WO2013091903A1 - Anti-crac channel antibodies - Google Patents
Anti-crac channel antibodies Download PDFInfo
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- WO2013091903A1 WO2013091903A1 PCT/EP2012/052641 EP2012052641W WO2013091903A1 WO 2013091903 A1 WO2013091903 A1 WO 2013091903A1 EP 2012052641 W EP2012052641 W EP 2012052641W WO 2013091903 A1 WO2013091903 A1 WO 2013091903A1
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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
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- 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
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- 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
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- 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 relates to antibodies that specifically bind to calcium release-activated calcium (CRAC) channels and methods of mediating T cell activation.
- CRAC calcium release-activated calcium
- Calcium (Ca 2+ ) is a second messenger in many eukaryotic cells.
- T cells T cells
- Ca 2+ signalling is essential for activation.
- Calcium release-activated calcium (CRAC) channels are store operated Ca 2+ entry channels in the plasma membrane. CRAC channels are highly selective for Ca 2+ and maintain a gating mechanism dependent on the depletion of endoplasmic reticulum Ca 2+ stores.
- Antigen recognition by a T lymphocyte initiates the release of Ca 2+ from endoplasmic reticulum (ER) stores. Depletion of Ca 2+ stores from the endoplasmic reticulum triggers the opening of CRAC channels. STIM1 (stromal interaction molecule 1 ) also mediates CRAC channel function. STIM1 is an ER Ca 2+ sensor that will activate CRAC channels during ER Ca 2+ depletion. Thus, during lymphocyte activation, intracellular Ca 2+ levels increase via the influx of Ca 2+ from ER stores as well as through CRAC channels in the plasma membrane.
- ER endoplasmic reticulum
- NFAT nuclear factor of activated T cells
- Ca 2+ - regulated transcription factor that is important in T cell activation.
- NFAT can be found in the cytoplasm of unactivated T cells. During activation, the influx of Ca 2+ results in NFAT
- Ion channels such as the CRAC channel have traditionally been targeted by small molecule modulators. Although small molecules can impair the targeted ion channels, the effect is often non-specific, because of a failure to discriminate between homologous species of a protein genus (i.e., impairing more than one kind of ion channel), and can also have diffuse side effects. Antibodies against ion channels have also been attempted as modulators. Most existing antibodies against ion channels do not perturb channel function and none of the known polyclonal antibodies produce complete channel block (Benham, Nat. Biotechnol. 2005; 23:
- the invention is in part based on the identification of an ion channel-specific antibody that specifically blocks the function of this ion channel. Distinct from deficiencies of small molecules and previous antibodies, an isolated antibody of the invention is ion channel specific, protein specific, and impairs the function of the channel. Specifically, the isolated antibody specifically binds a CRAC channel, more specifically ORAM , and reduces or inhibits the influx of calcium ions through the channel into T cells.
- the CRAC channel may be human CRAC channel.
- the present invention includes an anti-CRAC channel antibody that specifically binds to ORAM and inhibits the influx of calcium ions into a T cell.
- an anti-CRAC channel antibody of the invention reduces or inhibits translocation of NFAT from the cytoplasm to the nucleus.
- an anti-CRAC channel antibody of the invention reduces or inhibits pro-inflammatory cytokine production in T cells.
- an anti-CRAC channel antibody of the invention reduces or inhibits T cell proliferation. More specifically, a particularly useful anti-CRAC channel antibody according to the invention inhibits or decreases one or more effector functions such as IL-1 B, IL-2, IL-4, IL-5, IL-6, IL-13 and IFN-gamma production.
- an anti-CRAC channel antibody of the invention inhibits T cell proliferation.
- an anti-CRAC channel antibody of the invention inhibits T cell activation.
- an anti-CRAC channel antibody of the invention can inhibit T cell activation and effector function
- an anti-CRAC channel antibody may be administered to a subject in order to suppress an aberrant cell mediated immune response, such as that observed in undesirable inflammation, autoimmunity and graft versus host disease.
- An embodiment includes a method of administering an anti-CRAC channel antibody of the invention to inhibit graft versus host disease, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, or multiple schlerosis in a subject and/or to treat an autoimmune disorder in a subject and/or to treat chronic or other undesirable inflammation in a subject.
- Anti-CRAC channel antibodies of the invention include an antibody that binds the second extracellular loop of the Orail and inhibits or abrogates CRAC channel function.
- the anti-CRAC channel antibody is able to block the influx of calcium ions into T cells.
- the binding of the anti-CRAC channel antibody inhibits activation of T cells and also inhibits the translocation of NFAT from the cytoplasm to the nucleus.
- an anti-CRAC channel antibody is a monoclonal antibody (mAb) that is capable of binding the first or the second extracellular loop of CRAC.
- an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN; SEQ ID NO: 10) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence of amino acids 50 to 66
- HIYPGDGDTNYNGKFKG SEQ ID NO: 1 1
- SEQ ID NO: 9 SEQ ID NO: 9
- one, two or three of these amino acids may be substituted by a different amino acid
- a CDRH3 sequence of amino acids 99 to 107 GTTTVWDY; SEQ ID NO: 12
- the CDRH2 sequence is amino acids 50 to 58 (HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9.
- an anti-CRAC channel antibody comprises a light chain comprising a CDRL1 sequence of amino acids 24 to 40 (KSSQSLLNSRT; SEQ ID NO: 18) of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid; and/or a CDRL2 sequence of amino acids 56 to 62 (WASTRES; SEQ ID NO: 19) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid; and/or a CDRL3 sequence of amino acids 95 to 103 (KQSYNLPWT; SEQ ID NO: 20) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid.
- KQSYNLPWT KQSYNLPWT
- an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN; SEQ ID NO: 10) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid;
- a CDRH2 sequence of amino acids 50 to 66 HYPGDGDTNYNGKFKG; SEQ ID NO: 1 1 ) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and/or a CDRH3 sequence of amino acids 99 to 107 (GGTTVWDY; SEQ ID NO: 12) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and a light chain comprising a CDRL1 sequence of amino acids 24 to 40 (KSSQSLLNSRT; SEQ ID NO: 18) of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid; and/or a CDRL2 sequence of amino acids 56 to 62 (WASTRES; SEQ ID NO: 19) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid; and/or a CDRL3 sequence
- the CDRH2 sequence is amino acids 50 to 58 (HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9.
- an anti-CRAC channel antibody comprises SEQ ID NO: 9.
- an anti-CRAC channel antibody comprises SEQ ID NO: 17.
- an anti-CRAC channel antibody comprises a heavy chain variable domain having at least one of the following sequences: (a) a CDRH1 comprising SEQ ID NO: 10, optionally wherein one of these amino acids is substituted with a different amino acid or (b) a CDRH2 comprising SEQ ID NO: 52, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
- an anti-CRAC channel antibody comprises a CDRH2 that is amino acids 1 -9 of a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 58, SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 56, optionally wherein one of these amino acids is/are substituted with a different amino acid.
- an anti-CRAC channel antibody further comprises a CDRH3 selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 41 and SEQ ID NO: 50, optionally wherein one or two of these amino acids is substituted with a different amino acid.
- an anti-CRAC channel antibody further comprises a light chain variable domain, wherein the light chain variable domain comprises at least one of the following sequences (a) to (c): a) a CDRL1 comprising SEQ ID NO: 57, optionally wherein one or two of these amino acids is substituted with a different amino acid; b) a CDRL2 comprising SEQ ID NO: 19, optionally wherein one or two of these amino acids is substituted with a different amino acid; and c) a CDRL3 comprising SEQ ID NO: 55.
- an anti-CRAC channel antibody further comprises a CDRL1 having SEQ ID NO: 18, optionally wherein one or two of these amino acids is substituted with a different amino acid.
- an anti-CRAC channel antibody further comprises wherein CDRL1 comprising consensus sequence SEQ ID NO: 54, wherein CDRL1 is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 46, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
- an anti-CRAC channel antibody further comprises a CDRL3 selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 43 and SEQ ID NO: 47, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
- an anti-CRAC channel antibody binds an epitope comprising one or more residues selected from the group consisting of G12, Q13, P14, R15, P16, T17, S18, K19, P20, P21 , A22, S23, G24, A25, A26, A27, N28, V29, S30, T31 , S32, G33, I34, T35, P36, G37, Q38, A39 of SEQ ID NO: 3.
- an anti-CRAC channel antibody is a monoclonal antibody. In some embodiments, an anti-CRAC channel antibody is a polyclonal antibody. In some embodiments, an anti-CRAC channel antibody is selected from the group consisting of a chimeric antibody, an affinity matured antibody, a humanized antibody, and a human antibody. In some embodiments, an anti-CRAC channel antibody is an antibody fragment. In some embodiments, the antibody fragment is a Fab, Fab', F(ab') 2 , or scFv.
- the invention also provides a nucleic acid that encodes an anti-CRAC channel antibody of the invention, such as polynucleotides which encode an antibody light chain and/or an antibody heavy chain variable domain of the invention.
- a nucleic acid encodes an anti-CRAC channel antibody comprising polynucleotides of SEQ ID NOs: 14, 15, and 16, which encode CDRH1 , CDRH2, and CDRH3, respectively.
- a nucleic acid encodes an anti-CRAC channel antibody comprising polynucleotides of SEQ ID NOs: 18, 19 and 20, which encode CDRL1 , CDRL2 and CDRL3, respectively.
- the invention provides host cells comprising a nucleic acid or a vector of the invention.
- a vector can be of any type, for example a recombinant vector such as an expression vector. Any of a variety of host cells can be used.
- a host cell is a prokaryotic cell, for example, E. coli.
- a host cell is a eukaryotic cell, for example a mammalian cell such as Chinese Hamster Ovary (CHO) cell.
- a host cell is a plant cell, for example, tobacco.
- the invention also provides pharmaceutical compositions comprising an anti-
- CRAC channel antibody of the invention and a pharmaceutically acceptable carrier.
- Fig. 1 is a schematic depicting the ORAM subunit pore of the CRAC protein.
- ORAM has four transmembrane helices (M1 , M2, M3, and M4) that span the plasma membrane.
- ORAM also has two extracellular loops and one intracellular loop. Both the N- and C-termini are on the cytoplasmic side of the cell membrane.
- extracellular loops of ORAM are accessible to antibody binding domains.
- Figs. 2A and 2B are two graphs of ELISA results plotted as optical density (OD) at 450nm versus antibody dilution.
- Fig. 2 shows polyclonal antibody titers against peptides spanning the first (A) and second (B) extracellular loops of CRAC raised in Balb/c mice.
- A sera from six immunized mice A ( ⁇ ), B ( ⁇ ), C (A ), D (X), E ( * ), and F ( ⁇ ) were tested in an ELISA to detect binding to SEQ ID NO: 2 (first extracellular loop).
- In (B), sera from four immunized mice A ( ⁇ ), B ( ⁇ ), C ( A), and D (X) were tested in an ELISA to detect binding to SEQ ID NO: 3 (second extracellular loop).
- Fig. 3 is series of FACS dot plots with intensity of CFSE fluorescence indicating CRAC+ cells on the x-axis and fluorescence intensity on the y-axis indicating binding to CRAC channels.
- Fig. 3 shows that immunizing mice with the second extracellular loop of CRAC (Fig. 3D-3F), but not the first (Fig. 3A-3C), raises polyclonal titers against native CRAC expressed on the surface of cells.
- Fig. 4 is two FACS dot plots with intensity of CFSE fluorescence indicating
- Two different CRAC-overexpressing cell lines (Ba/F3, Fig. 4A, and Jurkat, Fig. 4B, CRAC+) were generated and hCRAC1415-10F8 (referred to as F8) was evaluated for binding on the untransfected line (CFSE low population) compared to the CRAC-overexpressing line (CFSE high population).
- Fig. 4 shows clone hCRAC1415-10F8 recognized CRAC on the surface of cell lines engineered to overexpress CRAC.
- Fig. 5 is an alignment between human ORAM and mouse ORAM .
- the top line is amino acids 49 to 301 of human ORAM (SEQ ID NO: 1 )
- the middle line is the alignment
- the bottom line is amino acids 54-304 of mouse ORAM (SEQ ID NO:25).
- the putative extracellular loops are shown underlined and in bold.
- Fig. 6 is two graphs of percent inhibition (compared to two different Ig control antibodies) versus antibody concentration. It shows that anti-CRAC channel antibody hCRAC1415-10F8 inhibits (A) NFAT translocation and (B) IL-2 production by Jurkat cells. Jurkat cells carrying an NFAT-luciferase reporter gene were stimulated with PHA-P. NFAT translocation and IL-2 production were measured at 4 hrs or 16 hrs post-stimulation, respectively.
- Fig. 7 is a graph of percent inhibition of T cell proliferation (normalized to untreated control) versus antibody concentration. It shows that anti-CRAC channel antibody hCRACI 415-10F8 inhibits proliferation of primary T cells stimulated with anti-CD3/CD28.
- Fig. 8 is two bar graphs depicting cytokine amount versus antibody amount in the extracellular medium of PBMC cell cultures. Fig. 8 shows that anti-CRAC channel antibody hCRAC1415-10F8 inhibits cytokine production of IL-4 (A) and IL-1 b (B) in stimulated PBMC cultures.
- Fig. 9 is a graph of cytokine inhibition versus antibody concentration or nonspecific immunosuppression (cyclosporine). It shows that anti-CRAC channel antibody hCRAC1415-10F8 (F8 antibody) inhibits T cell proliferation in a mixed lymphocyte reaction.
- the X-axis is the concentration of agents (cyclosporine A, F8 antibody, two separate lgG1 isotype controls, and no stimulator) used to test proliferation.
- the Y-axis is the percent of T cell proliferation compared to baseline (T cells stimulated in the absence of treatment).
- Fig. 10 is a graph of T cell proliferation by % of baseline versus concentration of antibody or control. It shows that anti-CRAC channel monoclonal antibodies inhibit proliferation of primary T cells stimulated with anti-CD3/CD28.
- Fig. 1 1 is a graph depicting concentration of IL-2 (pg/ml) versus concentration of anti-CRAC channel monoclonal antibody at 16 hours post-stimulation.
- Fig. 12 is a graph depicting concentration of IFN- ⁇ (pg/ml) versus
- Fig. 13 is a graph of T cell proliferation by % of baseline activation versus antibody concentration (nM).
- Anti-CRAC channel antibodies M-hCRAC1415- 10F8A2B3, M-hCRAC1415-14F74A1 , M-hCRAC1415-18F6A2 and M-hCRAC1415- 15F8A5B4-mlgG1 inhibited the proliferation of T cells isolated from RA patients and stimulated with SEB.
- Mouse lgG1 was a negative control.
- Fig. 14 is a series of graphs depicting data generated in a GVHD mouse model system.
- Fig. 14A is a Kaplan-Meier survival curve depicting the time to GVHD as well as the statistical analysis of the anti-CRAC channel monoclonal antibody (F8) versus the negative control (negative isotype antibody).
- Figs. 14B and 14C are bar graphs that quantify total human T cells by (B) the number of human CD4+ cells per 100 ⁇ _ of blood and (C) the number of human CD8+ cells per 100 ⁇ _ of blood.
- Figs. 14B and C depict the expansion of human T cells at different time points in the GVHD mouse model system.
- Fig. 14A is a Kaplan-Meier survival curve depicting the time to GVHD as well as the statistical analysis of the anti-CRAC channel monoclonal antibody (F8) versus the negative control (negative isotype antibody).
- Figs. 14B and 14C are bar graphs that quantify total human T cells
- Fig. 15 is a series of graphs depicting the effect of anti-CRAC channel monoclonal antibody hCRAC1415-10F8 on IL-2 secretion.
- Fig. 15A graphs concentration of IL-2 versus concentration of antibody and negative control.
- Fig. 15B graphs concentration of IL-2 versus concentration of positive control (CsA).
- Fig. 16 is a series of graphs depicting the effect of anti-CRAC channel monoclonal antibody hCRAC1415-10F8 on IFNy secretion.
- Fig. 16A graphs concentration of IFNy versus concentration of antibody and negative control.
- Fig. 16B graphs concentration of IFNy versus concentration of positive control (CsA).
- SEQ ID NO: 1 is the amino acid sequence for human ORAM , also known as CRAC channel membrane protein 1 ; GenBank accession no. AAH15369.2 Gl:
- SEQ ID NO: 2 is the amino acid sequence of the first extracellular loop of human ORAMwith an additional cysteine at the C-terminus to allow for coupling to carrier proteins.
- SEQ ID NO: 3 is the amino acid sequence of the second extracellular loop of human ORAMwith an additional cysteine at the C-terminus to allow for coupling to carrier proteins.
- SEQ ID NO: 4 is the human ORAI1 gene, which is found at 12q24.31 ;
- SEQ ID NO: 5 is a PCR reverse primer to amplify the heavy chain variable domain sequence.
- SEQ ID NO: 6 is a PCR reverse primer to amplify the heavy chain variable domain sequence.
- SEQ ID NO: 7 is a PCR reverse primer to amplify the light chain variable domain sequence.
- SEQ ID NO: 8 is a PCR reverse primer to amplify the heavy chain variable domain sequence.
- SEQ ID NO: 9 is the amino acid sequence for the heavy chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 10 is the amino acid sequence of CDRH1 of antibodies M- CRAC1415-10F8 A2B6C1 , M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M- hCRAC1415-15F3A6, M-hCRAC1415-15F45A1 , M-hCRAC1415-15F44A6, M- hCRAC1415-15F54A2, M-hCRAC1415-15F58A5B4 and M-hCRAC1415-14F74A1.
- SEQ ID NO: 1 1 is the amino acid sequence of CDRH2 of antibodies M-
- SEQ ID NO: 12 is the amino acid sequence of CDRH3 of antibody M- CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 13 is the nucleotide sequence encoding the heavy chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 14 is the nucleotide sequence encoding CDRH1 of antibody M- CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 15 is the nucleotide sequence encoding CDRH2 of antibody M- CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 16 is the nucleotide sequence encoding CDRH3 of antibody M-
- SEQ ID NO: 17 is the amino acid sequence for the light chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 18 is the amino acid sequence of CDRL1 of antibody M- CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 19 is the amino acid sequence of CDRL2 of antibodies M- CRAC1415-10F8 A2B6C1 , M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M- hCRAC1415-15F3A6, M-hCRAC1415-15F45A1 , M-hCRAC1415-15F44A6, M- hCRAC1415-15F54A2, M-hCRAC1415-15F58A5B4 and M- hCRAC1415-14F74A1.
- SEQ ID NO: 20 is the amino acid sequence of CDRL3 of antibody M-
- SEQ ID NO: 21 is the nucleotide sequence encoding the light chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 22 is the nucleotide sequence encoding CDRL1 of antibody M- CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 23 is the nucleotide sequence encoding CDRL2 of antibody M- CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 24 is the nucleotide sequence encoding CDRL3 of antibody M- CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 25 is the amino acid sequence for mouse ORAM , also known as ORAM ; GenBank accession no. BAF 47905.1 Gl: 126364336.
- SEQ ID NO: 26 is an N-linked glycosylation site where X can be any amino acid except proline.
- SEQ ID NO: 27 is an N-linked glycosylation site where X can be any amino acid except proline.
- SEQ ID NO: 28 is an alternative amino acid sequence of CDRH2 of antibody M-CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 29 is a substituted amino acid sequence of CDRH2 of antibody M-CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 30 is a substituted amino acid sequence of CDRH2 of antibody M-CRAC1415-10F8 A2B6C1 .
- SEQ ID NO: 31 is a peptide spanning the entire second extracellular loop
- SEQ ID NO: 32 is a peptide spanning part of the second extracellular loop (Peptide 1455).
- SEQ ID NO: 33 is a peptide spanning part of the second extracellular loop (Peptide 1454).
- SEQ ID NO: 34 is the heavy chain variable domain amino acid sequence of antibodies M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415- 15F3A6 and M-hCRAC1415-15F45A1 .
- SEQ ID NO: 35 is the CDRH2 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6 and M- hCRAC1415-15F45A1 .
- SEQ ID NO 36 is the CDRH3 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6 and M- hCRAC1415-15F45A1 .
- SEQ ID NO: 37 is the light chain variable domain amino acid sequence of antibodies M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415- 15F3A6 and M-hCRAC1415-15F45A1 .
- SEQ ID NO: 38 is the CDRL1 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6, M- hCRAC1415-15F45A1 , M-hCRAC1415-15F44A6, M-hCRAC1415-15F54A2 and M- hCRAC1415-14F74A1 .
- SEQ ID NO: 39 is the CDRL3 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6, M- hCRAC1415-15F45A1 and M-hCRAC-14F74A1 .
- SEQ ID NO: 40 is the heavy chain variable domain amino acid sequence of antibodies M-hCRAC1415-15F44A6 and M-hCRAC1415-15F54A2.
- SEQ ID NO: 41 is the CDRH3 amino acid sequence of antibodies M- hCRAC1415-15F44A6, M-hCRAC1415-15F54A2 and M-hCRAC1415-15F58A5B4.
- SEQ ID NO: 42 is the light chain variable domain amino acid sequence of antibodies M-hCRAC1415-15F44A6 and M-hCRAC1415-15F54A2.
- SEQ ID NO: 43 is the CDRL3 amino acid sequence of antibodies M- hCRAC1415-15F44A6 and M-hCRAC1415-15F54A2.
- SEQ ID NO: 44 is the heavy chain variable domain amino acid sequence for M-hCRAC1415-15F58A5B4.
- SEQ ID NO: 45 is the light chain variable domain amino acid sequence for M- hCRAC1415-15F58A5B4.
- SEQ ID NO: 46 is the CDRL1 amino acid sequence for M-hCRAC1415-
- SEQ ID NO: 47 is the CDRL3 amino acid sequence for M-hCRAC1415- 15F58A5B4.
- SEQ ID NO: 48 is the heavy chain variable domain amino acid sequence for M-hCRAC1415-14F74A1 .
- SEQ ID NO: 49 is the CDRH2 amino acid sequence for M-hCRAC1415- 14F74A1 .
- SEQ ID NO: 50 is the CDRH3 amino acid sequence for M-hCRAC1415- 14F74A1 .
- SEQ ID NO: 51 is the light chain variable domain amino acid sequence for M- hCRAC1415-14F74A1 .
- SEQ ID NO: 52 is a CDRH2 consensus sequence for an anti-CRAC channel antibody.
- SEQ ID NO: 53 is a IGHV1 -80 V H murine germline sequence.
- SEQ ID NO: 54 is a CDRL1 consensus sequence for an anti-CRAC channel antibody.
- SEQ ID NO: 55 is a CDRL3 consensus sequence for an anti-CRAC channel antibody.
- SEQ ID NO: 56 is a CDRH2 consensus sequence for an anti-CRAC channel antibody.
- SEQ ID NO: 57 is a CDRL1 consensus sequence for an anti-CRAC channel antibody.
- SEQ ID NO: 58 is the amino acid sequence of CDRH2 of antibodies M- hCRACI 415-15F44A6, M-hCRAC1415-15F54A2.
- SEQ ID NO: 59 is a IGKV8-21 V L murine germline sequence.
- SEQ ID NO: 60 is a portion of the IGKV8-21 V L germline sequence.
- amino acid as is commonly understood in the art, is meant to include the naturally occurring L a-amino acids or residues.
- the commonly used one and three letter abbreviations for naturally occurring amino acids are used herein (Lehninger, 1975, Biochemistry, 2d ed., pp. 71 -92, Worth Publishers, New York).
- a "modified” amino acid may be a D-amino acids as well as chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid.
- a "functional equivalent" of an amino acid, such as an analog or a mimetic may be used.
- analogs or mimetics of phenylalanine or proline would allow the same conformational restriction of the peptide compounds as natural Phe or Pro.
- Other examples of amino acids are listed by Roberts and Vellaccio, In: The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, Eds., Vol. 5 p 341 , Academic Press, Inc, N.Y. 1983, which is incorporated herein by reference.
- “Synthetic” amino acids are non-naturally occurring and include, for example, those where the naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acid) are replaced with other side chains, for instance with groups such as alkyi, lower alkyi, cyclic 4-, 5-, 6-, to 7-membered alkyi, amide, amide lower alkyi, amide di(lower alkyi), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered
- C-alpha-methyl amiono acids in particular C-alpha-methyl-leucine may be included in an engineered mimetic or library of such compounds.
- modified and synthetic amino acids include cinnamic acids, phenylglycine (Phg), 2,3-diaminobutyric acid (Dab), 2,4-diaminobutyric acid (gDab), 2,3-diaminopropionic acid (Dap), ⁇ - methylaspartate (MeAsp), cyclohexylalanine ( ⁇ -Cha), norleucine (Nle), norvaline (Nvl), isonipecotic acid (Ina), pipecolic acid (homoproline) (Pip or hPro),
- phenylacetic acids phenylpropanoic acids, 2-aminobutyric acid (Abu), sarcosine (Sar or N-methyl glycine), 6-aminohexanoic acid (Ahx), para-fluoro-Phenylalanine (p-F-Phe), ⁇ -amino-butyric acid (GABA), benzoic acids (such as p-aminobenzoic acid (PABA), etc.), hydrazinobenzoic acids, homophenylalanine (homophe or hPhe), ⁇ -cyanoAlanine ( ⁇ -cyano-Ala), methyl or ethyl aryl ethers of tyrosine (Tyr(Me) or Tyr(Et), respectively), aminoisobutyric acid (Aib, which is also known as ⁇ , ⁇ - dimethylglycine), S-methylcysteine (MeCys), ⁇ , ⁇ '-dimethyl-arginine
- antibody is, as commonly understood in the art, a protein that is capable of specifically binding to an antigen or a portion thereof and includes.
- the term includes full length antibodies of any isotype (that is, IgA, IgE, IgG, IgM and/or IgY) and any single chain thereof.
- antibody is used in the broadest sense and specifically includes monoclonal antibodies (including full length
- antibody refers to traditional antibodies, antibody fragments, antigen binding proteins, immunoadhesins, VHH domains (e.g., camelid antibody), etc.
- a full length antibody generally comprises two heavy chains and two light chains, each comprising a variable domain and a constant domain.
- Each variable domain contains a hypervariable region containing three CDRs or HVLs flanked by four segments of the framework region.
- the "framework region” contains all four segments FR1 , FR2, FR3, and FR4 that flank a set of three hypervariable regions (CDRs or HVLs).
- Antibodies can be chimeric, humanized, or human, for example, and can be antigen-binding fragments of these. Antibodies are generally produced by
- Antibody fragments comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof.
- antibody fragments include Fab, Fab', F(ab') 2 , F(ab)S, Fv fragments, single-chain Fv (scFv; see e.g.. Bird et al., Science 1988; 242:42S-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd
- VH and CHI domain typically the VH and CHI domain
- dAb typically a VH domain
- VH, VL, VhH, and V-NAR domains diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies such as bispecific antibodies, for example formed from antibody fragments; monovalent molecules comprising a single VH and a single VL chain; minibodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al..
- “Functional fragments” substantially retain binding to an antigen of the full-length antibody, and retain a biological activity.
- monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical and have identical structure and specificities except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single immunogenic determinant. Thus, 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.
- monoclonal antibodies 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 using the techniques described in Clackson et al.. 1991 , Nature 352:624-628 and Marks et al., 1991 , J. Mol. Biol. 222:581 -597, for example.
- Chain antibodies contain a portion of a heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and
- humanized antibody is a subset of chimeric antibodies.
- “Humanized” forms of nonhuman ⁇ e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin.
- humanized antibodies may be human immunoglobulins (recipient or acceptor antibody) in which variable domain hypervariable region residues of the recipient antibody are replaced by hypervariable region residues from a nonhuman species (donor antibody), such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
- donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
- the hypervariable regions can be complementarity-determining regions (CDRs) defined by sequence (see, for example Kabat 1991 , 1987, 1983), or hypervariable loops (HVLs) defined by structure (see for example, Chothia 1987), or both.
- the variable domain framework regions are derived from a consensus sequence variable domain, for example, containing at each residue an amino acid compiled as most abundant at that position in a class or subclass of human immunoglobulin variable domains, for example, in a Kabat compilation.
- variable domain framework region (FR) of the human immunoglobulin or consensus sequence is replaced with one or more corresponding residues of the nonhuman donor antibody and/or one or more amino acids of the donor antibody hypervariable regions is replaced with one or more corresponding human residues of the human recipient variable domain.
- one or more residues of the variable domain framework regions and/or hypervariable regions is a residue not found at the corresponding position in the recipient antibody or in the donor antibody.
- variable domain framework regions and hypervariable regions are generally made to further refine antibody performance, for example, improve binding affinity.
- the humanized antibodies used to produce the pan-specific antibodies described herein will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable region residues (CDRS or HVLs) correspond to those of a nonhuman immunoglobulin and all or substantially all of the framework region (FR) residues correspond to those of a human variable domain consensus sequence, and may include one or more amino acid substitutions.
- CDRS or HVLs hypervariable region residues
- FR framework region
- the number of amino acid substitutions in the human consensus framework region is typically no more than 10, and may be, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the heavy chain variable domain framework regions, and 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the light chain variable domain framework region.
- the humanized antibody optionally comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin (see Figure 1 ). For further details, see Jones et al., 1986, Nature 321 :522-525; Reichmann et al., 1988, Nature
- Human antibodies can be antibodies isolated from humans. There are also fully human antibodies produced originally in transgenic mice genetically modified to express human repertoire and immunized with the antigen of interest. See e.g., Bruggemann & Neuberger, Immunol. Today 1996; 17: 391 -397.
- human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
- the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences ⁇ e.g., mutations introduced by random or site-specific mutagenesis In vitro or by somatic mutation In vivo).
- human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, as these are described by other terms defined herein.
- a human antibody as is discussed in this paragraph may be a human monoclonal antibody.
- Such a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
- Human antibodies can be antibodies isolated from humans.
- human antibodies produced originally in, for example, transgenic mice (or other transgenic organisms) genetically modified to express human repertoire and immunized with the antigen of interest. See e.g., Bruggemann & Neuberger, Immunol. Today 1996; 17: 391 -397. Human antibodies may be isolated from sequence libraries built on selections of human germline sequences further diversified with natural and synthetic sequence diversity.
- an "Fv” fragment is an antibody fragment that contains a complete antigen recognition and binding site, and generally comprises a dimer of one heavy and one light chain variable domain in tight association that can be covalent in nature, for example in a single chain variable domain fragment (scFv). It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer.
- hypervariable regions or a subset thereof confer antigen binding specificity to the antibody.
- a single variable domain comprising only three
- hypervariable regions specific for an antigen has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site (Cai & Garen, Proc. Natl. Acad. Sci. USA, 93: 6280-6285, 1996).
- camelid antibodies that only have a heavy chain variable domain (VHH) can bind antigen (Desmyter et al., J. Biol. Chem., 277: 23645-23650, 2002; Bond et al., J. Mol. Biol. 2003; 332: 643-655).
- Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of antibody, where these domains are present in a single polypeptide chain.
- the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains that enables the scFv to form the desired structure for antigen binding.
- a “Fab” fragment includes a variable domain and a constant domain of the light chain and a variable domain and the first constant domain (C H 1 ) of the heavy chain.
- a Fab' fragment includes one or more cysteine carboxy terminal linkages to the heavy or light chains.
- F(ab') 2 antibody fragments comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known.
- diabodies refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H and V L ).
- V H heavy chain variable domain
- V L light chain variable domain
- Diabodies are described more fully, for example, in EP 404,097; WO 93/1 1 161 ; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6444-6448.
- linear antibodies refers to antibodies as described in Zapata et al., 1995, Protein Eng., 8(10): 1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (V H -CH1 -VH-C h 1 ) that, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
- a monobody can bind to an antigen in the absence of light chains and typically has three hypervariable regions, for example CDRs designated CDRH1 , CDRH2, and CDRH3.
- a heavy chain IgG monobody has two heavy chain antigen binding molecules connected by a disulfide bond.
- the heavy chain variable domain comprises one or more hypervariable regions, preferably a CDRH3 or HVL-H3 region.
- hypervariable region when used herein refers to the amino acid residues of an antibody that are responsible for antigen-binding.
- the hypervariable region comprises amino acid residues from a "complementarity-determining region” or "CDR" (defined by sequence as residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
- CDR complementarity-determining region
- HVL residues can include, 26-32 (L1 ), 50-52 (L2) and 91 -96 (L3) in the light chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain.
- phrases such as "Kabat position”, “Kabat residue”, and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains.
- the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
- a heavy chain variable domain may include amino acid insertions (residue 52a, 52b and 52c according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
- the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
- Framework region or "FR” residues are those variable domain residues flanking the hypervariable region residues as herein defined.
- a variable domain contains three hypervariable regions flanked by four sequences of the framework region (FR1 , FR2, FR3, and FR4).
- variable domain sequence refers to an artificial variable domain sequence comprising at each position the residue that is most abundant at that position in the variable domains of a group of antibodies of a particular class, for example, as described in the compilations of Kabat et al., Sequences of Proteins of Immunological Interest,. Public Health Service, National Institutes of Health,
- Polypeptide refers to a peptide or protein containing two or more amino acids linked by peptide bonds, and includes peptides, oligimers, proteins, and the like. Polypeptides can contain natural, modified, or synthetic amino acids. Polypeptides can also be modified naturally, such as by post-translational processing, or chemically, such as amidation, acylation, cross-linking, and the like.
- epitopope is a structure defined in the context of a molecular interaction between an "antigen binding polypeptide”, such as an antibody (Ab), and its corresponding "antigen” (Ag).
- antigen (Ag) refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag.
- Ag is used broadly and is generally intended to include target molecules that are specifically recognized by the Ab, thus including fragments or mimics of the molecule used in the immunization process for raising the Ab.
- epitope refers to the area or region on an Ag to which an Ab specifically binds.
- a protein epitope may comprise amino acid residues in the Ag that are directly involved in binding to a Ab (also called the immunodominant component of the epitope) and other amino acid residues, which are not directly involved in binding, such as amino acid residues of the Ag which are effectively blocked by the Ab (in other words, the amino acid residue is within the "solvent-excluded surface” and/or the "footprint” of the Ab).
- ORAM may comprise a number of different epitopes, which may include, without limitation, (1 ) linear peptide antigenic determinants, (2) conformational antigenic determinants which consist of one or more non-contiguous amino acids located near each other in the mature CRAC conformation; and (3) post-translational antigenic determinants which consist, either in whole or part, of molecular structures covalently attached to CRAC, such as carbohydrate groups.
- a conformational epitope is produced by spatially juxtaposed amino acids from different segments of one (or more) linear polypeptide chain(s).
- a linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain.
- an epitope may include other moieties, such as saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
- the epitope for a given antibody (Ab)/antigen (Ag) pair can be defined and characterized at different levels of detail using a variety of experimental and computational epitope mapping methods.
- the experimental methods include mutagenesis, X-ray crystallography, Nuclear Magnetic Resonance (NMR)
- the epitope for the interaction between the Ag and the Ab can be defined by the spatial coordinates defining the atomic contact points present in the Ag-Ab interaction, as well as information about their relative
- the epitope can be characterized by the spatial coordinates defining the atomic contacts between the Ag and Ab.
- the epitope can be characterized by the amino acid residues that it comprises as defined by a specific criterion such as the distance between atoms in the Ab and the Ag.
- the epitope can be characterized through function, e.g. by competition binding with other Abs.
- the epitope can also be defined more generically as comprising amino acid residues.
- epitope In the context of an X-ray derived crystal structure defined by spatial coordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag, the term epitope is herein, unless otherwise specified or contradicted by context, specifically defined as ORAM residues characterized by having a heavy atom (i.e. a non- hydrogen atom) within a distance of 4 A from a heavy atom in the Ab.
- a heavy atom i.e. a non- hydrogen atom
- Epitopes described on the amino acid level are said to be identical if they contain the same set of amino acid residues.
- Epitopes are said to overlap if at least one amino acid is shared by the epitopes.
- Epitopes are said to be separate (unique) if no amino acid residue is shared by the epitopes.
- Epitopes characterized by competition binding are said to be overlapping if the binding of the corresponding Ab's are mutually exclusive, i.e. binding of one Ab excludes simultaneous binding of the other Ab.
- the epitopes are said to be separate (unique) if the Ag is able to accommodate binding of both corresponding Ab's simultaneously.
- one or more antibodies do not have overlapping epitopes but can not bind simultaneously. Due to tertiary and quaternary structure of an antigen, one antibody may not be able to access its epitope due to previous binding of another antibody.
- paratope refers to the area or region on the Ab to which an Ag specifically binds, i.e. with which it makes physical contact to the Ag.
- an X-ray derived crystal structure defined by spatial coordinates of a complex between an Ab, such as a Fab fragment, and its Ag
- paratope is herein, unless otherwise specified or contradicted by context, specifically defined as Ag residues characterized by having a heavy atom (i.e. a non- hydrogen atom) within a distance of 4 A from a heavy atom in ORAM .
- the epitope and paratope for a given antibody (Ab)/antigen (Ag) pair may be identified by routine methods. For example, the general location of an epitope may be determined by assessing the ability of an antibody to bind to different fragments or variant ORAM polypeptides.
- the specific amino acids within ORAM that make contact with an antibody (epitope) and the specific amino acids in an antibody that make contact with ORAM (paratope) may also be determined using routine methods.
- the antibody and target molecule may be combined and the Ab/Ag complex may be crystallised.
- the crystal structure of the complex may be determined and used to identify specific sites of interaction between the antibody and its target.
- An antibody of the invention may have the ability to "compete" with another antibody of the invention for binding to ORAM or another appropriate target as described herein.
- Such cross-competing antibodies can be identified based on their ability to cross-compete with a known antibody of the invention in standard binding assays. For example, surface plasmon resonance (SPR), ELISA assays or flow cytometry may be used to demonstrate cross-competition. Such cross-competition may suggest that the two antibodies bind to identical, overlapping or similar epitopes.
- Epitope binning refers to the use of competitive binding assays to identify pairs of antibodies that are, or are not, capable of binding an antigen, such as
- mammal for purposes of treatment or therapy refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, and the like. Preferably, the mammal is human.
- a nucleic acid sequence or polynucleotide is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence. Often, "operably linked" DNA sequences are contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic
- oligonucleotide adaptors or linkers are used in accordance with conventional practice.
- Percent (%) amino acid sequence identity with respect to a polypeptide is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California.
- % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
- Percent (%) nucleic acid sequence identity is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a reference polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
- Primary refers to any of an order of mammals comprising humans, apes, monkeys, and related forms, such as lemurs and tarsiers.
- Purifying means increasing the degree of purity of a polypeptide in a composition by removing (completely or partially) at least one contaminant from the composition.
- a “purification step” may be part of an overall purification process resulting in an "essentially pure” composition.
- An essentially pure composition contains at least about 90% by weight of the polypeptide of interest, based on total weight of the composition, preferably at least about 95% by weight.
- an “isolated” antibody is one that has been identified and separated and/or recovered from at least one contaminant component of its natural environment that would interfere with diagnostic or therapeutic uses for the antibody, such as one or more of other antibodies, enzymes, hormones, and other proteinaceous or
- the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
- Isolated antibody includes the antibody in situ within recombinant cells since at least one contaminant component of the antibody's natural environment will not be present. Isolated antibody also includes the antibody in a therapeutic formulation or other
- contaminant component will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
- selective binds refers to the ability of an antibody to adhere to specific antigens and not adhere to other polypeptides.
- a "soluble" portion of a polypeptide refers to a portion that is soluble in water and lacks appreciable affinity for lipids (e.g., missing the
- transmembrane domain or the transmembrane and the cytoplasmic domains.
- a “variant" of a polypeptide refers to a polypeptide that contains an amino acid sequence that differs from a reference sequence.
- the reference sequence can be a full-length native polypeptide sequence or any other fragment of a full-length polypeptide sequence.
- the reference sequence is a variable domain heavy chain or variable domain light chain consensus sequence.
- a polypeptide variant generally has at least about 80% amino acid sequence identity with the reference sequence, usually at least 90, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even at least 99%. It should be noted, however, that to qualify as a "variant" such
- polypeptide sequence must differ from the reference sequence in at least one amino acid residue (which may be replaced or omitted).
- binding affinity is herein used as a measure of the strength of a non-covalent interaction between two molecules, e.g. and antibody, or fragment thereof, and an antigen.
- binding affinity is used to describe monovalent interactions (intrinsic activity).
- Binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determination of the dissociation constant (K D ).
- K D can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method (Biacore).
- the rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants k a (or k on ) and dissociation rate constant k d . (or k 0 fr), respectively.
- binding affinities associated with different molecular interactions may be compared by comparison of the K D values for the individual antibody/antigen complexes.
- specificity of an interaction may be assessed by determination and comparison of the K D value for the interaction of interest, e.g. a specific interaction between an antibody and an antigen, with the K D value of an interaction not of interest.
- the K D for the antibody with respect to the target will be 2-fold, preferably 5-fold, more preferably 10-fold less than K D with respect to the other, non- target molecule such as unrelated material or accompanying material in the environment. More preferably, the K D will be 50-fold less, such as 100-fold less, or 200-fold less; even more preferably 500-fold less, such as 1 ,000-fold less, or 10,000- fold less.
- this dissociation constant can be determined directly by well- known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (Byte 9:340-362, 1984).
- the K D may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman ⁇ Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993).
- Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis.
- the binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a BiacoreTM system.
- SPR Surface Plasmon Resonance
- a competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another ligand of that target, such as another antibody.
- the concentration at which 50% inhibition occurs is known as the Ki.
- the Ki is equivalent to K D .
- the Ki value will never be less than the K D , so measurement of Ki can conveniently be substituted to provide an upper limit for K D .
- An antibody of the invention may have a K D for its target of 1 x 10 "7 M or less, 1 x 10 "8 M or less, or 1 x 10 "9 M or less, or 1 x 10 "10 M or less, 1 x 10 "11 M or less, or 1 x 10 "12 M or less.
- An antibody that specifically binds its target may bind its target with a high affinity, that is, exhibiting a low K D as discussed above, and may bind to other, non- target molecules with a lower affinity.
- the antibody may bind to non- target molecules with a K D of 1 x 10 "6 M or more, more preferably 1 x 10 "5 M or more, more preferably 1 x 10 "4 M or more, more preferably 1 x 10 "3 M or more, even more preferably 1 x 10 "2 M or more.
- An antibody of the invention is preferably capable of binding to its target with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold 200-fold, 500-fold, 1 , 000-fold or 10,000-fold or greater than its affinity for binding to another non-target molecule.
- ORAM (also known as “CRACM1” and “Transmembrane protein 142A”) is a pore subunit of the CRAC channel. ORAM is an approximately 33 kDa transmembrane protein with the following sequence:
- ORAIl (SEQ ID NO: 1; Accession No. AAH15369.2 GL54035070).
- This protein has four helices (Ml, M2, M3, and M4) that span a cell's plasma membrane.
- the ORAIl possesses two extracellular loops of amino acids that separate the first and second transmembrane segments and the third and fourth transmembrane segments. Both the N- and C-termini are on the cytoplasmic side of the cell membrane.
- the extracellular loops of ORAIl are accessible to antibody binding domains. Evidence suggests that a tetramer of ORAIl forms the CRAC channel (Penna et al., Nature 2008;456: 116-20).
- ORAIl mediates Ca 2+ influx following depletion of intracellular Ca 2+ stores and channel activation by the Ca 2+ sensor, STIM1.
- ORAIl may be from any vertebrate, such as any mammal, such as a rodent (such as a mouse, rat or guinea pig), a lagomorph (such as a rabbit), an artiodactyl (such as a pig, cow, sheep or camel) or a primate (such as a monkey or human being).
- ORAIl is, preferably, human ORAIl .
- ORAIl may be translated from any naturally occurring genotype or allele that gives rise to a functional protein.
- Human ORAI1 also known as "CRACM1" and "TMEM142A
- Human ORAI1 is a gene at chromosome 12q24.31. (SEQ ID NO:4; Accession No. NG_007500.1 GL 171541812).
- ORAI1 has just 2 exons, nucleotides 1 to 496 and 14,487 to 15486 of SEQ ID NO:4.
- ORAM or fragments thereof can be used directly as an immunogen to generate antibodies.
- An antigen to be used for production of antibodies can be, for example, a soluble form of the full length polypeptide or a fragment thereof, such as a solubilized full length molecule or a fragment. Extracellular portions of ORAM were used as immunogens to generate mouse anti-CRAC channel antibodies.
- EVQLDADHDYPPGC SEQ ID NO:2; the first extracellular loop with a cysteine added to the C-terminus for coupling to KLH or BSA
- VWKFLPLKKQPGQPRPTSKPPASGAAANVSTSGITPGQAC SEQ ID NO:3; the second extracellular loop with a cysteine added to the C-terminus for coupling to BSA.
- An antibody or antibody fragment can be generated that binds to at least a portion of SEQ ID NO:2 and/or SEQ ID NO:3. Such an antibody could impede Ca 2+ influx and thereby suppress an immune response.
- An anti-CRAC channel antibody that impedes Ca 2+ influx can block T cell activation and/or decrease cytokine production (e.g., IL-2, IL-4, IL-10, etc.) in T cells.
- An anti-CRAC channel antibody can affect activation and/or cytokine production in both CD4+ and CD8+ T cells.
- an anti-CRAC channel antibody can modulate effector T cell responses.
- An antibody of the invention will have the ability to bind to a CRAC channel.
- an antibody of the invention will bind specifically to an extracellular loop of the ORAM . That is, an antibody of the invention will preferably bind to ORAM with greater binding affinity than that at which it binds to another molecule.
- An antibody of the invention may have the ability to bind or specifically bind ORAM as described herein, such as any target molecule as described herein.
- An antibody of the invention may have the ability to compete with another antibody of the invention for binding to ORAM or another appropriate target as described herein.
- an antibody of the invention may cross-compete with another antibody or antibodies described herein (such as anti-M-CRAC1415-10F8
- Such cross- competing antibodies can be identified based on their ability to cross-compete with a known antibody of the invention in standard binding assays.
- SPR e.g. by using a BiacoreTM system, ELISA assays or flow cytometry may be used to demonstrate cross-competition.
- cross-competition may suggest that two antibodies bind to identical, overlapping or similar epitopes.
- Anti-CRAC channel antibodies of the invention include an antibody that binds the second extracellular loop of the ORAM and inhibits or abrogates CRAC channel function.
- the anti-CRAC channel antibody is able to block the influx of calcium ions into T cells.
- the binding of the anti-CRAC channel antibody inhibits activation of T cells and also inhibits the translocation of NFAT from the cytoplasm to the nucleus.
- an anti-CRAC channel antibody comprises a monoclonal antibody (mAb) that is capable of binding the first or the second extracellular loop of ORAM .
- Multiple anti-CRAC channel antibodies generated by the methods of Example 9 comprised V H chains derived from mouse germline sequence IGHV1 -80 V H QVQLQQSGAELVKPGASVKISCKASGYAFSSYWMNVWKQRPGKGLEWIGQIYPGD GDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO: 53) .
- the antibodies generated in Example 9 also comprised highly similar CDRH1 and CDRH2 sequences.
- DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYW ASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNL (SEQ ID NO: 59) and comprised highly similar CDRL1 and CDRL2 and part of CDRL3 from the V K segment sequences but nearly all of these antibodies had a different portion of the CDRL3 segment from the J K segment. Again, without being bound by theory, this may indicate increased importance of CDRL1 and CDRL2 and the part of CDRL3 from the V K segment in binding CRAC channels.
- an anti-CRAC channel antibody is derived from the IGHV1 -80 V H germline sequence (SEQ ID NO: 53) and comprises a CDRH1 comprising SYWMN (SEQ ID NO: 10), wherein one of these amino acids is optionally substituted by a different amino acid; and a CDRH2 comprising
- SEQ ID NO: 52 XIYPGDXDTNYNGKFKG (SEQ ID NO: 52) or amino acids 1 -9 of SEQ ID NO: 52, wherein X is any amino acid, wherein one, two or three amino acids (not including X) may be substituted by a different amino acid.
- SEQ ID NO: 52 is the same as IGHV1 - 80 V H germline when X1 is Gin and X7 is Gly.
- the anti-CRAC channel antibody has a heavy chain derived from the IGHV1 -80 V H germline sequence (SEQ ID NO: 53) and also has a light chain derived from the IGKV8-21 V L germline sequence (SEQ ID NO: 59), and comprises a CDRL1 of KSSQSLXNSRT (SEQ ID NO: 57) or KSSQSLXNSRTRKNYLA (SEQ ID NO: 54), wherein X is a any amino acid, or wherein X is Leu or Phe, and wherein one, two or three amino acids (not including X) are optionally substituted with a different amino acid; and comprises a CDRL2 of WASTRES (SEQ ID NO: 19), wherein one or two of these amino acids is optionally substituted with a different amino acid.
- SEQ ID NOS: 54 and 57 are the same as IGKV8-21 V L germline when X is Leu.
- the anti-CRAC channel antibody further comprises a CDRL3
- an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence comprising SYWMN (SEQ ID NO: 10), wherein one of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence comprising XIYPGDXDTNYNGKFKG (SEQ ID NO: 52), wherein X is any amino acid, wherein one, two or three amino acids (not including X) may be substituted by a different amino acid; and/or a CDRH3 comprising QLGFRYAMDY (SEQ ID NO: 36), DHRDYYAMDY (SEQ ID NO: 41 ), SGRLRFAMDY (SEQ ID NO: 50) or GGTTWVDY (SEQ ID NO: 12).
- CDRH2 is SEQ ID NO: 52 and X1 is His or Gin.
- CDRH2 is SEQ ID NO: 52 and X7 is a nonpoiar amino acid.
- X7 is Gly or Ala.
- amino acids 1 -9 of SEQ ID NO: 52 are used when grafting CDRH2 onto a human framework to make a humanized antibody.
- CDRH2 is SEQ ID NO: 52 plus four additional amino acids on the C-terminal end: XIYPGDXDTNYNGKFKGKATL (SEQ ID NO: 56).
- CDRH2 is HIYPGDGDTNYNGKFKG (SEQ ID NO: 1 1 ), HIYPGDADTNYNGKFKG (SEQ ID NO: 35) or HIYPGDGDTNYNGKFKGKATL (SEQ ID NO: 49), or amino acids 1 -9 of any one of SEQ ID NOS: 52, 56, 1 1 , 35 or 49 when making a humanized antibody.
- an anti-CRAC channel antibody comprises a light chain comprising a CDRL1 sequence comprising KSSQSLXNSRT (SEQ ID NO: 57), wherein X is a any amino acid, wherein one, two or three amino acids (not including X) may be substituted with a different amino acid; and/or a CDRL2 sequence comprising
- WASTRES SEQ ID NO: 19
- one or two of these amino acids may be substituted with a different amino acid
- a CDRL3 sequence comprising
- CDRL1 is SEQ ID NO: 57 plus six additional amino acids on the C-terminal end: KSSQSLXNSRTRKNYLA (SEQ ID NO: 54). .
- CDRL1 is SEQ ID NO: 54 or 57 and X is a nonpoiar or aromatic amino acid. In some embodiments, CDRL1 is SEQ ID NO: 54 or 57 and X is Leu or Phe. In some embodiments, CDRL1 is KSSQSLLNSRT (SEQ ID NO: 18),
- CDRL3 is KQSYNLRT (SEQ ID NO: 39),
- an anti-CRAC channel antibody comprises a monoclonal antibody (mAb) that is capable of binding the first or the second extracellular loop of ORAM .
- an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN; SEQ ID NO: 10) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence of amino acids 50 to 66
- an anti-CRAC channel antibody comprises light chain comprising a CDRL1 sequence of amino acids 24 to 40
- KSSQSLLNSRT of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid
- CDRL2 sequence of amino acids 56 to 62 WASTRES; SEQ ID NO: 19
- CDRL3 sequence of amino acids 95 to 103 KQSYNLPWT
- an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence of amino acids 50 to 66 (HIYPGDGDTNYNGKFKG) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and/or a CDRH3 sequence of amino acids 99 to 107 (GGTTWVDY) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and a light chain comprising a CDRL1 sequence of amino acids 24 to 40 (KSSQSLLNSRT) of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid; and/or a CDRL2 sequence of amino acids 56 to 62 (WASTRES) of SEQ ID
- an anti-CRAC channel antibody comprises SEQ ID NO:9. In an embodiment, an anti-CRAC channel antibody comprises SEQ ID NO: 17.
- Embodiments of an anti-CRAC channel antibody includes can have, for example, at least 80%, at least about 85%, at least about 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the CDRs described herein.
- Such anti-CRAC channel antibodies are useful in methods of the invention.
- Monoclonal antibodies may be made using any number of methods including the hybridoma method first described by Kohler et a ⁇ ., 1975, Nature, 256:495. Monoclonal antibodies can also be made by recombinant DNA methods (see, for example, U.S. Patent No. 7,829,092).
- a mouse or other appropriate host animal such as a hamster
- lymphocytes that produce or are capable of producing antibodies that specifically bind the protein(s) used for immunization.
- lymphocytes may be immunized in vitro.
- lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
- the hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
- a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
- the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
- HGPRT or HPRT the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT- deficient cells.
- Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells.
- Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell
- Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
- the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
- RIA radioimmunoassay
- ELISA enzyme-linked immunosorbent assay
- the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al. 1980, Anal. Biochem. 107:220.
- Screening methods useful to identify anti-CRAC channel antibodies include, for example, ELISA, ECLA, and Biacore analysis. Such screening analysis permits selection of anti-CRAC channel antibodies that selectively bind CRAC- overexpressing cells.
- hybridoma cells that produce anti-CRAC channel antibodies of a desired specificity, affinity, and/or activity may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium.
- the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.
- the anti-CRAC monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
- affinity chromatography e.g., using protein A or protein G-Sepharose
- ion-exchange chromatography e.g., hydroxylapatite chromatography
- gel electrophoresis e.g., dialysis, etc.
- DNA encoding the anti-CRAC monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
- the hybridoma cells serve as a preferred source of such DNA.
- the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
- monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554. Clackson et al., Nature, 1991 , 352:624-628 and Marks et al., 1991 , J. Mol. Biol. 222:581 -597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., 1993, Nuc. Acids. Res. 21 :2265-2266). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of anti-CRAC monoclonal antibodies.
- phage display technology see, for example, McCafferty et al.,
- genes encoding antibody variable domains are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson et al., 1993, Current Opinion in Structural Biology 3:564-571 .
- DNA encoding an antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CL) sequences for the homologous murine
- immunoglobulin coding sequence can be fused with all or part of a sequence encoding a non-immunoglobulin polypeptide
- non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or can substitute for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
- one or more amino acid alterations are made to the amino acid sequence, as known. Alterations, even in the framework sequence, can improve affinity of antibodies.
- Humanized Antibodies Methods for humanizing nonhuman antibodies have been described in the art.
- a humanized antibody has one or more nonhuman amino acid residues introduced into its sequence. These nonhuman amino acid residues are often referred to as "import” or “donor” residues that are typically taken from an “import” or “donor” variable domain and introduced into a human “recipient” sequence.
- Humanization can be performed by substituting hypervariable region sequences (CDRs or HVLs) from nonhuman sources for the corresponding sequences of a human antibody. Accordingly, such "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
- humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
- the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and human anti-mouse antibody (HAMA) response when the antibody is intended for human therapeutic use.
- HAMA human anti-mouse antibody
- the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences.
- the human variable domain sequence which is closest to that of the rodent, is identified and the human variable domain framework region (FR) within it is accepted for the humanized antibody (Sims et al., 1993, J. Immunol. 151 :2296; Chothia et al., 1987, J. Mol. Biol., 196:901 ).
- Another method uses a particular framework region derived from a consensus sequence variable domain.
- the consensus sequence can be delineated by residues identified in a compilation of variable domain residues for known human antibodies in a particular subgroup of light or heavy chains.
- the same variable domain framework region may be used to produce several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285; Presta et al., 1993, J. Immunol. 151 :2623).
- Antibodies can be humanized with retention of high binding affinity for the antigen and other favorable biological properties.
- humanized antibodies are prepared by a process that includes analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, I.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
- variable domain framework region residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
- the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
- the variable domain framework regions of multiple humanized therapeutic antibodies can be identical to the sequences of a consensus sequence, the framework regions (FRs) of each humanized antibody sequence may differ from the FRs of the consensus sequence.
- Amino acid substitutions in the framework regions can be made, for example, to increase affinity to an antigen. See, for example, WO 98/45331 .
- Substituting mouse residues or alternative human residues can increase affinity of the antibody to the antigen.
- substitutions can be made to either the heavy or light chain variable domain. Most FR substitutions occur in the heavy chain variable domain framework region 3 (FR3).
- consensus framework regions can provide a basis for constructing a humanized antibody.
- a humanized antibody may be an antibody fragment, such as a Fab, optionally conjugated with one or more agents in order to generate an
- the humanized antibody may be a full length antibody, such as a full length lgG1 antibody.
- mice transgenic animals
- Mendez et al. (Nature Genet., 1997; 15: 146-156) have generated a line of transgenic mice designated as "XenoMouse ® II" that, when challenged with an antigen, generates high affinity fully human antibodies. This was achieved by germ- line integration of megabase human heavy chain and light chain loci into mice with deletion into endogenous J H segment as described above.
- the XenoMouse ® II harbors 1 ,020 kb of human heavy chain locus containing approximately 66 V H genes, complete D H and J H regions and three different constant regions ( ⁇ , ⁇ , and ⁇ ), and also harbors 800 kb of human ⁇ locus containing 32 VK genes, JK segments and CK genes.
- the antibodies produced in these mice closely resemble that seen in humans in all respects, including gene rearrangement, assembly, and repertoire.
- the human antibodies are preferentially expressed over endogenous antibodies due to deletion in endogenous J H segment that prevents gene rearrangement in the murine locus.
- Tc double trans-chromosomal mouse by introducing two individual human chromosome fragments (hCFs), one containing the entire Ig heavy chain locus (IgH, aboutl .5 Mb) and the other the entire kappa light chain locus (IgK,
- mice mounted antigen-specific human antibody response in the absence of mouse antibodies.
- the Tc technology may allow for the humanization of over megabase-sized, complex loci or gene clusters (such as those encoding T-cell receptors, major histocompatibility complex, P450 cluster etc) in mice or other animals.
- Another advantage of the method is the elimination of a need of cloning the large loci. This is a significant advantage since the cloning of over megabase-sized DNA fragments encompassing whole Ig loci remains difficult even with the use of yeast artificial chromosomes (Peterson et al., Trends Genet. 1997; 13: 61 -66;
- Gene shuffling can also be used to derive human antibodies from non-human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody.
- this method which is also called “epitope imprinting"
- either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras.
- HuMAb-Mouse ® mouse VDJ genes have been inactivated and replaced by human antibody genes.
- the HuMAb-Mouse ® contains unrearranged human antibody genes that encode both heavy and light chains.
- KM- Mouse ® a mouse that is the result of a cross between the HuMAb-Mouse ® and Kirin's TC MouseTM.
- This KM- Mouse ® had the human antibody repertoire of the HuMAb-Mouse HuMAb-Mouse ® and the KM- Mouse ® 's ability to produce all human antibody isotypes.
- Antibody Fragments In certain circumstances it may be advantageous to use antibody fragments, rather than whole antibodies in therapeutic treatments. The smaller size of the fragments allows for rapid clearance and/or may improve access to target cells.
- fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24: 107-1 17; and Brennan et al., 1985, Science, 229:81 ).
- fragments can be produced directly by recombinant host cells.
- Antibody fragments can be obtained using conventional synthetic, recombinant or protein engineering techniques, and the fragments can be screened for antigen-binding or other function in the same manner as are can be intact antibodies.
- Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing the facile production of large amounts of these fragments.
- Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al., 1992, Bio/Technology 10: 163-167).
- F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
- Fab and F(ab') 2 fragments with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046.
- Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
- An antibody of choice can be a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No.
- Fv and scFv are the only species of fragments with intact combining sites that are devoid of constant regions; thus, and are suitable for reduced nonspecific binding during in vivo use.
- scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra.
- the antibody fragment may also be a "linear antibody", e.g., as described in U.S. Patent 5,641 ,870 for example. Such linear antibody fragments may be monospecific or bispecific.
- Bispecific Antibodies are bispecific antibodies (aka “diabodies”).
- bispecific antibodies may bind to two different epitopes of a protein. Other such antibodies may combine a one antibody binding site with a binding site for another protein. Alternatively, one antibody arm may be combined with an arm that binds to a triggering molecule on a cell. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. See Tutt et al., 1991 , J. Immunol. 147:60.
- a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
- the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
- the preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino- terminal to the Fc region.
- the preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites.
- the multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains.
- the polypeptide chain(s) may comprise VD1 -(X1 ) n -VD2- (X2) n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1 .
- the polypeptide chain(s) may comprise: V H -C H 1 -flexible linker-V H -C H 1 -Fc region chain; or V H -CH1 -VH-C h 1 -FC region chain.
- the multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides.
- the multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
- the light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
- Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, and the like.
- Animals can be immunized against an antigen, an antigen cocktail,
- immunogenic conjugates or derivatives thereof by combining, for example, 100 g or 5 g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermal ⁇ at multiple sites.
- the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
- the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
- the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
- Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
- Amino Acid Sequence Variants Amino acid sequence modification(s) of anti-CRAC channel antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
- Amino acid sequence variants of anti-CRAC. antibodies are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the anti- CRAC channel antibody chains, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of an anti-CRAC channel antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
- the amino acid changes also may alter post-translational processes of an anti-CRAC channel antibody, such as changing the number or position of glycosylation sites.
- Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the anti-CRAC channel antibody that are preferred locations for mutagenesis.
- a residue or group of target residues can be identified (e.g., charged residues such as arg, asp, his, lys, and glu and replaced by a neutral or negatively charged amino acid, most preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen.
- Those amino acid locations e.g., charged residues such as arg, asp, his, lys, and glu and replaced by a neutral or negatively charged amino acid, most preferably alanine or polyalanine
- demonstrating functional sensitivity to substitutions are then refined by introducing further or others at, or for, the sites of substitution.
- site for introducing an amino acid sequence variation may be predetermined, the nature of a mutation per se need not be predetermined.
- alanine scanning or random mutagenesis is conducted at the target codon or region and expressed anti-CRAC channel antibody variants are screened for a desired activity (e.g., disruption of NFAT translocation to the nucleus, inhibition of T cell activation, inhibition of calcium influx in T cells, etc.).
- Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
- terminal insertions include an anti-CRAC channel antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag.
- Other insertional variants of an anti-CRAC channel antibody molecule include a fusion of an enzyme or a polypeptide which increases the serum half-life of the antibody to the N- or C-terminus of the anti-CRAC channel antibody.
- variants are an amino acid substitution variant. These variants have at least one amino acid residue in an anti-CRAC channel antibody molecule removed and a different residue inserted in its place. Sites of greatest interest for substitution mutagenesis include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1 , or as further described below in reference to amino acid classes, may be introduced and the products screened.
- Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
- Naturally occurring residues are divided into groups based on common side-chain properties: (1 ) hydrophobic: norleucine, met, ala, val, lei, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
- Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
- cysteine residue not involved in maintaining proper conformation of an- anti CRAC antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
- cysteine bond(s) may be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as a Fv fragment.
- substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
- a parent antibody e.g. a humanized or human antibody
- the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
- a convenient way for generating such substitution variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site.
- the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. inhibition of T cell activation) as herein disclosed.
- alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
- Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
- the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
- Glycosylation Variants are variants in which the glycosylation pattern of an antibody is altered. By altering is meant deleting one or more carbohydrate moieties found in the antibody, adding one or more carbohydrate moieties to the antibody, changing the composition of glycosylation (glycosylation pattern), the extent of glycosylation, etc.
- Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, Chem. Immunol. 1997; 65: 1 1 1 -128 ; Wright and Morrison, Trends Biotechnol. 1997; 15:26-32).
- immunoglobulin's can affect a protein's function (Boyd et al., Mol. Immunol. 1996; 32: 131 1 -1318), and the intramolecular interaction between portions of the
- glycoprotein can affect the conformation and presented three-dimensional surface of the glycoprotein.
- Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. For example, it has been reported that in a galactosylated IgG, the oligosaccharide moiety ' flips ' out of the inter-CH2 space and terminal N-acetylglucosamine residues become available to bind mannose binding protein (Malhotra et al., Nature Med. 1995; 1 :237-243).
- CMCL complement mediated lysis
- CHO cells with tetracycline-regulated expression of ⁇ (1 ,4)- ⁇ - acetylglucosaminyltransferase III (GnTIII), a glycosy transferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et al. Nature Biotech. 1999; 17: 176-180).
- N-linked refers to the attachment of the carbohydrate moiety to the side chain of an
- the tripeptide sequences asparagine-X-serine (SEQ ID NO:26) and asparagine-X-threonine (SEQ ID NO:27), where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
- SEQ ID NO:26 asparagine-X-serine
- SEQ ID NO:27 asparagine-X-threonine
- glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
- glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above- described tripeptide sequences (for N-linked glycosylation sites).
- the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
- removal of glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of the antibody.
- amino acid sequence is usually altered by altering the underlying nucleic acid sequence.
- Nucleic acid molecules encoding amino acid sequence variants of an anti- CRAC channel antibody are prepared by a variety of methods known in the art.
- These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-CRAC channel antibody.
- glycosylation may also be altered without altering the amino acid sequence or the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g. antibodies, as potential therapeutics is rarely the native cell, significant variations in the glycosylation pattern of the antibodies can be expected (see, e.g. Hse et al., J. Biol. Chem. 1997; 272:9062-9070). In addition to the choice of host cells, factors which affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like.
- glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and 5,278,299).
- Glycosylation, or certain types of glycosylation can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H).
- Endo H endoglycosidase H
- the recombinant host cell can be genetically engineered, e.g. make defective in processing certain types of polysaccharides.
- glycosylation structure of antibodies can be readily analyzed by
- oligosaccharides for analytical purposes are also known, and include, without limitation, enzymatic treatment, elimination using harsh alkaline environment to release mainly O-linked structures, and chemical methods using anhydrous hydrazine to release both N- and O-linked oligosaccharides.
- Suitable host cells for cloning or expressing the recombinant polypeptides, including monoclonal antibodies described herein are prokaryote, yeast, or higher eukaryote cells.
- Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis ⁇ e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P.
- Enterobacteriaceae such as Escherichia, e.
- E. coli 294 ATCC 31 ,446
- E. coli B E. coli X1776
- E. coli W31 10 ATCC 27,325.
- an antibody can be isolated from an E coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells.
- eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding.
- Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as
- Schizosaccharomyces pombe Kluyveromyces hosts such as, e.g., K. lactis, K.
- Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
- Suitable host cells for expression are derived from multicellular organisms.
- invertebrate cells include plant and insect cells.
- Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
- a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa caiifornica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
- Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
- Vertebrate cells in culture are also suitable hosts for expression.
- useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod.
- monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442);
- human lung cells W138, ATCC CCL 75
- human liver cells Hep G2, HB 8065
- mice mammary tumor MMT 060562, ATCC CCL51 ; TRI cells (Mather et al., 1982, Annals N. Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
- Host cells can be transformed with expression or cloning vectors for an anti- CRAC channel antibody and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
- Host cells used to produce an anti-CRAC channel antibody may be cultured in a variety of media.
- Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
- 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (e.g., gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary
- hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor
- salts such as sodium chloride, calcium, magnesium, and phosphate
- buffers such as HEPES
- nucleotides such as adenos
- an antibody When using recombinant techniques, an antibody can be produced
- particulate debris either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration.
- Carter et al., 1992, Bio/Technology 10: 163-167 describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and
- phenylmethylsulfonylfluoride over about 30 min.
- Cell debris can be removed by centrifugation.
- supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
- a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
- An antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
- the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
- Protein A can be used to purify antibodies that are based on human ⁇ 1 , ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., 1983, J. Immunol. Meth. 62: 1 -13). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., 1986, EMBO J. 5: 15671575).
- the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
- Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
- the antibody comprises a CH3 domain
- the Bakerbond ABXTM resin J. T. Baker, Phillipsburg, NJ is useful for purification.
- the mixture comprising an antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations ⁇ e.g., from about 0-0.25M salt).
- Anti-CRAC channel antibodies of the invention are capable of T-cell modulation. Such modulation includes inhibiting or reducing T cell activation, proinflammatory cytokine production (e.g., IL-1 B, IL-2, IL-4, IL-5, IL-6, IL-13, IFN- gamma), T cell proliferation, and NFAT translocation.
- Embodiments of the invention include administering an anti-CRAC channel antibody of the invention to a subject in need thereof to inhibit or reduce T cell activation, pro-inflammatory cytokine production (e.g., IL-1 B, IL-2, IL-4, IL-5, IL-6, IL-13, IFN-gamma), T cell proliferation, and NFAT translocation.
- Anti-CRAC channel antibodies of the invention may be administered to a subject with an immune disorder.
- An immune disorder can be an autoimmume disorder or disease and/or a chronic inflammatory disease.
- anti-CRAC channel antibodies of the invention may be administered to a subject with rheumatoid arthritis (RA).
- Anti-CRAC channel antibodies of the invention may be administered to a subject with inflammatory bowel disease (IBD), such as a subject with Crohn's disease (CD), such as a subject with ulcerative colitis (UC).
- IBD inflammatory bowel disease
- CD Crohn's disease
- UC ulcerative colitis
- Anti-CRAC channel antibodies of the invention may be administered to a subject with systemic lupus erythematosus (SLE).
- Anti-CRAC channel antibodies of the invention may be administered to a subject with psoriasis.
- Anti-CRAC channel antibodies of the invention may be administered to a subject with psoriatic arthritis (PA).
- Anti-CRAC channel antibodies of the invention may be administered to a subject with multiple sclerosis (MS). Such administration is expected to result in abatement of the t-cell component of the abnormal immune responses.
- RA Rheumatoid arthritis
- RA Rheumatoid arthritis
- inflammation of the joint which causes pain, stiffness, warmth, redness and swelling.
- This inflammation is a consequence of inflammatory cells invading the joints and these inflammatory cells release enzymes that may digest bone and cartilage.
- This inflammation can lead to severe bone and cartilage damage and to joint deterioration and severe pain amongst other physiologic effects.
- the involved joint can lose its shape and alignment, resulting in pain and loss of movement.
- rheumatoid arthritis There are several animal models for rheumatoid arthritis known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop an inflammatory arthritis that resembles human rheumatoid arthritis. Since CIA shares similar immunological and pathological features with RA, this makes it a suitable model for screening potential human anti-inflammatory compounds. Efficacy in this model is measured by decrease in joint swelling. Efficacy in RA in the clinic is measured by the ability to reduce symptoms in patients which is measured as a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein levels and levels of serum factors, such as anti-citrullinated protein antibodies.
- CIA collagen-induced arthritis
- IBD Inflammatory Bowel Disease
- UC ulcerative colitis
- CD Crohn's disease
- the inflammation is transmural, resulting in abscesses, fistulas and strictures, whereas in UC, the inflammation is typically confined to the mucosa.
- UC ulcerative colitis
- Efficacy in inflammatory bowel disease in the clinic may be measured as a reduction in CDAI score for CD which is scoring scale based on laboratory tests and a quality of life questionnaire.
- efficacy is mostly measured by increase in weight and also a disease activity index (DAI) which is a combination of stool consistency, weight and blood in stool.
- DAI disease activity index
- SLE Systemic lupus erythematosus
- the central mediator of disease in SLE is the production of autoantibodies against self-proteins/tissues and the subsequent generation of immune- mediated inflammation.
- Antibodies either directly or indirectly mediate inflammation.
- T lymphocytes are not thought to directly cause disease, they are required for auto-antibody production.
- the effects of SLE are systemic (kidney, lung,
- glomerulonephritis may result in some cases (i.e. lupus nephritis).
- chromosomal loci have been associated with the disease and may contribute towards different aspects of the disease, such as anti-dsDNA antibodies and
- Efficacy in SLE in human disease and in appropriate mouse models is measured by the ability of the therapeutic entity to decrease auto-antibodies (Eg: anti- dsDNA) and/or by decrease in renal pathology (enhanced kidney function), leading to amelioration of disease symptoms.
- Eg auto-antibodies
- renal pathology enhanced kidney function
- Psoriasis is a T-cell mediated inflammatory disorder of the skin that can cause considerable discomfort. It is a disease for which there is no cure and affects people of all ages. Although individuals with mild psoriasis can often control their disease with topical agents, more than one million patients worldwide require ultraviolet light treatments or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of ultraviolet radiation and the toxicities of many therapies limit their long-term use. Moreover, patients usually have recurrence of psoriasis, and in some cases rebound shortly after stopping immunosuppressive therapy.
- a recently developed model of psoriasis based on the transfer of CD4+ T cells mimics many aspects of human psoriasis and therefore can be used to identify compounds suitable for use in treatment of psoriasis (Davenport et al., Internat. Immunopharmacol. 2:653-672, 2002).
- Efficacy in this model is a measured by reduction in skin pathology using a scoring system.
- efficacy in patients is measured by a decrease in skin pathology.
- Psoriatic arthritis is a type of inflammatory arthritis that occurs in a subset of patients with psoriasis. In these patients, the skin pathology/symptoms are
- MS central nervous system
- myelin a key substance that serves as a nerve insulator and helps in the transmission of nerve signals. MS causes demyelinization of the white matter of the brain, with this process sometimes extending into the grey matter.
- Demyelinization is loss of myelin, which is composed of lipids and protein.
- myelin When myelin is damaged in MS, nerve fiber conduction is faulty or absent. Impaired bodily functions or altered sensations associated with those demyelinated nerve fibers give rise to the symptoms of MS.
- MS There are multiple sub-groups of MS, including relapsing remitting, primary progressive, and secondary progressive.
- Efficacy in mouse models is measured by a reduction in paralysis in the limbs.
- Efficacy in patients is measured by a reduced activity score in the brain as measured by MRI and also an improvement in muscle tone/movement.
- Anti-CRAC channel antibodies of the invention may be administered to a subject in order to attenuate graft versus host disease.
- GVHD is a complication that can occur after a bone marrow or stem cell transplant.
- transplanted T cells from the donor will attack the recipient's body as it recognizes the recipient as foreign material.
- Acute, or fulminant, GVHD usually occurs within the first 3 months after transplant. Whereas, chronic GVHD starts more than 3 months after transplant and can last for the rest of transplant recipient's life. Current treatments seek to suppress the immune response.
- Administration of anti-CRAC channel antibodies can be administered to attenuate the T cell response, thereby attenuating GVHD.
- An embodiment of the invention includes administering an anti-CRAC channel antibody to a patient in need thereof.
- the anti-CRAC channel antibody can be administered before or after transplant.
- the anti-CRAC channel antibody can be administered before or after rejection occurs.
- treatment refers to the medical therapy of any human or other animal subject in need thereof. Said subject is expected to have undergone physical examination by a medical or veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other animal subject.
- the timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject's health.
- said treatment may be palliative, symptomatic and/or curative.
- Preventative or “prophylactic” administration of antibodies of the invention is also contemplated, with prevention being defined as delaying or averting manifestation or aggravation of one or more symptoms of a disease or disorder.
- prophylactic, palliative, symptomatic and/or curative treatments may represent separate aspects of the invention.
- An antibody of the invention may be administered parenterally, such as intravenously, intramuscularly, or subcutaneously, inter alia.
- an antibody of the invention may be administered via a non-parenteral route, such as perorally or topically.
- An antibody of the invention may be administered
- An antibody of the invention may be administered therapeutically.
- anti-CRAC channel antibodies can be administered at a dosage that is efficacious for the treatment of that indication while minimizing toxicity and side effects.
- a therapeutically effective dosage for treating an autoimmune or inflammatory disease may be about 25 mg/dose, about 50 mg/dose, about 75 mg/dose, about 100 mg/dose, about 125mg/dose, or any dosage between those doses.
- a therapeutically effective dosage for treating an autoimmune or inflammatory disease may be between about 1 mg/kg body weight to about 1 1 mg/kg body weight, as well as any doses within that range.
- Dosing frequency may be, but is not limited to, biweekly, once weekly, bimonthly, once every other week, once every three weeks, once monthly or once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks.
- dosage and dosing frequency may change over the course of treatment. For example, a patient may begin with a high dose but be given lower maintenance doses. Similarly, a patient may begin with a certain dosing interval but have maintenance doses at less frequent intervals. In another embodiment, a patient's dosage may be
- Treatment may be continued as long as symptoms persist or until remission.
- Antibodies used in the methods of the invention are administered to a human patient in accord with methods known to medical practitioners, such as by
- intravenous administration e.g., as a bolus or by continuous infusion over a period of time
- subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, intrapulmonary, intracerobrospinal, intra-articular, intrasynovial, intrathecal, intralesional generally by intravenous or subcutaneous administration.
- a humanized anti-CRAC channel antibody is administered by
- Therapeutic formulations of an anti-CRAC channel antibody used in accordance with the present invention can be prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's).
- Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such
- chelating agents such as EDTA
- sugars such as sucrose, mannitol, trehalose or sorbitol
- salt-forming counter-ions such as sodium
- metal complexes e.g. Zn-protein complexes
- non-ionic surfactants such as
- Tween ® PluronicsTM or polyethylene glycol (PEG).
- Another formulation is a liquid multidose formulation comprising the anti-CRAC channel antibody at 40 mg/ml_, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 for storage at 2-8°C.
- Another anti-CRAC formulation can include 10mg/ml_ antibody in 9.0 mg/ml_ sodium chloride, 7.35 mg/ml_ sodium citrate dihydrate, 0.7mg/ml_ polysorbate 80, and Sterile Water for Injection, pH 6.5.
- Yet another aqueous pharmaceutical formulation comprises 10-30 mM sodium acetate from about pH 4.8 to about pH 5.5, preferably at pH5.5, polysorbate as a surfactant in an amount of about 0.01 -0.1 % v/v, trehalose at an amount of about 2-10% w/v, and benzyl alcohol as a preservative (U.S. 6, 171 ,586).
- Lyophilized formulations adapted for subcutaneous administration are described in WO97/04801 . Lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be
- a formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with
- the effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein or about from 1 to 99% of the heretofore employed dosages.
- Active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid
- hydrophobic polymers containing the antagonist which matrices are in the form of shaped articles, e.g. films, or microcapsules.
- sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
- copolymers of L-glutamic acid and ethyl-L-glutamate non-degradable ethylene-vinyl acetate
- degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
- the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
- peptides comprising the first extracellular loop (SEQ ID NO:2) and second extracellular loop (SEQ ID NO:3) were synthesized for use as immunogens.
- mice were immunized with peptides spanning the first or the second extracellular loop of ORAM together with different adjuvants.
- Anti-CRAC channel antibodies were raised with three immunizations with the peptide corresponding to the second extracellular loop.
- the second extracellular loop peptide was coupled to BSA through a C-terminal cysteine, which was added to the C-terminus of the extracellular loop during synthesis.
- Mice were immunized with 20 g protein mixed with complete Freund's adjuvant for the first injection. In the second and third immunizations, incomplete Freund's adjuvant was used.
- Eye-bleeds from mice were screened by ELISA for second extracellular loop peptide-specific polyclonal antibodies.
- the sera were screened for polyclonal titers that bound to native CRAC channels on the surface of cells by flow-cytometry (FACS).
- the first extracellular loop was also used as an immunogen using the same methods except that it was coupled to both BSA and KLH.
- Polyclonal titers were screened by ELISA for first extracellular loop peptide specificity and then screened for native CRAC specificity by FACS, also using the same methods as described above.
- mice with positive titers were boosted with 10 ⁇ g of soluble KLH- peptide conjugate by intravenous injection and sacrificed after three days. Spleens were removed aseptically and dispersed to a single cell suspension. Fusion of spleen cells and myeloma cells (P3X63Ag8.653, ATCC-# CRL 1580) was done by the electrofusion-method. The resulting hybridoma cells were seeded in microtiter plates and cultured at 37°C in 5% CO 2 . The tissue-culture medium was changed two times over a period of 10 days.
- FIG. 1 shows a representative polyclonal antibody titer from the sera of mice immunized with first and second extracellular loop peptides against the first and the second extracellular loop peptides, respectively.
- Figure 3 shows that serum samples from Balb/c mice immunized with the second extracellular loop peptide, but not the first extracellular loop peptide, demonstrated binding to CRAC-overexpressing cell lines.
- the first extracellular loop is the binding site of Ca 2+ ions, it did not raise CRAC-specific antibodies.
- a minimum of 10 Balb/c and 10 Medarex mice were immunized with the first extracellular loop peptide and no polyclonal titers specific for native CRAC on the surface of cells were observed.
- hybridomas were subcloned. Antibodies from hybridoma supernatants were purified over Protein A. Sixteen hybridoma clones were identified that exhibited binding to CRAC on the surface of cells. Screening the hybridoma supernatants derived from mice immunized with the second extracellular loop peptide identified clone anti-M- CRAC1415-10F8 A2B6C1 (also referred to herein as "F8") as a monoclonal antibody that bound CRAC on the surface of cell lines overexpressing CRAC ( Figure 4) using flow cytometry.
- the human Orail protein was overexpressed in both Jurkat cells (a human T- lymphoblastic cell line; ATCC TIB-152, Manassas, VA) and Ba/F3 cells (a murine bone marrow-derived pro-B-cell line; DSMZ -
- CRAC+ cell lines Ba/F3 cells do not express endogenous human CRAC (because Ba/F3 is a mouse cell line) and are referred to as CRAC- cell lines. Wild-type Jurkat do express endogenous human CRAC and so shRNA was used to completely abrogate endogenous expression of CRAC to create a CRAC- Jurkat line. Knockdown of CRAC expression in Jurkat cells was achieved through infection with lentiviral particles containing Orail shRNA (Santa Cruz Biotechnology, sc-76001 -V) and following manufacturer's protocols.
- CRAC positive (CRAC+) cell lines were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) using Invitrogen's CellTraceTM CFSE Cell Proliferation Kit (#C34554) according to the manufacturer's directions. After labeling with CFSE, cells were resuspended in DPBS including 5% fetal bovine serum (FBS) plus 10 pg/mL normal goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a cell final concentration of 4 x10 6 cells/mL.
- FBS fetal bovine serum
- IgG Jackson ImmunoResearch Laboratories, Inc., West Grove, PA
- CRAC negative (CRAC-) cell lines were also resuspended in DPBS/5% FBS plus 10 pg/mL normal goat IgG (Jackson ImmunoResearch Laboratories) at 4 x10 6 cells/mL, but were not labeled with CFSE. CRAC+ and CRAC- cells were mixed together in a 1 : 1 ratio.
- CRAC+/- cell line mixture was added to each well of a 96 well U-bottom BD Falcon plate and incubated for 30 minutes on ice with antibodies at the indicated final concentrations (1 pg/mL, 10 pg/mL, 30 pg/mL, and 50 pg/mL, see Fig. 4).
- the cell mixtures were washed with DPBS/5% FBS.
- Alexa Fluor ® 594 goat anti-mouse IgG Invitrogen A1 1020
- DPBS/5% FBS 10 pg/mL normal goat IgG was added to each well and incubated for 30 min on ice.
- the cells were again washed once with DPBS/5%FBS and were resuspended in 0.1 mL of DPBS/5% FBS for analysis on the Becton Dickinson LSRII SORP flow cytometer. Data were collected using the Becton Dickinson FACS Diva software, and analysis was completed using Tree Star's FlowJo analysis software.
- the untransfected cell lines (CRAC-) are CFSE low and the transfected, overexpressing cell lines (CRAC+) are CFSE high.
- Increased concentrations of F8 antibody demonstrate specific binding to the CFSE high (CRAC+) cells for both the Ba/F3 and Jurkat cell lines.
- the F8 antibody was specific to human CRAC since the F8 antibody did not bind to mouse CRAC (data not shown). Although human and mouse CRAC are highly similar, the sequences are less homologous in the 2 nd extracellular loop, as shown by a sequence alignment (Fig. 5).
- the heavy chain and light chain variable domain sequences of the F8 anti- CRAC channel antibody (M-CRAC1415-10F8 A2B6C1 ) were cloned and sequenced.
- cDNA was synthesized in a 5'- RACE reaction using the SMARTerTM RACE cDNA amplification kit (Clontech).
- GFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciences and cloned for sequencing using a Zero Blunt TOPO ® PCR Cloning Kit and chemically competent TOP10 E.coli (Invitrogen). Colony PCR was performed on selected colonies using an AmpliTaq ® Gold Master Mix from Applied Biosystems and
- M13uni/M13rev primers Colony PCR clean-up was performed using the ExoSAP-l enzyme mix (USB). Sequencing was performed at MWG Biotech, Martinsried Germany using M13uni(-21 )/M13rev(-29) sequencing primers. Sequences were analyzed and annotated using the VectorNTI program.
- a single unique murine kappa type light chain and a single unique murine heavy chain, subclass lgG1 were identified. Nucleic acid and amino acid sequences are listed below (leader peptide sequences have been omitted).
- Anti-M-CRAC1415-10F8 A2B6C1 's heavy chain variable domain amino acid sequence was identified to be the following (CDRs are underlined):
- CDRH2 HIYPGDGDTNYNGKFKG (SEQ ID NO: 1 1 );
- CDRH3 GGTTVWDY (SEQ ID NO: 12).
- Anti-M-CRAC1415-10F8 A2B6C1 's heavy chain variable domain nucleic acid sequence was identified as:
- GGT-3' (SEQ ID NO: 15);
- CDRH3 5' -G GAG GTACTAC G GTAGTAGTTG ACTAC-3' (SEQ ID NO: 16).
- Anti-M-CRAC1415-10F8 A2B6C1 's light chain variable domain amino acid sequence (signal peptide sequence omitted, CDRs underlined):
- CDRL1 KSSQSLLNSRT (SEQ ID NO: 18);
- CDRL2 WASTRES (SEQ ID NO: 19);
- CDRL3 KQSYNLPWT (SEQ ID NO: 20).
- Anti-M-CRAC1415-10F8 A2B6C1 's light chain variable domain nucleic acid sequence was identified as:
- CDRL1 5'-AAATCCAGTCAGAGTCTGCTCAACAGTAGAACC-3' (SEQ ID NO: 22); CDRL2: 5'-TGGGCATCCACTAGGGAATCT-3' (SEQ ID NO: 23); and
- CDRL3 5'-AAGCAATCTTATAATCTTCCGTGGACG-3' (SEQ ID NO: 24).
- CDRH2 Modifications Based on Oxford Molecular's AbM antibody modeling software, CDRH2 can also be defined as amino acids 50-58 of the heavy chain variable domain. Thus, CDRH2 can also be amino acids 50-58 (HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9.
- CDRH2 has a NG deamination site at amino acids 61 -62 of SEQ ID NO: 9.
- a deamination site may be substituted.
- amino acids 50- 66 of SEQ ID NO: 9 may be substituted to eliminate the site with a sequence of HIYPGDGDTNYAQKFKG (SEQ ID NO: 29) or HIYPGDGDTNYAQKFQG (SEQ ID NO: 30).
- NFAT can be found in the cytoplasm of unactivated T cells. During activation, the influx of Ca 2+ results in NFAT dephosphorylation by calcineurin, which fosters NFAT translocation to the nucleus and expression of genes involved in immune response. Measuring NFAT translocation in Jurkat cells after incubation with anti- CRAC channel antibodies therefore provides an indication of the ability of the antibodies to inhibit T cell activation.
- a Jurkat cell line comprising a NFAT luciferase reporter (NFAT-Luc) was resuspended in assay media (RPMI-1640 media containing 10% FBS and 2 pg/mL puromycin) to a concentration of 1 M/mL.
- the cells were plated (10 6 /well) in a BD Purecoat ® 96-well flat bottom plate. Plates were centrifuged for 3 m in at 2000 rpm with high brake. After centrifugation, the plates were flicked and 50 ⁇ _ of antibodies were added. Each of the antibodies was tested at 0.15, 0.3, 0.6, 1 .5, 3.0, 6.0, 12, 25, and 50 pg/mL. Each well was repeated in triplicate.
- the F8 antibody (see Fig. 6A), along with nine other antibodies (see Table 2), inhibited NFAT translocation from the cytoplasm to the nucleus to varying degrees. F8 antibody results were normalized to controls. Thus, incubation with the lgG1 negative control was normalized to 100% NFAT translocation to the nucleus.
- Anti-CRAC Channel Antibodies Inhibit IL-2 Production
- T-cells produce IL-2 during an immune response (i.e., activated T cells). Antigen binding to the TCR stimulates the production of IL-2. Activated CD4+ cells will produce and secrete IL-2, which is essential for the proliferation of both CD4+ and CD8+ cells. Measuring IL-2 production in Jurkat cells incubated with anti-CRAC channel antibodies provides an indication of their ability to inhibit T cell proinflammatory cytokine production and downstream effector functions, such as proliferation.
- Jurkat E6.1 cells (ATCC TIB-152) were harvested from culture, washed once with assay media (RPMI 1640 containing 10% FBS, 1X Gibco ® GlutaMAXTM, and 25 mM HEPES), and resuspended in assay media at 4 x 10 6 cells/mL. From the cell suspension, 25 ⁇ of cells were aliquoted to each well of 96 U-bottom plate (Becton Dickinson) except for the outer perimeter of wells that were left unfilled to create a media barrier. Anti-CRAC channel monoclonal antibodies and matched mouse lgG1 controls were diluted at 4X concentrations into assay media.
- assay media RPMI 1640 containing 10% FBS, 1X Gibco ® GlutaMAXTM, and 25 mM HEPES
- Antibodies were titered 1 :2 into assay media for final 4x concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 pg/mL. 25 ⁇ of antibody titration was added to 25 ⁇ of Jurkat E6.1 cells in a 96 well U-bottom plate. Antibodies were incubated with the cells for 30 min at 37°C in 5% C0 2 . PHA-P (Sigma Aldrich L8754) was diluted into assay media at a 2X concentration (15 pg/ml). 50 ⁇ of PHA-P dilution was added to each well on the 96 well U-bottom plate except for an unstimulated sample as a control. The media barrier was formed by adding 0.2 mL/well of assay media to each open well around the perimeter of the plate. The plate was then incubated at 37°C in 5% CO 2 . Cells were harvested at 16 h for cytokine analysis.
- the Millipore IL-2 Map Human Cytokine/Chemokine Immunoassay (Billerica, MA) was prepared according to the manufacturer's instructions. Cytokine data were collected using the BioRad BioPlex ® Reader and BioPlex ® Manager software.
- F8 antibody (see Fig. 6B), along with seven other antibodies (see Table 2), inhibited IL-2 production by Jurkat cells. F8 antibody results were normalized to controls. Thus, incubation with the lgG1 negative control was normalized to 100% IL- 2 production by the Jurkat cells. In duplicate, increasing concentrations of the F8 antibody inhibited IL-2 production (Fig. 6B), with an IC50 of 1 -4ug/ml_. Inhibition increased with higher F8 concentrations, including nearly 40-50% inhibition of IL-2 production by 50 pg/mL of F8 antibody, depending on the experiment. Figure 6B shows the complete titration curve using F8. Table 2 shows the percent inhibition of the other anti-CRAC mAbs at the 50ug/mL concentration.
- Production Anti-CRAC channel antibodies were shown to inhibit the activity of a T cell line in the previous examples, and so the antibodies were also evaluated for their ability to inhibit the effector functions of primary T cells upon stimulation. Effector functions of T cells include, but are not limited to, proliferation and cytokine production which were analyzed in this example. Total proliferation is measured by CFSE dilution. The florescence of CFSE, a green fluorescent dye that labels intracellular proteins, dilutes by half with each cell division.
- PBMC peripheral blood mononuclear cells
- PBMC were CFSE labeled with the Invitrogen CellTrace ® CFSE Cell
- Anti-CRAC channel antibodies and mouse lgG1 , K control monoclonal antibodies were diluted at 3X concentrations into assay media.
- the antibodies were titered 1 :2 into assay media for final 3X concentrations of 150, 75, 37.5, 18.75, 9.375, 4.6875, 2.343, 1 .1712 pg/ml in final volumes of .050 ml_.
- 2-Aminoethyl diphenylborinate (2-APB; Sigma Aldrich D9754) and Cyclosporin A (CsA; Sigma Aldrich 30024) were diluted into assay media at 3X concentrations of 300 ⁇ and 300 ng/mL respectively, which served as additional controls since both are calcium channel inhibitors.
- 0.050 ml_ of each of these inhibitors was aliquoted into separate wells of the 96 well U-bottom plate (BD Falcon 353077) in triplicate.
- 0.050 ml_ of CFSE labeled PBMC were aliquoted into their appropriate wells of 96 well U-bottom plate. Blocking antibodies and controls were incubated with cells for 1 hr at 37°C in 5% C0 2 .
- Anti-CD3 monoclonal UCHT1 (eBioscience 16-0038) and anti- CD28 costimulatory monoclonal antibody CD28.2 (eBioscience 16-0289) were diluted together to 3X concentrations of 30 ng/ml and 3 pg/rnl, respectively, in assay media. 0.050ml_ of the anti-CD3/CD28 dilution was added to all wells of the 96 well U-bottom plate, with the exception of the unstimulated sample.
- CD5-PE antibody PE conjugated mouse anti-human CD5 antibody (UCHT2) in FACS wash (2.5 ⁇ /.050 ml)
- UCHT2 PE conjugated mouse anti-human CD5 antibody
- FACS wash 2.5 ⁇ /.050 ml
- Cytokine/Chemokine Immunoassay (Cat# MPXHCYTO60KPMX14) was prepared as described in the protocol provided with the kit. IL-1 b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, 11-10, IL-12(p70), IL-13, GM-CSF, INF- ⁇ , MCP-1 , and TNF-a were analyzed.
- Cytokine/chemokine data were collected using the BioRad BioPlex ® Reader and BioPlex ® Manager software.
- the F8 antibody along with eight other anti-CRAC monoclonal antibodies (see Table 2), inhibited T cell proliferation upon stimulation with anti-CD3 and anti-CD28 to varying degrees. Results were normalized to controls (untreated cells). Thus, incubation without an antibody or with control IgG was 100% PBMC proliferation. As shown in Figure 7, increasing concentrations of the F8 antibody inhibited PBMC proliferation, with an IC50 of 1 ug/ml_. Inhibition increased with increased F8 concentrations, including nearly 90% inhibition of PBMC proliferation by 50 pg/mL of F8 antibody in some experiments.
- Increasing concentrations of the F8 antibody inhibited IL-4 and IL-1 b production (Fig. 8).
- a concentration of 50 pg/mL F8 antibody produced equal inhibition of IL-4 production compared to the positive controls, cyclosporine A (CsA) and 2-APB.
- the F8 antibody had a much greater inhibitory effect on IL-1 B production compared to lgG1 isotype control.
- the mixed-lymphocyte reaction (MLR) is an in vitro method for assaying T cell proliferation.
- CFSE a green fluorescent dye that labels intracellular proteins
- the stimulator is first inactivated (via mitomycin c or lethal x-irradiation) before being added to the MLR well. These inactivated cells merely provide foreign alloantigens to the responder population. Within 24-48 hours, the responder T cells have begun proliferating and within another 48 hours an expanding population of functional CTLs has been formed.
- CD4 (T H ) cells, dendritic cells and certain accessory cell types are all critical for the MLR to function.
- the primary MLR cells (cells obtained from an MLR which was performed in the absence of Test Compound) are labeled with CFSE, a green fluorescent dye which is imported into living cells where it is acted upon by an enzyme and then reacts with cellular proteins, the number of cell divisions experienced over time by the labeled cells is reflected in the reduction of green label associated with each cell.
- PBMC from two donors were cultured in RPMI-1640 containing Glutamax ® ,
- HEPES HEPES
- 10% human serum Invitrogen #34005100.
- the PBMC from the two donors were labeled separately with CFSE and resuspended in growth media at 2M/ml_.
- 50 ⁇ _ of each donor's cells was added to a 96-well U-bottom plate, for a total of 200,000/well.
- 100 ⁇ _ of F8 antibody at 2X final concentration a commercially bought mouse lgG1 antibody (eBio #16-4714-85), an internally developed mouse lgG1 antibody, or 100 ng/mL of cyclosporine A (Sigma, cat #C-3662) were added to the cells.
- the lgG1 antibodies were used as negative isotype controls.
- the F8 antibody along with 10 other anti-CRAC channel antibodies, inhibited T cell proliferation to varying degrees.
- Fig. 9 indicates that T cell incubation with 3 pg/mL to 50 pg/mL of F8 antibody inhibited proliferation of CD4+ cells. A similar effect is seen on CD8+ cells.
- the F8 antibody at concentrations of 1 .56 pg/mL to 0.15 pg/mL did not inhibit the CD4+ cells as the data were similar to baseline
- GQPRPTSKPPASGAAANVSTSGITPGQA (SEQ ID NO: 32), has the N terminal 1 1 amino acids removed compared to 1415.
- Peptide 1454 with the sequence
- PASGAAANVSTSGITPGQA (SEQ ID NO: 33), has an additional nine amino acids removed from the N terminus compared to 1455. All three peptides were used in ELISA to measure binding by anti-CRAC channel antibodies. Nunc ® immunoplates were coated with 1 pg/ml of indicated peptide in PBS and incubated overnight at 4°C. Plates were blocked with blocking buffer (PBS with 0.05% Tween ® -20) for 15 min and were washed with PBS/0.05%Tween ® -20. Each of the anti-CRAC channel antibodies were added and the plates were incubated for 1 hour at room temperature.
- cDNA was synthesized in a 5'- RACE reaction using the SMARTerTM RACE cDNA amplification kit (Clontech Laboratories, Mountain View, CA). Subsequent target amplification of heavy chain and light chain sequences was performed by PCR using Phusion ® Hot Start polymerase (Finnzymes) and the universal primer mix (UPM) included a forward primer in the SMARTerTM RACE kit.
- Two different reverse primers with the following sequences were used independently for heavy chain (VH domain) amplification: 5'-CCCTTGACCAGGCATCCCAG-3' (SEQ ID NO: 5) and
- GFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciences and cloned for sequencing using a Zero Blunt TOPO ® PCR Cloning Kit and chemically competent TOP10 E.coli (Invitrogen). Colony PCR was performed on selected colonies using an AmpliTaq ® Gold Master Mix from Applied Biosystems and
- a single unique murine kappa type light chain and a single unique murine heavy chain for each antibody were identified. Amino acid sequences are listed below (leader peptide sequences have been omitted).
- Anti-M-CRAC1415-15F58A5B4's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
- CDRH2 HIYPGDGDTNYNGKFKG (SEQ ID NO: 1 1 )
- CDRH3 DHRDYYAMDY (SEQ ID NO: 41 )
- Anti-M- CRAC1415-15F58A5B4's light chain variable domain amino acid sequence (signal peptide sequence omitted, CDRs underlined):
- CDRL2 WASTRES (SEQ ID NO: 19)
- CDRL3 SQSYNLRT (SEQ ID NO: 47)
- Anti-M-CRAC1415-15F44A6's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
- CDRH3 DHRDYYAMDY (SEQ ID NO: 41 )
- Anti-M- CRAC1415-15F44A6's light chain variable domain amino acid sequence (signal peptide sequence omitted, CDRs underlined):
- CDRL2 WASTRES (SEQ ID NO: 19)
- CDRL3 KQSYDLTRS (SEQ ID NO: 43)
- M-hCRAC1415-15F54A2 chain subclass lgG2a/k
- M-hCRAC1415-15F54A2 chain subclass lgG2a/k
- Anti-M-CRAC1415-17F6A2's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
- Anti-M-CRAC1415-17F6A2's light chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
- CDRL1 KSSQSLLNSRTRKNYLA (SEQ ID NO: 38)
- CDRL2 WASTRES (SEQ ID NO: 19)
- CDRL3 KQSYNLRT (SEQ ID NO: 39)
- M-hCRAC1415-17F1 A4B6 Three additional hybridomas, designated M- hCRAC1415-15F3A6 and M-hCRAC1415-15F45A1 (all with chain subclass lgG1 /k), also each produced a monoclonal antibody with the same heavy and light chain variable domain amino acid sequences and the same CDRs as 17F6A2.
- Anti-M-CRAC1415-14F74A1 's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
- Anti-M-CRAC1415-14F74A1 's light chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
- CDRL1 KSSQSLLNSRTRKNYLA (SEQ ID NO: 38)
- CDRL2 WASTRES (SEQ ID NO: 19)
- CDRL3 KQSYNLRT (SEQ ID NO: 39)
- anti-CRAC channel antibodies had been shown to inhibit the activity of a T cell line (see, e.g. , Examples 4-5), the antibodies were also evaluated for their ability to inhibit the effector functions of primary T cells upon stimulation (as in Example 6) in multiple donor samples. Effector functions of T cells include, but are not limited to, proliferation and cytokine production which were analyzed in this example. Total proliferation is measured by CFSE dilution. The florescence of CFSE, a green fluorescent dye that labels intracellular proteins, dilutes by half with each cell division.
- PBMC peripheral blood mononuclear cells
- PBMC were CFSE labeled with the Invitrogen CellTrace ® CFSE Cell
- Anti-CRAC channel antibodies and mouse lgG1 , K control monoclonal antibody were diluted at 4X concentrations of 1332nM into assay media.
- the antibodies were titrated in triplicate 1 :2 into assay media in a 96 well U-bottom plate (BD Falcon 353077) for final 4X concentrations of 1332, 666, 333, 166.5, 83.25, 41 .625, 20.8125, 10.40625nM in final volumes of .050 mL.
- An additional .050mls of assay media was added to all wells, bringing the volume to 0.1 ml/well.
- 0.050 mL of CFSE labeled PBMC was added to all wells of 96 well U-bottom plate. Antibodies and controls were incubated with cells for 1 hr at 37°C in 5% C0 2 . Anti-CD3 monoclonal UCHT1 (eBioscience 16-0038) and anti-CD28 costimulatory monoclonal antibody CD28.2 (eBioscience 16-0289) were diluted together to 4X concentrations of 40 ng/ml and 4 pg/rnl, respectively, in assay media. 0.050mL of the anti-CD3/CD28 dilution were added to all wells on the 96 well U-bottom plate, with the exception of the unstimulated sample, bringing the final volume to .2mls/well.
- Live/Dead ® Fixable Far Red Dead Cell Stain (Invitrogen, Cat# L10120) was prepared by adding 0.050 ml DMSO to one vial of Far Red Dead Cell Stain to create a stock solution, which was then diluted 1 ⁇ of stock stain solution per 1 ml DPBS.
- 0.050 ml of AVID stain was added to each well of 96 well U-bottom plate containing CFSE labeled cells and then incubated at room temperature for 20 minutes. After incubation, 0.15 ml cold FACS Wash (D-PBS + 5%FBS) was added to each well. Plates were then centrifuged 1500 rpm for 5min, and supernatants were aspirated.
- CD5-PE/CD4 APC-eFluor 780 antibodies PE conjugated mouse anti-human CD5 antibody (UCHT2) + APC- eFluor 780 conjugated mouse anti human CD4 antibody (RPA-T4) in FACS wash (2.5 Ml+2.5ul/.050 ml) was added to each well of 96 well plate containing AVID/CFSE labeled cells. The cells were incubated on ice for 30 minutes.
- Cytokine/Chemokine Immunoassay (Cat# MPXHCYTO60KPMX14) was prepared as described in the protocol provided with the kit. IL-1 b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, 11-10, IL-12(p70), IL-13, GM-CSF, IFN- ⁇ , MCP-1 , and TNF-a were analyzed.
- Cytokine/chemokine data were collected using the BioRad BioPlex ® Reader and BioPlex ® Manager software.
- Results Three representative anti-CRAC antibodies (10F8A2B3, 14F74A1 , and 17F6A2) were evaluated for their ability to inhibit T cell proliferation upon stimulation with anti-CD3 and anti-CD28. Results were normalized to activated, but not antibody treated, cell controls. Thus, incubation without an antibody was 100% T cell proliferation, otherwise referred to as baseline activation. As shown in Figure 10, increasing concentrations of all three anti-CRAC antibodies tested inhibited PBMC proliferation. Inhibition increased with increased F8 concentrations, including nearly 90% inhibition of T cell proliferation by 166nM of some antibodies. In this
- the anti-CRAC antibodies were better at suppressing T cell proliferation than the positive control, 100ng/ml_ cyclosporine A.
- Anti-CRAC channel antibodies were evaluated for their ability to inhibit the effector functions of primary T cells isolated from rheumatoid arthritis (RA) patients.
- RA PBMC Frozen human RA patient peripheral blood mononuclear cells (RA PBMC) (Astarte Biologies; Redmond, WA) were thawed and removed directly to 13 mL of assay media (RPMI 1640, 10% FBS, 1X GlutaMAXTM, and 25 mM HEPES). RA PBMC was centrifuged for 10min at 1 100 rpm. The cell pellet was aspirated and resuspended in 10 mL of pre-warmed assay media.
- assay media RPMI 1640, 10% FBS, 1X GlutaMAXTM, and 25 mM HEPES
- RA PBMC was CFSE labeled with the Invitrogen CellTrace ® CFSE Cell Proliferation Kit following manufacturer's instructions. After labeling, the cells were re-suspended at 2 x 10 6 cells/mL in assay media described above in Example 2.
- Anti-CRAC channel antibodies and mouse lgG1 , K control monoclonal antibody were diluted at 4X concentrations of 1332nM into assay media.
- the antibodies were titrated in triplicate 1 :2 into assay media in a 96 well U-bottom plate (BD Falcon 353077) for final 4X concentrations of 1332, 666, 333, 166.5, 83.25, 41 .625, 20.8125, 10.40625nM in final volumes of .050 ml_.
- An additional .050mls of assay media was added to all wells, bringing the volume to .1 ml/well.
- 0.050 ml_ of CFSE labeled RA patient PBMC was added to all wells of 96 well U-bottom plate. Antibodies and controls were incubated with cells for 1 hr at 37°C in 5% C0 2 . Staphylococcal enterotoxin B, SEB (Sigma Aldrich S4881 -1 mg) was diluted at a 4X concentration of 5ng/ml in assay media. 0.050ml_ of SEB dilution was added to all wells on the 96 well U-bottom plate, with the exception of the unstimulated sample, bringing the final volume to
- Cells were incubated at 37°C in 5% CO2 for 6 days. Cells were harvested from the culture at day 6. Plates were centrifuged at 1500rpm for 5min. 0.075 ml of supernatant were removed to a separate 96 well plate for 72 hr cytokine analysis. Remaining supernatant was removed by flicking the plate. Cells were resuspended in 0.2 ml D-PBS per well. The plate was centrifuged at 1500 rpm for 5m in and supernatants were then aspirated.
- Live/Dead ® Fixable Far Red Dead Cell Stain (Invitrogen, Cat# L10120) was prepared by adding 0.050 ml DMSO to one vial of Far Red Dead Cell Stain to create a stock solution, which was then diluted 1 ⁇ of stock stain solution per 1 ml DPBS.
- 0.050 ml of AVID stain was added to each well of 96 well U-bottom plate containing CFSE labeled cells and then incubated at room temperature for 20 minutes. After incubation, 0.15 ml cold FACS Wash (D-PBS + 5%FBS) was added to each well. Plates were then centrifuged 1500 rpm for 5min, and supernatants were aspirated.
- the cells were then washed and then 0.050 ml CD3-PE/CD4 APC-eFluor 780 antibodies (PE conjugated mouse anti-human CD3 antibody (HIT3a) + APC- eFluor 780 conjugated mouse anti human CD4 antibody (RPA-T4) in FACS wash (2.5 Ml+2.5ul/.050 ml) was added to each well of 96 well plate containing AVID/CFSE labeled cells. The cells were incubated on ice for 30 minutes. Following incubation, 0.15 ml FACS wash was added to each well and then centrifuged at 1500 rpm for 5 minutes.
- HIT3a PE conjugated mouse anti-human CD3 antibody
- RPA-T4 APC-eFluor 780 conjugated mouse anti human CD4 antibody
- GVHD Graft Versus Host Disease
- humanized mice Transfer of human peripheral blood mononuclear cells to immunodeficient mice (hereinafter “humanized mice”) initiates human T cell expansion and results in a T cell mediated graft versus host disease (GvHD) which affects several major organs and can be measured as weight loss.
- GvHD T cell mediated graft versus host disease
- Treatment of humanized mice with an antibody targeting the human CRAC channel can therefore be used to elucidate whether such a compound has inhibitory effects in vivo on human T cell expansion and T cell mediated tissue inflammation.
- mice Humanized mice. Using Ficoll-Paque (GE Healthcare) gradients and Leucosep-tubes (Greiner Bio-One) human PBMCs were purified from buffy coats donated by healthy human donors. Female NOD.scid IL-2 receptor common gamma chain (IL-2Rgc) " _ mice (Taconic) were injected i.v. with 20x10 6 human PBMCs. Following injection of cells all animals were weighed and randomized into groups. Treatments with mouse anti-human CRAC1415-10F8 antibodies or matching isotype control antibodies (mouse IgGl k anti-trinitrophenyl (TNP)) were initiated on day 0 by intraperitoneal administration of 10mg/kg. Treatment was continued 3 times per week for the duration of the study. Assessment of animal health and weight was performed 3 times per week and mice experiencing more than 20 percent weight loss or impaired health with simultaneous detection of substantial human cell expansion in the blood were graded as having GvHD.
- IL-2Rgc
- staining with the following fluorochrome-conjugated antibodies was performed for 20 minutes in 100 ⁇ _ staining volume: Anti-human (h)CD45-FITC, anti-human CD4-PerCP, anti-human CD8- Pacific Blue, anti-human CD19-PECy7 (all BD Biosciences), anti-mouse (m)CD45-Pacific Orange (Caltag) and anti-human CD3-Qdot 655 (Invitrogen). Staining with LIVF DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen) was included to exclude dead cells.
- Human T cells were gated as live and single lymphocytes based on FSC/SSC properties and Near- IR staining with the following surface maker phenotype: mCD45 " hCD45 + CD3 + CD19 " CD4 + /CD8 + . Cells were washed once following staining and transferred to BD TruCount tubes (BD Biosciences) for absolute cell counting and analyzed on a LSRII flow cytometer using FACSDiva software (BD Biosciences).
- mice were used to determine the effect of CRAC channel blockade in vivo on human T cell expansion and human T cell mediated GvHD.
- Figure 14A shows a Kaplan-Meier survival curve of time to GvHD in groups of mice post injection of human PBMCs and following treatment from day 0 with either mouse anti-hCRAC1415-10F8 antibody or a mouse anti-TNP isotype control antibody.
- Anti-CRAC significantly (p ⁇ 0.01 ) postponed the time to GvHD compared to the isotype control. Numbers in brackets indicate number of surviving mice per total number of mice in each group at study termination. A group of mice not injected with PBMCs were included as reference.
- Figures 14B and 14C show the absolute number of human CD4 + and CD8 + T cells in 100 ⁇ of mouse blood taken at the indicated time points. Notably, anti-CRAC showed a significant reduction in both CD4 + and CD8 + T cell numbers in blood compared to the isotype control at all time points analysed.
- anti-CRAC channel monoclonal antibody shows in vivo proof of concept by significantly delaying GvHD and human T cell expansion in the humanized mouse model of GVHD.
- Anti-CRAC channel antibodies were shown to inhibit the activity of primary T cells isolated from peripheral blood of healthy individuals and rheumatoid arthritis patients (RA) in the previous examples, and so one of the lead antibodies was also evaluated for its ability to inhibit the effector functions of synovial T cells derived from rheumatoid arthritis patients. Effector functions of T cells include, but are not limited to, IL2 and IFN- ⁇ secretion which were analyzed in this example. The levels of secreted cytokines were measured by commercial ELISA kits.
- RA SFMC synovial fluid mononuclear cells
- penicillin/streptomycin at 1x10 6 /ml.
- Anti-CRAC channel antibody 10F8.mlgG1 , mouse lgG1 , K control monoclonal antibody and cyclosporine A (Sigma catalog number C1832, dissolved in 100% DMSO) were diluted at 2X concentrations of 1332 nM into assay media.
- One representative anti-CRAC antibody (10F8) was evaluated for its ability to inhibit the CD3 and CD28-costimulated cytokine secretion of SFMC from RA patients.
- 10F8 antibody inhibited IL-2 secretion in dose-dependent and complete manner, but not its isotype control, mouse IgGl
- 10F8 antibody also inhibited IFN- ⁇ secretion in a dose-dependent manner with the maximum 82% effect at the highest concentration tested.
- Similar results were achieved in another experiment with a second RA patient sample that tested positive control cyclosporine A's ability to inhibit cytokine secretion.
- the anti-CRAC antibody was similar or better at suppressing cytokines than the cyclosporine A in both experiments (see Figures 15B and 16B).
- the antibody of any one of embodiments 1 -5, or the fragment thereof, wherein the antibody is a monoclonal antibody (mAb). 7. The antibody of any one of embodiments 1 -6, or the fragment thereof, wherein the antibody is selected from the group consisting of a linear antibody, a single chain antibody, a monobody, and a multispecific antibody. 8. The antibody of any one of embodiments 1 -7, or the fragment thereof, wherein the fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , Fv, and scFv.
- mAb monoclonal antibody
- a CDRH2 comprising amino acids 1 -9 of SEQ ID NO: 52, optionally wherein one of these amino acids is substituted with a different amino acid.
- CDRH2 is amino acids 1 -9 of a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 58, SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 56, optionally wherein one of these amino acids is substituted with a different amino acid.
- a CDRL1 comprising SEQ ID NO: 57, optionally wherein one or two of these amino acids is substituted with a different amino acid;
- a CDRL2 comprising SEQ ID NO: 19, optionally wherein one or two of these amino acids is substituted with a different amino acid
- a CDRL3 comprising SEQ ID NO: 55.
- CDRL1 comprises SEQ ID NO: 18, optionally wherein one or two of these amino acids is substituted with a different amino acid.
- CDRL1 comprises consensus sequence SEQ ID NO: 54 and is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 46, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
- CDRL3 selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 43 and SEQ ID NO: 47, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
- a CDRL1 comprising SEQ ID NO: 18;
- a CDRL2 comprising SEQ ID NO: 19
- a CDRL3 comprising SEQ ID NO: 20.
- a CDRL1 comprising SEQ ID NO: 38;
- a CDRL3 comprising SEQ ID NO: 39. cording to any one of embodiments 1 -15 or 70-71 comprising: a) a CDRH1 comprising SEQ ID NO: 10;
- a CDRL1 comprising SEQ ID NO: 38;
- a CDRL3 comprising SEQ ID NO: 43. cording to any one of embodiments 1 -15 or 70-71 comprising: a) a CDRH1 comprising SEQ ID NO: 10;
- a CDRL1 comprising SEQ ID NO: 46;
- a CDRL3 comprising SEQ ID NO: 47. cording to any one of embodiments 1 -15 or 70-71 comprising: a) a CDRH1 comprising SEQ ID NO: 10;
- the antibody of any one of embodiments 1 -37 or 70-71 , or the fragment thereof, wherein in the antibody is a humanized antibody.
- polynucleotide encodes a heavy chain variable domain having human framework regions. 45. The polynucleotide of any one of embodiments 41 -44, wherein the
- polynucleotide encodes a light chain variable domain having human framework regions.
- An expression vector comprising the polynucleotide of any one of embodiments 41 -45 operably linked to expression control elements such that the antibody of any of embodiments 1 -40 may be expressed.
- a recombinant host cell comprising the expression vector of embodiment 46.
- a method of producing an anti-CRAC channel antibody comprising culturing a host cell of any one of embodiments 47-49 in conditions appropriate for expressing the nucleic acid, and isolating the antibody.
- autoimmune or inflammatory disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, multiple sclerosis and graft versus host disease.
- a method of treating an autoimmune or inflammatory disease in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71.
- the autoimmune or inflammatory disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, multiple sclerosis and graft versus host disease.
- a method for modulating T-cells in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71 .
- a method for inhibiting T cell activation in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71 .
- a method for inhibiting NFAT translocation in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71 .
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Abstract
The invention relates to antibodies that specifically bind to calcium release- activated calcium (CRAC) channels and inhibit the influx of calcium through CRAC channels. Such antibodies can modulate T cell activation and function.
Description
ANTI-CRAC CHANNEL ANTIBODIES
Cross-Reference to Related Applications
This application claims priority to U.S. Provisional Application 61 /579,272, filed December 22, 201 1 .
Field of the Invention
The present invention relates to antibodies that specifically bind to calcium release-activated calcium (CRAC) channels and methods of mediating T cell activation.
INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
The Sequence Listing, entitled "SEQUENCE LISTING", is 57,233 bytes, was created on February 2, 2012 and is incorporated herein by reference.
BACKGROUND
Calcium (Ca2+) is a second messenger in many eukaryotic cells. In T cells,
Ca2+ signalling is essential for activation. Calcium release-activated calcium (CRAC) channels are store operated Ca2+ entry channels in the plasma membrane. CRAC channels are highly selective for Ca2+ and maintain a gating mechanism dependent on the depletion of endoplasmic reticulum Ca2+ stores.
Antigen recognition by a T lymphocyte initiates the release of Ca2+ from endoplasmic reticulum (ER) stores. Depletion of Ca2+ stores from the endoplasmic reticulum triggers the opening of CRAC channels. STIM1 (stromal interaction molecule 1 ) also mediates CRAC channel function. STIM1 is an ER Ca2+ sensor that will activate CRAC channels during ER Ca2+ depletion. Thus, during lymphocyte activation, intracellular Ca2+ levels increase via the influx of Ca2+ from ER stores as well as through CRAC channels in the plasma membrane.
NFAT (nuclear factor of activated T cells) is a Ca2+- regulated transcription factor that is important in T cell activation. NFAT can be found in the cytoplasm of unactivated T cells. During activation, the influx of Ca2+ results in NFAT
dephosphorylation by calcineurin, which fosters NFAT translocation to the nucleus and expression of genes important to the immune response.
Recently, Feske et al. discovered a subset of severe combined immune deficiency (SCID) patients, wherein affected patients were homozygous for a single missense mutation in ORAI1 (Nature, 2006; 441 : 179-185). Heterozygous family members were not affected. The missense mutation C271 T produces the change Arg91 Trp in the first transmembrane domain of ORAM . This single missense mutation causes loss of CRAC-channel function, and thereby, a failure in T cell activation and immunity.
Ion channels such as the CRAC channel have traditionally been targeted by small molecule modulators. Although small molecules can impair the targeted ion channels, the effect is often non-specific, because of a failure to discriminate between homologous species of a protein genus (i.e., impairing more than one kind of ion channel), and can also have diffuse side effects. Antibodies against ion channels have also been attempted as modulators. Most existing antibodies against ion channels do not perturb channel function and none of the known polyclonal antibodies produce complete channel block (Benham, Nat. Biotechnol. 2005; 23:
1234-1235). There is a need for an ion channel specific, protein specific antibody that inhibits the function of the particular ion channel. To date, there are no public data demonstrating modulation of CRAC channel activity by any antibody.
SUMMARY
The invention is in part based on the identification of an ion channel-specific antibody that specifically blocks the function of this ion channel. Distinct from deficiencies of small molecules and previous antibodies, an isolated antibody of the invention is ion channel specific, protein specific, and impairs the function of the channel. Specifically, the isolated antibody specifically binds a CRAC channel, more specifically ORAM , and reduces or inhibits the influx of calcium ions through the channel into T cells. The CRAC channel may be human CRAC channel.
Accordingly, the present invention includes an anti-CRAC channel antibody that specifically binds to ORAM and inhibits the influx of calcium ions into a T cell. In one embodiment, an anti-CRAC channel antibody of the invention reduces or inhibits translocation of NFAT from the cytoplasm to the nucleus. In another embodiment, an anti-CRAC channel antibody of the invention reduces or inhibits pro-inflammatory
cytokine production in T cells. In another embodiment, an anti-CRAC channel antibody of the invention reduces or inhibits T cell proliferation. More specifically, a particularly useful anti-CRAC channel antibody according to the invention inhibits or decreases one or more effector functions such as IL-1 B, IL-2, IL-4, IL-5, IL-6, IL-13 and IFN-gamma production. In another embodiment, an anti-CRAC channel antibody of the invention inhibits T cell proliferation. In another embodiment, an anti-CRAC channel antibody of the invention inhibits T cell activation.
Since an anti-CRAC channel antibody of the invention can inhibit T cell activation and effector function, an anti-CRAC channel antibody may be administered to a subject in order to suppress an aberrant cell mediated immune response, such as that observed in undesirable inflammation, autoimmunity and graft versus host disease. An embodiment includes a method of administering an anti-CRAC channel antibody of the invention to inhibit graft versus host disease, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, or multiple schlerosis in a subject and/or to treat an autoimmune disorder in a subject and/or to treat chronic or other undesirable inflammation in a subject.
Anti-CRAC channel antibodies of the invention include an antibody that binds the second extracellular loop of the Orail and inhibits or abrogates CRAC channel function. The anti-CRAC channel antibody is able to block the influx of calcium ions into T cells. As a result, the binding of the anti-CRAC channel antibody inhibits activation of T cells and also inhibits the translocation of NFAT from the cytoplasm to the nucleus. In an embodiment, an anti-CRAC channel antibody is a monoclonal antibody (mAb) that is capable of binding the first or the second extracellular loop of CRAC. In an embodiment, an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN; SEQ ID NO: 10) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence of amino acids 50 to 66
(HIYPGDGDTNYNGKFKG; SEQ ID NO: 1 1 ) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and/or a CDRH3 sequence of amino acids 99 to 107 (GGTTVWDY; SEQ ID NO: 12) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid. In another embodiment, the CDRH2 sequence is amino acids
50 to 58 (HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9. In an embodiment, an anti-CRAC channel antibody comprises a light chain comprising a CDRL1 sequence of amino acids 24 to 40 (KSSQSLLNSRT; SEQ ID NO: 18) of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid; and/or a CDRL2 sequence of amino acids 56 to 62 (WASTRES; SEQ ID NO: 19) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid; and/or a CDRL3 sequence of amino acids 95 to 103 (KQSYNLPWT; SEQ ID NO: 20) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid. In an embodiment, an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN; SEQ ID NO: 10) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid;
and/or a CDRH2 sequence of amino acids 50 to 66 (HIYPGDGDTNYNGKFKG; SEQ ID NO: 1 1 ) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and/or a CDRH3 sequence of amino acids 99 to 107 (GGTTVWDY; SEQ ID NO: 12) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and a light chain comprising a CDRL1 sequence of amino acids 24 to 40 (KSSQSLLNSRT; SEQ ID NO: 18) of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid; and/or a CDRL2 sequence of amino acids 56 to 62 (WASTRES; SEQ ID NO: 19) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid; and/or a CDRL3 sequence of amino acids 95 to 103 (KQSYNLPWT; SEQ ID NO: 20) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid. In another embodiment, the CDRH2 sequence is amino acids 50 to 58 (HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9. In an embodiment, an anti-CRAC channel antibody comprises SEQ ID NO: 9. In embodiment, an anti-CRAC channel antibody comprises SEQ ID NO: 17.
In an embodiment, an anti-CRAC channel antibody comprises a heavy chain variable domain having at least one of the following sequences: (a) a CDRH1 comprising SEQ ID NO: 10, optionally wherein one of these amino acids is substituted with a different amino acid or (b) a CDRH2 comprising SEQ ID NO: 52, optionally wherein
one, two or three of these amino acids is substituted with a different amino acid. In an embodiment, an anti-CRAC channel antibody comprises a CDRH2 that is amino acids 1 -9 of a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 58, SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 56, optionally wherein one of these amino acids is/are substituted with a different amino acid. In an embodiment, an anti-CRAC channel antibody further comprises a CDRH3 selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 41 and SEQ ID NO: 50, optionally wherein one or two of these amino acids is substituted with a different amino acid. In an embodiment, an anti-CRAC channel antibody further comprises a light chain variable domain, wherein the light chain variable domain comprises at least one of the following sequences (a) to (c): a) a CDRL1 comprising SEQ ID NO: 57, optionally wherein one or two of these amino acids is substituted with a different amino acid; b) a CDRL2 comprising SEQ ID NO: 19, optionally wherein one or two of these amino acids is substituted with a different amino acid; and c) a CDRL3 comprising SEQ ID NO: 55. In an embodiment, an anti-CRAC channel antibody further comprises a CDRL1 having SEQ ID NO: 18, optionally wherein one or two of these amino acids is substituted with a different amino acid. In another embodiment, an anti-CRAC channel antibody further comprises wherein CDRL1 comprising consensus sequence SEQ ID NO: 54, wherein CDRL1 is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 46, optionally wherein one, two or three of these amino acids is substituted with a different amino acid. In an
embodiment, an anti-CRAC channel antibody further comprises a CDRL3 selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 43 and SEQ ID NO: 47, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
In an embodiment, an anti-CRAC channel antibody binds an epitope comprising one or more residues selected from the group consisting of G12, Q13, P14, R15, P16, T17, S18, K19, P20, P21 , A22, S23, G24, A25, A26, A27, N28, V29, S30, T31 , S32, G33, I34, T35, P36, G37, Q38, A39 of SEQ ID NO: 3.
In some embodiments, an anti-CRAC channel antibody is a monoclonal antibody. In some embodiments, an anti-CRAC channel antibody is a polyclonal antibody. In some embodiments, an anti-CRAC channel antibody is selected from the
group consisting of a chimeric antibody, an affinity matured antibody, a humanized antibody, and a human antibody. In some embodiments, an anti-CRAC channel antibody is an antibody fragment. In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, or scFv.
The invention also provides a nucleic acid that encodes an anti-CRAC channel antibody of the invention, such as polynucleotides which encode an antibody light chain and/or an antibody heavy chain variable domain of the invention. In an embodiment, a nucleic acid encodes an anti-CRAC channel antibody comprising polynucleotides of SEQ ID NOs: 14, 15, and 16, which encode CDRH1 , CDRH2, and CDRH3, respectively. In another embodiment, a nucleic acid encodes an anti-CRAC channel antibody comprising polynucleotides of SEQ ID NOs: 18, 19 and 20, which encode CDRL1 , CDRL2 and CDRL3, respectively.
In one aspect, the invention provides host cells comprising a nucleic acid or a vector of the invention. A vector can be of any type, for example a recombinant vector such as an expression vector. Any of a variety of host cells can be used. In one embodiment, a host cell is a prokaryotic cell, for example, E. coli. In one embodiment, a host cell is a eukaryotic cell, for example a mammalian cell such as Chinese Hamster Ovary (CHO) cell. In one embodiment, a host cell is a plant cell, for example, tobacco.
The invention also provides pharmaceutical compositions comprising an anti-
CRAC channel antibody of the invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 is a schematic depicting the ORAM subunit pore of the CRAC protein. ORAM has four transmembrane helices (M1 , M2, M3, and M4) that span the plasma membrane. ORAM also has two extracellular loops and one intracellular loop. Both the N- and C-termini are on the cytoplasmic side of the cell membrane. The
extracellular loops of ORAM are accessible to antibody binding domains.
Figs. 2A and 2B are two graphs of ELISA results plotted as optical density (OD) at 450nm versus antibody dilution. Fig. 2 shows polyclonal antibody titers against peptides spanning the first (A) and second (B) extracellular loops of CRAC raised in Balb/c mice. In (A), sera from six immunized mice A (♦), B (■), C (A ), D (X),
E (*), and F (·) were tested in an ELISA to detect binding to SEQ ID NO: 2 (first extracellular loop). In (B), sera from four immunized mice A (♦), B (■), C ( A), and D (X) were tested in an ELISA to detect binding to SEQ ID NO: 3 (second extracellular loop).
Fig. 3 is series of FACS dot plots with intensity of CFSE fluorescence indicating CRAC+ cells on the x-axis and fluorescence intensity on the y-axis indicating binding to CRAC channels. Fig. 3 shows that immunizing mice with the second extracellular loop of CRAC (Fig. 3D-3F), but not the first (Fig. 3A-3C), raises polyclonal titers against native CRAC expressed on the surface of cells.
Fig. 4 is two FACS dot plots with intensity of CFSE fluorescence indicating
CRAC+ cells on the x-axis and fluorescence intensity on the y-axis indicating specific binding with CRAC. Two different CRAC-overexpressing cell lines (Ba/F3, Fig. 4A, and Jurkat, Fig. 4B, CRAC+) were generated and hCRAC1415-10F8 (referred to as F8) was evaluated for binding on the untransfected line (CFSE low population) compared to the CRAC-overexpressing line (CFSE high population). Fig. 4 shows clone hCRAC1415-10F8 recognized CRAC on the surface of cell lines engineered to overexpress CRAC.
Fig. 5 is an alignment between human ORAM and mouse ORAM . The top line is amino acids 49 to 301 of human ORAM (SEQ ID NO: 1 ), the middle line is the alignment, and the bottom line is amino acids 54-304 of mouse ORAM (SEQ ID NO:25). The putative extracellular loops are shown underlined and in bold.
Fig. 6 is two graphs of percent inhibition (compared to two different Ig control antibodies) versus antibody concentration. It shows that anti-CRAC channel antibody hCRAC1415-10F8 inhibits (A) NFAT translocation and (B) IL-2 production by Jurkat cells. Jurkat cells carrying an NFAT-luciferase reporter gene were stimulated with PHA-P. NFAT translocation and IL-2 production were measured at 4 hrs or 16 hrs post-stimulation, respectively.
Fig. 7 is a graph of percent inhibition of T cell proliferation (normalized to untreated control) versus antibody concentration. It shows that anti-CRAC channel antibody hCRACI 415-10F8 inhibits proliferation of primary T cells stimulated with anti-CD3/CD28.
Fig. 8 is two bar graphs depicting cytokine amount versus antibody amount in the extracellular medium of PBMC cell cultures. Fig. 8 shows that anti-CRAC channel antibody hCRAC1415-10F8 inhibits cytokine production of IL-4 (A) and IL-1 b (B) in stimulated PBMC cultures.
Fig. 9 is a graph of cytokine inhibition versus antibody concentration or nonspecific immunosuppression (cyclosporine). It shows that anti-CRAC channel antibody hCRAC1415-10F8 (F8 antibody) inhibits T cell proliferation in a mixed lymphocyte reaction. The X-axis is the concentration of agents (cyclosporine A, F8 antibody, two separate lgG1 isotype controls, and no stimulator) used to test proliferation. The Y-axis is the percent of T cell proliferation compared to baseline (T cells stimulated in the absence of treatment).
Fig. 10 is a graph of T cell proliferation by % of baseline versus concentration of antibody or control. It shows that anti-CRAC channel monoclonal antibodies inhibit proliferation of primary T cells stimulated with anti-CD3/CD28.
Fig. 1 1 is a graph depicting concentration of IL-2 (pg/ml) versus concentration of anti-CRAC channel monoclonal antibody at 16 hours post-stimulation.
Fig. 12 is a graph depicting concentration of IFN-γ (pg/ml) versus
concentration of anti-CRAC channel monoclonal antibody at 72 hours post- stimulation.
Fig. 13 is a graph of T cell proliferation by % of baseline activation versus antibody concentration (nM). Anti-CRAC channel antibodies M-hCRAC1415- 10F8A2B3, M-hCRAC1415-14F74A1 , M-hCRAC1415-18F6A2 and M-hCRAC1415- 15F8A5B4-mlgG1 inhibited the proliferation of T cells isolated from RA patients and stimulated with SEB. Mouse lgG1 was a negative control.
Fig. 14 is a series of graphs depicting data generated in a GVHD mouse model system. Fig. 14A is a Kaplan-Meier survival curve depicting the time to GVHD as well as the statistical analysis of the anti-CRAC channel monoclonal antibody (F8) versus the negative control (negative isotype antibody). Figs. 14B and 14C are bar graphs that quantify total human T cells by (B) the number of human CD4+ cells per 100 μΙ_ of blood and (C) the number of human CD8+ cells per 100 μΙ_ of blood. Figs. 14B and C depict the expansion of human T cells at different time points in the GVHD mouse model system.
Fig. 15 is a series of graphs depicting the effect of anti-CRAC channel monoclonal antibody hCRAC1415-10F8 on IL-2 secretion. Fig. 15A graphs concentration of IL-2 versus concentration of antibody and negative control. Fig. 15B graphs concentration of IL-2 versus concentration of positive control (CsA).
Fig. 16 is a series of graphs depicting the effect of anti-CRAC channel monoclonal antibody hCRAC1415-10F8 on IFNy secretion. Fig. 16A graphs concentration of IFNy versus concentration of antibody and negative control. Fig. 16B graphs concentration of IFNy versus concentration of positive control (CsA). Brief Description of the Sequence Identification Listing
SEQ ID NO: 1 is the amino acid sequence for human ORAM , also known as CRAC channel membrane protein 1 ; GenBank accession no. AAH15369.2 Gl:
54035070.
SEQ ID NO: 2 is the amino acid sequence of the first extracellular loop of human ORAMwith an additional cysteine at the C-terminus to allow for coupling to carrier proteins.
SEQ ID NO: 3 is the amino acid sequence of the second extracellular loop of human ORAMwith an additional cysteine at the C-terminus to allow for coupling to carrier proteins.
SEQ ID NO: 4 is the human ORAI1 gene, which is found at 12q24.31 ;
GenBank accession no. NG_007500.1 Gl: 171541812.
SEQ ID NO: 5 is a PCR reverse primer to amplify the heavy chain variable domain sequence.
SEQ ID NO: 6 is a PCR reverse primer to amplify the heavy chain variable domain sequence.
SEQ ID NO: 7 is a PCR reverse primer to amplify the light chain variable domain sequence.
SEQ ID NO: 8 is a PCR reverse primer to amplify the heavy chain variable domain sequence.
SEQ ID NO: 9 is the amino acid sequence for the heavy chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 10 is the amino acid sequence of CDRH1 of antibodies M- CRAC1415-10F8 A2B6C1 , M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M- hCRAC1415-15F3A6, M-hCRAC1415-15F45A1 , M-hCRAC1415-15F44A6, M- hCRAC1415-15F54A2, M-hCRAC1415-15F58A5B4 and M-hCRAC1415-14F74A1.
SEQ ID NO: 1 1 is the amino acid sequence of CDRH2 of antibodies M-
CRAC1415-10F8 A2B6C1 , and M-hCRAC1415-15F58A5B4.
SEQ ID NO: 12 is the amino acid sequence of CDRH3 of antibody M- CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 13 is the nucleotide sequence encoding the heavy chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 14 is the nucleotide sequence encoding CDRH1 of antibody M- CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 15 is the nucleotide sequence encoding CDRH2 of antibody M- CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 16 is the nucleotide sequence encoding CDRH3 of antibody M-
CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 17 is the amino acid sequence for the light chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 18 is the amino acid sequence of CDRL1 of antibody M- CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 19 is the amino acid sequence of CDRL2 of antibodies M- CRAC1415-10F8 A2B6C1 , M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M- hCRAC1415-15F3A6, M-hCRAC1415-15F45A1 , M-hCRAC1415-15F44A6, M- hCRAC1415-15F54A2, M-hCRAC1415-15F58A5B4 and M- hCRAC1415-14F74A1.
SEQ ID NO: 20 is the amino acid sequence of CDRL3 of antibody M-
CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 21 is the nucleotide sequence encoding the light chain variable domain of antibody M-CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 22 is the nucleotide sequence encoding CDRL1 of antibody M- CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 23 is the nucleotide sequence encoding CDRL2 of antibody M- CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 24 is the nucleotide sequence encoding CDRL3 of antibody M- CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 25 is the amino acid sequence for mouse ORAM , also known as ORAM ; GenBank accession no. BAF 47905.1 Gl: 126364336.
SEQ ID NO: 26 is an N-linked glycosylation site where X can be any amino acid except proline.
SEQ ID NO: 27 is an N-linked glycosylation site where X can be any amino acid except proline.
SEQ ID NO: 28 is an alternative amino acid sequence of CDRH2 of antibody M-CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 29 is a substituted amino acid sequence of CDRH2 of antibody M-CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 30 is a substituted amino acid sequence of CDRH2 of antibody M-CRAC1415-10F8 A2B6C1 .
SEQ ID NO: 31 is a peptide spanning the entire second extracellular loop
(Peptide 1415).
SEQ ID NO: 32 is a peptide spanning part of the second extracellular loop (Peptide 1455).
SEQ ID NO: 33 is a peptide spanning part of the second extracellular loop (Peptide 1454).
SEQ ID NO: 34 is the heavy chain variable domain amino acid sequence of antibodies M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415- 15F3A6 and M-hCRAC1415-15F45A1 .
SEQ ID NO: 35 is the CDRH2 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6 and M- hCRAC1415-15F45A1 .
SEQ ID NO 36 is the CDRH3 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6 and M- hCRAC1415-15F45A1 .
SEQ ID NO: 37 is the light chain variable domain amino acid sequence of antibodies M-hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415- 15F3A6 and M-hCRAC1415-15F45A1 .
SEQ ID NO: 38 is the CDRL1 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6, M- hCRAC1415-15F45A1 , M-hCRAC1415-15F44A6, M-hCRAC1415-15F54A2 and M- hCRAC1415-14F74A1 .
SEQ ID NO: 39 is the CDRL3 amino acid sequence of antibodies M- hCRAC1415-17F1A4B6, M-hCRAC1415-17F6A2, M-hCRAC1415-15F3A6, M- hCRAC1415-15F45A1 and M-hCRAC-14F74A1 .
SEQ ID NO: 40 is the heavy chain variable domain amino acid sequence of antibodies M-hCRAC1415-15F44A6 and M-hCRAC1415-15F54A2.
SEQ ID NO: 41 is the CDRH3 amino acid sequence of antibodies M- hCRAC1415-15F44A6, M-hCRAC1415-15F54A2 and M-hCRAC1415-15F58A5B4.
SEQ ID NO: 42 is the light chain variable domain amino acid sequence of antibodies M-hCRAC1415-15F44A6 and M-hCRAC1415-15F54A2.
SEQ ID NO: 43 is the CDRL3 amino acid sequence of antibodies M- hCRAC1415-15F44A6 and M-hCRAC1415-15F54A2.
SEQ ID NO: 44 is the heavy chain variable domain amino acid sequence for M-hCRAC1415-15F58A5B4.
SEQ ID NO: 45 is the light chain variable domain amino acid sequence for M- hCRAC1415-15F58A5B4.
SEQ ID NO: 46 is the CDRL1 amino acid sequence for M-hCRAC1415-
15F58A5B4.
SEQ ID NO: 47 is the CDRL3 amino acid sequence for M-hCRAC1415- 15F58A5B4.
SEQ ID NO: 48 is the heavy chain variable domain amino acid sequence for M-hCRAC1415-14F74A1 .
SEQ ID NO: 49 is the CDRH2 amino acid sequence for M-hCRAC1415- 14F74A1 .
SEQ ID NO: 50 is the CDRH3 amino acid sequence for M-hCRAC1415- 14F74A1 .
SEQ ID NO: 51 is the light chain variable domain amino acid sequence for M- hCRAC1415-14F74A1 .
SEQ ID NO: 52 is a CDRH2 consensus sequence for an anti-CRAC channel antibody.
SEQ ID NO: 53 is a IGHV1 -80 VH murine germline sequence.
SEQ ID NO: 54 is a CDRL1 consensus sequence for an anti-CRAC channel antibody.
SEQ ID NO: 55 is a CDRL3 consensus sequence for an anti-CRAC channel antibody.
SEQ ID NO: 56 is a CDRH2 consensus sequence for an anti-CRAC channel antibody.
SEQ ID NO: 57 is a CDRL1 consensus sequence for an anti-CRAC channel antibody.
SEQ ID NO: 58 is the amino acid sequence of CDRH2 of antibodies M- hCRACI 415-15F44A6, M-hCRAC1415-15F54A2.
SEQ ID NO: 59 is a IGKV8-21 VL murine germline sequence.
SEQ ID NO: 60 is a portion of the IGKV8-21 VL germline sequence.
DESCRIPTION
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "amino acid", as is commonly understood in the art, is meant to include the naturally occurring L a-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein (Lehninger, 1975, Biochemistry, 2d ed., pp. 71 -92, Worth Publishers, New York). A "modified" amino acid may be a D-amino acids as well as chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. A "functional equivalent" of an amino acid, such as an analog or a mimetic, may be used. For example, analogs or mimetics of phenylalanine or proline would allow the same conformational restriction of the peptide compounds
as natural Phe or Pro. Other examples of amino acids are listed by Roberts and Vellaccio, In: The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, Eds., Vol. 5 p 341 , Academic Press, Inc, N.Y. 1983, which is incorporated herein by reference. "Synthetic" amino acids are non-naturally occurring and include, for example, those where the naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acid) are replaced with other side chains, for instance with groups such as alkyi, lower alkyi, cyclic 4-, 5-, 6-, to 7-membered alkyi, amide, amide lower alkyi, amide di(lower alkyi), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered
heterocyclic. Other examples of synthetic amino acids are naphthylalanine, L- hydroxypropylglycine, L-3,4-dihydroxyphenylalanyl, alpha-am ino acids such as L- alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha-methyl-alanyl, beta amino- acids such as beta-alanine, and isoquinolyl. In other aspects C-alpha-methyl amiono acids, in particular C-alpha-methyl-leucine may be included in an engineered mimetic or library of such compounds. Other exemplary modified and synthetic amino acids include cinnamic acids, phenylglycine (Phg), 2,3-diaminobutyric acid (Dab), 2,4-diaminobutyric acid (gDab), 2,3-diaminopropionic acid (Dap), β- methylaspartate (MeAsp), cyclohexylalanine (β-Cha), norleucine (Nle), norvaline (Nvl), isonipecotic acid (Ina), pipecolic acid (homoproline) (Pip or hPro),
phenylacetic acids, phenylpropanoic acids, 2-aminobutyric acid (Abu), sarcosine (Sar or N-methyl glycine), 6-aminohexanoic acid (Ahx), para-fluoro-Phenylalanine (p-F-Phe), γ-amino-butyric acid (GABA), benzoic acids (such as p-aminobenzoic acid (PABA), etc.), hydrazinobenzoic acids, homophenylalanine (homophe or hPhe), β-cyanoAlanine (β-cyano-Ala), methyl or ethyl aryl ethers of tyrosine (Tyr(Me) or Tyr(Et), respectively), aminoisobutyric acid (Aib, which is also known as α,α- dimethylglycine), S-methylcysteine (MeCys), Ν,Ν'-dimethyl-arginine ((Me)2Arg), hydroxyProline (Hyp), citruline (Cit), homolysine (homoLys or hLys), 5- aminopentanoic acid or aminovaleric acid (5-Ava), (S)-3-Benzo[b]thiophen-3-yl- aminopropanoic acid (L-BBTA), pyroglutamic acid (pGlu), and the like.
The term "antibody" is, as commonly understood in the art, a protein that is capable of specifically binding to an antigen or a portion thereof and includes. The term includes full length antibodies of any isotype (that is, IgA, IgE, IgG, IgM and/or
IgY) and any single chain thereof. The term "antibody" is used in the broadest sense and specifically includes monoclonal antibodies (including full length
monoclonal antibodies), multispecific antibodies {e.g., bispecific antibodies), and antibody fragments that exhibit a desired biological activity or function. The term antibody refers to traditional antibodies, antibody fragments, antigen binding proteins, immunoadhesins, VHH domains (e.g., camelid antibody), etc.
A full length antibody generally comprises two heavy chains and two light chains, each comprising a variable domain and a constant domain. Each variable domain contains a hypervariable region containing three CDRs or HVLs flanked by four segments of the framework region. As used herein, the "framework region" contains all four segments FR1 , FR2, FR3, and FR4 that flank a set of three hypervariable regions (CDRs or HVLs).
Antibodies can be chimeric, humanized, or human, for example, and can be antigen-binding fragments of these. Antibodies are generally produced by
immunizing an animal with an antigen, and can be produced by recombinant technology, or by synthesis of the amino acid sequence, for example. "Antibody fragments" comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, F(ab)S, Fv fragments, single-chain Fv (scFv; see e.g.. Bird et al., Science 1988; 242:42S-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd
(typically the VH and CHI domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies such as bispecific antibodies, for example formed from antibody fragments; monovalent molecules comprising a single VH and a single VL chain; minibodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al.. Protein Eng 1997; 10:949-57); camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, where the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. "Functional fragments" substantially retain binding to an antigen of the full-length antibody, and retain a biological activity.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual
antibodies of the population are identical and have identical structure and specificities except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single immunogenic determinant. Thus, 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. For example, monoclonal antibodies 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 using the techniques described in Clackson et al.. 1991 , Nature 352:624-628 and Marks et al., 1991 , J. Mol. Biol. 222:581 -597, for example.
"Chimeric" antibodies (immunoglobulins) contain a portion of a heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and
Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81 :6851 -6855).
The term "humanized antibody", as used herein, is a subset of chimeric antibodies. "Humanized" forms of nonhuman {e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For example, humanized antibodies may be human immunoglobulins (recipient or acceptor antibody) in which variable domain hypervariable region residues of the recipient antibody are replaced by hypervariable region residues from a nonhuman species (donor antibody), such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
The hypervariable regions can be complementarity-determining regions (CDRs) defined by sequence (see, for example Kabat 1991 , 1987, 1983), or hypervariable loops (HVLs) defined by structure (see for example, Chothia 1987), or both. In some embodiments, the variable domain framework regions are derived from a consensus sequence variable domain, for example, containing at each residue an amino acid compiled as most abundant at that position in a class or subclass of human immunoglobulin variable domains, for example, in a Kabat compilation. In some instances, one or more amino acids of the variable domain framework region (FR) of the human immunoglobulin or consensus sequence is replaced with one or more corresponding residues of the nonhuman donor antibody and/or one or more amino acids of the donor antibody hypervariable regions is replaced with one or more corresponding human residues of the human recipient variable domain. In some instances, one or more residues of the variable domain framework regions and/or hypervariable regions is a residue not found at the corresponding position in the recipient antibody or in the donor antibody.
Modifications to the amino acid sequence of the variable domain framework regions and hypervariable regions are generally made to further refine antibody performance, for example, improve binding affinity. In general, the humanized antibodies used to produce the pan-specific antibodies described herein will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable region residues (CDRS or HVLs) correspond to those of a nonhuman immunoglobulin and all or substantially all of the framework region (FR) residues correspond to those of a human variable domain consensus sequence, and may include one or more amino acid substitutions. In some embodiments, the number of amino acid substitutions in the human consensus framework region is typically no more than 10, and may be, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the heavy chain variable domain framework regions, and 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the light chain variable domain framework region. The humanized antibody optionally comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin (see Figure 1 ). For further details, see Jones et al., 1986, Nature 321 :522-525; Reichmann et al., 1988, Nature
332:323-329; and Presta, 1992, Cur Op. Struct. Biol. 2:593-596.
Human antibodies can be antibodies isolated from humans. There are also fully human antibodies produced originally in transgenic mice genetically modified to express human repertoire and immunized with the antigen of interest. See e.g., Bruggemann & Neuberger, Immunol. Today 1996; 17: 391 -397.
The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences {e.g., mutations introduced by random or site-specific mutagenesis In vitro or by somatic mutation In vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, as these are described by other terms defined herein. Such a human antibody as is discussed in this paragraph may be a human monoclonal antibody. Such a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Human antibodies can be antibodies isolated from humans. There are also fully human antibodies produced originally in, for example, transgenic mice (or other transgenic organisms) genetically modified to express human repertoire and immunized with the antigen of interest. See e.g., Bruggemann & Neuberger, Immunol. Today 1996; 17: 391 -397. Human antibodies may be isolated from sequence libraries built on selections of human germline sequences further diversified with natural and synthetic sequence diversity.
An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site, and generally comprises a dimer of one heavy and one light chain variable domain in tight association that can be covalent in nature, for example in a single chain variable domain fragment (scFv). It is in this configuration that the three hypervariable regions of each variable domain interact to define an
antigen-binding site on the surface of the VH-VL dimer. Collectively, the six
hypervariable regions or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain comprising only three
hypervariable regions specific for an antigen has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site (Cai & Garen, Proc. Natl. Acad. Sci. USA, 93: 6280-6285, 1996). For example, naturally occurring camelid antibodies that only have a heavy chain variable domain (VHH) can bind antigen (Desmyter et al., J. Biol. Chem., 277: 23645-23650, 2002; Bond et al., J. Mol. Biol. 2003; 332: 643-655).
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, 1994, In: The Pharmacology of Monoclonal Antibodies, Vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.
A "Fab" fragment includes a variable domain and a constant domain of the light chain and a variable domain and the first constant domain (CH1 ) of the heavy chain. A Fab' fragment includes one or more cysteine carboxy terminal linkages to the heavy or light chains. F(ab')2 antibody fragments comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known.
The term "diabodies" refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two variable domains on the same chain, the variable domains are forced to pair with
complementary domains of another chain, creating two antigen-binding sites.
Diabodies are described more fully, for example, in EP 404,097; WO 93/1 1 161 ; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6444-6448.
The expression "linear antibodies" refers to antibodies as described in Zapata et al., 1995, Protein Eng., 8(10): 1057-1062. Briefly, these antibodies contain a pair
of tandem Fd segments (VH-CH1 -VH-Ch1 ) that, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
The term "monobody" as used herein, refers to an antigen binding molecule with a heavy chain variable domain and no light chain variable domain. A monobody can bind to an antigen in the absence of light chains and typically has three hypervariable regions, for example CDRs designated CDRH1 , CDRH2, and CDRH3. A heavy chain IgG monobody has two heavy chain antigen binding molecules connected by a disulfide bond. The heavy chain variable domain comprises one or more hypervariable regions, preferably a CDRH3 or HVL-H3 region.
The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity-determining region" or "CDR" (defined by sequence as residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )) and/or those residues from a "hypervariable loop" (defined by structure and differing for each antibody; see, for example: Chothia and Lesk, 1987, J. Mol. Biol. 196:901 -917). In one example, HVL residues can include, 26-32 (L1 ), 50-52 (L2) and 91 -96 (L3) in the light chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain.
Phrases such as "Kabat position", "Kabat residue", and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include amino acid insertions (residue 52a, 52b and 52c according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by
alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
"Framework region" or "FR" residues are those variable domain residues flanking the hypervariable region residues as herein defined. In general, a variable domain contains three hypervariable regions flanked by four sequences of the framework region (FR1 , FR2, FR3, and FR4).
The term "consensus sequence", as used herein, refers to an artificial variable domain sequence comprising at each position the residue that is most abundant at that position in the variable domains of a group of antibodies of a particular class, for example, as described in the compilations of Kabat et al., Sequences of Proteins of Immunological Interest,. Public Health Service, National Institutes of Health,
Bethesda, Md. (5th Ed, 1991 ); (4th Ed 1987); (3rd Ed 1983). The consensus variable domain sequences do not have any known antibody binding specificity or affinity.
"Polypeptide" refers to a peptide or protein containing two or more amino acids linked by peptide bonds, and includes peptides, oligimers, proteins, and the like. Polypeptides can contain natural, modified, or synthetic amino acids. Polypeptides can also be modified naturally, such as by post-translational processing, or chemically, such as amidation, acylation, cross-linking, and the like.
The term "epitope", as used herein, is a structure defined in the context of a molecular interaction between an "antigen binding polypeptide", such as an antibody (Ab), and its corresponding "antigen" (Ag). The term antigen (Ag) refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag. Herein, Ag is used broadly and is generally intended to include target molecules that are specifically recognized by the Ab, thus including fragments or mimics of the molecule used in the immunization process for raising the Ab.
Generally, "epitope" refers to the area or region on an Ag to which an Ab specifically binds. A protein epitope may comprise amino acid residues in the Ag that are directly involved in binding to a Ab (also called the immunodominant component of the epitope) and other amino acid residues, which are not directly involved in binding, such as amino acid residues of the Ag which are effectively blocked by the Ab (in other words, the amino acid residue is within the "solvent-excluded surface"
and/or the "footprint" of the Ab). The term epitope herein includes both types of binding sites in any particular region of CRAC that specifically binds to an anti-CRAC channel antibody, or another CRAC channel-specific agent according to the invention, unless otherwise stated (e.g., in some contexts the invention relates to antibodies that bind directly to particular amino acid residues). ORAM may comprise a number of different epitopes, which may include, without limitation, (1 ) linear peptide antigenic determinants, (2) conformational antigenic determinants which consist of one or more non-contiguous amino acids located near each other in the mature CRAC conformation; and (3) post-translational antigenic determinants which consist, either in whole or part, of molecular structures covalently attached to CRAC, such as carbohydrate groups. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of one (or more) linear polypeptide chain(s). A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include other moieties, such as saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
The epitope for a given antibody (Ab)/antigen (Ag) pair can be defined and characterized at different levels of detail using a variety of experimental and computational epitope mapping methods. The experimental methods include mutagenesis, X-ray crystallography, Nuclear Magnetic Resonance (NMR)
spectroscopy, Hydrogen deuterium exchange Mass Spectrometry (HX-MS) and various competition binding methods; methods that are well known in the art. As each method relies on a unique principle, the description of an epitope is linked to the method by which it has been determined. Thus, depending on the epitope mapping method employed, the epitope for a given Ab/Ag pair will be defined differently.
At its most detailed level, the epitope for the interaction between the Ag and the Ab can be defined by the spatial coordinates defining the atomic contact points present in the Ag-Ab interaction, as well as information about their relative
contributions to the binding thermodynamics. At a less detailed level, the epitope can be characterized by the spatial coordinates defining the atomic contacts between the Ag and Ab. At an even less detailed level the epitope can be characterized by the amino acid residues that it comprises as defined by a specific criterion such as the distance between atoms in the Ab and the Ag. At a further less detailed level the
epitope can be characterized through function, e.g. by competition binding with other Abs. The epitope can also be defined more generically as comprising amino acid residues.
In the context of an X-ray derived crystal structure defined by spatial coordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag, the term epitope is herein, unless otherwise specified or contradicted by context, specifically defined as ORAM residues characterized by having a heavy atom (i.e. a non- hydrogen atom) within a distance of 4 A from a heavy atom in the Ab.
From the fact that descriptions and definitions of epitopes, dependent on the epitope mapping method used, are obtained at different levels of detail, it follows that comparison of epitopes for different Abs on the same Ag can similarly be conducted at different levels of detail.
Epitopes described on the amino acid level, e.g. determined from an X-ray structure, are said to be identical if they contain the same set of amino acid residues. Epitopes are said to overlap if at least one amino acid is shared by the epitopes. Epitopes are said to be separate (unique) if no amino acid residue is shared by the epitopes.
Epitopes characterized by competition binding are said to be overlapping if the binding of the corresponding Ab's are mutually exclusive, i.e. binding of one Ab excludes simultaneous binding of the other Ab. The epitopes are said to be separate (unique) if the Ag is able to accommodate binding of both corresponding Ab's simultaneously. There are instances when one or more antibodies do not have overlapping epitopes but can not bind simultaneously. Due to tertiary and quaternary structure of an antigen, one antibody may not be able to access its epitope due to previous binding of another antibody.
The term "paratope" refers to the area or region on the Ab to which an Ag specifically binds, i.e. with which it makes physical contact to the Ag.
In the context of an X-ray derived crystal structure, defined by spatial coordinates of a complex between an Ab, such as a Fab fragment, and its Ag, the term paratope is herein, unless otherwise specified or contradicted by context, specifically defined as Ag residues characterized by having a heavy atom (i.e. a non- hydrogen atom) within a distance of 4 A from a heavy atom in ORAM .
The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair may be identified by routine methods. For example, the general location of an epitope may be determined by assessing the ability of an antibody to bind to different fragments or variant ORAM polypeptides. The specific amino acids within ORAM that make contact with an antibody (epitope) and the specific amino acids in an antibody that make contact with ORAM (paratope) may also be determined using routine methods. For example, the antibody and target molecule may be combined and the Ab/Ag complex may be crystallised. The crystal structure of the complex may be determined and used to identify specific sites of interaction between the antibody and its target.
An antibody of the invention may have the ability to "compete" with another antibody of the invention for binding to ORAM or another appropriate target as described herein. Such cross-competing antibodies can be identified based on their ability to cross-compete with a known antibody of the invention in standard binding assays. For example, surface plasmon resonance (SPR), ELISA assays or flow cytometry may be used to demonstrate cross-competition. Such cross-competition may suggest that the two antibodies bind to identical, overlapping or similar epitopes.
"Epitope binning" refers to the use of competitive binding assays to identify pairs of antibodies that are, or are not, capable of binding an antigen, such as
ORAM , simultaneously. Families of antibodies (or bins) that have the same, or overlapping, binding specificity can then be used to help define specific epitopes on ORAM . Epitope binning experiments provide evidence that antigenically distinct epitopes are present. However, by themselves, they do not identify, or "map" the epitope to a specific amino acid sequence or location on ORAM . Competition for binding can be evaluated for any pair of antibodies or fragments. Favourable properties of a family (or bin) of antibodies can be correlated with binding to a specific epitope defined in terms of the antibody bin.
"Mammal" for purposes of treatment or therapy refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, and the like. Preferably, the mammal is human.
A nucleic acid sequence or polynucleotide is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. Often,
"operably linked" DNA sequences are contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic
oligonucleotide adaptors or linkers are used in accordance with conventional practice.
"Percent (%) amino acid sequence identity" with respect to a polypeptide is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California.
For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y,
where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
"Percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a reference polypeptide-encoding nucleic acid sequence, after aligning the sequences
and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
"Primate" as used herein refers to any of an order of mammals comprising humans, apes, monkeys, and related forms, such as lemurs and tarsiers.
"Purifying" means increasing the degree of purity of a polypeptide in a composition by removing (completely or partially) at least one contaminant from the composition. A "purification step" may be part of an overall purification process resulting in an "essentially pure" composition. An essentially pure composition contains at least about 90% by weight of the polypeptide of interest, based on total weight of the composition, preferably at least about 95% by weight.
An "isolated" antibody is one that has been identified and separated and/or recovered from at least one contaminant component of its natural environment that would interfere with diagnostic or therapeutic uses for the antibody, such as one or more of other antibodies, enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one contaminant component of the antibody's natural environment will not be present. Isolated antibody also includes the antibody in a therapeutic formulation or other
administration form because at least one contaminant component will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
The term "selectively binds" as used herein, refers to the ability of an antibody to adhere to specific antigens and not adhere to other polypeptides.
A "soluble" portion of a polypeptide, as used herein, refers to a portion that is soluble in water and lacks appreciable affinity for lipids (e.g., missing the
transmembrane domain or the transmembrane and the cytoplasmic domains).
A "variant" of a polypeptide refers to a polypeptide that contains an amino acid sequence that differs from a reference sequence. The reference sequence can be a full-length native polypeptide sequence or any other fragment of a full-length polypeptide sequence. In some embodiments, the reference sequence is a variable domain heavy chain or variable domain light chain consensus sequence. A polypeptide variant generally has at least about 80% amino acid sequence identity with the reference sequence, usually at least 90, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even at least 99%. It should be noted, however, that to qualify as a "variant" such
polypeptide sequence must differ from the reference sequence in at least one amino acid residue (which may be replaced or omitted).
The term "binding affinity" is herein used as a measure of the strength of a non-covalent interaction between two molecules, e.g. and antibody, or fragment thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).
Binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determination of the dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or kon) and dissociation rate constant kd. (or k0fr), respectively. KD is related to ka and kd through the equation KD = kd / ka.
Following the above definition binding affinities associated with different molecular interactions, e.g. comparison of the binding affinity of different antibodies for a given antigen, may be compared by comparison of the KD values for the individual antibody/antigen complexes.
Similarly, the specificity of an interaction may be assessed by determination and comparison of the KD value for the interaction of interest, e.g. a specific interaction between an antibody and an antigen, with the KD value of an interaction not of interest.
Typically, the KD for the antibody with respect to the target will be 2-fold, preferably 5-fold, more preferably 10-fold less than KD with respect to the other, non- target molecule such as unrelated material or accompanying material in the environment. More preferably, the KD will be 50-fold less, such as 100-fold less, or 200-fold less; even more preferably 500-fold less, such as 1 ,000-fold less, or 10,000- fold less.
The value of this dissociation constant can be determined directly by well- known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (Byte 9:340-362, 1984). For example, the KD may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman {Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system.
A competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another ligand of that target, such as another antibody. The concentration at which 50% inhibition occurs is known as the Ki. Under ideal conditions, the Ki is equivalent to KD. The Ki value will never be less than the KD, so measurement of Ki can conveniently be substituted to provide an upper limit for KD.
An antibody of the invention may have a KD for its target of 1 x 10"7M or less, 1 x 10"8M or less, or 1 x 10"9M or less, or 1 x 10"10M or less, 1 x 10"11M or less, or 1 x 10"12M or less.
An antibody that specifically binds its target may bind its target with a high affinity, that is, exhibiting a low KD as discussed above, and may bind to other, non- target molecules with a lower affinity. For example, the antibody may bind to non-
target molecules with a KD of 1 x 10"6 M or more, more preferably 1 x 10"5 M or more, more preferably 1 x 10"4 M or more, more preferably 1 x 10"3 M or more, even more preferably 1 x 10"2 M or more. An antibody of the invention is preferably capable of binding to its target with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold 200-fold, 500-fold, 1 , 000-fold or 10,000-fold or greater than its affinity for binding to another non-target molecule.
Calcium-Release Activated Calcium Channel
ORAM (also known as "CRACM1" and "Transmembrane protein 142A") is a pore subunit of the CRAC channel. ORAM is an approximately 33 kDa transmembrane protein with the following sequence:
1 mhpepappps rsspelppsg gsttsgsrrs rrrsgdgepp gapppppsav typdwigqsy 61 sevmslnehs mqalswrkly lsraklkass rtsallsgfa mvamvevqld adhdyppgll 121 iafsacttvl vavhlfalmi stcilpniea vsnvhnlnsv kesphermhr hielawafst 181 vigtllflae vllcwvkfl plkkqpgqpr ptskppasga aanvstsgit pgqaaaiast 241 timvpfglif ivfavhfyrs lvshktdrqf qelnelaefa rlqdqldhrg dhpltpgshy 301 a
(SEQ ID NO: 1; Accession No. AAH15369.2 GL54035070). This protein has four helices (Ml, M2, M3, and M4) that span a cell's plasma membrane. Thereby, the ORAIl possesses two extracellular loops of amino acids that separate the first and second transmembrane segments and the third and fourth transmembrane segments. Both the N- and C-termini are on the cytoplasmic side of the cell membrane. The extracellular loops of ORAIl are accessible to antibody binding domains. Evidence suggests that a tetramer of ORAIl forms the CRAC channel (Penna et al., Nature 2008;456: 116-20). ORAIl mediates Ca2+ influx following depletion of intracellular Ca2+ stores and channel activation by the Ca2+ sensor, STIM1. In terms of the current invention, ORAIl may be from any vertebrate, such as any mammal, such as a rodent (such as a mouse, rat or guinea pig), a lagomorph (such as a rabbit), an artiodactyl (such as a pig, cow, sheep or camel) or a primate (such as a monkey or human being). ORAIl is, preferably, human ORAIl . ORAIl may be translated from any naturally occurring genotype or allele that gives rise to a functional protein.
Human ORAI1 (also known as "CRACM1" and "TMEM142A) is a gene at chromosome 12q24.31. (SEQ ID NO:4; Accession No. NG_007500.1 GL 171541812).
ORAI1 has just 2 exons, nucleotides 1 to 496 and 14,487 to 15486 of SEQ ID NO:4.
ORAM or fragments thereof can be used directly as an immunogen to generate antibodies. An antigen to be used for production of antibodies can be, for example, a soluble form of the full length polypeptide or a fragment thereof, such as a solubilized full length molecule or a fragment. Extracellular portions of ORAM were used as immunogens to generate mouse anti-CRAC channel antibodies.
EVQLDADHDYPPGC (SEQ ID NO:2; the first extracellular loop with a cysteine added to the C-terminus for coupling to KLH or BSA) and
VWKFLPLKKQPGQPRPTSKPPASGAAANVSTSGITPGQAC (SEQ ID NO:3; the second extracellular loop with a cysteine added to the C-terminus for coupling to BSA). An antibody or antibody fragment can be generated that binds to at least a portion of SEQ ID NO:2 and/or SEQ ID NO:3. Such an antibody could impede Ca2+ influx and thereby suppress an immune response. An anti-CRAC channel antibody that impedes Ca2+ influx can block T cell activation and/or decrease cytokine production (e.g., IL-2, IL-4, IL-10, etc.) in T cells. An anti-CRAC channel antibody can affect activation and/or cytokine production in both CD4+ and CD8+ T cells.
Thereby, an anti-CRAC channel antibody can modulate effector T cell responses.
Anti-CRAC Channel Antibodies
An antibody of the invention will have the ability to bind to a CRAC channel.
Preferably, an antibody of the invention will bind specifically to an extracellular loop of the ORAM . That is, an antibody of the invention will preferably bind to ORAM with greater binding affinity than that at which it binds to another molecule. An antibody of the invention may have the ability to bind or specifically bind ORAM as described herein, such as any target molecule as described herein.
An antibody of the invention may have the ability to compete with another antibody of the invention for binding to ORAM or another appropriate target as described herein. For example, an antibody of the invention may cross-compete with another antibody or antibodies described herein (such as anti-M-CRAC1415-10F8
A2B6C1 , anti-M-CRAC1415-17F1A4B6, anti-M-CRAC1415-17F6A2, anti-M-
CRAC1415-15F3A6, anti-M-CRAC1415-15F44A6, anti-M-CRAC1415-15F45A1 , anti- M-CRAC1415-15F54A2, anti-M-CRAC1415-15F58A5B4 and anti-M-CRAC1415- 14F74A1 antibodies) described herein for binding to ORAM , or to a suitable fragment or variant of ORAM that is bound by the antibodies described herein. Such cross- competing antibodies can be identified based on their ability to cross-compete with a known antibody of the invention in standard binding assays. For example, SPR e.g. by using a Biacore™ system, ELISA assays or flow cytometry may be used to demonstrate cross-competition. Such cross-competition may suggest that two antibodies bind to identical, overlapping or similar epitopes.
Anti-CRAC channel antibodies of the invention include an antibody that binds the second extracellular loop of the ORAM and inhibits or abrogates CRAC channel function. The anti-CRAC channel antibody is able to block the influx of calcium ions into T cells. As a result, the binding of the anti-CRAC channel antibody inhibits activation of T cells and also inhibits the translocation of NFAT from the cytoplasm to the nucleus. In an embodiment, an anti-CRAC channel antibody comprises a monoclonal antibody (mAb) that is capable of binding the first or the second extracellular loop of ORAM .
Multiple anti-CRAC channel antibodies generated by the methods of Example 9 (below) comprised VH chains derived from mouse germline sequence IGHV1 -80 VH QVQLQQSGAELVKPGASVKISCKASGYAFSSYWMNVWKQRPGKGLEWIGQIYPGD GDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO: 53). This is surprising because different antibodies are typically derived from different germlines since VH-D-J recombinations are generated at random from the genome. The antibodies generated in Example 9 also comprised highly similar CDRH1 and CDRH2 sequences. Without being bound by theory, multiple selection of IGHV1 -80 VH and conservation of the CDRH1 and CDRH2 regions may indicate the importance of this region for binding the CRAC channel. Additionally, nearly all of the antibodies generated in Example 9 had different CDRH3 segments, which, without being bound by theory, may indicate a decreased importance of CDRH3 (derived from the D segment) in binding. This would be surprising because CDRH3 is more frequently the most important binding site for specificity.
The same principle is true for the light chains generated for anti-CRAC channel antibodies by the methods of Example 9. Each of these antibodies comprised VL chains derived from the IGKV8-21 VL germ line sequence
DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYW ASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNL (SEQ ID NO: 59) and comprised highly similar CDRL1 and CDRL2 and part of CDRL3 from the VK segment sequences but nearly all of these antibodies had a different portion of the CDRL3 segment from the JK segment. Again, without being bound by theory, this may indicate increased importance of CDRL1 and CDRL2 and the part of CDRL3 from the VK segment in binding CRAC channels.
Therefore, in an embodiment, an anti-CRAC channel antibody is derived from the IGHV1 -80 VH germline sequence (SEQ ID NO: 53) and comprises a CDRH1 comprising SYWMN (SEQ ID NO: 10), wherein one of these amino acids is optionally substituted by a different amino acid; and a CDRH2 comprising
XIYPGDXDTNYNGKFKG (SEQ ID NO: 52) or amino acids 1 -9 of SEQ ID NO: 52, wherein X is any amino acid, wherein one, two or three amino acids (not including X) may be substituted by a different amino acid. SEQ ID NO: 52 is the same as IGHV1 - 80 VH germline when X1 is Gin and X7 is Gly. In an embodiment, the anti-CRAC channel antibody has a heavy chain derived from the IGHV1 -80 VH germline sequence (SEQ ID NO: 53) and also has a light chain derived from the IGKV8-21 VL germline sequence (SEQ ID NO: 59), and comprises a CDRL1 of KSSQSLXNSRT (SEQ ID NO: 57) or KSSQSLXNSRTRKNYLA (SEQ ID NO: 54), wherein X is a any amino acid, or wherein X is Leu or Phe, and wherein one, two or three amino acids (not including X) are optionally substituted with a different amino acid; and comprises a CDRL2 of WASTRES (SEQ ID NO: 19), wherein one or two of these amino acids is optionally substituted with a different amino acid. SEQ ID NOS: 54 and 57 are the same as IGKV8-21 VL germline when X is Leu. In an embodiment, the anti-CRAC channel antibody further comprises a CDRL3 having consensus sequence
[K/S]QSY[N/D]L[P/T/-][W/R][T/S] (SEQ ID NO: 55), wherein KQSYNL (SEQ ID NO: 60) is the VK part of the germline.
In an embodiment, an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence comprising SYWMN (SEQ ID NO: 10), wherein one
of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence comprising XIYPGDXDTNYNGKFKG (SEQ ID NO: 52), wherein X is any amino acid, wherein one, two or three amino acids (not including X) may be substituted by a different amino acid; and/or a CDRH3 comprising QLGFRYAMDY (SEQ ID NO: 36), DHRDYYAMDY (SEQ ID NO: 41 ), SGRLRFAMDY (SEQ ID NO: 50) or GGTTWVDY (SEQ ID NO: 12). In some embodiments, CDRH2 is SEQ ID NO: 52 and X1 is His or Gin. In some embodiments, CDRH2 is SEQ ID NO: 52 and X7 is a nonpoiar amino acid. In some embodiments, X7 is Gly or Ala. In some embodiments, amino acids 1 -9 of SEQ ID NO: 52 are used when grafting CDRH2 onto a human framework to make a humanized antibody. In some embodiments, CDRH2 is SEQ ID NO: 52 plus four additional amino acids on the C-terminal end: XIYPGDXDTNYNGKFKGKATL (SEQ ID NO: 56). In some embodiments, CDRH2 is HIYPGDGDTNYNGKFKG (SEQ ID NO: 1 1 ), HIYPGDADTNYNGKFKG (SEQ ID NO: 35) or HIYPGDGDTNYNGKFKGKATL (SEQ ID NO: 49), or amino acids 1 -9 of any one of SEQ ID NOS: 52, 56, 1 1 , 35 or 49 when making a humanized antibody. In an embodiment, an anti-CRAC channel antibody comprises a light chain comprising a CDRL1 sequence comprising KSSQSLXNSRT (SEQ ID NO: 57), wherein X is a any amino acid, wherein one, two or three amino acids (not including X) may be substituted with a different amino acid; and/or a CDRL2 sequence comprising
WASTRES (SEQ ID NO: 19), wherein one or two of these amino acids may be substituted with a different amino acid; and/or a CDRL3 sequence comprising
[K/S]QSY[N/D]L[P/T/-][W/R][T/S] (SEQ ID NO: 55), wherein X is any amino acid. In some embodiments, CDRL1 is SEQ ID NO: 57 plus six additional amino acids on the C-terminal end: KSSQSLXNSRTRKNYLA (SEQ ID NO: 54). . In some
embodiments, CDRL1 is SEQ ID NO: 54 or 57 and X is a nonpoiar or aromatic amino acid. In some embodiments, CDRL1 is SEQ ID NO: 54 or 57 and X is Leu or Phe. In some embodiments, CDRL1 is KSSQSLLNSRT (SEQ ID NO: 18),
KSSQSLLNSRTRKNYLA (SEQ ID NO: 38) or KSSQSLFNSRTRKNYLA (SEQ ID NO: 46). In some embodiments, CDRL3 is KQSYNLRT (SEQ ID NO: 39),
KQSYDLTRS (SEQ ID NO: 43), or SQSYNLRT (SEQ ID NO: 47) or KQSYNLPWT (SEQ ID NO: 20).
In an embodiment, an anti-CRAC channel antibody comprises a monoclonal antibody (mAb) that is capable of binding the first or the second extracellular loop of ORAM . In an embodiment, an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN; SEQ ID NO: 10) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence of amino acids 50 to 66
(HIYPGDGDTNYNGKFKG) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and/or a CDRH3 sequence of amino acids 99 to 107 (GGTTWVDY) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid. In another embodiment, the CDRH2 sequence is amino acids 50 to 58 (HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9. In an embodiment, an anti-CRAC channel antibody comprises light chain comprising a CDRL1 sequence of amino acids 24 to 40
(KSSQSLLNSRT) of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid; and/or a CDRL2 sequence of amino acids 56 to 62 (WASTRES; SEQ ID NO: 19) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid; and/or a CDRL3 sequence of amino acids 95 to 103 (KQSYNLPWT) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid. In an embodiment, an anti-CRAC channel antibody comprises a heavy chain comprising a CDRH1 sequence of amino acids 31 to 35 (SYWMN) of SEQ ID NO: 9, wherein one of these amino acids may be substituted by a different amino acid; and/or a CDRH2 sequence of amino acids 50 to 66 (HIYPGDGDTNYNGKFKG) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and/or a CDRH3 sequence of amino acids 99 to 107 (GGTTWVDY) of SEQ ID NO: 9, wherein one, two or three of these amino acids may be substituted by a different amino acid; and a light chain comprising a CDRL1 sequence of amino acids 24 to 40 (KSSQSLLNSRT) of SEQ ID NO: 17, wherein one, two or three of these amino acids may be substituted with a different amino acid; and/or a CDRL2 sequence of amino acids 56 to 62 (WASTRES) of SEQ ID NO: 17, wherein one or two of these amino acids may be substituted with a different amino acid; and/or a CDRL3 sequence of amino acids 95 to 103 (KQSYNLPWT) of SEQ ID NO: 17,
wherein one or two of these amino acids may be substituted with a different amino acid. In another embodiment, the CDRH2 sequence is amino acids 50 to 58
(HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9. In an embodiment, an anti-CRAC channel antibody comprises SEQ ID NO:9. In an embodiment, an anti-CRAC channel antibody comprises SEQ ID NO: 17.
Embodiments of an anti-CRAC channel antibody includes can have, for example, at least 80%, at least about 85%, at least about 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the CDRs described herein. Such anti-CRAC channel antibodies are useful in methods of the invention.
Antibody Production
Monoclonal Antibodies. Monoclonal antibodies may be made using any number of methods including the hybridoma method first described by Kohler et a\., 1975, Nature, 256:495. Monoclonal antibodies can also be made by recombinant DNA methods (see, for example, U.S. Patent No. 7,829,092).
In a hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind the protein(s) used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT- deficient cells.
Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell
Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., X63- Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, 1984, J. Immunol. 133:3001 ; and Brodeur et a\., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al. 1980, Anal. Biochem. 107:220.
Screening methods useful to identify anti-CRAC channel antibodies include, for example, ELISA, ECLA, and Biacore analysis. Such screening analysis permits selection of anti-CRAC channel antibodies that selectively bind CRAC- overexpressing cells.
Once hybridoma cells that produce anti-CRAC channel antibodies of a desired specificity, affinity, and/or activity are identified, clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.
The anti-CRAC monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g.,
using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
DNA encoding the anti-CRAC monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., 1993, Curr. Opinion in Immunol. 5:256-262 and Pluckthun, 1992, Immunol. Revs. 130: 151 -188.
In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554. Clackson et al., Nature, 1991 , 352:624-628 and Marks et al., 1991 , J. Mol. Biol. 222:581 -597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., 1993, Nuc. Acids. Res. 21 :2265-2266). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of anti-CRAC monoclonal antibodies.
Alternatively, phage display technology (see, for example, McCafferty et al.,
1990, Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable domain gene repertoires from non- immunized donors. According to this technique, genes encoding antibody variable domains are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the
functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson et al., 1993, Current Opinion in Structural Biology 3:564-571 .
DNA encoding an antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CL) sequences for the homologous murine
sequences (U.S. Patent No. 4,816,567; and Morrison, et al., 1984, Proc. Nat'l Acad. Sci. USA 81 :6851 ). Alternatively, the immunoglobulin coding sequence can be fused with all or part of a sequence encoding a non-immunoglobulin polypeptide
(heterologous polypeptide). The non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or can substitute for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
To generate an antibody mutant, one or more amino acid alterations (e.g. substitution, deletion, insertion) are made to the amino acid sequence, as known. Alterations, even in the framework sequence, can improve affinity of antibodies.
Humanized Antibodies. Methods for humanizing nonhuman antibodies have been described in the art. Preferably, a humanized antibody has one or more nonhuman amino acid residues introduced into its sequence. These nonhuman amino acid residues are often referred to as "import" or "donor" residues that are typically taken from an "import" or "donor" variable domain and introduced into a human "recipient" sequence. Humanization can be performed by substituting hypervariable region sequences (CDRs or HVLs) from nonhuman sources for the corresponding sequences of a human antibody. Accordingly, such "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 nonhuman species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and human anti-mouse antibody (HAMA) response when the antibody is intended for human therapeutic use. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human variable domain sequence, which is closest to that of the rodent, is identified and the human variable domain framework region (FR) within it is accepted for the humanized antibody (Sims et al., 1993, J. Immunol. 151 :2296; Chothia et al., 1987, J. Mol. Biol., 196:901 ). Another method uses a particular framework region derived from a consensus sequence variable domain. The consensus sequence can be delineated by residues identified in a compilation of variable domain residues for known human antibodies in a particular subgroup of light or heavy chains. The same variable domain framework region may be used to produce several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285; Presta et al., 1993, J. Immunol. 151 :2623).
Antibodies can be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process that includes analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, I.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, variable domain framework region residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
Although the variable domain framework regions of multiple humanized therapeutic antibodies can be identical to the sequences of a consensus sequence, the framework regions (FRs) of each humanized antibody sequence may differ from the FRs of the consensus sequence. Amino acid substitutions in the framework regions can be made, for example, to increase affinity to an antigen. See, for example, WO 98/45331 . Substituting mouse residues or alternative human residues can increase affinity of the antibody to the antigen. Substitutions can be made to either the heavy or light chain variable domain. Most FR substitutions occur in the heavy chain variable domain framework region 3 (FR3). Thus, consensus framework regions can provide a basis for constructing a humanized antibody.
A humanized antibody may be an antibody fragment, such as a Fab, optionally conjugated with one or more agents in order to generate an
immunoconjugate. Alternatively, the humanized antibody may be a full length antibody, such as a full length lgG1 antibody.
Human Antibodies. Besides isolating antibodies from humans, it is now possible to produce transgenic animals (e.g. mice) that are capable, upon
immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggemann & Neuberger, Immunol. Today 1996; 17: 391 -397.
Mendez et al. (Nature Genet., 1997; 15: 146-156) have generated a line of transgenic mice designated as "XenoMouse® II" that, when challenged with an antigen, generates high affinity fully human antibodies. This was achieved by germ- line integration of megabase human heavy chain and light chain loci into mice with deletion into endogenous JH segment as described above. The XenoMouse® II harbors 1 ,020 kb of human heavy chain locus containing approximately 66 VH genes, complete DH and JH regions and three different constant regions (μ, δ, and γ), and
also harbors 800 kb of human κ locus containing 32 VK genes, JK segments and CK genes. The antibodies produced in these mice closely resemble that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. The human antibodies are preferentially expressed over endogenous antibodies due to deletion in endogenous JH segment that prevents gene rearrangement in the murine locus.
Tomizuka et al. (Proc. Natl. Acad. Sci. USA 2000; 97: 722-727) described generation of a double trans-chromosomal (Tc) mouse by introducing two individual human chromosome fragments (hCFs), one containing the entire Ig heavy chain locus (IgH, aboutl .5 Mb) and the other the entire kappa light chain locus (IgK,
.about.2 Mb) into a mouse strain whose endogenous IgH and IgK loci were
inactivated. These mice mounted antigen-specific human antibody response in the absence of mouse antibodies. The Tc technology may allow for the humanization of over megabase-sized, complex loci or gene clusters (such as those encoding T-cell receptors, major histocompatibility complex, P450 cluster etc) in mice or other animals. Another advantage of the method is the elimination of a need of cloning the large loci. This is a significant advantage since the cloning of over megabase-sized DNA fragments encompassing whole Ig loci remains difficult even with the use of yeast artificial chromosomes (Peterson et al., Trends Genet. 1997; 13: 61 -66;
Jacobovits, Curr. Biol. 1994; 4: 761 -763). Moreover, the constant region of the human IgH locus is known to contain sequences difficult to clone (Kang and Cox, Genomics 1996; 35: 189-195).
Gene shuffling can also be used to derive human antibodies from non-human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called "epitope imprinting", either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, i.e. the epitope governs (imprints) the choice of the human chain partner. When the process is
repeated in order to replace the remaining non-human chain, a human antibody is obtained (PCT WO 93/06213). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin.
Commercially, Medarex, Inc. has also developed a transgenic mouse that expresses human antibodies. In the HuMAb-Mouse®, mouse VDJ genes have been inactivated and replaced by human antibody genes. The HuMAb-Mouse® contains unrearranged human antibody genes that encode both heavy and light chains.
Medarex has also collaborated with the pharmaceutical division of Kirin Brewery Co., Ltd., to produce the KM- Mouse®, a mouse that is the result of a cross between the HuMAb-Mouse® and Kirin's TC Mouse™. This KM- Mouse® had the human antibody repertoire of the HuMAb-Mouse HuMAb-Mouse® and the KM- Mouse®'s ability to produce all human antibody isotypes.
Antibody Fragments. In certain circumstances it may be advantageous to use antibody fragments, rather than whole antibodies in therapeutic treatments. The smaller size of the fragments allows for rapid clearance and/or may improve access to target cells.
Various techniques have been developed for the production of antibody fragments. Traditionally, fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24: 107-1 17; and Brennan et al., 1985, Science, 229:81 ). However, fragments can be produced directly by recombinant host cells. Antibody fragments can be obtained using conventional synthetic, recombinant or protein engineering techniques, and the fragments can be screened for antigen-binding or other function in the same manner as are can be intact antibodies. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing the facile production of large amounts of these fragments. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio/Technology 10: 163-167). In another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other
techniques for the production of antibody fragments will be apparent to the skilled practitioner. An antibody of choice can be a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571 ,894; and U.S. Patent No. 5,587,458. Fv and scFv are the only species of fragments with intact combining sites that are devoid of constant regions; thus, and are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Patent 5,641 ,870 for example. Such linear antibody fragments may be monospecific or bispecific.
Bispecific Antibodies. Bispecific antibodies (aka "diabodies") are
antibodies that have binding specificities for at least two different epitopes.
Exemplary bispecific antibodies may bind to two different epitopes of a protein. Other such antibodies may combine a one antibody binding site with a binding site for another protein. Alternatively, one antibody arm may be combined with an arm that binds to a triggering molecule on a cell. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. See Tutt et al., 1991 , J. Immunol. 147:60.
Multivalent Antibodies. A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino- terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1 -(X1 )n-VD2- (X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or
polypeptide, and n is 0 or 1 . For instance, the polypeptide chain(s) may comprise: VH-CH1 -flexible linker-VH-CH1 -Fc region chain; or VH-CH1 -VH-Ch1 -FC region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
Polyclonal antibodies. Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, and the like.
Animals can be immunized against an antigen, an antigen cocktail,
immunogenic conjugates, or derivatives thereof by combining, for example, 100 g or 5 g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermal^ at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
Amino Acid Sequence Variants. Amino acid sequence modification(s) of anti-CRAC channel antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of anti-CRAC. antibodies are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the anti- CRAC channel antibody chains, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues
within the amino acid sequences of an anti-CRAC channel antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of an anti-CRAC channel antibody, such as changing the number or position of glycosylation sites.
Alanine scanning mutagenesis, as described by Cunningham and Wells (Science 1989; 244:1081 -1085) is a useful method for identification of certain residues or regions of the anti-CRAC channel antibody that are preferred locations for mutagenesis. A residue or group of target residues can be identified (e.g., charged residues such as arg, asp, his, lys, and glu and replaced by a neutral or negatively charged amino acid, most preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. Those amino acid locations
demonstrating functional sensitivity to substitutions are then refined by introducing further or others at, or for, the sites of substitution. While the site for introducing an amino acid sequence variation may be predetermined, the nature of a mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or region and expressed anti-CRAC channel antibody variants are screened for a desired activity (e.g., disruption of NFAT translocation to the nucleus, inhibition of T cell activation, inhibition of calcium influx in T cells, etc.).
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-CRAC channel antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of an anti-CRAC channel antibody molecule include a fusion of an enzyme or a polypeptide which increases the serum half-life of the antibody to the N- or C-terminus of the anti-CRAC channel antibody.
Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in an anti-CRAC channel antibody molecule removed and a different residue inserted in its place. Sites of greatest interest for substitution mutagenesis include hypervariable regions, but FR alterations are also
contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1 , or as further described below in reference to amino acid classes, may be introduced and the products screened.
Table 1
Original Residue Exemplary Substitution Preferred Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gin; asn lys
Asn (N) gin; his; lys; arg gin
Asp (D) glu glu
Cys (C) ser ser
Gin (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gin; lys; arg arg lie (I) leu; val; met; ala; phe;norleucine leu
Leu (L) norleucine; ile; val;met; ala; phe ile
Lys (K) arg; gin; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; ala; norleucine leu
Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1 ) hydrophobic: norleucine, met, ala, val, lei, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
Any cysteine residue not involved in maintaining proper conformation of an- anti CRAC antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as a Fv fragment.
Another type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitution variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. inhibition of T cell activation) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between an antibody and ORAM . Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
Glycosylation Variants. Glycosylation variants of antibodies are variants in which the glycosylation pattern of an antibody is altered. By altering is meant deleting one or more carbohydrate moieties found in the antibody, adding one or more carbohydrate moieties to the antibody, changing the composition of glycosylation (glycosylation pattern), the extent of glycosylation, etc.
Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, Chem. Immunol. 1997; 65: 1 1 1 -128 ; Wright and Morrison, Trends Biotechnol. 1997; 15:26-32). The oligosaccharide side chains of the
immunoglobulin's can affect a protein's function (Boyd et al., Mol. Immunol. 1996; 32: 131 1 -1318), and the intramolecular interaction between portions of the
glycoprotein can affect the conformation and presented three-dimensional surface of the glycoprotein. Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. For example, it has been reported that in a galactosylated IgG, the oligosaccharide moiety 'flips' out of the inter-CH2 space and terminal N-acetylglucosamine residues become available to bind mannose binding protein (Malhotra et al., Nature Med. 1995; 1 :237-243).
Removal by glycopeptidase of the oligosaccharides from CAMPATH-1 H (a
recombinant humanized murine monoclonal lgG1 antibody which recognizes the CDw52 antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO) cells resulted in a complete reduction in complement mediated lysis (CMCL) (Boyd et al., Mol. Immunol. 1996; 32: 131 1 -1318), while selective removal of sialic acid residues using neuraminidase resulted in no loss of DMCL. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1 ,4)-Ν- acetylglucosaminyltransferase III (GnTIII), a glycosy transferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et al. Nature Biotech. 1999; 17: 176-180).
Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine (SEQ ID NO:26) and asparagine-X-threonine (SEQ ID NO:27), where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate
moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked
glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above- described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of the antibody.
The amino acid sequence is usually altered by altering the underlying nucleic acid sequence. Nucleic acid molecules encoding amino acid sequence variants of an anti- CRAC channel antibody are prepared by a variety of methods known in the art.
These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-CRAC channel antibody.
The glycosylation (including glycosylation pattern) of antibodies may also be altered without altering the amino acid sequence or the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g. antibodies, as potential therapeutics is rarely the native cell, significant variations in the glycosylation pattern of the antibodies can be expected (see, e.g. Hse et al., J. Biol. Chem. 1997; 272:9062-9070). In addition to the choice of host cells, factors which affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335;
5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H). In addition, the recombinant host cell can be genetically engineered, e.g. make defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.
The glycosylation structure of antibodies can be readily analyzed by
conventional techniques of carbohydrate analysis, including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharide compositional analysis, sequential enzymatic digestion, and HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge. Methods for releasing oligosaccharides for analytical purposes are also known, and include, without limitation, enzymatic treatment, elimination using harsh alkaline environment to release mainly O-linked structures, and chemical methods using anhydrous hydrazine to release both N- and O-linked oligosaccharides.
Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the recombinant polypeptides, including monoclonal antibodies described herein are prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis {e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P.
aeruginosa, and Streptomyces. Examples of cloning hosts include, but are not limited to, E. coli 294 (ATCC 31 ,446), E. coli B, E. coli X1776 (ATCC 31 ,537), and E. coli W31 10 (ATCC 27,325). These examples are illustrative rather than limiting.
Production in E. coli is fast and cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. 5,648,237 (Carter et. al.), U.S. 5,789, 199 (Joly et al.), and U.S. 5,840,523 (Simmons et al.) which describes a translation initiation region (TIR) and signal sequences for optimizing expression and secretion. After expression, an antibody can be isolated from an E coli cell paste in
a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding.
Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as
Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K.
fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24, 178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K . thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;
Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for expression are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa caiifornica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
Vertebrate cells in culture (tissue culture) are also suitable hosts for expression. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216);
mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251 ; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442);
human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);
mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells (Mather et al., 1982, Annals N. Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells can be transformed with expression or cloning vectors for an anti- CRAC channel antibody and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
Host cells used to produce an anti-CRAC channel antibody may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., 1979, Meth. Enz. 58:44, Barnes et al. , 1980, Anal. Biochem. 102:255, U.S. Pat. Nos. 4,767,704; 4,657,866;
4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (e.g., gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary
supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
Purification of Antibody
When using recombinant techniques, an antibody can be produced
intracellularly, in the periplasmic space, or directly secreted into the medium. If an antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology 10: 163-167 describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and
phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where an antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
An antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1 , γ2, or γ4 heavy chains (Lindmark et al., 1983, J. Immunol. Meth. 62: 1 -13). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., 1986, EMBO J. 5: 15671575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin Sepharose® chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE,
and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step(s), the mixture comprising an antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations {e.g., from about 0-0.25M salt).
Methods of Treatment
Anti-CRAC channel antibodies of the invention are capable of T-cell modulation. Such modulation includes inhibiting or reducing T cell activation, proinflammatory cytokine production (e.g., IL-1 B, IL-2, IL-4, IL-5, IL-6, IL-13, IFN- gamma), T cell proliferation, and NFAT translocation. Embodiments of the invention include administering an anti-CRAC channel antibody of the invention to a subject in need thereof to inhibit or reduce T cell activation, pro-inflammatory cytokine production (e.g., IL-1 B, IL-2, IL-4, IL-5, IL-6, IL-13, IFN-gamma), T cell proliferation, and NFAT translocation.
Anti-CRAC channel antibodies of the invention may be administered to a subject with an immune disorder. An immune disorder can be an autoimmume disorder or disease and/or a chronic inflammatory disease. Thus, anti-CRAC channel antibodies of the invention may be administered to a subject with rheumatoid arthritis (RA). Anti-CRAC channel antibodies of the invention may be administered to a subject with inflammatory bowel disease (IBD), such as a subject with Crohn's disease (CD), such as a subject with ulcerative colitis (UC). Anti-CRAC channel antibodies of the invention may be administered to a subject with systemic lupus erythematosus (SLE). Anti-CRAC channel antibodies of the invention may be administered to a subject with psoriasis. Anti-CRAC channel antibodies of the invention may be administered to a subject with psoriatic arthritis (PA). Anti-CRAC channel antibodies of the invention may be administered to a subject with multiple sclerosis (MS). Such administration is expected to result in abatement of the t-cell component of the abnormal immune responses.
Rheumatoid arthritis (RA) is a systemic disease that affects the entire body and is one of the most common forms of arthritis. It is characterized by inflammation
of the joint, which causes pain, stiffness, warmth, redness and swelling. This inflammation is a consequence of inflammatory cells invading the joints and these inflammatory cells release enzymes that may digest bone and cartilage. As a result this inflammation can lead to severe bone and cartilage damage and to joint deterioration and severe pain amongst other physiologic effects. The involved joint can lose its shape and alignment, resulting in pain and loss of movement.
There are several animal models for rheumatoid arthritis known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop an inflammatory arthritis that resembles human rheumatoid arthritis. Since CIA shares similar immunological and pathological features with RA, this makes it a suitable model for screening potential human anti-inflammatory compounds. Efficacy in this model is measured by decrease in joint swelling. Efficacy in RA in the clinic is measured by the ability to reduce symptoms in patients which is measured as a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein levels and levels of serum factors, such as anti-citrullinated protein antibodies.
Inflammatory Bowel Disease (IBD) is a disease that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms. It primarily causes abdominal pain, diarrhea (which may be bloody), vomiting, or weight loss, but may also cause complications outside of the gastrointestinal tract such as skin rashes, arthritis, inflammation of the eye, tiredness, and lack of concentration. Patients with IBD can be divided into two major classes, those with ulcerative colitis (UC) and those with Crohn's disease (CD). While CD generally involves the ileum and colon, it can affect any region of the intestine but is often discontinuous (focused areas of disease spread throughout the intestine), UC always involves the rectum (colonic) and is more continuous. In CD, the inflammation is transmural, resulting in abscesses, fistulas and strictures, whereas in UC, the inflammation is typically confined to the mucosa. There is no known pharmaceutical or surgical cure for Crohn's disease, whereas some patients with UC can be cured by surgical removal of the colon.
Treatment options are restricted to controlling symptoms, maintaining remission and preventing relapse. Efficacy in inflammatory bowel disease in the clinic may be measured as a reduction in CDAI score for CD which is scoring scale based on laboratory tests and a quality of life questionnaire. In animal models, efficacy is mostly
measured by increase in weight and also a disease activity index (DAI) which is a combination of stool consistency, weight and blood in stool.
Systemic lupus erythematosus (SLE) is an immune-complex related disorder characterized by chronic IgG antibody production directed at ubiquitous self antigens such as anti-dsDNA. The central mediator of disease in SLE is the production of autoantibodies against self-proteins/tissues and the subsequent generation of immune- mediated inflammation. Antibodies either directly or indirectly mediate inflammation. Although T lymphocytes are not thought to directly cause disease, they are required for auto-antibody production. The effects of SLE are systemic (kidney, lung,
musculoskeletal system, mucocutaneous, eye, central nervous system cardiovascular system, gastrointestinal tract, bone marrow, blood), rather than localized to a specific organ, although glomerulonephritis may result in some cases (i.e. lupus nephritis).
Multiple chromosomal loci have been associated with the disease and may contribute towards different aspects of the disease, such as anti-dsDNA antibodies and
glomerulonephritis. Efficacy in SLE in human disease and in appropriate mouse models is measured by the ability of the therapeutic entity to decrease auto-antibodies (Eg: anti- dsDNA) and/or by decrease in renal pathology (enhanced kidney function), leading to amelioration of disease symptoms.
Psoriasis is a T-cell mediated inflammatory disorder of the skin that can cause considerable discomfort. It is a disease for which there is no cure and affects people of all ages. Although individuals with mild psoriasis can often control their disease with topical agents, more than one million patients worldwide require ultraviolet light treatments or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of ultraviolet radiation and the toxicities of many therapies limit their long-term use. Moreover, patients usually have recurrence of psoriasis, and in some cases rebound shortly after stopping immunosuppressive therapy. A recently developed model of psoriasis based on the transfer of CD4+ T cells mimics many aspects of human psoriasis and therefore can be used to identify compounds suitable for use in treatment of psoriasis (Davenport et al., Internat. Immunopharmacol. 2:653-672, 2002). Efficacy in this model is a measured by reduction in skin pathology using a scoring system. Similarly, efficacy in patients is measured by a decrease in skin pathology.
Psoriatic arthritis is a type of inflammatory arthritis that occurs in a subset of patients with psoriasis. In these patients, the skin pathology/symptoms are
accompanied by joint swelling, similar to that seen in rheumatoid arthritis. It features patchy, raised, red areas of skin inflammation with scaling. Psoriasis often affects the tips of the elbows and knees, the scalp, the navel, and around the genital areas or anus. Approximately 10% of patients who have psoriasis also develop an associated inflammation of their joints.
Multiple sclerosis is a disease of the central nervous system (CNS) marked by numbness, weakness, loss of muscle coordination and problems with vision, speech, and bladder control. MS is an autoimmune disease in which the body's immune system attacks myelin, a key substance that serves as a nerve insulator and helps in the transmission of nerve signals. MS causes demyelinization of the white matter of the brain, with this process sometimes extending into the grey matter.
Demyelinization is loss of myelin, which is composed of lipids and protein. When myelin is damaged in MS, nerve fiber conduction is faulty or absent. Impaired bodily functions or altered sensations associated with those demyelinated nerve fibers give rise to the symptoms of MS. There are multiple sub-groups of MS, including relapsing remitting, primary progressive, and secondary progressive. Mouse models
representing some of these subtypes are available for testing efficacy of potential therapies. Efficacy in mouse models is measured by a reduction in paralysis in the limbs. Efficacy in patients is measured by a reduced activity score in the brain as measured by MRI and also an improvement in muscle tone/movement.
Anti-CRAC channel antibodies of the invention may be administered to a subject in order to attenuate graft versus host disease. GVHD is a complication that can occur after a bone marrow or stem cell transplant. In GVHD, transplanted T cells from the donor will attack the recipient's body as it recognizes the recipient as foreign material. Acute, or fulminant, GVHD usually occurs within the first 3 months after transplant. Whereas, chronic GVHD starts more than 3 months after transplant and can last for the rest of transplant recipient's life. Current treatments seek to suppress the immune response. Administration of anti-CRAC channel antibodies can be administered to attenuate the T cell response, thereby attenuating GVHD.
An embodiment of the invention includes administering an anti-CRAC channel antibody to a patient in need thereof. The anti-CRAC channel antibody can be administered before or after transplant. The anti-CRAC channel antibody can be administered before or after rejection occurs.
The term "treatment", as used herein, refers to the medical therapy of any human or other animal subject in need thereof. Said subject is expected to have undergone physical examination by a medical or veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other animal subject. The timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject's health. Thus, said treatment may be palliative, symptomatic and/or curative. "Preventative" or "prophylactic" administration of antibodies of the invention is also contemplated, with prevention being defined as delaying or averting manifestation or aggravation of one or more symptoms of a disease or disorder. In terms of the present invention, prophylactic, palliative, symptomatic and/or curative treatments may represent separate aspects of the invention.
An antibody of the invention may be administered parenterally, such as intravenously, intramuscularly, or subcutaneously, inter alia. Alternatively, an antibody of the invention may be administered via a non-parenteral route, such as perorally or topically. An antibody of the invention may be administered
prophylactically. An antibody of the invention may be administered therapeutically.
Depending on the indication to be treated and factors relevant to the dosing that a physician of skill in the field would be familiar with (such as the mode of action of the antibody, half life and formulation), anti-CRAC channel antibodies can be administered at a dosage that is efficacious for the treatment of that indication while minimizing toxicity and side effects. For example, a therapeutically effective dosage for treating an autoimmune or inflammatory disease may be about 25 mg/dose, about 50 mg/dose, about 75 mg/dose, about 100 mg/dose, about 125mg/dose, or any dosage between those doses. In another embodiment, a therapeutically effective dosage for treating an autoimmune or inflammatory disease may be between about 1 mg/kg body weight to about 1 1 mg/kg body weight, as well as any doses within that
range. A physician skilled in the field would be familiar with determining dosing frequencies. Dosing frequency may be, but is not limited to, biweekly, once weekly, bimonthly, once every other week, once every three weeks, once monthly or once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. Those of skill in the art will appreciate that dosage and dosing frequency may change over the course of treatment. For example, a patient may begin with a high dose but be given lower maintenance doses. Similarly, a patient may begin with a certain dosing interval but have maintenance doses at less frequent intervals. In another embodiment, a patient's dosage may be
determined based on fluctuation in symptoms at different stages in the treatment. Treatment may be continued as long as symptoms persist or until remission.
Antibodies used in the methods of the invention are administered to a human patient in accord with methods known to medical practitioners, such as by
intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, intrapulmonary, intracerobrospinal, intra-articular, intrasynovial, intrathecal, intralesional, generally by intravenous or subcutaneous administration. In an embodiment, a humanized anti-CRAC channel antibody is administered by
intravenous infusion with 0.9% sodium chloride solution as an infusion vehicle.
Pharmaceutical Formulations. Therapeutic formulations of an anti-CRAC channel antibody used in accordance with the present invention can be prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as
Tween®, Pluronics™ or polyethylene glycol (PEG).
Another formulation is a liquid multidose formulation comprising the anti-CRAC channel antibody at 40 mg/ml_, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 for storage at 2-8°C. Another anti-CRAC formulation can include 10mg/ml_ antibody in 9.0 mg/ml_ sodium chloride, 7.35 mg/ml_ sodium citrate dihydrate, 0.7mg/ml_ polysorbate 80, and Sterile Water for Injection, pH 6.5. Yet another aqueous pharmaceutical formulation comprises 10-30 mM sodium acetate from about pH 4.8 to about pH 5.5, preferably at pH5.5, polysorbate as a surfactant in an amount of about 0.01 -0.1 % v/v, trehalose at an amount of about 2-10% w/v, and benzyl alcohol as a preservative (U.S. 6, 171 ,586). Lyophilized formulations adapted for subcutaneous administration are described in WO97/04801 . Lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be
administered subcutaneously to the mammal to be treated herein.
A formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with
complementary activities that do not adversely affect each other. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein or about from 1 to 99% of the heretofore employed dosages.
Active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid
hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
The invention may be better understood with reference to the following examples that are exemplary and do not serve to limit the invention in any way.
Examples
The invention is further described by reference to the following examples that are exemplary in nature and are not intended to limit the scope of the invention in any way.
Example 1
Generation of anti-CRAC Channel Antibodies and Primary Screening for
Binding
To develop functional antibodies against human ORAM , peptides comprising the first extracellular loop (SEQ ID NO:2) and second extracellular loop (SEQ ID NO:3) were synthesized for use as immunogens.
Methods
Immunization. Balb/C mice were immunized with peptides spanning the first or the second extracellular loop of ORAM together with different adjuvants.
Anti-CRAC channel antibodies were raised with three immunizations with the peptide corresponding to the second extracellular loop. The second extracellular loop peptide was coupled to BSA through a C-terminal cysteine, which was added to the C-terminus of the extracellular loop during synthesis. Mice were immunized with 20 g protein mixed with complete Freund's adjuvant for the first injection. In the second and third immunizations, incomplete Freund's adjuvant was used. Ten days after the last immunization, eye-bleeds from mice were screened by ELISA for second extracellular loop peptide-specific polyclonal antibodies. In addition, the sera were screened for polyclonal titers that bound to native CRAC channels on the surface of cells by flow-cytometry (FACS).
The first extracellular loop was also used as an immunogen using the same methods except that it was coupled to both BSA and KLH. Polyclonal titers were screened by ELISA for first extracellular loop peptide specificity and then screened for native CRAC specificity by FACS, also using the same methods as described above.
Screening of polyclonal sera by direct ELISA assay. Nunc® immunoplates were coated with 1 pg/ml of first or second extracellular loop peptide in PBS and incubated overnight at 4°C. Plates were blocked with blocking buffer (PBS with 0.05% Tween®-20) for 15min and were washed with PBS/0.05% Tween®-20. Sera were added in threefold dilutions with a starting dilution of 1 :100, and the plates were incubated for 1 hour at room temperature. After another five washes, wells were incubated with HRP-conjugated F(ab')2 goat anti-mouse IgG (Jackson
ImmunoResearch) for one hour. Plates were washed and developed with TMB- substrate (Kem-EN-Tec) as described by the manufacturer. Absorbance at 450 nm was measured on an ELISA-reader.
Screening of polyclonal sera by flow-cytometry (FACS). Sera (1 :50 and 1 :100 dilution) from immunized mice were incubated in 96-wells plates for 30 min on ice with either with BAF-3 cells engineered to overexpress CRAC or with wild-type BAF-3 cells. Samples were washed three times with RPMI media, and incubated with
APC-conjugated F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch) for 30 min on ice. Samples were then washed 3 times with PBS, resuspended in PBS
containing 1 % PFA and analyzed by flow-cytometry (FACSarray®, BD Biosciences).
Fusion. Mice with positive titers were boosted with 10 μg of soluble KLH- peptide conjugate by intravenous injection and sacrificed after three days. Spleens were removed aseptically and dispersed to a single cell suspension. Fusion of spleen cells and myeloma cells (P3X63Ag8.653, ATCC-# CRL 1580) was done by the electrofusion-method. The resulting hybridoma cells were seeded in microtiter plates and cultured at 37°C in 5% CO2. The tissue-culture medium was changed two times over a period of 10 days.
Direct ELISA assay. Nunc® immunoplates were coated with 1 pg/ml of extracellular loop 2 peptide in PBS and incubated overnight at 4°C. Plates were blocked with blocking buffer (PBS with 0.05% Tween®-20) for 15 min and were washed with PBS/0.05%Tween®-20. Culture supernatants from the hybridoma cells were added and the plates were incubated for 1 hour at room temperature. After another five washes, wells were incubated with HRP-conjugated F(ab')2 goat anti- mouse IgG (Jackson ImmunoResearch) for one hour. Plates were washed and developed with TMB-substate (Kem-EN-Tec) as described by the manufacturer. Absorbance at 450 nm was measured on an ELISA-reader.
Flow-cytometry assay. Tissue-culture supernatants (100μΙ) from the fusions were incubated with a mixture of BAF-3 labeled with cell tracker (Molecular Probes cat#C34551 ) and BAF-3 ORAM cells (105 cells, 1 : 1 ratio) in 96-wells plates for 30 min on ice. Samples were washed three times with PBS and incubated with APC- conjugated F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch) in RPMI1640 for 30 min on ice. Samples were washed 3 times with RPMI1640, resuspended in PBS containing 1 % PFA. and analyzed by flow-cytometry (FACSarray®).
Results
ELISA with first and second extracellular loop peptide was used as a primary screen to evaluate sera from immunized mice for binding of polyclonal antibody titer to the first and second extracellular loop peptides. In parallel, flow cytometry confirmed binding native CRAC on cells.
Figure 2 shows a representative polyclonal antibody titer from the sera of mice immunized with first and second extracellular loop peptides against the first and the second extracellular loop peptides, respectively. These results demonstrate that both antigens generated antibody titer that recognized the respective first and second extracellular loop peptides.
Figure 3 shows that serum samples from Balb/c mice immunized with the second extracellular loop peptide, but not the first extracellular loop peptide, demonstrated binding to CRAC-overexpressing cell lines. Notably, although the first extracellular loop is the binding site of Ca2+ ions, it did not raise CRAC-specific antibodies. A minimum of 10 Balb/c and 10 Medarex mice were immunized with the first extracellular loop peptide and no polyclonal titers specific for native CRAC on the surface of cells were observed.
Example 2
FACS Analysis of F8 binding to CRAC Overexpressinq Cell Lines
Splenocytes from mice immunized with the second extracellular loop peptide and demonstrating anti-CRAC channel titers were fused and the resulting
hybridomas were subcloned. Antibodies from hybridoma supernatants were purified over Protein A. Sixteen hybridoma clones were identified that exhibited binding to CRAC on the surface of cells. Screening the hybridoma supernatants derived from mice immunized with the second extracellular loop peptide identified clone anti-M- CRAC1415-10F8 A2B6C1 (also referred to herein as "F8") as a monoclonal antibody that bound CRAC on the surface of cell lines overexpressing CRAC (Figure 4) using flow cytometry.
Methods
The human Orail protein was overexpressed in both Jurkat cells (a human T- lymphoblastic cell line; ATCC TIB-152, Manassas, VA) and Ba/F3 cells (a murine bone marrow-derived pro-B-cell line; DSMZ - Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany), using standard protocols. These engineered lines are hereafter referred to as CRAC+ cell lines.
Ba/F3 cells do not express endogenous human CRAC (because Ba/F3 is a mouse cell line) and are referred to as CRAC- cell lines. Wild-type Jurkat do express endogenous human CRAC and so shRNA was used to completely abrogate endogenous expression of CRAC to create a CRAC- Jurkat line. Knockdown of CRAC expression in Jurkat cells was achieved through infection with lentiviral particles containing Orail shRNA (Santa Cruz Biotechnology, sc-76001 -V) and following manufacturer's protocols.
CRAC positive (CRAC+) cell lines were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) using Invitrogen's CellTrace™ CFSE Cell Proliferation Kit (#C34554) according to the manufacturer's directions. After labeling with CFSE, cells were resuspended in DPBS including 5% fetal bovine serum (FBS) plus 10 pg/mL normal goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a cell final concentration of 4 x106 cells/mL. CRAC negative (CRAC-) cell lines were also resuspended in DPBS/5% FBS plus 10 pg/mL normal goat IgG (Jackson ImmunoResearch Laboratories) at 4 x106 cells/mL, but were not labeled with CFSE. CRAC+ and CRAC- cells were mixed together in a 1 : 1 ratio. The
CRAC+/- cell line mixture was added to each well of a 96 well U-bottom BD Falcon plate and incubated for 30 minutes on ice with antibodies at the indicated final concentrations (1 pg/mL, 10 pg/mL, 30 pg/mL, and 50 pg/mL, see Fig. 4). The cell mixtures were washed with DPBS/5% FBS. Alexa Fluor® 594 goat anti-mouse IgG (Invitrogen A1 1020) in DPBS/5% FBS plus 10 pg/mL normal goat IgG was added to each well and incubated for 30 min on ice. The cells were again washed once with DPBS/5%FBS and were resuspended in 0.1 mL of DPBS/5% FBS for analysis on the Becton Dickinson LSRII SORP flow cytometer. Data were collected using the Becton Dickinson FACS Diva software, and analysis was completed using Tree Star's FlowJo analysis software.
Results
The data indicate that antibody F8 recognizes and binds cellular CRAC on the surface of cell lines engineered to overexpress CRAC (Fig. 4). The untransfected cell lines (CRAC-) are CFSE low and the transfected, overexpressing cell lines (CRAC+) are CFSE high. Increased concentrations of F8 antibody demonstrate
specific binding to the CFSE high (CRAC+) cells for both the Ba/F3 and Jurkat cell lines.
Additionally, the F8 antibody was specific to human CRAC since the F8 antibody did not bind to mouse CRAC (data not shown). Although human and mouse CRAC are highly similar, the sequences are less homologous in the 2nd extracellular loop, as shown by a sequence alignment (Fig. 5).
Example 3
Cloning and sequencing of mouse anti-M-CRAC1415-10F8 A2B6C1
The heavy chain and light chain variable domain sequences of the F8 anti- CRAC channel antibody (M-CRAC1415-10F8 A2B6C1 ) were cloned and sequenced.
Methods
Total RNA was extracted from hybridoma cells using the RNeasy®-Mini Kit
(Qiagen) according to the manufacturer's directions (All kits and reagents in this example were used according to the manufacturer's instructions unless otherwise noted) and used as template for cDNA synthesis. cDNA was synthesized in a 5'- RACE reaction using the SMARTer™ RACE cDNA amplification kit (Clontech
Laboratories, Mountain View, CA). Subsequent target amplification of heavy chain and light chain sequences was performed by PCR using Phusion® Hot Start polymerase (Finnzymes) and the universal primer mix (UPM) included a forward primer in the SMARTer™ RACE kit. Two different reverse primers with the following sequences were used independently for heavy chain (VH domain) amplification: 5'-CCCTTGACCAGGCATCCCAG-3' (SEQ ID NO: 5) and
5'-CTTGCCATTGAGCCAGTCCTGGTGCATGATGG-3' (SEQ ID NO: 6). Two different reverse primers with the following sequences were used independently for light chain amplification: 5'-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3' (SEQ ID NO: 7) and 5'- GTTGTTCAAGAAGCACACGACTG-3' (SEQ ID NO: 8).
PCR products were separated by gel electrophoresis and extracted using the
GFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciences and cloned for sequencing using a Zero Blunt TOPO® PCR Cloning Kit and chemically
competent TOP10 E.coli (Invitrogen). Colony PCR was performed on selected colonies using an AmpliTaq® Gold Master Mix from Applied Biosystems and
M13uni/M13rev primers. Colony PCR clean-up was performed using the ExoSAP-l enzyme mix (USB). Sequencing was performed at MWG Biotech, Martinsried Germany using M13uni(-21 )/M13rev(-29) sequencing primers. Sequences were analyzed and annotated using the VectorNTI program.
Results
A single unique murine kappa type light chain and a single unique murine heavy chain, subclass lgG1 were identified. Nucleic acid and amino acid sequences are listed below (leader peptide sequences have been omitted).
Anti-M-CRAC1415-10F8 A2B6C1 's heavy chain variable domain amino acid sequence was identified to be the following (CDRs are underlined):
1 QVQLQQSGAE LVRPGSSVKI SCKASGYAFR SYWMNWVKQR PGQGLEWIGH 51 IYPGDGDTNY NGKFKGKATL TADKSSSTAY MQLSSLTSED SAVYLCGRGG 101 TTWVDYWGQ GTTLTVSS (SEQ ID NO: 9).
CDRH1 : SYWMN (SEQ ID NO: 10);
CDRH2: HIYPGDGDTNYNGKFKG (SEQ ID NO: 1 1 ); and
CDRH3: GGTTVWDY (SEQ ID NO: 12).
Anti-M-CRAC1415-10F8 A2B6C1 's heavy chain variable domain nucleic acid sequence (signal peptide sequence omitted) was identified as:
5'-CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCA GTGAAGATTTCCTGCAAGGCTTCTGGCTATGCATTCAGGAGCTACTGGATGAAC TG G GTGAAG C AG AG G C CTG G AC AG GGTCTTG AGTG G ATTG G AC ATATTTATC CT G G AG ATG GTG ATACTAACTAC AATG G AAAGTTC AAG G GTAAAG C C AC ACTG ACT G C AG AC AAATC CTC C AG C AC AG C CTAC ATG C AG CTC AG C AGC CTAAC ATCTG AG GACTCTGCGGTCTATTTGTGTGGAAGGGGAGGTACTACGGTAGTAGTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3' (SEQ ID NO: 13).
Nucleotides from SEQ ID NO: 13 coding for:
CDRH1 : 5'-AGCTACTGGATGAAC-3' (SEQ ID NO: 14);
C D RH2 : 5' -C ATATTTATC CTG GAG ATG GTG ATACTAACTAC AATG G AAAGTTC AAG
GGT-3' (SEQ ID NO: 15);
CDRH3: 5' -G GAG GTACTAC G GTAGTAGTTG ACTAC-3' (SEQ ID NO: 16).
Anti-M-CRAC1415-10F8 A2B6C1 's light chain variable domain amino acid sequence (signal peptide sequence omitted, CDRs underlined):
1 DIVMSQSPSS LAVSAGEKVT MSCKSSQSLL NSRTRKNYLA WYQQKPGQSP 51 KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKQSYNL 101 PWTFGGGTKL EIKR (SEQ ID NO: 17)
CDRL1 : KSSQSLLNSRT (SEQ ID NO: 18);
CDRL2: WASTRES (SEQ ID NO: 19); and
CDRL3: KQSYNLPWT (SEQ ID NO: 20).
Anti-M-CRAC1415-10F8 A2B6C1 's light chain variable domain nucleic acid sequence (signal peptide sequence omitted) was identified as:
5'-GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGA AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAA AGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGA TCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGT GGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAGGACCT GGCAGTTTATTACTGCAAGCAATCTTATAATCTTCCGTGGACGTTCGGTGGAGG CACCAAGCTGGAAATCAAACGG-3' (SEQ ID NO: 21 ).
Nucleotides from SEQ ID NO: 21 coding for:
CDRL1 : 5'-AAATCCAGTCAGAGTCTGCTCAACAGTAGAACC-3' (SEQ ID NO: 22); CDRL2: 5'-TGGGCATCCACTAGGGAATCT-3' (SEQ ID NO: 23); and
CDRL3: 5'-AAGCAATCTTATAATCTTCCGTGGACG-3' (SEQ ID NO: 24).
CDRH2 Modifications
Based on Oxford Molecular's AbM antibody modeling software, CDRH2 can also be defined as amino acids 50-58 of the heavy chain variable domain. Thus, CDRH2 can also be amino acids 50-58 (HIYPGDGDT; SEQ ID NO: 28) of SEQ ID NO: 9.
Deamidation of asparagines in regions important for activity (e.g., CDRs) can affect efficacy in some polypeptides. On first examination, CDRH2 has a NG deamination site at amino acids 61 -62 of SEQ ID NO: 9. During a humanization process, such a deamination site may be substituted. For example, amino acids 50- 66 of SEQ ID NO: 9 may be substituted to eliminate the site with a sequence of HIYPGDGDTNYAQKFKG (SEQ ID NO: 29) or HIYPGDGDTNYAQKFQG (SEQ ID NO: 30).
Example 4
Anti-CRAC Channel Antibodies Inhibit NFAT Translocation
NFAT can be found in the cytoplasm of unactivated T cells. During activation, the influx of Ca2+ results in NFAT dephosphorylation by calcineurin, which fosters NFAT translocation to the nucleus and expression of genes involved in immune response. Measuring NFAT translocation in Jurkat cells after incubation with anti- CRAC channel antibodies therefore provides an indication of the ability of the antibodies to inhibit T cell activation.
Methods
A Jurkat cell line comprising a NFAT luciferase reporter (NFAT-Luc) was resuspended in assay media (RPMI-1640 media containing 10% FBS and 2 pg/mL puromycin) to a concentration of 1 M/mL. The cells were plated (106/well) in a BD Purecoat® 96-well flat bottom plate. Plates were centrifuged for 3 m in at 2000 rpm with high brake. After centrifugation, the plates were flicked and 50 μΙ_ of antibodies were added. Each of the antibodies was tested at 0.15, 0.3, 0.6, 1 .5, 3.0, 6.0, 12, 25, and 50 pg/mL. Each well was repeated in triplicate. Controls of no treatment and 2- aminoethoxydiphenyl borate (2-APB, an inositol 1 ,4,5-triphosphate (IP3) receptor inhibitor that inhibits NFAT) were also included. The plates were incubated at 37°C
in 5% CO2 for 1 hour. Media were removed, and each reaction was incubated at 37°C in 5% CO2 for 4 and 16 h in after the addition of the stimulators phorbol 12- myristate 13-acetate (PMA) (40ng/ml_) and thapsigargin (1 μΜ/mL) in assay media. Once the incubation period is finished, plates were removed from the incubator and 100 μί Steady-Glo® reagent (Promega Corp., Madison, Wl) was added to each well and mixed. Luminescence was read on a FlexStation® 3 microplate reader with 1 second integration time, top read and analyzed with SoftMax® Pro Data Acquisition & Analysis Software (Molecular Devices, Sunnyvale, CA). Figure 6A shows a representative experiment with F8 and mouse lgG1 isotype controls #1 and #2. Table 2 shows the activity of all monoclonal antibodies specific for native CRAC in this assay. The value corresponds to percent inhibition compared to control at 50ug/ml_. Table 2 details the results in functional studies for a number of anti-CRAC antibodies demonstrating different levels of activity. Either IC50 or the % inhibition compared to control is shown in the columns as indicated. NA=IC50 was
undefinable; ND=not done.
Table 2
Results
The F8 antibody (see Fig. 6A), along with nine other antibodies (see Table 2), inhibited NFAT translocation from the cytoplasm to the nucleus to varying degrees. F8 antibody results were normalized to controls. Thus, incubation with the lgG1 negative control was normalized to 100% NFAT translocation to the nucleus.
Increasing concentrations of the F8 antibody inhibited NFAT translocation to the nucleus (Fig. 6A), with an IC50 of 1 -4ug/mL. Inhibition increased with higher F8 concentrations, including nearly 30-45% inhibition of NFAT translocation by 50 pg/mL of F8 antibody, depending on the experiment.
Example 5
Anti-CRAC Channel Antibodies Inhibit IL-2 Production
T-cells produce IL-2 during an immune response (i.e., activated T cells). Antigen binding to the TCR stimulates the production of IL-2. Activated CD4+ cells will produce and secrete IL-2, which is essential for the proliferation of both CD4+ and CD8+ cells. Measuring IL-2 production in Jurkat cells incubated with anti-CRAC channel antibodies provides an indication of their ability to inhibit T cell proinflammatory cytokine production and downstream effector functions, such as proliferation.
Methods
Jurkat E6.1 cells (ATCC TIB-152) were harvested from culture, washed once with assay media (RPMI 1640 containing 10% FBS, 1X Gibco® GlutaMAX™, and 25 mM HEPES), and resuspended in assay media at 4 x 106 cells/mL. From the cell suspension, 25 μί of cells were aliquoted to each well of 96 U-bottom plate (Becton Dickinson) except for the outer perimeter of wells that were left unfilled to create a media barrier. Anti-CRAC channel monoclonal antibodies and matched mouse lgG1 controls were diluted at 4X concentrations into assay media. Antibodies were titered 1 :2 into assay media for final 4x concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 pg/mL. 25 μί of antibody titration was added to 25 μί of Jurkat E6.1 cells in a 96 well U-bottom plate. Antibodies were incubated with the cells for 30 min at 37°C in 5% C02. PHA-P (Sigma Aldrich L8754) was diluted into assay media at a 2X concentration (15 pg/ml). 50 μί of PHA-P dilution was added to each well on the 96 well U-bottom plate except for an unstimulated sample as a control. The media barrier was formed by adding 0.2 mL/well of assay media to each open well around the perimeter of the plate. The plate was then incubated at 37°C in 5% CO2. Cells were harvested at 16 h for cytokine analysis.
For cytokine analysis, the Millipore IL-2 Map Human Cytokine/Chemokine Immunoassay (Billerica, MA) was prepared according to the manufacturer's instructions. Cytokine data were collected using the BioRad BioPlex® Reader and BioPlex® Manager software.
Results
The F8 antibody (see Fig. 6B), along with seven other antibodies (see Table 2), inhibited IL-2 production by Jurkat cells. F8 antibody results were normalized to controls. Thus, incubation with the lgG1 negative control was normalized to 100% IL- 2 production by the Jurkat cells. In duplicate, increasing concentrations of the F8 antibody inhibited IL-2 production (Fig. 6B), with an IC50 of 1 -4ug/ml_. Inhibition increased with higher F8 concentrations, including nearly 40-50% inhibition of IL-2 production by 50 pg/mL of F8 antibody, depending on the experiment. Figure 6B shows the complete titration curve using F8. Table 2 shows the percent inhibition of the other anti-CRAC mAbs at the 50ug/mL concentration.
Example 6
Anti-CRAC Channel Antibodies Inhibit T Cell Proliferation and Cytokine
Production Anti-CRAC channel antibodies were shown to inhibit the activity of a T cell line in the previous examples, and so the antibodies were also evaluated for their ability to inhibit the effector functions of primary T cells upon stimulation. Effector functions of T cells include, but are not limited to, proliferation and cytokine production which were analyzed in this example. Total proliferation is measured by CFSE dilution. The florescence of CFSE, a green fluorescent dye that labels intracellular proteins, dilutes by half with each cell division.
Methods
Frozen human donor peripheral blood mononuclear cells (PBMC) (Research Blood Components; Boston, MA) were thawed and removed directly to 13 mL of assay media (RPMI 1640, 10% FBS, 1X GlutaMAX™, and 25 mM HEPES). PBMC were centrifuged for 10 min at 1 100 rpm. The cell pellet was aspirated and
resuspended in 10 mL of pre-warmed assay media.
PBMC were CFSE labeled with the Invitrogen CellTrace® CFSE Cell
Proliferation Kit following manufacturer's instructions. After labeling, the cells were re-suspended at 4 x 106 cells/mL in assay media described above in Example 2.
Anti-CRAC channel antibodies and mouse lgG1 , K control monoclonal
antibodies (eBioscience 16-4714) were diluted at 3X concentrations into assay media. The antibodies were titered 1 :2 into assay media for final 3X concentrations of 150, 75, 37.5, 18.75, 9.375, 4.6875, 2.343, 1 .1712 pg/ml in final volumes of .050 ml_. 2-Aminoethyl diphenylborinate (2-APB; Sigma Aldrich D9754) and Cyclosporin A (CsA; Sigma Aldrich 30024) were diluted into assay media at 3X concentrations of 300 μΜ and 300 ng/mL respectively, which served as additional controls since both are calcium channel inhibitors. 0.050 ml_ of each of these inhibitors was aliquoted into separate wells of the 96 well U-bottom plate (BD Falcon 353077) in triplicate. 0.050 ml_ of CFSE labeled PBMC were aliquoted into their appropriate wells of 96 well U-bottom plate. Blocking antibodies and controls were incubated with cells for 1 hr at 37°C in 5% C02. Anti-CD3 monoclonal UCHT1 (eBioscience 16-0038) and anti- CD28 costimulatory monoclonal antibody CD28.2 (eBioscience 16-0289) were diluted together to 3X concentrations of 30 ng/ml and 3 pg/rnl, respectively, in assay media. 0.050ml_ of the anti-CD3/CD28 dilution was added to all wells of the 96 well U-bottom plate, with the exception of the unstimulated sample.
Cells were incubated at 37°C in 5% C02 for 3 days. 0.030 ml of supernatant were harvested at 16 hours and removed to a separate 96 well plate for cytokine analysis. Samples were stored at -80°C for future analysis.
Cells and remaining supernatant was removed from the culture at day 3. Plates were centrifuged at 1500rpm for 5m in. 0.075 ml of supernatant were removed to a separate 96 well plate for 72 hr cytokine analysis. Remaining supernatant was removed by flicking the plate. Cells were resuspended in 0.2 ml D-PBS per well. The plate was centrifuged at 1500 rpm for 5min and supernatants were then aspirated.
One vial of Live/Dead® Fixable Aqua Dead Cell Stain (Invitrogen, Cat# L34957) was prepared by adding 0.050 ml DMSO to one vial of Aqua Dead Cell
Stain to create a stock solution, which was then diluted 5 μΙ of stock stain solution per 1 ml DPBS. 0.050 ml of AVID stain was added to each well of 96 well U-bottom plate containing CFSE labeled cells and then incubated at room temperature for 20 minutes. After incubation, 0.15 ml cold FACS Wash (D-PBS + 5%FBS) was added to each well. Plates were then centrifuged 1500 rpm for 5min, and supernatants were aspirated. The cells were then washed and then 0.050 ml CD5-PE antibody (PE conjugated mouse anti-human CD5 antibody (UCHT2) in FACS wash (2.5 μΙ/.050
ml)) was added to each well of 96 well plate containing AVID/CFSE labeled cells. The cells were incubated on ice for 30 minutes.
Following incubation, 0.15 ml FACS wash was added to each well and then centrifuged at 1500 rpm for 5 minutes. Supernatants were aspirated, and the cells were washed once with 0.2 ml FACS wash per well and centrifuged at 1500 rpm for 5 minutes. Supernatants were aspirated, and the cells were resuspended in 0.1 ml FACS Wash per well for analysis on a BD LSRII Special SORP flow cytometer. Data were collected using the BD FACS Diva software, and analysis was completed using Tree Star's FlowJo analysis software.
For cytokine analysis, a Millipore 14-plex Milliplex Map Human
Cytokine/Chemokine Immunoassay (Cat# MPXHCYTO60KPMX14) was prepared as described in the protocol provided with the kit. IL-1 b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, 11-10, IL-12(p70), IL-13, GM-CSF, INF-γ, MCP-1 , and TNF-a were analyzed.
Cytokine/chemokine data were collected using the BioRad BioPlex® Reader and BioPlex® Manager software.
Results
The F8 antibody, along with eight other anti-CRAC monoclonal antibodies (see Table 2), inhibited T cell proliferation upon stimulation with anti-CD3 and anti-CD28 to varying degrees. Results were normalized to controls (untreated cells). Thus, incubation without an antibody or with control IgG was 100% PBMC proliferation. As shown in Figure 7, increasing concentrations of the F8 antibody inhibited PBMC proliferation, with an IC50 of 1 ug/ml_. Inhibition increased with increased F8 concentrations, including nearly 90% inhibition of PBMC proliferation by 50 pg/mL of F8 antibody in some experiments. At 50 pg/mL of F8 antibody, the inhibition of PBMC proliferation is greater comparable to that seen with the positive controls, 10Ong/mL cyclosporine A and 10OuM 2-APB. Results with the other anti-CRAC monoclonal antibodies can be seen in Table 2.
Similar results were achieved when cytokine levels in the culture supernatants were examined, as exemplified by IL-4 (at 16 h) and IL-1 b (at 72 h) in Figure 8.
Increasing concentrations of the F8 antibody inhibited IL-4 and IL-1 b production (Fig. 8). A concentration of 50 pg/mL F8 antibody produced equal inhibition of IL-4
production compared to the positive controls, cyclosporine A (CsA) and 2-APB. A concentration of 50 pg/mL F8 antibody produced greater inhibition of IL-1 B
production compared to cyclosporine A but slightly less inhibition of IL-1 B production compared to 2-APB. The F8 antibody had a much greater inhibitory effect on IL-1 B production compared to lgG1 isotype control.
Example 7
Mixed Lymphocyte Reaction
In order to assess the ability of the anti-CRAC channel antibody F8 to inhibit T cell activation, T cell proliferation in the presence of the F8 antibody was
investigated using a mixed lymphocyte reaction. The mixed-lymphocyte reaction (MLR) is an in vitro method for assaying T cell proliferation. When allogeneic
(different HLA haplotype) populations of PBMC are cultured together, TH cell populations are activated and proliferate. Total proliferation is measured by CFSE dilution. The florescence of CFSE, a green fluorescent dye that labels intracellular proteins, dilutes by half with each cell division.
One population— the stimulator— is first inactivated (via mitomycin c or lethal x-irradiation) before being added to the MLR well. These inactivated cells merely provide foreign alloantigens to the responder population. Within 24-48 hours, the responder T cells have begun proliferating and within another 48 hours an expanding population of functional CTLs has been formed. CD4 (TH) cells, dendritic cells and certain accessory cell types are all critical for the MLR to function.
If the primary MLR cells (cells obtained from an MLR which was performed in the absence of Test Compound) are labeled with CFSE, a green fluorescent dye which is imported into living cells where it is acted upon by an enzyme and then reacts with cellular proteins, the number of cell divisions experienced over time by the labeled cells is reflected in the reduction of green label associated with each cell.
Methods
PBMC from two donors were cultured in RPMI-1640 containing Glutamax®,
HEPES, and 10% human serum (Invitrogen #34005100). The PBMC from the two donors were labeled separately with CFSE and resuspended in growth media at
2M/ml_. 50 μΙ_ of each donor's cells was added to a 96-well U-bottom plate, for a total of 200,000/well. 100 μΙ_ of F8 antibody at 2X final concentration, a commercially bought mouse lgG1 antibody (eBio #16-4714-85), an internally developed mouse lgG1 antibody, or 100 ng/mL of cyclosporine A (Sigma, cat #C-3662) were added to the cells. The lgG1 antibodies were used as negative isotype controls. Each condition was performed in triplicate and incubated for six days at 37°C in 5% CO2. On day 6, cells were harvested by centrifuging each plate at 2000 rpm for 3 minutes. Each plate was flicked and resuspended in AVID solution. Cells were washed once in PBS and surface stained for CD3 (BD#560365), CD4 (BD#555349), and CD8 (Biolegend#301032). The cells were washed twice in DPBS and resuspended in 1 % PFA in PBS. The fixed cells were acquired on LSRII SORP flow cytometer.
Results
The F8 antibody, along with 10 other anti-CRAC channel antibodies, inhibited T cell proliferation to varying degrees. Fig. 9 indicates that T cell incubation with 3 pg/mL to 50 pg/mL of F8 antibody inhibited proliferation of CD4+ cells. A similar effect is seen on CD8+ cells. The F8 antibody at concentrations of 1 .56 pg/mL to 0.15 pg/mL did not inhibit the CD4+ cells as the data were similar to baseline
(unstimulated cells) and the negative control (both lgG1 isotype controls) in this experiment. The F8 antibody at a concentration of 50 pg/mL inhibited T cell proliferation by at least 60%. Table 2 shows the activity of the other 10 anti-CRAC monoclonal antibodies. A positive control, 100ng/ml_ cyclosporine A, routinely inhibited T cell proliferation by 80%. Example 8
Epitope Definition for Functional anti-CRAC channel antibodies
To more finely map the epitope within the second extracellular loop to which the anti-CRAC channel antibodies bind, an initial series of overlapping peptides were designed that span the entire second extracellular loop. Additional overlapping peptides and alanine scanning mutagenesis may allow further delineation of the epitope.
Methods
In addition to the peptide spanning the entire second extracellular loop
(referred to as 1415; residues homologous with mouse shown underlined below), two additional peptides were synthesized. Peptide 1455, with the sequence
GQPRPTSKPPASGAAANVSTSGITPGQA (SEQ ID NO: 32), has the N terminal 1 1 amino acids removed compared to 1415. Peptide 1454, with the sequence
PASGAAANVSTSGITPGQA (SEQ ID NO: 33), has an additional nine amino acids removed from the N terminus compared to 1455. All three peptides were used in ELISA to measure binding by anti-CRAC channel antibodies. Nunc® immunoplates were coated with 1 pg/ml of indicated peptide in PBS and incubated overnight at 4°C. Plates were blocked with blocking buffer (PBS with 0.05% Tween®-20) for 15 min and were washed with PBS/0.05%Tween®-20. Each of the anti-CRAC channel antibodies were added and the plates were incubated for 1 hour at room temperature. After another five washes wells were incubated with HRP-conjugated F(ab')2 goat anti- mouse IgG (Jackson ImmunoResearch) for one hour. Plates were washed and developed with TMB-substrate (Kem-EN-Tec) as described by the manufacturer. Absorbance at 450 nm was measured on an ELISA-reader. Results
As shown in Table 3 below, all of the anti-CRAC channel antibodies tested bind to peptides 1415 and 1455, but not 1454. This indicates that the antibodies do not require the first 1 1 amino acids of the second extracellular loop for binding. It also suggests that the nine amino missing from 1454 play a role in antibody binding to CRAC. In Table 3, (+) indicates binding was detected, (-) indicates binding could not be detected.
1415 WVKFLPLKKQPGQPRPTSKPPASGAAANVSTSGITPGQA (SEQ ID NO : 31) 1455 GQPRPTSKPPASGAAANVSTSGITPGQA (SEQ ID NO : 32) 1454 PASGAAANVSTSGITPGQA (SEQ ID NO : 33)
Table 3
Antibody 1415 1455 1454
10F8 + + -
14F74A1 + + -
15F3A6 + + -
15F32A2 + + -
15F44A6 + + -
15F45A1 + + -
15F54A2 + + -
15F58A5 + + -
15F60A1 + + -
17F1A4 + + -
17F6A2 + + -
Example 9
Cloning and Sequencing Further Mouse Anti-CRAC Channel Antibodies
The heavy chain and light chain variable domain sequences of the following anti-CRAC channel antibodies were cloned and sequenced:
Total RNA was extracted from hybridoma cells using the RNeasy®-Mini Kit (Qiagen) according to the manufacturer's directions (All kits and reagents in this
example were used according to the manufacturer's instructions unless otherwise noted) and used as template for cDNA synthesis. cDNA was synthesized in a 5'- RACE reaction using the SMARTer™ RACE cDNA amplification kit (Clontech Laboratories, Mountain View, CA). Subsequent target amplification of heavy chain and light chain sequences was performed by PCR using Phusion® Hot Start polymerase (Finnzymes) and the universal primer mix (UPM) included a forward primer in the SMARTer™ RACE kit. Two different reverse primers with the following sequences were used independently for heavy chain (VH domain) amplification: 5'-CCCTTGACCAGGCATCCCAG-3' (SEQ ID NO: 5) and
5'-CTTGCCATTGAGCCAGTCCTGGTGCATGATGG-3' (SEQ ID NO: 6).
Two different reverse primers with the following sequences were used independently for light chain amplification:
5'-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3' (SEQ ID NO: 7) and 5'- GTTGTTCAAGAAGCACACGACTG-3' (SEQ ID NO: 8).
PCR products were separated by gel electrophoresis and extracted using the
GFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciences and cloned for sequencing using a Zero Blunt TOPO® PCR Cloning Kit and chemically competent TOP10 E.coli (Invitrogen). Colony PCR was performed on selected colonies using an AmpliTaq® Gold Master Mix from Applied Biosystems and
M13uni/M13rev primers. Colony PCR clean-up was performed using the ExoSAP-IT® enzyme mix (USB). Sequencing was performed at MWG Biotech, Martinsried Germany using M13uni(-21 )/M13rev(-29) sequencing primers. Sequences were analyzed and annotated using the VectorNTI program. Results
A single unique murine kappa type light chain and a single unique murine heavy chain for each antibody were identified. Amino acid sequences are listed below (leader peptide sequences have been omitted).
Anti-M-CRAC1415-15F58A5B4's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
1 QVQLQQSGAE LVRPGSSVKI SCKASGYVFS SYWMNWVKQR PGQGLEWIGH
51 IYPGDGDTNY NGKFKGEATL TADKSSSTAY MQLSSLTSED SAVYFCARDH 101 RDYYAMDYWG QGTSVTVSS (SEQ ID NO: 44)
CDRH1 : SYWMN (SEQ ID NO: 10)
CDRH2: HIYPGDGDTNYNGKFKG (SEQ ID NO: 1 1 )
CDRH3: DHRDYYAMDY (SEQ ID NO: 41 )
Anti-M- CRAC1415-15F58A5B4's light chain variable domain amino acid sequence (signal peptide sequence omitted, CDRs underlined):
1 DTMMSQSPSS LAVSAGEKVT MSCKSSQSLF NSRTRKNYLA WYQQKPGQSP 51 KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCSQSYNL 101 RTFGGGTKLE ITR (SEQ ID NO: 45) CDRL1 : KSSQSLFNSRTRKNYLA (SEQ ID NO: 46)
CDRL2: WASTRES (SEQ ID NO: 19)
CDRL3: SQSYNLRT (SEQ ID NO: 47)
Anti-M-CRAC1415-15F44A6's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
1 QVQLQQSGAE LVRPGSSVKI SCKASGYAFS SYWMNWVKQR PGQGLEWMGQ 51 IYPGDGDTNY NGKFKGKATL TADKSSSTAY MQLSSLTSED SAVFFCARVS 101 RFAVAMDYWG QGTSVTVSS (SEQ ID NO: 40)
CDRH1 : SYWMN (SEQ ID NO: 10)
CDRH2: QIYPGDGDTNYNGKFKG (SEQ ID NO: 58)
CDRH3: DHRDYYAMDY (SEQ ID NO: 41 )
Anti-M- CRAC1415-15F44A6's light chain variable domain amino acid sequence (signal peptide sequence omitted, CDRs underlined):
1 DIVMSQSPSS LAVSAGEKVT MSCKSSQSLL NSRTRKNYLA WYQQKPGQSP
51 KLLIYWASTR ESGVPDRFTG SESGTNFTLT ISSVQAEDLA VYYCKQSYDL
101 TRSFGGGTKL EIKR (SEQ ID NO: 42)
CDRL1 KSSQSLLNSRTRKNYLA (SEQ ID NO: 38)
CDRL2 WASTRES (SEQ ID NO: 19)
CDRL3 KQSYDLTRS (SEQ ID NO: 43)
An additional hybridoma, designated M-hCRAC1415-15F54A2 (chain subclass lgG2a/k) also produced a monoclonal antibody with the same heavy and light chain variable domain amino acid sequences and the same CDRs as 15F44A6.
Anti-M-CRAC1415-17F6A2's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
1 QVQLQQSGAE LVRPGSSVKI SCKASGYAFS SYWMNWVKQR PGQGLEWIGH 51 IYPGDADTNY NGKFKGKATL TADKSSSTAY MHLSSLTSED SAVYFCSRQL
101 GFRYAMDYWG QGTSVTVSS (SEQ ID NO: 34)
CDRH1 : SYWMN (SEQ ID NO: 10)
CDRH2: HIYPGDADTNYNGKFKG (SEQ ID NO: 35)
CDRH3: QLGFRYAMDY (SEQ ID NO: 36)
Anti-M-CRAC1415-17F6A2's light chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
1 DIVMSQSPSS LAVSAGEKVT MSCKSSQSLL NSRTRKNYLA WYQQKPGQSP
51 KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKQSYNL 101 RTFGGGTKLE IKR (SEQ ID NO: 37)
CDRL1 : KSSQSLLNSRTRKNYLA (SEQ ID NO: 38)
CDRL2: WASTRES (SEQ ID NO: 19)
CDRL3: KQSYNLRT (SEQ ID NO: 39)
Three additional hybridomas, designated M-hCRAC1415-17F1 A4B6, M- hCRAC1415-15F3A6 and M-hCRAC1415-15F45A1 (all with chain subclass lgG1 /k), also each produced a monoclonal antibody with the same heavy and light chain variable domain amino acid sequences and the same CDRs as 17F6A2.
Anti-M-CRAC1415-14F74A1 's heavy chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
1 QVQLQQSGAE LVRPGSSVKI SCKASGYAFS SYWMNWVKQR PGQGLEWIGH 51 IYPGDGDTNY NGKFKGKATL TADKSSSTAY MQLSGLTSED SAVYFCARSG 101 RLRFAMDYWG QGTSVTVSS (SEQ ID NO: 48)
CDRH1 : SYWMN (SEQ ID NO: 10)
CDRH2: HIYPGDGDTNYNGKFKGKATL (SEQ ID NO: 49)
CDRH3: SGRLRFAMDY (SEQ ID NO: 50)
Anti-M-CRAC1415-14F74A1 's light chain variable domain amino acid sequence was identified to be the following (signal peptide omitted, CDRs are underlined):
1 DIVMSQSPSS LAVSAGEKVT MSCKSSQSLL NSRTRKNYLA WYQQKPGQSP 51 KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKQSYNL
101 RTFGGGTKLE IQR (SEQ ID NO: 51)
CDRL1 : KSSQSLLNSRTRKNYLA (SEQ ID NO: 38)
CDRL2: WASTRES (SEQ ID NO: 19)
CDRL3: KQSYNLRT (SEQ ID NO: 39)
Example 10
Anti-CRAC Channel Monoclonal Antibodies Inhibit T Cell Proliferation and
Cytokine Production
Since anti-CRAC channel antibodies had been shown to inhibit the activity of a T cell line (see, e.g. , Examples 4-5), the antibodies were also evaluated for their ability to inhibit the effector functions of primary T cells upon stimulation (as in
Example 6) in multiple donor samples. Effector functions of T cells include, but are not limited to, proliferation and cytokine production which were analyzed in this example. Total proliferation is measured by CFSE dilution. The florescence of CFSE, a green fluorescent dye that labels intracellular proteins, dilutes by half with each cell division.
Methods
Frozen human donor peripheral blood mononuclear cells (PBMC) (Research Blood Components; Boston, MA) were thawed and removed directly to 13 mL of assay media (RPMI 1640, 10% FBS, 1X GlutaMAX™, and 25 mM HEPES). PBMC were centrifuged for 10min at 1 100 rpm. The cell pellet was aspirated and
resuspended in 10 mL of pre-warmed assay media.
PBMC were CFSE labeled with the Invitrogen CellTrace® CFSE Cell
Proliferation Kit following manufacturer's instructions. After labeling, the cells were re-suspended at 2 x 106 cells/mL in assay media described above in Example 2.
Anti-CRAC channel antibodies and mouse lgG1 , K control monoclonal antibody (Biolegend 400124) were diluted at 4X concentrations of 1332nM into assay media. The antibodies were titrated in triplicate 1 :2 into assay media in a 96 well U-bottom plate (BD Falcon 353077) for final 4X concentrations of 1332, 666, 333, 166.5, 83.25, 41 .625, 20.8125, 10.40625nM in final volumes of .050 mL. An additional .050mls of assay media was added to all wells, bringing the volume to 0.1 ml/well. To the antibody titrations, 0.050 mL of CFSE labeled PBMC was added to all wells of 96 well U-bottom plate. Antibodies and controls were incubated with cells for 1 hr at 37°C in 5% C02. Anti-CD3 monoclonal UCHT1 (eBioscience 16-0038) and anti-CD28 costimulatory monoclonal antibody CD28.2 (eBioscience 16-0289) were diluted together to 4X concentrations of 40 ng/ml and 4 pg/rnl, respectively, in assay media. 0.050mL of the anti-CD3/CD28 dilution were added to all wells on the 96 well U-bottom plate, with the exception of the unstimulated sample, bringing the final volume to .2mls/well.
Cells were incubated at 37°C in 5% C02 for 3 days. 0.050 ml of supernatant were harvested at 16 hours and removed to a separate 96 well plate for cytokine analysis. Samples were stored at -80°C for future analysis.
Cells and remaining supernatant was removed from the culture at day 3. Plates were centrifuged at 1500rpm for 5min. 0.075 ml of supernatant were removed to a separate 96 well plate for 72 hr cytokine analysis. Remaining supernatant was removed by flicking the plate. Cells were resuspended in 0.2 ml D-PBS per well. The plate was centrifuged at 1500 rpm for 5min and supernatants were then aspirated.
One vial of Live/Dead® Fixable Far Red Dead Cell Stain (Invitrogen, Cat# L10120) was prepared by adding 0.050 ml DMSO to one vial of Far Red Dead Cell Stain to create a stock solution, which was then diluted 1 μΙ of stock stain solution per 1 ml DPBS. 0.050 ml of AVID stain was added to each well of 96 well U-bottom plate containing CFSE labeled cells and then incubated at room temperature for 20 minutes. After incubation, 0.15 ml cold FACS Wash (D-PBS + 5%FBS) was added to each well. Plates were then centrifuged 1500 rpm for 5min, and supernatants were aspirated. The cells were then washed and then 0.050 ml CD5-PE/CD4 APC-eFluor 780 antibodies (PE conjugated mouse anti-human CD5 antibody (UCHT2) + APC- eFluor 780 conjugated mouse anti human CD4 antibody (RPA-T4) in FACS wash (2.5 Ml+2.5ul/.050 ml) was added to each well of 96 well plate containing AVID/CFSE labeled cells. The cells were incubated on ice for 30 minutes.
Following incubation, 0.15 ml FACS wash was added to each well and then centrifuged at 1500 rpm for 5 minutes. Supernatants were aspirated, and the cells were washed once with 0.2 ml FACS wash per well and centrifuged at 1500 rpm for 5 minutes. Supernatants were aspirated, and the cells were resuspended in 0.1 ml FACS Wash per well for analysis on a BD LSRII Special SORP flow cytometer. Data were collected using the BD FACS Diva software, and analysis was completed using Tree Star's FlowJo analysis software.
For cytokine analysis, a Millipore 14-plex Milliplex Map Human
Cytokine/Chemokine Immunoassay (Cat# MPXHCYTO60KPMX14) was prepared as described in the protocol provided with the kit. IL-1 b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, 11-10, IL-12(p70), IL-13, GM-CSF, IFN-γ, MCP-1 , and TNF-a were analyzed.
Cytokine/chemokine data were collected using the BioRad BioPlex® Reader and BioPlex® Manager software.
Results
Three representative anti-CRAC antibodies (10F8A2B3, 14F74A1 , and 17F6A2) were evaluated for their ability to inhibit T cell proliferation upon stimulation with anti-CD3 and anti-CD28. Results were normalized to activated, but not antibody treated, cell controls. Thus, incubation without an antibody was 100% T cell proliferation, otherwise referred to as baseline activation. As shown in Figure 10, increasing concentrations of all three anti-CRAC antibodies tested inhibited PBMC proliferation. Inhibition increased with increased F8 concentrations, including nearly 90% inhibition of T cell proliferation by 166nM of some antibodies. In this
experiment, the anti-CRAC antibodies were better at suppressing T cell proliferation than the positive control, 100ng/ml_ cyclosporine A.
Similar results were achieved when cytokine levels in the culture supernatants were examined, as exemplified by IL-2 (at 16 h) and IFNy (at 72 h) in Figures 1 1 and 12, respectively. Increasing concentrations of the three anti-CRAC antibodies inhibited IL-2 and IFNy production (Figures 1 1 and 12, respectively). The anti-CRAC antibodies had a much greater inhibitory effect on cytokine production compared to lgG1 isotype control and, at certain concentrations, the positive control, 100ng/mL of cyclosporine A.
Example 11
Anti-CRAC Channel Antibodies Inhibit Activation of T Cells Isolated from
Rheumatoid Arthritis Patients
Anti-CRAC channel antibodies were evaluated for their ability to inhibit the effector functions of primary T cells isolated from rheumatoid arthritis (RA) patients. Methods
Frozen human RA patient peripheral blood mononuclear cells (RA PBMC) (Astarte Biologies; Redmond, WA) were thawed and removed directly to 13 mL of assay media (RPMI 1640, 10% FBS, 1X GlutaMAX™, and 25 mM HEPES). RA PBMC was centrifuged for 10min at 1 100 rpm. The cell pellet was aspirated and resuspended in 10 mL of pre-warmed assay media.
RA PBMC was CFSE labeled with the Invitrogen CellTrace® CFSE Cell Proliferation Kit following manufacturer's instructions. After labeling, the cells were
re-suspended at 2 x 106 cells/mL in assay media described above in Example 2.
Anti-CRAC channel antibodies and mouse lgG1 , K control monoclonal antibody (Biolegend 400124) were diluted at 4X concentrations of 1332nM into assay media. The antibodies were titrated in triplicate 1 :2 into assay media in a 96 well U-bottom plate (BD Falcon 353077) for final 4X concentrations of 1332, 666, 333, 166.5, 83.25, 41 .625, 20.8125, 10.40625nM in final volumes of .050 ml_. An additional .050mls of assay media was added to all wells, bringing the volume to .1 ml/well. To the antibody titrations, 0.050 ml_ of CFSE labeled RA patient PBMC was added to all wells of 96 well U-bottom plate. Antibodies and controls were incubated with cells for 1 hr at 37°C in 5% C02. Staphylococcal enterotoxin B, SEB (Sigma Aldrich S4881 -1 mg) was diluted at a 4X concentration of 5ng/ml in assay media. 0.050ml_ of SEB dilution was added to all wells on the 96 well U-bottom plate, with the exception of the unstimulated sample, bringing the final volume to
0.2mls/well.
Cells were incubated at 37°C in 5% CO2 for 6 days. Cells were harvested from the culture at day 6. Plates were centrifuged at 1500rpm for 5min. 0.075 ml of supernatant were removed to a separate 96 well plate for 72 hr cytokine analysis. Remaining supernatant was removed by flicking the plate. Cells were resuspended in 0.2 ml D-PBS per well. The plate was centrifuged at 1500 rpm for 5m in and supernatants were then aspirated.
One vial of Live/Dead® Fixable Far Red Dead Cell Stain (Invitrogen, Cat# L10120) was prepared by adding 0.050 ml DMSO to one vial of Far Red Dead Cell Stain to create a stock solution, which was then diluted 1 μΙ of stock stain solution per 1 ml DPBS. 0.050 ml of AVID stain was added to each well of 96 well U-bottom plate containing CFSE labeled cells and then incubated at room temperature for 20 minutes. After incubation, 0.15 ml cold FACS Wash (D-PBS + 5%FBS) was added to each well. Plates were then centrifuged 1500 rpm for 5min, and supernatants were aspirated. The cells were then washed and then 0.050 ml CD3-PE/CD4 APC-eFluor 780 antibodies (PE conjugated mouse anti-human CD3 antibody (HIT3a) + APC- eFluor 780 conjugated mouse anti human CD4 antibody (RPA-T4) in FACS wash (2.5 Ml+2.5ul/.050 ml) was added to each well of 96 well plate containing AVID/CFSE labeled cells. The cells were incubated on ice for 30 minutes.
Following incubation, 0.15 ml FACS wash was added to each well and then centrifuged at 1500 rpm for 5 minutes. Supernatants were aspirated, and the cells were washed once with 0.2 ml FACS wash per well and centrifuged at 1500 rpm for 5 minutes. Supernatants were aspirated, and the cells were resuspended in 0.1 ml FACS Wash per well for analysis on a BD LSRII Special SORP flow cytometer. Data were collected using the BD FACS Diva software, and analysis was completed using Tree Star's FlowJo analysis software.
Results
Four anti-CRAC antibodies (10F8A2B3, 14F74A1 , 17F6A2, and 15F58A5B4) were evaluated for their ability to inhibit the proliferation of T cells isolated from RA patients and stimulated with SEB. The 15F58A5B4 binding domain was engineered on a mouse lgG1 isotype such that all antibodies tested in this experiment were of the mouse lgG1 isotype. All four of the antibodies tested inhibited proliferation to varying degress. Results were normalized to activated, but untreated, controls. Thus, incubation without an antibody or with control IgG was 100% T cell proliferation. As shown in Figure 13, inhibition increased with increased antibody concentrations, including nearly 90% inhibition of PBMC proliferation by 41 nM of 14F74A.
Example 12
In Vivo Proof of Concept in Humanized Mouse Model of GVHD
A Graft Versus Host Disease (GVHD) mouse model system was used to evaluate in vivo proof of concept for anti-CRAC channel monoclonal antibodies.
Transfer of human peripheral blood mononuclear cells to immunodeficient mice (hereinafter "humanized mice") initiates human T cell expansion and results in a T cell mediated graft versus host disease (GvHD) which affects several major organs and can be measured as weight loss. Treatment of humanized mice with an antibody targeting the human CRAC channel can therefore be used to elucidate whether such a compound has inhibitory effects in vivo on human T cell expansion and T cell mediated tissue inflammation.
Methods
Humanized mice. Using Ficoll-Paque (GE Healthcare) gradients and Leucosep-tubes (Greiner Bio-One) human PBMCs were purified from buffy coats donated by healthy human donors. Female NOD.scid IL-2 receptor common gamma chain (IL-2Rgc)" _ mice (Taconic) were injected i.v. with 20x106 human PBMCs. Following injection of cells all animals were weighed and randomized into groups. Treatments with mouse anti-human CRAC1415-10F8 antibodies or matching isotype control antibodies (mouse IgGl k anti-trinitrophenyl (TNP)) were initiated on day 0 by intraperitoneal administration of 10mg/kg. Treatment was continued 3 times per week for the duration of the study. Assessment of animal health and weight was performed 3 times per week and mice experiencing more than 20 percent weight loss or impaired health with simultaneous detection of substantial human cell expansion in the blood were graded as having GvHD.
FACS analysis of blood samples. Blood samples were taken once weekly throughout the study and at time of GvHD to monitor human cell expansion. 100μΙ_ of blood was drawn from each mouse into EDTA tubes by submandibular-vein bleeding. Red blood cells were lysed with ACK buffer for 7 min. at RT, samples were washed once and stained with anti-mouse CD16/32 purified (Fc Block, BD Biosciences) for 10 min. cold. Hereafter, staining with the following fluorochrome-conjugated antibodies was performed for 20 minutes in 100μΙ_ staining volume: Anti-human (h)CD45-FITC, anti-human CD4-PerCP, anti-human CD8-Pacific Blue, anti-human CD19-PECy7 (all BD Biosciences), anti-mouse (m)CD45-Pacific Orange (Caltag) and anti-human CD3-Qdot 655 (Invitrogen). Staining with LIVF DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen) was included to exclude dead cells. Human T cells were gated as live and single lymphocytes based on FSC/SSC properties and Near- IR staining with the following surface maker phenotype: mCD45"hCD45+CD3+CD19" CD4+/CD8+. Cells were washed once following staining and transferred to BD TruCount tubes (BD Biosciences) for absolute cell counting and analyzed on a LSRII flow cytometer using FACSDiva software (BD Biosciences).
Statistics. For evaluation of statistical differences between anti-CRAC and the isotype control; Kaplan-Meier survival analysis and Mantel-Cox Log-Rank test was used in analysis of GvHD and Mann-Whitney's U-test was used in analysis of
human T cells in blood. Bar plots show mean ± SEM and a p-value <0.05 was considered statistically significant, **p<0.01 , ***p<0.001 .
Results
Humanized mice were used to determine the effect of CRAC channel blockade in vivo on human T cell expansion and human T cell mediated GvHD.
Figure 14A shows a Kaplan-Meier survival curve of time to GvHD in groups of mice post injection of human PBMCs and following treatment from day 0 with either mouse anti-hCRAC1415-10F8 antibody or a mouse anti-TNP isotype control antibody. Anti-CRAC significantly (p<0.01 ) postponed the time to GvHD compared to the isotype control. Numbers in brackets indicate number of surviving mice per total number of mice in each group at study termination. A group of mice not injected with PBMCs were included as reference.
Figures 14B and 14C show the absolute number of human CD4+ and CD8+ T cells in 100μΙ of mouse blood taken at the indicated time points. Notably, anti-CRAC showed a significant reduction in both CD4+ and CD8+ T cell numbers in blood compared to the isotype control at all time points analysed.
Therefore, anti-CRAC channel monoclonal antibody (F8) shows in vivo proof of concept by significantly delaying GvHD and human T cell expansion in the humanized mouse model of GVHD.
Example 13
Anti-CRAC channel antibody inhibits the effector function of T cells in synovial fluid of rheumatoid arthritis patients
Anti-CRAC channel antibodies were shown to inhibit the activity of primary T cells isolated from peripheral blood of healthy individuals and rheumatoid arthritis patients (RA) in the previous examples, and so one of the lead antibodies was also evaluated for its ability to inhibit the effector functions of synovial T cells derived from rheumatoid arthritis patients. Effector functions of T cells include, but are not limited to, IL2 and IFN-γ secretion which were analyzed in this example. The levels of secreted cytokines were measured by commercial ELISA kits.
Methods
Fresh human RA patient synovial fluid mononuclear cells (RA SFMC) were separated from the knee joint fluids (Beijing University People's Hospital, PRC) by centrifugation for 10 min at 2000 rpm. The cell pellet was aspirated and resuspended in pre-warmed assay media (RPM I 1640, 10% heat inactivated FBS, 1 %
penicillin/streptomycin) at 1x106/ml.
Anti-CRAC channel antibody 10F8.mlgG1 , mouse lgG1 , K control monoclonal antibody and cyclosporine A (Sigma catalog number C1832, dissolved in 100% DMSO) were diluted at 2X concentrations of 1332 nM into assay media. Following a serial titration in duplicate 1 :2 (mAbs) or 1 :3 (CsA) in a 96 well deep-well plate (Nunc 278743) for final 2X concentrations ranging from 0.8 to 666 nM (mAbs: 666, 333, 166.5, 83.25, 41.63, 20.8, 10.4, 5.2 and 2.6 nM; CsA: 666, 222, 74, 24, 8, 2.6 and 0.8 nM) in final volume of 1 .5 mL/well, 0.1 ml_ of which were transferred to a dry 96 well U-bottom assay plate (Corning costar 3799) that had been pre-coated, to all wells except the edge, with anti-CD3 mAb (BD Pharmingen catalog number 555336) at 0.3 ug/mL and anti-CD28 mAb (BD Pharmingen catalog number 555725) at 3 ug/mL in sterile PBS overnight. Then the assay plate was added with 0.1 mL of RA patient SFMC at 105 cell/well, bringing the final volume to 0.2mL/well.
Cell cultures were incubated at 37°C in 5% C02 for 40 hours. 120 mL supernatants were harvested from each well of the culture plate for cytokine analysis with IL-2 and IFN-γ ELISA kits (eBioscience catalog number 88-7026-88 and 88- 7316-88 respectively). According to manufacturer's suggestions, the testing supernatants were 1/2 diluted for cytokine measurement.
Results
One representative anti-CRAC antibody (10F8) was evaluated for its ability to inhibit the CD3 and CD28-costimulated cytokine secretion of SFMC from RA patients. As shown in Figure 15A, 10F8 antibody inhibited IL-2 secretion in dose-dependent and complete manner, but not its isotype control, mouse IgGl As shown in Figure 16A, 10F8 antibody also inhibited IFN-γ secretion in a dose-dependent manner with the maximum 82% effect at the highest concentration tested. Similar results were
achieved in another experiment with a second RA patient sample that tested positive control cyclosporine A's ability to inhibit cytokine secretion. The anti-CRAC antibody was similar or better at suppressing cytokines than the cyclosporine A in both experiments (see Figures 15B and 16B).
EXEMPLARY EMBODIMENTS
1 . An antibody, or a fragment thereof, that specifically binds a CRAC channel and inhibits calcium flux through the CRAC channel.
2. The antibody of embodiment 1 , wherein the antibody, or the fragment thereof, specifically binds the second extracellular loop of ORAM . 3. The antibody of embodiment 1 or 2, wherein the CRAC channel is a human CRAC channel.
4. The antibody of any one of embodiments 1 -3, or the fragment thereof, wherein the human CRAC channel comprises ORAM having an amino acid sequence of SEQ ID NO: 3.
5. The antibody of any one of embodiments 1 -4, or the fragment thereof, wherein the antibody binds an epitope comprising one or more residues selected from the group consisting of G12, Q13, P14, R15, P16, T17, S18, K19, P20, P21 , A22, S23, G24, A25, A26, A27, N28, V29, S30, T31 , S32, G33, I34, T35, P36, G37, Q38, A39 of SEQ ID NO: 3.
6. The antibody of any one of embodiments 1 -5, or the fragment thereof, wherein the antibody is a monoclonal antibody (mAb).
7. The antibody of any one of embodiments 1 -6, or the fragment thereof, wherein the antibody is selected from the group consisting of a linear antibody, a single chain antibody, a monobody, and a multispecific antibody. 8. The antibody of any one of embodiments 1 -7, or the fragment thereof, wherein the fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, and scFv.
9. The antibody of any one of embodiments 1 -8, or the fragment thereof, comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises at least one of the following sequences (a) to (b):
a) a CDRH1 comprising SEQ ID NO: 10, optionally wherein one of these amino acids is substituted with a different amino acid; and
b) a CDRH2 comprising amino acids 1 -9 of SEQ ID NO: 52, optionally wherein one of these amino acids is substituted with a different amino acid.
10. The antibody according to embodiment 9, or the fragment thereof, wherein CDRH2 is amino acids 1 -9 of a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 58, SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 56, optionally wherein one of these amino acids is substituted with a different amino acid.
1 1. The antibody according to embodiment 9 or 10, or the fragment thereof, further comprising a CDRH3 selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 41 and SEQ ID NO: 50, optionally wherein one or two of these amino acids is substituted with a different amino acid.
12. The antibody of any one of embodiments 1 -1 1 or 70, or the fragment thereof, further comprising a light chain variable domain, wherein the light chain variable domain comprises at least one of the following sequences (a) to (c):
a) a CDRL1 comprising SEQ ID NO: 57, optionally wherein one or two of these amino acids is substituted with a different amino acid;
b) a CDRL2 comprising SEQ ID NO: 19, optionally wherein one or two of these amino acids is substituted with a different amino acid; and
c) a CDRL3 comprising SEQ ID NO: 55.
13. The antibody according to embodiment 12, or the fragment thereof, wherein CDRL1 comprises SEQ ID NO: 18, optionally wherein one or two of these amino acids is substituted with a different amino acid.
14. The antibody according to any one of embodiments 12-13, or the fragment thereof, wherein CDRL1 comprises consensus sequence SEQ ID NO: 54 and is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 46, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
15. The antibody according to embodiment 12, or the fragment thereof, wherein CDRL3 selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 43 and SEQ ID NO: 47, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
16. The antibody according to any one of embodiments 1 -15 or 70-71 comprising:
a) a CDRH1 comprising SEQ ID NO: 10;
b) a CDRH2 comprising amino acids 1 -9 of SEQ ID NO: 1 1 ; and c) a CDRH3 comprising SEQ ID NO: 12.
17. The antibody according to embodiment 16 further comprising:
a) a CDRL1 comprising SEQ ID NO: 18;
b) a CDRL2 comprising SEQ ID NO: 19; and c) a CDRL3 comprising SEQ ID NO: 20.
18. The antibody according to any one of embodiments 1 -15 or 70-71 comprising:
a) a CDRH1 comprising SEQ ID NO: 10;
b) a CDRH2 comprising amino acids 1 -9 of SEQ ID NO: 35; and c) a CDRH3 comprising SEQ ID NO: 36.
cording to embodiment 18 further comprising:
a) a CDRL1 comprising SEQ ID NO: 38;
b) a CDRL2 comprising SEQ ID NO: 19; and
c) a CDRL3 comprising SEQ ID NO: 39. cording to any one of embodiments 1 -15 or 70-71 comprising: a) a CDRH1 comprising SEQ ID NO: 10;
b) a CDRH2 comprising amino acids 1 -9 of SEQ ID NO: 1 1 ; and c) a CDRH3 comprising SEQ ID NO: 41. cording to embodiment 20 further comprising:
a) a CDRL1 comprising SEQ ID NO: 38;
b) a CDRL2 comprising SEQ ID NO: 19; and
c) a CDRL3 comprising SEQ ID NO: 43. cording to any one of embodiments 1 -15 or 70-71 comprising: a) a CDRH1 comprising SEQ ID NO: 10;
b) a CDRH2 comprising amino acids 1 -9 of SEQ ID NO: 1 1 ; and c) a CDRH3 comprising SEQ ID NO: 41. cording to embodiment 22 further comprising:
a) a CDRL1 comprising SEQ ID NO: 46;
b) a CDRL2 comprising SEQ ID NO: 19; and
c) a CDRL3 comprising SEQ ID NO: 47. cording to any one of embodiments 1 -15 or 70-71 comprising: a) a CDRH1 comprising SEQ ID NO: 10;
b) a CDRH2 comprising amino acids 1 -9 of SEQ ID NO: 49; and c) a CDRH3 comprising SEQ ID NO: 50. cording to embodiment 24 further comprising:
a) a CDRL1 comprising SEQ ID NO: 38;
b) a CDRL2 comprising SEQ ID NO: 19; and
c) a CDRL3 comprising SEQ ID NO: 39.
26. The antibody of any one of embodiments 1 -25 or 70-71 , or the fragment thereof, wherein the heavy chain variable domain comprises a human framework region.
27. The antibody of any one of embodiments 1 1 -26 or 70-71 , or the fragment thereof, wherein the light chain variable domain comprises a human framework region.
28. The antibody of any one of embodiments 1 -17, 19, 21 , 23, 25-27 or 70-71 , or the fragment thereof, wherein the heavy chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 9.
29. The antibody of any one of embodiments 1 -15, 17-19, 21 , 23, 25-27 or 70-71 , or the fragment thereof, wherein the heavy chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 34. 30. The antibody of any one of embodiments 1 -15, 17, 19-21 , 23, 25-27 or 70-71 , or the fragment thereof, wherein the heavy chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 40.
31. The antibody of any one of embodiments 1 -15, 17, 19, 21 -23, 25-27 or 70-71 , or the fragment thereof, wherein the heavy chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 44.
32. The antibody of any one of embodiments 1 -15, 17, 19, 21 , 23-27 or 70-71 , or the fragment thereof, wherein the heavy chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 48. 33. The antibody of any one of embodiments 1 -18, 20, 22, 24, 26-28 or 70-71 or the fragment thereof, wherein the light chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 17.
34. The antibody of any one of embodiments 1 -16, 18-20, 22, 24, 26-27 or 70-71 , or the fragment thereof, wherein the light chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 37.
35. The antibody of any one of embodiments 1 -16, 18, 20-22, 24, 26-27 or 70-71 , or the fragment thereof, wherein the light chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98%
sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 42.
36. The antibody of any one of embodiments 1 -16, 18, 20, 22-24, 26-27 or 70-71 , or the fragment thereof, wherein the light chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 45.
37. The antibody of any one of embodiments 1 -16, 18, 20, 22, 24-27 or 70-71 , or the fragment thereof, wherein the light chain variable domain has a sequence identity selected from the group of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and 100% sequence identity, to SEQ ID NO: 51 .
38. The antibody of any one of embodiments 1 -37 or 70-71 , or the fragment thereof, wherein the antibody is a human antibody.
39. The antibody of any one of embodiments 1 -37 or 70-71 , or the fragment thereof, wherein in the antibody is a humanized antibody.
40. An isolated antibody that competes for binding with any one of the antibodies of embodiments 1 to 39 or 70-71 .
41. An isolated nucleic acid encoding the antibody of any of embodiments 1 -40 or 70- 71. 42. The isolated polynucleotide of embodiment 41 encoding a polypeptide
comprising SEQ ID NO: 14 encoding a CDRH1 , SEQ ID NO: 15 encoding a CDRH2, and SEQ ID NO: 16 encoding a CDRH3.
43. The polynucleotide of any one of embodiments 41 -42, further comprising SEQ ID NO: 22 encoding a CDRL1 , SEQ ID NO: 23 encoding a CDRL2, and SEQ ID NO: 24 encoding a CDRL3.
44. The polynucleotide of any one of embodiments 41 -43, wherein the
polynucleotide encodes a heavy chain variable domain having human framework regions. 45. The polynucleotide of any one of embodiments 41 -44, wherein the
polynucleotide encodes a light chain variable domain having human framework regions.
46. An expression vector comprising the polynucleotide of any one of embodiments 41 -45 operably linked to expression control elements such that the antibody of any of embodiments 1 -40 may be expressed.
47. A recombinant host cell comprising the expression vector of embodiment 46. 48. The host cell of embodiment 47, wherein the host cell is eukaryotic.
49. The host cell of embodiment 47, wherein the host cell is mammalian.
50. A method of producing an anti-CRAC channel antibody, the method comprising culturing a host cell of any one of embodiments 47-49 in conditions appropriate for expressing the nucleic acid, and isolating the antibody.
51. An antibody produced by the process of embodiment 50. 52. A pharmaceutical composition comprising the antibody of any one of
embodiments 1 -40, 51 or 70-71 and a pharmaceutically acceptable carrier, diluent or excipient.
53. The antibody of any one of embodiments 1 -40, 51 or 70-71 , wherein the antibody is an IgG antibody. 54. The antibody of embodiment 53, wherein the IgG antibody is of an lgG1 , lgG2, lgG3, or lgG4 antibody.
55. An antibody according to any one of embodiments 1 -40, 51 or 70-71 for use as a medicament.
56. An antibody according to any one of embodiments 1 -40, 51 or 70-71 for the manufacture of a medicament for the treatment of an autoimmune disease.
57. An antibody according to any one of embodiments 1 -40, 51 or 70-71 for the treatment of an autoimmune or inflammatory disease.
58. The antibody of any one of embodiments 55-57, wherein the autoimmune or inflammatory disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, multiple sclerosis and graft versus host disease.
59. The antibody of any one of embodiments 55-58, wherein T cell activation, T cell proliferation, N FAT translocation or pro-inflammatory cytokine production is inhibited or reduced.
60. A method of treating an autoimmune or inflammatory disease in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71. 61. The method of embodiment 60, wherein the autoimmune or inflammatory disease is selected from the group consisting of inflammatory bowel disease,
rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, multiple sclerosis and graft versus host disease.
62. The method of embodiment 61 , wherein T cell activation, T cell proliferation, NFAT translocation or pro-inflammatory cytokine production is inhibited or reduced.
63. A method for modulating T-cells in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71 .
64. A method for inhibiting T cell activation in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71 . 65. A method for inhibiting NFAT translocation in a patient comprising administering to the patient an effective amount of the antibody of any one of embodiments 1 -40, 51 or 70-71 .
66. The method of embodiment 65, wherein pro-inflammatory cytokine production is reduced or inhibited.
67. The method of embodiment 66, wherein production of one or more of IL-1 B, IL-2, IL-4, IL-5, IL-6, IL-13 and IFN-γ is inhibited or reduced.
68. The method of embodiment 65, wherein T cell proliferation is inhibited or reduced.
69. An antibody according to any one of embodiments 1 -41 , 50 or 70-71 for reducing or inhibiting T cell activation, T cell proliferation, NFAT translocation or proinflammatory cytokine production.
70. The antibody of embodiment 9, wherein the heavy chain variable domain comprises sequences (a) and (b).
71. The antibody of embodiment 12, wherein the light chain variable domain comprises sequences (a) and (b).
All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.
Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly con-tradicted by context.
The terms "a" and "an" and "the" and similar referents as used in the context of de-scribing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.
The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or
embodiment of the invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.
Claims
1 . A monoclonal antibody (mAb), or a fragment thereof, that specifically binds a human CRAC channel and inhibits calcium flux through the CRAC channel.
2. The antibody of claim 1 , or the fragment thereof, that specifically binds the second extracellular loop of the human CRAC channel.
3. The antibody of any one of claims 1 -2, or the fragment thereof, wherein the human CRAC channel comprises ORAM having an amino acid sequence of SEQ ID
NO: 3 and wherein the antibody binds an epitope comprising one or more residues selected from the group consisting of G12, Q13, P14, R15, P16, T17, S18, K19, P20, P21 , A22, S23, G24, A25, A26, A27, N28, V29, S30, T31 , S32, G33, I34, T35, P36, G37, Q38, A39 of SEQ ID NO: 3.
4. The antibody of any one of claims 1 -3, or the fragment thereof, comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises at least one of the following sequences:
a) a CDRH1 comprising SEQ ID NO: 10, optionally wherein one of these amino acids is substituted with a different amino acid; and
b) a CDRH2 comprising SEQ ID NO: 52, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
5. The antibody of claim 4, or the fragment thereof, wherein CDRH2 is amino acids 1 -9 of a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ
ID NO: 58, SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 56, optionally wherein one of these amino acids is/are substituted with a different amino acid.
6. The antibody of claim 4 or 5, or the fragment thereof, further comprising a CDRH3 selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 41 and SEQ ID
NO: 50, optionally wherein one or two of these amino acids is substituted with a different amino acid.
7. The antibody of any one of claims 1 -6, or the fragment thereof, further comprising a light chain variable domain, wherein the light chain variable domain comprises at least one of the following sequences (a) to (c):
a) a CDRL1 comprising SEQ ID NO: 57, optionally wherein one or two of these amino acids is substituted with a different amino acid;
b) a CDRL2 comprising SEQ ID NO: 19, optionally wherein one or two of these amino acids is substituted with a different amino acid; and
c) a CDRL3 comprising SEQ ID NO: 55.
8. The antibody of claim 7, or the fragment thereof, wherein CDRL1 comprises SEQ ID NO: 18, optionally wherein one or two of these amino acids is substituted with a different amino acid.
9. The antibody of any one of claims 7-8, or the fragment thereof, wherein CDRL1 comprises consensus sequence SEQ ID NO: 54 and is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 46, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
10. The antibody of claim 7, or the fragment thereof, wherein CDRL3 selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 43 and SEQ ID NO: 47, optionally wherein one, two or three of these amino acids is substituted with a different amino acid.
1 1. The antibody of any one of claims 1 -10, or the fragment thereof, wherein the antibody is a human antibody or a humanized antibody.
12. An expression vector comprising a polynucleotide encoding the antibody of any one of claims 1 -1 1 operably linked to expression control elements such that the antibody of any of embodiments 1 -1 1 may be expressed.
13. An antibody according to any one of claims 1 -1 1 for use as a medicament.
14. An antibody according to any one of claims 1 -1 1 for the treatment of an autoimmune or inflammatory disease, wherein the autoimmune or inflammatory disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, multiple sclerosis and graft versus host disease.
15. An antibody according to any one of claims 1 -1 1 for reducing or inhibiting T cell activation, T cell proliferation, NFAT translocation or pro-inflammatory cytokine production.
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WO2020176844A1 (en) * | 2019-02-28 | 2020-09-03 | Board Of Regents, The University Of Texas System | Compositions for treating osteosarcoma and methods of use |
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