WO2015015489A1 - Anticorps convenant au traitement du diabète et de maladies autoimmunes - Google Patents

Anticorps convenant au traitement du diabète et de maladies autoimmunes Download PDF

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WO2015015489A1
WO2015015489A1 PCT/IL2014/050684 IL2014050684W WO2015015489A1 WO 2015015489 A1 WO2015015489 A1 WO 2015015489A1 IL 2014050684 W IL2014050684 W IL 2014050684W WO 2015015489 A1 WO2015015489 A1 WO 2015015489A1
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antibody
nkp46
diabetes
seq
cells
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PCT/IL2014/050684
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English (en)
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Angel Porgador
Ofer Mandelboim
Shlomo Nedvetzki
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Biolinerx Ltd.
Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
B.G. Negev Technologies & Applications Ltd.
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Publication of WO2015015489A1 publication Critical patent/WO2015015489A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention in some embodiments thereof, relates to novel NKp46 antibodies and, more particularly, but not exclusively, to the use of same for preventing and/or treating diabetes and autoimmune diseases.
  • Type 1 diabetes is a multifactorial autoimmune disease in which insulin- producing beta cells in pancreatic islets are destroyed by autoreactive T cells. Mononuclear cells infiltrate the pancreatic islets of Langerhans during a variable period of clinically silent inflammation (insulitis), and eventually T cells destroy insulin- producing beta cells. Full-blown type 1 diabetes ensues when most beta cells are destroyed and the pancreas ceases to produce enough insulin. Exogenous insulin must then be administered for life. To date, the only treatment that frees T1D patients from their insulin dependency is allogeneic transplantation of islets obtained from deceased donors.
  • Type 2 diabetes is characterized by insulin resistance which may be combined with reduced insulin secretion.
  • the defective responsiveness to insulin is believed to involve the insulin receptor.
  • hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver.
  • As the disease progresses impairment of insulin secretion occurs, and therapeutic replacement of insulin is often required.
  • Beta cell destruction also occurs in type 2 diabetes, and it has been proposed that one contributing factor is increased beta cell apoptosis.
  • T1D Diabetes, especially T1D, is considered to be a T cell mediated disease.
  • NK cells a link between the development of diabetes and NK cells has been suggested [Poirot L. et al., Proc Natl Acad Sci U S A. (2004) 101(21):8102-7].
  • Poirot et al. disclose that the proportion and number of NK cells, and the timing of their entry to the pancreas correlate with the severity of T1D in transgenic NOD mice. It has also been disclosed that depletion of NK cells in transgenic NOD mice models of accelerated T1D significantly inhibits diabetes development.
  • the molecular mechanisms of NK cell involvement in T ID are still largely unknown.
  • NK cells recognize target cells through a diverse array of activating receptors and a delicate balance between inhibitory and activating signals tightly regulates their activation.
  • the killing mediated by NK cells involves several activating receptors, such as the natural cytotoxicity receptors (NCRs) NKp30, NKp44, NKp46 and NKG2D.
  • NCRs natural cytotoxicity receptors
  • NKp30, NKp44 and NKp46 are expressed almost exclusively on NK cells, whereas NKG2D is expressed in additional types of lymphocytes such as CD8+ T cells.
  • NKp46 NCR-1 in mice
  • the NKp46 cellular ligands are still unknown.
  • the human NKp46 receptor has multiple isoforms including isoform a (GenBank Accession No. CAA04714); isoform b (GenBank Accession No. CAA06872); isoform c (GenBank Accession No. CAA06873), and isoform d (GenBank Accession No. CAA06874).
  • the NKp46 receptor comprises two extracellular Ig-like domains of the C2 type, a transmembrane portion and an intracellular segment.
  • NKp46Dl a Dl domain
  • NKp46D2 a D2 domain
  • NKp46D2 a D2 domain
  • NKp46 is directly involved in the killing of ⁇ -cells by using a chimeric molecule composed of the extracellular portion of NKp46 fused to human IgGl and showed the existence of NKp46 ligand(s) on pancreas ⁇ -cells. Moreover, injection of soluble NKp46 proteins into non-obese diabetic mice during the early phase of insulitis and the prediabetic stage prevented the development of T 1 D .
  • U.S. Patent Application Publication No. 2012/0076753 discloses the use of the natural cytotoxicity receptor NKp46 for preventing and treating diabetes, including type 1 and type 2 diabetes.
  • U.S. Patent Application Publication No. 2012/0076753 provides compositions comprising a fragment of the extracellular region of NKp46, an antibody specific for the extracellular region of NKp46, or a combination thereof, for preventing the onset and progression of diabetes.
  • U.S. Patent Application Publication No. 2008/0274047 discloses methods of treating immunoproliferative and autoimmune disorders (e.g. type 1 diabetes) using antibodies which bind NK cell receptors (e.g. antibodies directed against human NKp46), particularly to deplete cells involved in the immunoproliferative pathology.
  • U.S. 2008/0274047 further discloses that injection of anti-human NKp46 antibodies into transgenic mice expressing human NKp46 resulted in depletion of NK cells in blood, spleen, liver and lung.
  • U.S. Patent Application Publication No. 2007/0231813 discloses methods and compositions to evaluate the therapeutic response and/or side effects of a subject to a therapeutic composition comprising therapeutic antibodies (preferably antibodies or proteins comprising Fc portions of the G4 subclass) in situations where target neutralization is desired without depletion of a target cell.
  • the composition may specifically bind an NK receptor inter alia NKp46 and the subject may have juvenile onset diabetes.
  • U.S. Patent Application Publication No. 2004/0038339 discloses a multifunctional polypeptide comprising (a) a first domain comprising a binding site specifically recognizing an extracellular epitope of the NKG2D receptor complex; and (b) a second domain having receptor or ligand function, wherein said receptor or ligand function may be an antigen binding site of an antibody or fragment thereof directed against e.g. NKp46.
  • the composition may be used for treating autoimmune diseases, e.g. insulin-dependent diabetes mellitus, wherein elimination of the subpopulation of immune cells that causes the disease is desired.
  • an isolated antibody comprising an antigen recognition region which comprises six complementarity determining region (CDR) amino acid sequences as set forth in: (i) SEQ ID NOs: 4, 6, 8, 12, 14 and 16; (ii) SEQ ID NOs: 20, 22, 24, 28, 30 and 32; or (iii) SEQ ID NOs: 36, 38, 40, 44, 46 and 48.
  • CDR complementarity determining region
  • a pharmaceutical composition comprising as an active ingredient the isolated antibody of some embodiments of the invention and a pharmaceutically acceptable carrier.
  • a method for preventing or treating diabetes or an autoimmune disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of some embodiments of the invention.
  • an article of manufacture comprising the isolated antibody of some embodiments of the invention being packaged in a packaging material and identified in print, in or on the packaging material for use in the treatment of diabetes.
  • an isolated polynucleotide encoding the antibody of some embodiments of the invention.
  • a method of producing an antibody specific for the extracellular region of NKp46 comprising: (a) immunizing a mouse with a NKp46-Ig fusion polypeptide and NCRl-Ig so as to activate antibody producing cells; (b) generating hybridomas from spleen cells of the mouse of step (a) comprising fusing the spleen cells with myeloma cells; (c) isolating RNA from positively selected hybridomas of step (b) and reverse transcribing the RNA into cDNA; and (d) cloning the cDNA of step (c) into a vector; thereby producing the antibody specific for the extracellular region of NKp46.
  • the antibody is an antibody fragment.
  • the antibody is selected from the group consisting of a Fab fragment, an Fv fragment and a single chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is humanized. According to some embodiments of the invention, the antibody is a chimeric antibody.
  • the diabetes is type 1 diabetes.
  • the diabetes is type 2 diabetes.
  • the therapeutically effective amount results in an increase in blood insulin levels of the subject following the administering.
  • the therapeutically effective amount results in reduction in pancreatic beta cell destruction in the subject following the administering.
  • the article of manufacture further comprising insulin.
  • the nucleic acid sequence of the isolated polynucleotide is as set forth in: (i) SEQ ID NOs: 3, 5, 7, 11, 13 and 15; (ii) SEQ ID NOs: 19, 21, 23, 27, 29 and 31; or (iii) SEQ ID NOs: 35, 37, 39, 43, 45 and 47.
  • step (b) of the method is effected by fusing the spleen cells with the myeloma cells at a ratio of 10: 1 in the presence of PEG.
  • FIGs. 1A-D are photographs depicting ELISA screening results of fusion no. 68 hybridomas' antibodies on NKp46-D2-Ig coated plates. Blue wells indicate positive binding to NKp46-D2-Ig.
  • FIG. 2 is a table summarizing ELISA screening results of fusion no. 68 hybridomas' antibodies on plates coated with human Ig, NKp46-Dl-Ig, NKp46-D2-Ig, NKp46-Ig, mouse NCRl-Ig and human LIRl-Ig. Blue highlight indicates specific binding to human NKp46-D2-Ig and full length human NKp46-Ig.
  • FIG. 3 is a table summarizing flow cytometry analysis results of fusion no. 68 hybridomas' antibodies positive staining on NK92-NKp46 cells (NK92 cells over- expressing NKp46) as compared to natural killer 92 (NK92) cell line. Numbers illustrate mean fluorescence intensity. Of note, hybridoma clone numbers 80.2, 35.4, 67.3, 117.4 and 104.1 bind NK92-NKp46 cells with a significantly higher intensity as compared to NK92 cells.
  • FIGs. 4A-N are graphs illustrating flow cytometry analysis results of fusion no.
  • FIGs. 5A-B are photographs depicting analysis of total RNA isolated from positive hybridomas.
  • the isolated total RNA of the sample was run alongside a DNA marker Marker III (TIANGEN, Cat. No: MD103) on a 1.5 % agarose/GelRedTM gel electrophoresis.
  • Figure 5A illustrates the DNA marker Marker III (lane weights in Kb);
  • Figure 5B Lane M illustrates DNA marker Marker III and Lane R illustrates Total RNA of the hybridoma sample.
  • FIG. 6 is a photograph depicting antibody fragment amplification.
  • Four microliters PCR products of each sample were run alongside the DNA marker Marker III on a 1.5 % agarose/GelRedTM gel electrophoresis. The PCR products were purified.
  • Lane M illustrates the DNA marker Marker III; Lane 1 illustrates VH; and Lane 2 illustrates VL.
  • FIGs. 7A-B, 8A-B and 9A-B depict sequencing of antibody fragments from hybridomas with insertions of VH and VL genes. Ten clones from each hybridoma with insertions of VH and VL genes were sent for sequencing of the antibody fragments. The VH and VL genes were found nearly identical. Their consensus sequence was assumed to be the sequence of the antibody produced by the hybridoma antibody.
  • Figures 7A-B illustrate Hybridoma 68.80.2 Sequences
  • Figures 8A-B illustrate Hybridoma 68.117.4 Sequences
  • Figures 9A-B illustrate Hybridoma 68.104.1.31 Sequences.
  • the present invention in some embodiments thereof, relates to novel NKp46 antibodies and, more particularly, but not exclusively, to the use of same for preventing and/or treating diabetes and autoimmune diseases.
  • NK cells kill their targets by using a limited set of NK killer receptors. Amongst these receptors is the NKp46 receptor which plays a critical role in the mechanism of killing targeted cells. However the NKp46 cellular ligands are still unknown.
  • mice injected with low doses of streptozotocin (LDST) in the absence of NKp46 and NKp46-deficient mice had less development of type 1 diabetes (T1D) induced by injection of a LDST.
  • LDST streptozotocin
  • NKp46 is directly involved in the killing of ⁇ -cells by using a chimeric molecule composed of the extracellular portion of NKp46 fused to human IgGl and showed the existence of NKp46 ligand(s) on pancreas ⁇ -cells. Further findings demonstrate that human ⁇ cells are killed in an NKp46-dependent manner and the recognition to the ligand expressed on ⁇ cells is confined to domain 2 (NKp46-D2) and not domain 1 (NKp46-Dl) [Gur C et al., J Immunol. (2011) 187(6):3096-103 and U.S. Patent Application No. 20120076753].
  • mice were immunized with a combination of a NKp46-D2 peptide and a NCR1 peptide (see the Examples sections which follow).
  • This vaccination approach maximizes the generation of antibodies which bind both human NKp46-D2 and the mouse NCR1-D2. Consequently three specific antibodies were generated, each comprising six unique CDR sequences, and all of which specifically bind NKp46.
  • These novel anti-NKp46 antibodies are contemplated for the treatment of disease in cases in which inhibition of NKp46 is warranted (e.g. in diabetes).
  • an isolated antibody comprising an antigen recognition region which comprises six complementarity determining region (CDR) amino acid sequences.
  • CDR complementarity determining region
  • the amino acid sequences are as set forth in SEQ ID NOs: 4, 6, 8, 12, 14 and 16.
  • SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 are arranged in a sequential order (N>C, as CDRs 1-3, respectively) on a heavy chain of the antibody
  • SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16 are arranged in a sequential order (N>C, as CDRs 1-3, respectively) on a light chain of the antibody.
  • the amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the peptide set forth in SEQ ID NOs: 4, 6, 8, 12, 14 or 16, wherein the peptide is capable of binding NKp46 (e.g. the D2 region of human NKp46).
  • NKp46 e.g. the D2 region of human NKp46
  • amino acid sequences are as set forth in SEQ ID Nos: 20, 22, 24, 28, 30 and 32.
  • SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24 are arranged in a sequential order (N>C, as CDRs 1-3, respectively) on a heavy chain of the antibody, while SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32 are arranged in a sequential order (N>C, as CDRs 1-3, respectively) on a light chain of the antibody.
  • the amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the peptide set forth in SEQ ID NOs: 20, 22, 24, 28, 30 or 32, wherein the peptide is capable of binding NKp46 (e.g. the D2 region of human NKp46).
  • NKp46 e.g. the D2 region of human NKp46
  • amino acid sequences are as set forth in SEQ ID Nos: 36, 38, 40, 44, 46 and 48.
  • SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 are arranged in a sequential order (N>C, as CDRs 1-3, respectively) on a heavy chain of the antibody, while SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48 are arranged in a sequential order (N>C, as CDRs 1-3, respectively) on a light chain of the antibody.
  • the amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the peptide set forth in SEQ ID NOs: 36, 38, 40, 44, 46 or 48, wherein the peptide is capable of binding NKp46 (e.g. the D2 region of human NKp46).
  • NKp46 e.g. the D2 region of human NKp46
  • Homology e.g., percent homology, identity + similarity
  • Homology comparison software including for example, the BlastP or TBLASTN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters, when starting from a polypeptide sequence; or the tBLASTX algorithm (available via the NCBI) such as by using default parameters, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
  • NCBI National Center of Biotechnology Information
  • default parameters for tBLASTX include: Max target sequences: 100; Expected threshold: 10; Word size: 3; Max matches in a query range: 0; Scoring parameters: Matrix - BLOSUM62; filters and masking: Filter - low complexity regions.
  • isolated refers to at least partially separated from the natural environment e.g., from a cell.
  • antibody refers to an intact antibody molecule and the phrase “antibody fragment” refers to a functional fragment thereof, such as Fab, F(ab') 2 , and Fv that are capable of binding to macrophages.
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain
  • Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
  • two Fab' fragments are obtained per antibody molecule
  • (iii) (Fab') 2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • i fragment of an antibody molecule that can be obtained by treating whole antibody with the enzyme pepsin without
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries, as is known in the art, for example using techniques such as those described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597, and further described hereinbelow.
  • Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11: 1271-77 (1993); and U.S. Patent No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • the CDR amino acid sequences are as set forth in SEQ ID NOs: 4, 6, 8, 12, 14 and 16.
  • the CDR amino acid sequences are as set forth in SEQ ID NOs: 20, 22, 24, 28, 30 and 32.
  • the CDR amino acid sequences are as set forth in SEQ ID NOs: 36, 38, 40, 44, 46 and 48.
  • humanized antibodies are preferably used.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • the antibodies are produced essentially as described hereinbelow. Specifically, mice are first immunized with a NKp46-D2-Ig fusion polypeptide and a NCRl-Ig fusion polypeptide and screened for generation of anti-NKp46 specific antibodies. Next hybridomas are generated by fusing spleen cells of mice generating a high titer of specific anti-NKp46 antibodies with myeloma cells (e.g. NSO myeloma cells at a 10: 1 ratio, respectively, in the presence of PEG). Hybridomas are then screened for specific binding to human NKp46. Total RNA is isolated from positively selected hybridomas and reverse transcribed into cDNA. The cDNA is then cloned into an expression vector which is subsequently introduced into a host cell.
  • myeloma cells e.g. NSO myeloma cells at a 10: 1 ratio, respectively, in the presence of PEG.
  • the ratio of spleen cells producing anti-NKp46 antibodies and myeloma cells may be 25: 1, 20: 1, 15: 1, 10: 1, 5: 1 or 2: 1, as can be determined by one of ordinary skill in the art.
  • Any expression vector may be used in accordance with the present invention.
  • Exemplary mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • prokaryotic or eukaryotic cells may be used as host-expression systems to express the polypeptides of some embodiments of the invention.
  • Exemplary cells which may be used include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence, mammalian cells or other protein expression systems.
  • Mammalian expression systems can also be used to express the polypeptides of some embodiments of the invention.
  • the expression vector (also referred to herein as a "nucleic acid construct") of some embodiments of the invention may further include additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • polypeptides of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the nucleic acid sequence comprises SEQ ID Nos: 3, 5, 7, 11, 13 and 15.
  • the nucleic acid sequence comprises SEQ ID Nos: 19, 21, 23, 27, 29 and 31.
  • the nucleic acid sequence comprises SEQ ID Nos: 35, 37, 39, 43, 45 and 47.
  • nucleic acid constructs may also be used for in-vivo use where they are administered to a subject in need thereof (e.g., diabetic), as further described hereinbelow.
  • antibodies and antibody fragments generated according to the teachings of the present invention serve as inhibitors of NKp46.
  • NKp46 refers to any human or non-human homolog, ortholog or isoform of the human natural cytotoxicity receptor known as NKp46, including for example those having GenBank Accession Nos. NP_001138929.1, NP_001138930.1, NP_001229285.1, NP_001229286.1 or NP_004820.1.
  • the antibodies of the present invention bind the D2 region of human NKp46.
  • the antibody is a bi-specific antibody.
  • Bi- specific antibodies can be e.g. monoclonal antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities can be for NKp46D2 and the other one is for any other antigen, for example a different NK receptor e.g. NKG2D.
  • Methods of generating bi-specific antibodies are disclosed for example, in Suresh et al (Methods in Enzymology 121:210 (1986)).
  • the antibody of the present invention binds
  • NKp46 and inhibits its receptor activity without substantially (e.g., less than 10 %) killing, destroying or eliminating the cell which bears the specific antigen which is recognized by the particular antibody (e.g. the antibody is a "non-depleting antibody” or a "non-cytotoxic antibody”).
  • antibodies may be tested for NKp46 inhibitory activity and binding affinities.
  • Appropriate immunoassays for detecting specific antibody to NKp46 or specific antibody to D2 of NKp46 are known in the art and may be readily used for detecting antibodies according to the present invention.
  • Suitable immunoassays include for example, radioimmunoassays, (RIA), fluorescent immunoassays, (FIA), enzyme-linked immunosorbant assays (ELISA), "sandwich” immunoassays, gel diffusion precipitation reactions, immunodiffusion assays, precipitation reactions, agglutination assays and Immunoelectrophoresis assays [see for example, Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1999)].
  • detection of anti-NKp46 antibody can be carried out using surface plasmon resonance, in which NKp46 bound to an appropriate solid substrate is exposed to the specific antibody. Binding of the antibody to NKp46 on the solid substrate results in a change in the intensity of surface plasmon resonance that can be detected qualitatively or quantitatively by an appropriate instrument, e.g., a BiacoreTM apparatus.
  • One specific use for the antibodies of the present invention is for preventing or treating diabetes in a subject in need thereof.
  • Diabetes refers to a disease resulting either from an absolute deficiency of insulin (type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type 2 diabetes), i.e., impaired insulin action, in an organism.
  • the diabetic patient thus has absolute or relative insulin deficiency, and may display, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine, excessive discharge of urine (polyuria), increased thirst (polydipsia) and increased hunger (polyphagia). Symptoms may develop quite rapidly (e.g. within weeks or months) in type 1 diabetes, particularly in children.
  • Diabetes as used herein encompasses any stage or type of diabetes, including, but not limited to, type 1 diabetes mellitus, type 2 diabetes mellitus, metabolic syndrome, insulin deficiency syndrome, overt diabetes, pre-diabetes, Latent autoimmune diabetes of adults (LAD A), maturity onset diabetes of the young (MODY 1-11) and permanent neonatal diabetes mellitus.
  • LAD A Latent autoimmune diabetes of adults
  • MODY 1-11 maturity onset diabetes of the young
  • permanent neonatal diabetes mellitus permanent neonatal diabetes mellitus.
  • NK cells may be involved in the etiology of autoimmune diseases. Specifically, NK cells may be involved in direct killing of tissue cells, which could lead to acceleration of autoimmunity. As NK cells kill their targets by using NK killer receptors, including the NKp46 receptor, inhibition of NKp46 using the novel antibodies of the present invention may be beneficial for the prevention or treatment of various autoimmune diseases.
  • antibodies of the present invention may further be used for preventing or treating autoimmune diseases in a subject in need thereof.
  • autoimmune disease refers to a disease where the body's immune system attacks its own cells or tissues.
  • Autoimmune diseases include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
  • autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S 135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S 132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Klin Klin Klin Klinschr 2000 Aug 25;112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S.
  • autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189).
  • autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome.
  • Diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S 125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J.
  • autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 ( 1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), inflammatory bowel disease (IBD) including Crohn's disease, ileitis and ulcerative colitis.
  • chronic inflammatory intestinal diseases Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 ( 1): 16
  • celiac disease Listroenterol Hepatol. 2000 Jan;23 ( 1): 16
  • celiac disease Listroenterol Hepatol. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122
  • IBD inflammatory bowel disease
  • autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
  • autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al, Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun;l 1 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).
  • autoimmune neurological diseases include, but are not limited to, multiple sclerosis (MS, Cross AH. et al, J Neuroimmunol 2001 Jan 1;112 (1-2): 1), Alzheimer's disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.
  • MS multiple sclerosis
  • MS Cross AH. et al, J Neuroimmunol 2001 Jan 1;112 (1-2): 1
  • Alzheimer's disease Oron L. et al, J Neural Transm Suppl. 1997;49:77
  • myasthenia gravis Infante AJ. And Kraig E, Int Rev Immuno
  • autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
  • autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1 (2): 140).
  • autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et ah, Lupus 1998;7 Suppl 2:S 107-9).
  • autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et ah, Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et ah, Ann N Y Acad Sci 1997 Dec 29;830:266).
  • autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et ah, Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et ah, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun;169: 107).
  • the autoimmune disease comprises multiple sclerosis (MS).
  • the autoimmune comprises inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • treating refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition or keeping a disease, disorder or medical condition from occurring in a subject who may be at risk for the disease disorder or condition, but has not yet been diagnosed as having the disease disorder or condition.
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a disease, disorder or condition.
  • the term "preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
  • the term "subject” refers to an animal, preferably a mammal, most preferably a human being, including both young and old human beings of both genders who suffer from or are predisposed to diabetes.
  • the antibody of the present invention is administered to the subject at a stage of type 1 diabetes comprising pre-insulitis, early insulitis, pre-diabetes and/or overt diabetes.
  • the antibody of the present invention is administered to the subject at a stage of type 2 diabetes comprising hyperinsulinemia, pre-diabetes and/or overt diabetes.
  • the antibody of the present invention is administered to a subject with an autoimmune disease at disease onset, during disease remission, during an acute stage of the disease or during a chronic stage of the disease.
  • Diagnosis of diabetes or of an autoimmune disease is known to one of skill in the art.
  • tests may be used to diagnose diabetes and pre-diabetes by measuring glucose and insulin levels (e.g. blood or urine levels), including for example, fasting plasma glucose (FPG) test, oral glucose tolerance test (OGTT), random plasma glucose (RPG) test, AlC test and serum insulin level test.
  • glucose and insulin levels e.g. blood or urine levels
  • FPG fasting plasma glucose
  • OGTT oral glucose tolerance test
  • RPG random plasma glucose
  • the antibody of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the antibody accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient (antibody) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., diabetes) or prolong the survival of the subject being treated. According to one embodiment, the therapeutically effective amount results in an increase in blood insulin levels and/or in reduction of blood glucose levels (e.g. to normal levels) of the subject following administration of the antibody.
  • a therapeutically effective amount means an amount of active ingredient (antibody) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., diabetes) or prolong the survival of the subject being treated.
  • the therapeutically effective amount results in an increase in blood insulin levels and/or in reduction of blood glucose levels (e.g. to normal levels) of the subject following administration of the antibody.
  • the therapeutically effective amount results in reduction in pancreatic beta cell destruction in the subject following administration of the antibody.
  • Measurement of pancreatic cell mass may be used according to any method known in the art, as for example, by a biopsy, using magnetic resonance imaging (MRI) or using nuclear imaging techniques.
  • measuring pancreatic cell mass may be carried out by using non-invasive imaging using agents that permit visualization of changes in ⁇ -cell mass e.g. using near-infrared fluorescent ⁇ -cell imaging agent or using a radioisotope-labeled fluorescent ⁇ -cell imaging agent as taught in Reiner et al., Proc Natl Acad Sci USA (2011) 108(31): 12815-20; or using a ⁇ -cell-specific monoclonal antibody IC2, modified with a radioisotope chelator for nuclear imaging as taught in Moore et al. Diabetes (2001), Vol. 50(10): 2231-2236.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • the therapeutically effective amount or dose can be estimated from animal models (e.g. STZ diabetic mice) to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • animal models e.g. STZ diabetic mice
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition (e.g. diabetes).
  • an indicated condition e.g. diabetes
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the article of manufacture may further comprise another therapeutic composition for diabetes, e.g. insulin.
  • insulin e.g. insulin
  • the antibodies or fragments thereof can be packaged in one container while insulin may be packaged in a second container both for therapeutic treatment.
  • the insulin composition may comprise any type of insulin known for therapeutics.
  • insulin of the invention may include rapid-acting insulin (e.g. which typically starts working within a few minutes and lasts for a couple of hours), regular- or short-acting insulin (e.g. which typically takes about 30 minutes to work fully and lasts for 3 to 6 hours, intermediate- acting insulin (e.g. which typically takes 2 to 4 hours to work fully and its effect can last for up to 18 hours), or long-acting insulin (e.g. for which there are typically no peak levels in the bloodstream, and can keep working for an entire day).
  • rapid-acting insulin e.g. which typically starts working within a few minutes and lasts for a couple of hours
  • regular- or short-acting insulin e.g. which typically takes about
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • NKp46-Ig2 and NCRl-Igl were generated as previously described [Gazit R et al., Nat Immunol. (2006) 7(5): 517- 23; Mandelboim O et al., Nature. (2001) 409(6823): 1055-60].
  • Truncated fusion proteins of NKp46Dl-Ig (including the leader peptide 1-21 and residues 1-100) and NKp46D2-Ig (residues 101-235) were generated by polymerase chain reaction (PCR) amplification and cloned into a mammalian expression vector containing the Fc portion of human IgGl as previously described [Mandelboim O et al., Nature. (2001) 409(6823): 1055-60].
  • NKp46D2-Ig which lacks its original leader peptide sequence
  • a methionine start codon was added and the PCR- amplified fragment of NKp46D2 was cloned in frame with the leader peptide of CD5.
  • Sequencing of the constructs revealed that all cDNAs were in frame with the human Fc genomic DNA and were identical to the reported sequences.
  • the production of the fusion proteins in COS cells was previously described [Arnon TI et al., Blood. (2004) 103(2):664-72].
  • NKp46D2 Chinese hamster ovary (CHO) cells
  • the NKp46D2-Ig fragment was cloned into the pcDNA 3.1 vector. After recloning, the highest protein-producing clone was adapted for special serum-free medium (CHO- SFM II; Gibco, Grand Island, NY), followed by optimization for growth in large-scale cultures. Supernatants were collected and purified on protein-G columns using fast- protein liquid chromatography (FPLC).
  • FPLC fast- protein liquid chromatography
  • mice were injected intraperitonealy (IP) with 40 ⁇ g of the antigen NKp46-D2-Ig in Complete Freund's Adjuvant (CFA). Two weeks later mice were injected with the antigens NKp46-D2-Ig and NCRl-Ig respectively every other week in incomplete Freund's adjuvant. After 4 injections mice were injected with boost of the antigen NKp46-D2-Ig in adjuvant. After 2-5 days mice were bled and were screened using ELISA for antibodies in the serum against NKp46-D2-Ig and NCRl-Ig.
  • IP intraperitonealy
  • CFA Complete Freund's Adjuvant
  • Spleen cells (approximately 10 cells) from high titer mice were fused with NSO myeloma cell line at a ratio of 10: 1 respectively in the present of 50 % PEG.
  • Mature clones were screened by ELISA and approximately 130 hybridomas were detected for producing antibodies binding to NKp46-D2-Ig, none of them were detected for antibodies binding to antigen NCRl-Ig ( Figures 1A-D).
  • the hybridomas' antibodies were screened by ELISA for binding to human Ig, NKp46-Dl-Ig, NKp46-D2-Ig, NKp46-Ig, mouse NCRl-Ig & human LIRl-Ig ( Figure 2).
  • NK92-NKp46 natural killer 92
  • Figure 3 Staining of NK92-NKp46 cells was carried out using FACS analysis. In short, cells were harvested, washed and incubated with mAb supernatant followed by washing and incubation with fluorescent anti-mouse commercial Abs. Washed cells were then analyzed in FACSCantoTM II flow cytometer.

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Abstract

L'invention concerne un anticorps qui a été isolé et qui comprend un site de combinaison avec l'antigène comprenant six séquences d'acides aminés de région déterminant la complémentarité ou "CDR" (Complementarity Determining Region) telles que définies dans (i) SEQ ID NO: 4, 6, 8, 12, 14 et 16, dans (ii) SEQ ID NO: 20, 22, 24, 28, 30 et 32, ou dans (iii) SEQ ID NO: 36, 38, 40, 44, 46 et 48. L'invention concerne également, d'une part des procédés de production correspondants, d'autre part une composition pharmaceutique comprenant cet anticorps, et enfin des utilisations de l'anticorps et de cette composition.
PCT/IL2014/050684 2013-07-30 2014-07-28 Anticorps convenant au traitement du diabète et de maladies autoimmunes WO2015015489A1 (fr)

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WO2018047154A1 (fr) 2016-09-07 2018-03-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Anticorps anti-nkp46 et leur utilisation thérapeutique
WO2018138032A3 (fr) * 2017-01-24 2018-10-04 Innate Pharma Agents de liaison nkp46
CN108779175A (zh) * 2015-12-28 2018-11-09 依奈特制药公司 NKp46结合蛋白的可变区
WO2019190952A1 (fr) * 2018-03-29 2019-10-03 Lan Bo Chen Anticorps monoclonaux qui se lient à ssea4 et leurs utilisations
US11141463B2 (en) 2016-07-11 2021-10-12 The National Institute for Biotechnology in the Negev Ltd. Fusion proteins with extended serum half life
WO2024026284A3 (fr) * 2022-07-25 2024-04-18 Interius Biotherapeutics, Inc. Polypeptides mutés, compositions les comprenant et leurs utilisations

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