were assessed using the ISRE reporter gene assay as described in Example 2.
• the cynomolgus (SEQ ID NO: 217) and murine TLR3 cDNAs (SEQ ID NO: 161) were amplified from whole blood and cloned into the pCEP4 vector (Clontech) , and expressed as described above.
• mAb 15EVQ had IC50s of 4.18 ⁇ g/ml and 1.74 ⁇ g/ml in the cyno NF- ⁇ and ISRE assays, respectively, compared to IC50s of 0.44 and 0.65 ⁇ g/ml in the human TLR3 NF-kB and ISRE assays, respectively.
• Isotype control antibodies had no effect in these assays.
• TLR3 antibody antagonists protect from acute lethal viral infections
• Example 14 descirbes prophylactic treatment (dosed on days -1, 4, 8, and 12) with TLR3 antibody antagonists against influenza A infection. This example demonstrates that therapeutic dosing of TLR3 antibody antagonists (day 3 after influenza A infection after the onset of clinical symptoms) are efficacious in enhancing survival.
• influenza A virus challenge model was used as a model of acute lethal viral infection as described in Example 14, except that dosing of animals with mAb 5249 was done 3 days post infection with influenza A, and the animals dosed were 8 weeks old.
• Anti-mouse IgGl isotype control mAb was from BioLegend. The animals were dosed days 3, 7 and 11 post- infections with influenza A.
• the human TLR3 extracellular domain was crystallized in complex with Fabs of mAb 15EVQ, mAb 12QVQ/QSV and mAb cl068.
• TLR3 ECD amino acids 1-703 of SEQ ID NO: 2
• TLR3 ECD 4 mg was mixed with 2.4 mg of each Fab and incubated at 4 °C for 3.5 h, corresponding to a molar ratio of 1 TLR3 ECD: 1.1 Fab.
• the complex was purified by anion exchange chromatography on a MonoQ 5/50 GL column (GE Healthcare, Piscataway, NJ) , equilibrated with 20 mM Tris pH 8.5, 10% glycerol (buffer A) and eluted with 20 mM Tris pH 8.5, 10% glycerol, 1 M NaCl (buffer B) .
• MMS Microseed-Matrix Screening
• Crystallization screening was performed using the Oryx4 automatic protein crystallization robot (Douglas Instruments) by dispensing components in the following volume ratio: 1 protein solution: 0.25 seed stock: 0.75 reservoir solution. Crystals diffracting to -10-A resolution grew from 0.1 M Na acetate pH 4.5, 2.9 M (NH 4 ) 2 S0 4 , 5% methyl-pentane-diol (MPD) and 0.1 M Na acetate pH 4.5, 26% PEG3350, 1 M LiCl.
• MMS with the above conditions was combined with additive screening using selected components of the Hampton Additive Screen HR2-428 (Hampton Research, Aliso Viejo, CA) in the following volume ratio: 1 protein solution: 0.125 seed stock: 0.2 additive solution: 0.675 reservoir solution.
• X-ray quality crystals of the TLR3 ECD complexed with the Fabs, which diffract to ⁇ 5-A resolution, were obtained after applying a combination of MMS and Additive screening from a solution containing 0.1 M Na acetate pH 4.5, 28% PEG 3350, 1 M LiCl, and 30 mM Gly-Gly-Gly.
• a crystal size -1.0 x 0.5 x 0.1 mm 3
• a synthetic mother liquor 0.1 M Na acetate, pH 4.5, 28% PEG 3350, 1 M LiCl, 16% glycerol
• flash frozen in the stream of nitrogen at 100 K.
• X-ray diffraction data were collected and processed using a Rigaku MicroMaxTM-007HF microfocus X-ray generator equipped with an OsmicTM VariMaxTM confocal optics, Saturn 944 CCD detector, and an X-streamTM 2000 cryocooling system (Rigaku,
• the crystal structure of the TLR3 ECD - Fab 15EVQ - Fab 12QVQ/QSV - Fab cl068 was determined by molecular replacement using Phaser (Read, Acta Crystallogr. D. Biol. Crystallogr. 57:1373-1382, 2001).
• the search models were TLR3 ECD
• the structure was refined as rigid body domains (each V or C domain) for the Fabs and 13 rigid segments (Definitions used in the refinement: 30-60, 61-108,109-156,157-206,207-257,258- 307, 308-363, 364-415, 416-464, 465-514, 515-570, 571-618, 619-687) for the TLR3 ECD with one B factor for each Fab rigid body and a single B for the entire TLR3 ECD.
• TLS Translation/ Libration/ Screw refinement was introduced for each of the Fab rigid bodies and TLR3 ECD was divided into 2 TLS segments at residue 330 of SEQ ID NO: 2. Glycan density was visible for 10 of the 15 N-glycosylation sites.
• Carbohydrate models from the crystal structure of the human TLR3 extracellular domain Choe et al . , Science
• TLR3 ECD The density for a short missing segment in TLR3 ECD (residues 337-342 of SEQ ID NO: 2) was visible after rigid body refinement, and it was filled in with the corresponding segment from the TLR3 extracellular structure 2a0z (Bell et al., Proc. Natl. Acad. Sci. (USA) 102:10976-10980, 2005, PDB strucutre ID: 2a0z) .
• the C-terminus of TLR3 ECD contained additional density that matches that of 2a0z.
• TLR3 ECD model was a hybrid between the TLR3 sturctures lziw and 2a0z and refined as 13 rigid body segments (amino acid range: 30-60,61-108,109- 156, 157-206, 207-257, 258-307, 308-363, 364-415, 416-464, 465- 514, 515-570, 571-618, 619-687) .
• the LCDR3 of Fab 12QVQ/QSV apparently adopted different conformation from its free form.
• Multi-start simulated annealing was carried out with standard parameters in PHENIX.
• the models of this LCDR3 were visually inspected in the electron density map and the "best-matching" conformation was grafted onto the original model.
• the refinement process was monitored by Rf ree against 5% of the reflections set aside prior to initiating the calculations . In the final round, one B factor for each residue was included.
• Model inspection and manual rebuilding of the elbow regions of the Fabs and side chains at the protein-protein interfaces were done using COOT (Emsley et al . , Acta Crystallogr. D. Biol. Crystallogr. 60:2126-32, 2004).
• the final R cryst and R free were 26.8% and 30.0%, respectively, for all 15,792 independent reflections to 5.0 A.
• the refinement statistics are given in Tables 13 and 14.
• the overall molecular structure of the complex is shown in Figure 28.
• the structural model for TLR3 ECD includes all residues from 30 to 687 of huTLR3 (SEQ ID NO: 2) .
• the TLR3 ECD molecule is very similar to the previously reported structures in overall topology (rmsd of 0.79 A for lziw, 613 Ca's, and 1.37 A for 2a0z, 595 Ca's).
• the Fab structures are all identical to their respective unbound forms except for LCDR3 of Fab 12QVQ/QSV as described in Methods as well as the elbow regions and some side chains at TLR3 ECD/Fab interfaces.
• Values for highest resolution shell are in ()'s.
• R o r y i : - ⁇ "F obs ! - !F csk .]
• conformational epitope was composed of residues from the TLR3 LRRs 3-7 (amino acids 100-221 of SEQ ID NO: 2.
• the binding of Fab 12QVQ/QSV buried approximately 928 A 2 and 896 A 2 on the antigen and antibody, respectively.
• TLR3 SEQ ID NO: 2
• epitope residues S115, D116, K117, A120, K139, N140, N141, V144, K145, T166, Q167, V168, S188, E189, D192, A195, and A219.
• Fab 12QVQ/QSV the crystal structure identified following paratope residues: light chain (SEQ ID NO: 211) : G28, S29, Y30, Y31, E49, D50, Y90, D91, and D92.
• Heavy chain SEQ ID NO: 214) : N32, Q54, R56, S57, K58, Y60, Y104, P105, F106, and Y107.
• Fab 15EVQ and Fab cl068 bound non-overlapping epitopes spanning LRRs 15-23 (amino acids 406-635 of SEQ ID NO: 2) near the C-terminus ( Figure 28) .
• Fab 15EVQ buried 1080 A 2 and 1064 A 2 on the antigen and antibody, respectively, whereas Fab cl068 buried 963 A 2 and 914 A 2 on the antigen and antibody, respectively.
• the epitope for Fab 15EVQ covers residues K416, K418, L440, N441, E442, Y465, N466, K467, Y468, R488, R489, A491, K493, N515, N516, N517, H539, N541, S571, L595 and K619 of TLR3 shown in SEQ ID NO: 2.
• the crystal structure identified following paratope residues light chain (SEQ ID NO: 41) : Q27, Y32, N92, T93, L94, and S95.
• Heavy chain (SEQ ID NO: 216) W33, F50, D52, D55, Y57, N59, P62, E99, Y101, Y104, and D106.
• Heavy chain T30, T31, Y32, W33, H35, E50, N52, N54, N55, R57, N59, V99, M102, 1103, and T104.
• the mAb 15EVQ epitope contains TLR3 residues N517, H539 and N541, which overlap with the C-terminal dsRNA binding site (Bell et al . , Proc . Natl. Acad. Sci . USA,
• both mAb 12QVQ/QSV and Fab cl068 can bind to a signaling unit (SU) without disrupting its function. Sterically, it is unlikely that the two Fab fragments of a mAb molecule would be able to bind
• Binding of TLR3 to dsRNA is not limited to the signaling unit defined by the dsRNA:TLR3 complex (Liu, et al . , Science, 320: 379-81, 2008) . It is possible that clustering of multiple SUs can lead to enhancement of signaling or that efficient
• TLR3 signaling requires this clustering.
• the positioning of mAb 12QVQ/QSV and mAb cl068 can block the clustering and result in neutralization of TLR3 activity.
• the maximal neutralization effects of antibodies would therefore be dependent upon the degree of separation of SUs due to antibody binding.
• mAb 12QVQ/QSV would cause larger separation than mAb cl068, and this could translate to greater potency of mAb 12QVQ/QSV. This is consistent with observations that mAb cl068 and mAb 15EVQ can lead to -50% and 100% TLR3 neutralization at saturation concentrations, respectively, and mAb 12QVQ/QSV exhibits intermediate activity.
• TLR3 signaling model in which the dsRNA:TLR3 signaling units cluster to achieve efficient signaling.
• mAb 12QVQ/QSV and mAb cl068 and also define a class of antibodies that can partially modulate TLR3 signaling without interfering with ligand binding or receptor dimerization .
• TL 3 deficiency improves lipid profiles and liver steatosis
• TLR3 _ ⁇ mice were obtained from Dr. Richard A. Flavell (Yale University) and described previously (Shulman, J. Clin. Invest. 106:171-6, 2000). Both TLR3 _/" and wild type (WT) control mice (C57BL/6) were fed either normal chow or high fat diet (HFD) (Purina TestDiet #58126) consisting of 60.9% kcal fat and 20.8% kcal carbohydrates. Mice were maintained on a 12:12-h light-dark cycle, with water and food ad libitum. The weight of each mouse was measured weekly; the data were presented as means ⁇ SD. Liver samples were taken for RNA isolation and histological analysis.
• HFD high fat diet
• Plasma levels of total cholesterol (TC) , HDL, LDL, and triglycerides (TG) were measured using a clinical chemistry instrument (Alfa Wassermann Diagnostic Technologies, West Caldwell, NJ) .
• Free fatty acid (FFA) levels were determined using a NEFA kit (Wako Chemicals, Richmond, VA) . Sample preparation and assays were performed according to the manufacturer's recommendation.
• TaqMan probes and primers were purchased from Life Technologies, Carlsbad, CA.
• Total of 35 genes involved in lipid and glucose metabolism as well as inflammation were examed for gene expression analysis. The relative
• livers were isolated and wet weight was determined. Liver samples were fixed in 10% neutral buffered formalin, processed for routine paraffin sections, and HE stained. The level of liver adiposity was determined by histological evaluation. The statistical analysis was performed using Mann-Whitney Test.
• TLR3 _ ⁇ mice on chow diet had significantly decreased plasma TG but similar TC, HDL, LDL, (Table 15) and FFA levels compared to WT controls.
• WT control animals showed elevated plasma TC, HDL, and LDL levels, while TLR3 _ ⁇ mice were partially protected (Table 15) .
• Liver weights (normalized to respective body weight) were reduced -16%, p ⁇ 0.05 in TLR3 _/" mice fed HFD compared to those of WT animals on HFD. Histological analysis indicated that TLR3 _ ⁇ animals on HDF also had significantly reduced lipid
• LXRa and PPAR5 were up-regulated (30%, p ⁇ 0.05 and 186%, p ⁇ 0.001, respectively) in TLR3 _/" mice fed HFD when compared to WT animals, consistent with lower lipid levels in TLR3 deficient mice.
• LXRa target genes ABCA1 and SREBP1 were also upregulated (71%, p ⁇ 0.01 and 131%, p ⁇ 0.001, respectively) in these animals.
• Cross-talk between TLR signaling and LXR has been reported (Castrillo et al . , Mol. Cell. 4:805-15, 2003). Upregulation of ABCA1, a cholesterol transporter, suggests improved cholesterol transport in HFD fed TLR3 _ ⁇ mice .

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