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WO2017160599A1 - Utilisation d'antagonistes de cd300b pour traiter un sepsis et un choc septique - Google Patents

Utilisation d'antagonistes de cd300b pour traiter un sepsis et un choc septique Download PDF

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WO2017160599A1
WO2017160599A1 PCT/US2017/021654 US2017021654W WO2017160599A1 WO 2017160599 A1 WO2017160599 A1 WO 2017160599A1 US 2017021654 W US2017021654 W US 2017021654W WO 2017160599 A1 WO2017160599 A1 WO 2017160599A1
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cd300b
antibody
lps
tlr4
antagonist
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John Ernest COLIGAN
Oliver H. VOSS
Konrad Jerzy KRZEWSKI
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This relates to the field of treating and preventing sepsis and septic shock, specifically to the use of CD300b antagonists to treat and/or prevent septic shock and sepsis.
  • the innate immune system is the first line of host defense against invading pathogens (Iwasaki and Medzhitov, 2015, Nat Immunol 16, 343-353).
  • LPS present in gram-negative bacteria membranes, causes strong immune responses following detection by Toll-like receptor (TLR)4 on immune cells (Iwasaki and Medzhitov, 2015, supra).
  • TLR Toll-like receptor
  • Activation of immune cells, including macrophages ( ⁇ ) and dendritic cells (DC) results in the release of pro-inflammatory cytokines, like TNFoc, IL-6, and IL-12, and clearance of infectious organisms.
  • IL-10 an antiinflammatory cytokine
  • IL-10 an antiinflammatory cytokine
  • host tissue damage Saraiva and O'Garra, 2010, Nat Rev Immunol 10, 170-181.
  • Excessive immune cell activation leads to a more severe immunopathology, such as septic shock and, subsequently, death (Hotchkiss et al., 2013, Nat Rev Immunol 13, 862-874; Iwasaki and Medzhitov, 2015, supra).
  • TLR4-dependent LPS recognition is initiated by LPS binding to CD 14 (Wright et al., 1990, Science 249, 1431-1433) with subsequent transfer to the TLR4/MD-2 complex (Shimazu et al., 1999, J Exp Med 189, 1777-1782).
  • These adaptor molecules promote signaling via the p38-, Jun-, ERK1/2-MAPK and TBK1- ⁇ signaling cascades leading to the activation of transcription factors like NFKB, AP-1 and IRF3, which promote the expression of cytokine-encoding genes.
  • TREMl Bochon et al., 2001, Nature 410, 1103-1107
  • TREM2 Trinbull et al., 2006, J Immunol 177, 3520-3524
  • CD209 Nagaoka et al., 2005, Int Immunol 17, 827-836
  • CD1 lb Li et al., 2014, Nat Commun 5, 3039
  • human (h)CD300a Nakahashi-Oda et al., 2012, J Exp Med 209, 1493-1503
  • mouse (m)CLM4 Totsuka et al., 2014, Nat Commun 5, 4710)
  • mCD300b CCM7
  • Sepsis is a systemic inflammatory syndrome caused by infection that results in tissue damage, multisystem organ failure and, subsequently, death (Cohen, 2002, Nature 420, 885-891).
  • current therapies to treat sepsis remain ineffective and all clinical trials based on neutralization of specific inflammatory cytokines have failed, highlighting the need for new treatments (Wenzel and Edmond, 2012, N Engl J Med 366, 2122-2124).
  • CD300b is involved in regulating the immune response to bacterial infection. It is disclosed herein that CD300b is a novel LPS binding receptor, and the mechanism underlying CD300b augmentation of septic shock is elucidated. CD300b-expressing macrophages were identified as the key cell type augmenting septic shock. It was demonstrated that CD300b/DAP12 associates with TLR4/CD14 upon LPS binding, promoting MyD88/TIRAP dissociation from the complex and the recruitment and activation of Syk and PI3K. This results in the activation of AKT, which subsequently leads to a reduced production of the anti-inflammatory cytokine IL-10 by
  • CD300b also enhances TLR4/CD14-TRIF-IRF3 signaling responses, resulting in elevated IFN- ⁇ levels. This provides new therapeutic strategies for the treatment and/or prevention of sepsis and septic shock.
  • a pharmaceutical composition that includes a CD300b antagonist, for use in treating or preventing the development of sepsis or septic shock in a subject.
  • the pharmacological composition can optionally include interleukin (IL)-IO, a TLR4 antagonist (such as, but not limited to, an antibody that specifically binds TLR4, or an antigen binding fragment thereof), a CD 14 antagonist (such as, but not limited to, an antibody that specifically binds CD14, or an antigen binding fragment thereof), a Programmed Death (PD)-l antagonist (such as, but not limited to, an antibody that specifically binds PD-1, Programmed Death Ligand (PD-L)l or PD-L2, or an antigen binding fragment thereof), a cytotoxic T cell T-lymphocyte associated protein (CTLA)-4 antagonist (such as, but not limited to, an antibody that specifically binds CTLA-4, or an antigen binding fragment thereof), and/or a B and T cell attenuator (BTLA) antagonist (such as,
  • IL
  • methods for treating or preventing the development of septic shock or sepsis in a subject.
  • the methods include administering to the subject a therapeutically effective amount of a CD300b antagonist.
  • the methods can optionally include administering a therapeutically effective amount of IL-10, a TLR4 antagonist, a CD 14 antagonist, a PD-1 antagonist, a CTLA-4 antagonist, and/or a BTLA antagonist.
  • the CD300b antagonist, the TLR4 antagonist, the CD 14 antagonist, the PD-1 antagonist, the CTLA-4 antagonist, and/or the BTLA antagonist is an antibody or an antigen binding fragment thereof.
  • FIGS. 1A-1G LPS is a ligand for CD300b.
  • (1 A-1C) Sensorgrams of mCD300b-Fcy (1 A), hCD300b-Fcy (lB), mCD300d-Fcy, mCD300a-Fcy, mCD300f-Fcy, and NITR-FCY (IC) protein binding to immobilized LPS (E.coli 0111:B4) over the indicated times. Binding was initiated at 60 s and the dissociation phase begun at 240 s and is expressed in resonance units (RU).
  • RU resonance units
  • mCD300d/FcRy- (3 ⁇ 4, mCD300f- ( ⁇ ), EV-expressing L929 cells ( ⁇ ) were incubated with FITC- labeled LPS from E. coli or S. minnesota (10 ⁇ g/ml) for 1 h at 37°C (IE) or 4°C (IF). Binding was analyzed by flow cytometry and expressed as mean fluorescence intensity (MFI). (1G)
  • mCD300b/DAP12-expressing L929 cells were incubated with FITC-labeled LPS from E. coli (10 ⁇ g/ml) for 1 h, and mixed with increasing concentrations of unlabeled LPS from E. coli, S.
  • FIGS. 3A-3C Neutralization of IL-10 augments the pathogenesis of LPS-induced sepsis and septic shock.
  • (3 A) Anti-IL-10- or control Ab-treated WT and Cd300b ⁇ ' ⁇ mice (n 12) were i.p. injected with a toxic dose of LPS (37 mg/kg). Survival was monitored every 6 h for 7 days.
  • the graphs in (3C) show mean values + SEM from 5 mice per group, NS, not significant; *p ⁇ 0.05, and **p ⁇ 0.01.
  • FIGS. 4A-4C CD300b-expressing macrophages are responsible for augmenting the pathogenesis of LPS-induced sepsis and septic shock.
  • 4B H&E staining of lung tissues from PBS- and CkMBP-liposome-injected WT and Cd300b ⁇ ' ⁇ mice after LPS treatment. Data in (4B) are a representative from 5 mice per group.
  • (4C) Serum cytokine concentrations measured from PBS- and C MBP-liposome-injected WT and Cd300b ⁇ ' ⁇ mice after LPS treatment.
  • the graphs in (4C) show mean values + SEM from 5 mice per group, NS, not significant; *p ⁇ 0.05, and **p ⁇ 0.01.
  • FIGS. 5A-5C CD300b-expressing ⁇ increase the severity of sepsis and septic shock by altering the levels of cytokines produced.
  • 5A ⁇ from WT or Cd300b ⁇ ' ⁇ mice were stimulated with 2 ⁇ g/ml of LPS for various lengths of time. Cytokine levels were assessed by flow cytometry. No differences in cytokine levels between diluent control-treated WT and Cd300b ⁇ '- ⁇ was observed.
  • FIGS. 6A-6I LPS binding by CD300b promotes the association with TLR4- MyD88/TIRAP complex and dampens the production of IL-10 via DAP12-Syk-PI3K recruitment.
  • 6A ⁇ from WT, Cd300b ⁇ ' ⁇ , Cdl4 ⁇ ' ⁇ or TLR4 ' mice were lysed and analyzed by immunoblotting with the indicated Abs. GAPDH was used as a loading control.
  • DSP dithiobissuccinimidyl propionate
  • Samples were immunoprecipitated with anti-CD300b or anti-IgG isotype control Ab and analyzed by immunoblotting with the indicated Abs.
  • (6C-6D) ⁇ from WT or Cd300b ⁇ ' ⁇ mice were stimulated with LPS (2 ⁇ g/ml) for various lengths of time. Reactions were immunoprecipitated with anti-CD300b (6C), anti-TLR4 (6D) or anti-IgG isotype control Ab (6C-6D), and then analyzed by immunoblotting with the indicated Abs.
  • (6E) ⁇ from WT were pretreated for 12 h with anti-IgG isotype control Ab, anti-CD300b Ab, anti-CD 14 Ab or both anti-CD300b and anti-CD 14 Abs before the addition of LPS (2 ⁇ g/ml).
  • Cell lysates were immunoprecipitated with anti-TLR4 or anti-IgG isotype control Ab and samples were analyzed by immunoblotting with the indicated Abs.
  • (6F-6G) ⁇ from WT, Cd300b '-, or Cd300f mice were stimulated with LPS (2 ⁇ g/ml) for various lengths of time and cell lysates were analyzed for the levels of phosphorylated (6F) or total protein expression (6G) by immunoblotting with the indicated Ab.
  • (6H) ⁇ from WT or Cd300b A mice were treated with p38 inhibitor SB203580 (10 ⁇ ) and/or the ERK1/2 inhibitor PD98059 (25 ⁇ ) for 1 h prior to stimulation with LPS (2 ⁇ g/ml) for additional 2 h.
  • the levels of p38 and ERK1/2 phosphorylation were analyzed by immunoblotting with the indicated Ab.
  • Data in (6A-6H) are a representative of three experiments. Error bars represent SEM from three experiments (I), *p ⁇ 0.05, and **p ⁇ 0.01.
  • FIGS. 7A-7J CD300b recognizes LPS via the lipid A core structure related to FIG. 1.
  • FIGS. 8A-8N CD300b binds LPS or E. coli but not other TLR or NOD ligands and CLP-treated WT but not Cd300b ⁇ ' ⁇ mice have a higher bacterial burden, related to Figure 1 and 2.
  • 8A-8B mCD300b/DAP12- (MFI: 144) or clonal cell lines expressing different levels of cell surface mCD300b- (Clone 1, MFI: 34; Clone 2, MFI: 86; Clone 3, MFI: 138) were incubated with FrrC-labeled LPS from E. coli (10 ⁇ g/ml) for 1 h at 4°C. Binding was analyzed by flow cytometry and expressed as MFI.
  • mCD300b/DAP12-, mCD300b- and EV-expressing L929 cells were lysed and the expression level of CD300b and DAP12 (overexpressed and endogenous) was assessed by immunoblotting with the indicated Abs. GAPDH served as loading control.
  • 8D- 8E mCD300b/DAP12- and EV-expressing L929 cells were incubated with rhodamine-labeled PAM3CSK4, Poly(I:C) or MDP (10 ⁇ g/ml) for 1 h at 37 °C (8D) or 4°C (8E). Binding was analyzed by flow cytometry and displayed as MFI.
  • FIGS. 9A-9J CD300b/TLR4 co-expressing macrophages were depleted in vivo using dichloromethylene biphosphate (ChMBP)-encapsulated liposomes, while selective reduction of CD300b-expressing neutrophils or NK cells does not augment the pathogenesis of septic shock, related to FIG. 4.
  • ChoMBP dichloromethylene biphosphate
  • cDC conventional DC
  • pDC plasmacytoid DC
  • ⁇ macrophages
  • eosinophils CDllb hi CDllc Ly6G lo SSC hi
  • neutrophils CDl lb hi CDllc Ly6G hi SSC 10
  • FoB follicular B cells
  • MzB marginal zone B cells
  • TrB transitional B cells (B220 + CD21 lo/ CD23 ); CD4 + and CD8 + T cells.
  • Relative copy number (RCN) of murine Cd300b and TLR4 after normalization with GAPDH The graph in (9 A) shows mean values + SD from two experiments.
  • (9B) Flow cytometry analysis of CD300b and TLR4 expression on WT and Cd300b ⁇ ⁇ differentiated from the bone marrow or isolated from the peritoneal cavities or organ tissues. The dot plots shown in (9B) are representative of three experiments.
  • FIGS. 10A-10I Recognition of LPS by CD300b/TLR4 co-expression macrophages but not dendritic cells modulates TLR4-mediated cytokine responses in a CD300b/CD14 dependent manner, related to FIGS. 5 and 6.
  • 10A Flow cytometry analysis of CD300b and TLR4 expression on WT dendritic cells differentiated from the bone marrow (BMDC) or DC isolated from the peritoneal cavity.
  • BMDC bone marrow
  • Cd300b ' mice were stimulated with 2 ⁇ g/ml of LPS for various lengths of time. Cytokine levels were assessed by flow cytometry.
  • Binding was analyzed by flow cytometry and displayed as percentage of LPS binding after considering LPS- binding from WT ⁇ as 100%.
  • FIGS. 11A-11J Recognition of LPS by CD300b-expressing macrophages regulates efferocytosis and TLR4-MyD88- and TLR4-TRIF-dependent inflammatory cytokine responses, related to Figure 6.
  • 11 A-l 1C ⁇ from WT mice were co-cultured with pHrodo- labeled apoptotic cells (AC) at a ratio of 1:2 ( ⁇ : ⁇ ) in presence or absence of LPS (2 ⁇ g/ml) for various lengths of time.
  • AC pHrodo- labeled apoptotic cells
  • FIG. 12 Schematic diagram of CD300b function.
  • CD300b is a PS-receptor that under physiological conditions regulates efferocytosis in a DAP12-dependent manner (Murakami et al., 2014, Cell Death Differ 21, 1746-1757), thus maintaining cellular homeostasis (left).
  • CD300b Upon acute bacterial infection, CD300b binds LPS, and forms a complex with TLR4/CD14.
  • CD300b- dependent recruitment of DAP12, Syk and PI3K promotes the formation of a
  • CD300b/DAP12/TLR4-Syk-PI3K signaling complex results in the PI3K-mediated phosphorylation of AKT and, likely, in alteration of PtdIns(4,5)P2 levels through PtdIns(3,4,5)P3 synthesis (Kagan and Medzhitov, 2006, supra; Patel and Mohan, 2005, Immunol Res 31, 47-55) that could facilitate the dissociation of MyD88/TIRAP from the complex, leading to a reduced activation of the MEK1/2-ERK1/2-NFKB signaling cascade and lower IL-10 production.
  • CD300b plays a role in TLR4/CD14-TRIF signaling by increasing the IFN- ⁇ response. Since TRIF signaling originates subsequent to endocytosis and DAP12 facilitates TLR4 endocytosis and TRIF signaling (Zanoni et al., 2011, Cell 147, 868-880), CD300b likely also regulates endocytic signaling of the TLR4 complex (through DAP12 recruitment). Thus, CD300b links TLR4/CD 14-TRIF-IRF3 signaling and the DAP12-Syk-PI3K cascade.
  • DAP12, Syk, and PI3K are not recruited to the TLR4 complex, resulting in sustained association of MyD88/TIRAP, which leads to an elevated ERK1/2 activation, prolonged activation of NFKB and presumably AP-1, thereby promoting an enhanced IL-10 response.
  • the lack of DAP12 recruitment likely affects TLR4 internalization, resulting in a reduced activation of TRIF-IRF3 pathway. Consequently, the reduced pro-inflammatory cytokine response and elevated IL-10 levels allow for the subsequent survival from an acute infection, and excessive LPS exposure (right). Dashed grey lines indicate dampened/inhibited signaling, solid black lines indicate activated signaling, and question marks indicate putative processes or pathways.
  • FIGS. 13A-13B Efficacy assessment of an anti-CD300b antibody therapy using a preventive treatment model in acute septic mice.
  • R&D anti-CD300b
  • R&D anti-IgG control
  • LPS 37 ⁇ g/g
  • Mouse survival was monitored every 6 hours for 7 days and results of the endpoint study were graphed using a Kaplan-Meier survival plot.
  • an anti-CD300b Ab treatment according to a preventive protocol showed that administration of anti-CD300b Ab significantly prolonged the survival of septic WT mice as compared to anti-IgG isotype control antibody treated mice (bottom line).
  • B Serum cytokine concentrations measured from anti-IgG (left bar) and anti-CD300b Ab (right bar) treated WT mice.
  • Applying an anti-CD300b Ab treatment according to a preventive protocol showed that anti- CD300b Ab treated animals showed significant lower pro-inflammatory cytokine levels and increased IL-10 concentration.
  • FIGS. 14A-14B Efficacy assessment of an anti-CD300b antibody therapy using a therapeutic treatment model in acute septic mice.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • sequence Listing is submitted as an ASCII text file [Sequence_Listing, March 9, 2017, 5,986 bytes], which is incorporated by reference herein. In the accompanying sequence listing:
  • SEQ ID Nos: 1-8 are the amino acid sequence of framework regions.
  • SEQ ID Nos: 9-10 are the amino acid sequences of interleukin (IL)-10 molecules.
  • SEQ ID Nos: 11-12 are the nucleic acid sequence of primers. DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
  • the CD300 receptor family is composed of type I transmembrane proteins with a single IgV-like extracellular domain that can transmit either activating or inhibitory signals (Borrego, 2013, Blood 121, 1951-1960).
  • the orthologous mouse family has a variety of names, including CMRF-like molecules (CLM) (Borrego, 2013, supra). The human nomenclature is used herein for both species.
  • CD300b predominantly expressed on myeloid cells, contains a short intracellular tail and gains activation potential by association with the immunoreceptor tyrosine-based activating motif (ITAM)-bearing adaptor molecule, DNAX activating protein of 12 kDa (DAP12) (Yamanishi et al., 2008, Blood 111, 688-698).
  • CD300b functions as an activating receptor by recognizing outer membrane-exposed phosphatidylserine (PS) to promote the phagocytosis of apoptotic cells (AC) via the DAP12 signaling pathway (Murakami et al., 2014, supra).
  • PS outer membrane-exposed phosphatidylserine
  • CD300b cross-linking of CD300b induces the release of inflammatory cytokines from mast cells (Yamanishi et al., 2008, supra) and Cd300b ' mice were found to be less prone to LPS-induced lethal inflammation than wild-type (WT) mice (Yamanishi et al., 2012, J Immunol 189, 1773-1779).
  • LPS as a ligand for CD300b and that CD300b expression significantly enhances endotoxemia- and peritonitis-induced lethality, which correlates with an increased pro-inflammatory (TNFoc and IFNy) cytokine response and reduced levels of IL-10.
  • Syk-PI3K kinase cascade promotes the dissociation of MyD88/TIRAP from the complex and AKT phosphorylation, leading to a limited production of IL-10 via an AKT-mediated inhibition of the MEK1/2-ERK1/2-NFKB signaling cascade.
  • CD300b activates the TLR4/CD14-TRIF-IRF3 signaling pathway, resulting in enhanced IFN- ⁇ production.
  • a previously unidentified LPS-induced signaling complex (CD300b/DAP12/TLR4-Syk-PI3K) is identified that effectively amplifies both the TLR4- MyD88- and TLR4/CD14-TRIF-mediated inflammatory responses, leading to increased mortality from sepsis.
  • CD300b and DAP12 were identified as important molecules regulating the TLR4 pathway.
  • clinical intervention, targeting these molecules can be used to regulate LPS-induced TLR4 signaling, and thus treat or prevent septic shock.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects.
  • Antibody A polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope (e.g., an antigen, such as a tumor or viral antigen or a fragment thereof) on another protein of interest, such as Cd300b, PD-1 or CTLA-4.
  • an epitope e.g., an antigen, such as a tumor or viral antigen or a fragment thereof
  • an epitope e.g., an antigen, such as a tumor or viral antigen or a fragment thereof
  • an epitope e.g., an antigen, such as a tumor or viral antigen or a fragment thereof
  • an epitope e.g., an antigen, such as a tumor or viral antigen or a fragment thereof
  • an epitope e.g., an antigen, such as a tumor or viral antigen or a fragment thereof
  • immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.
  • an immunoglobulin typically has a heavy and light chain.
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains").
  • the heavy and the light chain variable regions specifically bind the antigen.
  • Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs". The extent of the framework region and CDRs has been defined (see, Kabat et al., Sequences of Proteins of
  • the Kabat database is now maintained online.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well- known schemes, including those described by Kabat et al. ("Sequences of Proteins of
  • CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located.
  • VH CDR3 is the CDR3 from the variable region of the heavy chain of the antibody in which it is found
  • VL CDRl is the CDR1 from the variable region of the light chain of the antibody in which it is found.
  • Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.
  • Heavy chain CDRs are sometimes referred to as LCDR 1 , LCDR2, and LCDR3.
  • VH refers to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.
  • VL refers to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
  • a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected, or a progeny thereof.
  • Monoclonal antibodies are produced by known methods, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas.”
  • Monoclonal antibodies include chimeric, humanized and fully human monoclonal antibodies. In some examples monoclonal antibodies are isolated from a subject. The amino acid sequences of isolated monoclonal antibodies can be determined. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988).)
  • a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic)
  • the non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical.
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No. 5,585,089).
  • a “neutralizing antibody” is an antibody that interferes with any of the biological activities of its target polypeptide, such as a CD300b polypeptide, a PD-1 polypeptide, a BTLA polypeptide, or a CTLA-4 polypeptide.
  • a neutralizing antibody can interfere with the ability of a CD300b polypeptide to induce an activity.
  • the neutralizing antibody can reduce the ability of a CD300b polypeptide to bind LPS by about 50%, about 70%, about 90% or more. Any standard assay to measure immune responses, including those described herein, may be used to assess potentially neutralizing antibodies.
  • B- and T-lymphocyte attenuator A protein also known as CD272.
  • BTLA expression is induced during activation of T cells, and BTLA remains expressed on Thl cells.
  • BTLA interacts with a B7 homolog, B7H4, and plays a role in T-Cell inhibition via interaction with tumor necrosis family receptors.
  • BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM).
  • HVEM herpes virus entry mediator
  • a specific, non-limiting BTLA amino acid sequence, and an mRNA sequence encoding BTLA is provided in GENBANK® Accession No.
  • CD300b also known as LMIR5, CD300LB, CLM-7, and IREM-3, is a 26 kDa- 32 kDa glycoprotein member of the immunoglobulin superfamily.
  • the mouse form, often called CLM-7, consists of a 140 amino acid (aa) extracellular domain (ECD) with one Ig-like V-type domain, a 21 aa transmembrane segment, and a 31 aa cytoplasmic domain (Martinez-Barriocanal et al, 2006, J Immunol 177, 2819-2830).
  • mouse CLM-7 shares 51% and 86% aa sequence identity with human and rat CLM-7, respectively.
  • the transmembrane segment contains a positively charged lysine, which enables the association of CLM-7 with DAP12, DAP10, and potentially other adaptor proteins.
  • the cytoplasmic domain of human CD300b contains a phosphorylable tyrosine motif, while that of CLM-7 does not.
  • CD300b is expressed on the surface of myeloid lineage cells (Yamanishi et al., 2008, supra;
  • CD300b forms noncovalent ds-homodimers and cis- heterodimers with other CD300 family proteins, and the composition of these dimers affects the cellular response (Martinez-Barriocanal et al, 2010, J Biol Chem 28, 41781-41794).
  • Antibody cross-linking of CD300b induces mast cell granule release and cytokine production as well as its tyrosine phosphorylation of CD300b (in humans) (Yamanishi et al. (2008) Blood 111:688).
  • CD300b recognizes phoshatidylserine (PS), a ligand exposed on the outer membrane of apoptotic cells, to regulate phagocytosis of apoptotic cells but does not, as previously suggested, directly recognize TIM1 or TIM4 (Murakami et al., 2014, supra; Yamanishi et al, 2010, J Exp Med 207, 1501-1511).
  • PS phoshatidylserine
  • CD14 A glycoprotein composed of about 356 amino acids and anchored through glycosylphosphatidylinositol (GPI) on membranes of monocytes, macrophages, dendritic cells, neutrophils and some B cells.
  • GPI glycosylphosphatidylinositol
  • Human CD14 is known as an LPS receptor for endotoxins of gram- negative bacteria (Wright et al., 1990, supra), which receives LPS from LBP (LPS binding protein), and subsequently leads to the transfer of LPS to the TLR4/MD-2 complex resulting in inflammatory cytokine production.
  • Myeloid cells like macrophages, that express membrane-bound CD 14 (hereinafter, also referred to as "mCD14”) are activated by a complex of LPS/LBP and soluble CD14 (hereinafter, also referred to as "sCD14”) to induce production of inflammatory cytokines (Hailman et al., 1996, J. Immunol. 156, 4384-4390).
  • Human CD14 includes both mCD14 and sCD14 forms.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments
  • cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • Constant amino acid substitutions are those substitutions that do not substantially affect or decrease an activity of a polypeptide, such as an antibody that binds CD300b, or any polypeptide antagonist.
  • Specific, non-limiting examples of a conservative substitution include the following examples: ginal Residue Conservative Substitutions
  • conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the polypeptide binds with the same affinity as the unsubstituted (parental) polypeptide.
  • Non-conservative substitutions are those that reduce the ability of the polypeptide.
  • a polypeptide that consists essentially of a specified amino acid sequence if it does not include any additional amino acid residues.
  • the polypeptide can include additional non-peptide components, such as labels (for example, fluorescent, radioactive, or solid particle labels), sugars or lipids.
  • additional non-peptide components such as lipids, sugars or labels.
  • Cytokine The term "cytokine” is used as a generic name for a diverse group of soluble proteins and peptides that act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Examples of cytokines include, but are not limited to, tumor necrosis factor a (TNFa), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 12 (IL-12), macrophage inflammatory protein 2 (MIP-2), KC, and interferon- ⁇ (IFN- ⁇ ).
  • TNFa tumor necrosis factor a
  • IL-6 interleukin 6
  • IL-10 interleukin 10
  • IL-12 interleukin 12
  • MIP-2 macrophage inflammatory protein 2
  • KC and interferon- ⁇
  • CTL-4 Cytotoxic T-lymphocyte-Associated Protein 4
  • CTLA4 is a member of the immunoglobulin superfamily.
  • CTLA4 is a protein receptor that functions as an immune checkpoint, and thus downregulates immune responses.
  • CTLA4 is constitutively expressed in regulatory T cells (Tregs) and is upregulated in conventional T cells after activation.
  • CLTA4 binds CD80 or CD86 on the surface of antigen-presenting cells, and is an inhibitor of T cells.
  • Specific non-limiting examples of a CTLA protein and an mRNA encoding CTLLA are disclosed, for example, in GENBANK® Accession No. NM_001037631, October 7, 2016, incorporated herein by reference.
  • Degenerate variant A polynucleotide encoding a peptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in this disclosure as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is unchanged.
  • Expression Control Sequences Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct gene reading frame to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
  • a promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see e.g., Bitter et al., 1987, Methods in Enzymology 153, 516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.
  • promoters derived from the genome of mammalian cells such as the metallothionein promoter
  • mammalian viruses such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences.
  • Gram-Negative Bacteria Those bacteria having a plurality of exterior membranes, a distinctive outer membrane component of which is lipopolysaccharide (LPS) capable of binding to CD300b, and a mammalian host's native CD14 receptors, thereby inducing disease etiology and symptoms characteristic of microbe infection.
  • Typical gram-negative species include but are not limited to those most commonly associated with sepsis and septic shock in humans, e.g., as reported in the HANDBOOK OF ENDOTOXINS, 1 : 187-214, eds. R. Proctor and E. Rietschel, Elsevier, Amsterdam (1984).
  • Septic shock is commonly caused by gram- negative endotoxin-producing bacteria such as Escherichia coli, Klebsiella pneumoniae, Proteus species, Pseudomonas aeruginosa and Salmonella.
  • Gram-Positive Bacteria Bacteria characterized by a preponderance of peptidoglycans relative to LPS molecules in their membranes, which are capable of inducing disease etiology and symptoms characteristic of microbe infection, similar to those described for gram- negative species.
  • Heterologous Originating from separate genetic sources or species.
  • a polypeptide that is heterologous is derived from a different cell or tissue type, or a different species from the recipient, and is cloned into a cell that normally does not express that polypeptide.
  • mouse (or human) CD300b cloned in a fibroblast cell line that does not express CD300b generates a heterologous CD300b protein.
  • an antibody that specifically binds to a protein of interest will not specifically bind to a heterologous protein.
  • Host cells Cells in which a vector can be propagated and its DNA expressed.
  • the cell may be prokaryotic or eukaryotic.
  • the cell can be mammalian, such as a human cell.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.
  • Immune response A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • the response is specific for a particular antigen (an "antigen-specific response").
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • the response is a B cell response, and results in the production of specific antibodies.
  • Inhibiting or treating a disease Inhibiting a disease, such as septic shock, refers to inhibiting the full development of a disease.
  • inhibiting a disease refers to lessening symptoms of septic shock, such as preventing the development of multi-organ failure or circulatory failure in a person who is known to be septic, or lessening a sign or symptom of septic shock, such as fever.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the disease, such as septic shock, such as reducing fever or stabilizing blood pressure in a subject with septic shock.
  • Isolated An "isolated" biological component (such as a nucleic acid, antibody, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule.
  • Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • Linker sequence is an amino acid sequence that covalently links two polypeptides or fragments thereof, or polypeptide and an effector molecule, usually in order to provide freedom of movement to the linked components.
  • linker sequences can be inserted between them.
  • Linker sequences which are generally between 2 and 25 amino acids in length, are well known in the art and include, but are not limited to, the glycine(4)- serine spacer (GGGGS x3) described by
  • Lymphocytes A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects, such as primates (e.g., baboons), cats, dogs, cows, sheep, horses, and rodents (e.g., mice and rats).
  • primates e.g., baboons
  • cats dogs, cows, sheep, horses
  • rodents e.g., mice and rats
  • Oligonucleotide A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
  • ORF Open reading frame
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence, such as a sequence that encodes a polypeptide.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • the disclosed peptides include synthetic embodiments of peptides described herein.
  • analogs non-peptide organic molecules
  • derivatives chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences
  • variants homologs
  • Each polypeptide of this disclosure is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.
  • Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein can be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR1R2 wherein Ri and R2 are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring.
  • Amino groups of the peptide can be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or can be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.
  • a pharmaceutically-acceptable acid addition salt such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts
  • Hydroxyl groups of the peptide side chains may be converted to C1-C16 alkoxy or to a Ci- Ci6 ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C 2 - C 4 alkylenes.
  • Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • Peptidomimetic and organomimetic embodiments are envisioned, whereby the three- dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of a polypeptide having measurable or enhanced ability to generate an immune response.
  • a pharmacophore is an idealized three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD).
  • compositions and formulations suitable for pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions such as powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • a “therapeutically effective amount” is a quantity of a composition or a cell to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit septic shock, reduce fever, or prevent multi-organ failure in a subject infected with a gram-negative bacteria.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve an in vitro effect.
  • Polynucleotide refers to a polymeric form of nucleotide at least 10 bases in length.
  • a recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • the term includes single- and double- stranded forms of DNA.
  • Polypeptide Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
  • a polypeptide can be between 5 and 25 amino acids in length. In one embodiment, a polypeptide is from about 10 to about 20 amino acids in length. In yet another embodiment, a polypeptide is from about 11 to about 18 amino acids in length. With regard to polypeptides, the word “about” indicates integer amounts. Thus, in one example, a polypeptide "about” 11 amino acids in length is from 10 to 12 amino acids in length. Similarly, a polypeptide "about” 18 amino acids in length is from about 17 to about 19 amino acids in length.
  • a polypeptide "about" a specified number of residues can be one amino acid shorter or one amino acid longer than the specified number.
  • a fusion polypeptide includes the amino acid sequence of a first polypeptide and a second different polypeptide (for example, a heterologous polypeptide), and can be synthesized as a single amino acid sequence.
  • Preventing or treating a disease refers to inhibiting the partial or full development of a disease, for example sepsis or septic shock, such as in a person with a bacterial infection or at risk of a bacterial infection.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as sepsis or septic shock, after it has begun to develop.
  • treatment refers to ameliorating symptoms such as, but not limited to, ague, sweating, fever, and changes in blood pressure, or increasing organ function.
  • a probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule.
  • Primers are short nucleic acids, preferably DNA oligonucleotides, of about 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example by polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides.
  • probes and primers can be selected that comprise about 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
  • PD- 1 molecules are members of the immunoglobulin gene superfamily.
  • the human PD-1 has an extracellular region containing an immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region including an
  • immunoreceptor tyrosine-based inhibitory motif (Ishida et al, EMBO J. 11:3887, 1992; Shinohara et al, Genomics 23:704, 1994; U.S. Patent No. 5,698,520,incorporated herein by reference). These features also define a larger family of molecules, called the immunoinhibitory receptors, which also includes gp49B, PIR-B, and the killer inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol. Today 18:286). Without being bound by theory, it is believed that the tyrosyl phosphorylated ⁇ motif of these receptors interacts with the S112-domain containing phosphatase, which leads to inhibitory signals.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • PD-1 is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line undergoing activation-induced apoptosis. PD-1 is expressed on T cells, B cells, and macrophages.
  • the ligands for PD-1 are the B7 family members PD-ligand 1 (PD-Ll, also known as B7-H1) and PD-L2 (also known as B7-DC).
  • PD-1 is expressed on activated T cells, B cells, and monocytes.
  • Experimental data implicates the interactions of PD- 1 with its ligands in down regulation of central and peripheral immune responses.
  • proliferation in wild-type T cells but not in PD-1 -deficient T cells is inhibited in the presence of PD-Ll.
  • PD-1 -deficient mice exhibit an autoimmune phenotype.
  • An exemplary amino acid sequence of human PD-1 is set forth in Ishida et al., EMBO J. 11:3887, 1992; Shinohara et al. Genomics 23:704,1994; U.S. Pat. No. 5,698,520):
  • PD-1 binds two ligands, PD-Ll and PD-L2, both of which are human PD-1 ligand polypeptides, that are members of the B7 family of polypeptides.
  • PD-1 antagonists include agents that reduce the expression or activity of a PD ligand 1 (PD- Ll) or a PD ligand 2 (PD-L2) or reduces the interactions between PD-1 and PD-Ll or PD-L2.
  • exemplary compounds include antibodies (such as an anti-PD-1 antibody, an anti-PD-Ll antibody, and an anti-PD-L2 antibody), RNAi molecules (such as anti-PD-1 RNAi molecules, anti-PD-Ll RNAi, and an anti-PD-L2 RNAi), antisense molecules (such as an anti-PD-1 antisense RNA, an anti-PD-Ll antisense RNA, and an anti-PD-L2 antisense RNA), dominant negative proteins (such as a dominant negative PD-1 protein, a dominant negative PD-Ll protein, and a dominant negative PD-L2 protein), see, for example, PCT Publication No. 2008/083174, incorporated herein by reference.
  • polypeptides disclosed herein can be purified (and/or synthesized) by any of the means known in the art (see, e.g., Guide to Protein Purification, ed. Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982).
  • Substantial purification denotes purification from other proteins or cellular components.
  • a substantially purified protein is at least about 60%, 70%, 80%, 90%, 95%, 98% or 99% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.
  • a purified nucleic acid is one in which the nucleic acid is more enriched than the nucleic acid in its natural environment within a cell.
  • a nucleic acid or cell preparation is purified such that the nucleic acid or cell represents at least about 60% (such as, but not limited to, 70%, 80%, 90%, 95%, 98% or 99%) of the total nucleic acid or cell content of the preparation, respectively.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of at least two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant polypeptide has an amino acid sequence that is not naturally occurring or that is made by two otherwise separated segments of an amino acid sequence.
  • nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, GC v. AT content), and nucleic acid type (for example, RNA versus DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
  • Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed.
  • optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
  • NCBI National Center for Biotechnology Information
  • blastp blastn
  • blastx blastx
  • tblastn tblastx
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • Septic shock or sepsis Sepsis is a disease which has infectious cause and which shows the pathology of systemic inflammatory response syndrome (SIRS). Initial symptoms found include ague, sweating, fever, and decrease in the blood pressure. When various inflammatory mediators and blood coagulation factors increase in the whole body, disturbance in the microcirculation occurs, and this results in the worsening of the pathological conditions; septic shock includes organ perfusion abnormalities, and multiple organ dysfunction, which can result in death.
  • SIRS systemic inflammatory response syndrome
  • LPS gram-negative bacteria and lipoteichoic acid
  • gram-positive bacteria for example, LPS of gram-negative bacteria and lipoteichoic acid (LTA) in gram-positive bacteria, that are recognized by leukocytes (monocytes/macrophages and neutrophils) or vascular endothelial cells, which in turn causes production of various inflammatory mediators.
  • LTA lipoteichoic acid
  • CD14 and TLRs which are pattern recognition molecules in the innate immune system play an important role in such activation of the target cell by the bacterial constituent components.
  • endotoxic shock Septic shock involved with gram-negative bacteria.
  • endotoxic shock A significant portion of the peripheral responses occurring during septic shock are initiated by endotoxin (also referred to herein as "LPS"), an outer- membrane component of gram- negative bacteria which is released upon the death or multiplication of the bacteria.
  • endotoxin also referred to herein as "LPS”
  • LPS endotoxin
  • septic shock refers to septic shock involved with either gram-negative and/or gram-positive bacteria.
  • Therapeutically effective amount A quantity of a specific substance, such as a CD300b antagonist, sufficient to achieve a desired effect in a subject being treated.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in bone) that has been shown to achieve a desired effect, such as preventing or treating sepsis.
  • transduced A transduced cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transduction encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • TLR Toll-like Receptors
  • TLRs recognize conserved motifs found in various pathogens and mediate defense responses. Triggering of the TLR pathway leads to the activation of NF- ⁇ and subsequent regulation of immune and inflammatory genes.
  • the TLRs and members of the interleukin (IL)-1 receptor family share a conserved stretch of about 200 amino acids known as the TIR domain.
  • TLRs associate with a number of cytoplasmic adaptor proteins containing TIR domains including MyD88 (myeloid differentiation factor), MAL/TIRAP (MyD88-adaptor- like/TIR-associated protein), TRIF (Toll-receptor-associated activator of interferon) and TRAM (Toll-receptor associated molecule).
  • TLRs are 4- and 6-kb transcripts that are most abundant in placenta and pancreas.
  • TLR activity includes activation of NFKB. Activation of TLRs can result in increased production of TNFa, IL- ⁇ , IL-6, IL-8, IL-12, RANTES, MIP-la, and ⁇ -1 ⁇ .
  • TLR4 recognizes LPS, which is a glycolipid located in the outer membrane of gram- negative bacteria. In macrophages, LPS is transferred to the TLR4-MD2 complex by LBP and CD 14. LPS binding induces the formation of a receptor multimer composed of two copies of the TLR4-MD2-LPS complex. TLR4 dimerization leads to the subsequent recruitment of the adapter proteins MyD88 and TRIF (Toll/interleukin-1 receptor domain containing adaptor protein inducing interferon ⁇ ). The latter mediates activation of interferon regulatory factor (IRF) 3 and IRF7, leading to enhanced expression of interferons.
  • IRF interferon regulatory factor
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker gene and other genetic elements known in the art.
  • Vectors include plasmid vectors, including plasmids for expression in gram-negative and gram-positive bacterial cell. Exemplary vectors include those for expression in E. coli and Salmonella.
  • Vectors also include viral vectors, such as, but are not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors. Vectors also include vectors for expression in yeast cells.
  • CD300b antagonists are of use in treating, preventing, or delaying the development of, sepsis and septic shock.
  • the CD300b antagonist can be, for example, a soluble receptor, an antibody that specifically binds CD300b, or an inhibitor nucleic acid molecule (RNAi), such as, but not limited to, an siRNA or an shRNA.
  • RNAi inhibitor nucleic acid molecule
  • the CD33b antagonist can inhibit the interaction of CD300b with LPS, CD14 and/or TLR4.
  • the CD300b antagonist can be an antibody, such as an antibody.
  • Antibodies that specifically bind CD300b are commercially available.
  • An exemplary nucleic acid sequence encoding human CD300b is provided in GENBANK® Accession No. NM_174892.3 (January 5, 2016), and an exemplary amino acid sequence of human CD300b is provided in GENBANK® Accession No. NP_777552.3 (January 5, 2016), which are both incorporated by reference herein.
  • polyclonal mouse antibodies to human CD300b are available from R&D.
  • Mouse monoclonal antibodies are available from R&D systems, MAB2580 and Novus Biologicals, Antibody
  • Antibodies that specifically bind CD300b are of use in the methods disclosed herein.
  • Antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies, and immunoglobulin (Ig) fusion proteins.
  • Polyclonal anti-CD300b antibodies can be prepared by one of skill in the art, such as by immunizing a suitable subject (such as a veterinary subject) with a
  • CD300b immunogen The anti-CD300b antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized CD300b polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • CD300b interacts with LPS, CD14, and/or TLR4.
  • an antibody of use specifically binds CD300b and inhibits the interaction of CD300b with LPS, CD14 and/or TLR4.
  • the antibody molecules that specifically bind CD300b can be isolated from a mammal (such as from serum) and further purified by techniques known to one of skill in the art. For example, antibodies can be purified using protein A chromatography to isolate IgG antibodies.
  • Antibody-producing cells can be obtained from a subject and used to prepare monoclonal antibodies by standard techniques (see Kohler and Milstein Nature 256:495 49, 1995; Brown et al., J. Immunol. 127:539 46, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985; Gefter, M. L. et al. (1977) Somatic Cell Genet. 3:231 36; Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, N.Y. (1980); Kozbor et al. Immunol.
  • an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with CD300b, and the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that specifically binds to the polypeptide of interest.
  • an immortal cell line (such as a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with a CD300b peptide with an immortalized mouse cell line.
  • a mouse myeloma cell line is utilized that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • myeloma cell lines can be used as a fusion partner according to standard techniques, including, for example, P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines, which are available from the American Type Culture Collection (ATCC),
  • ATCC American Type Culture Collection
  • HAT-sensitive mouse myeloma cells can be fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused (and unproductively fused) myeloma cells. Hybridoma cells producing a monoclonal antibody of interest can be detected, for example, by screening the hybridoma culture supematants for the production antibodies that bind a CD300b polypeptide, such as by using an immunological assay (such as an enzyme-linked immunosorbant assay (ELISA) or radioimmunoassay (RIA).
  • an immunological assay such as an enzyme-linked immunosorbant assay (ELISA) or radioimmunoassay (RIA).
  • a monoclonal antibody that specifically binds CD300b can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (such as an antibody phage display library) with CD300b to isolate immunoglobulin library members that specifically bind the polypeptide.
  • Library members can be selected that have particular activities, such as inhibiting the interaction of CD300b with LPS, CD 14 and/or TLR4. Kits for generating and screening phage display libraries are
  • the sequence of the specificity determining regions of each CDR is determined. Residues outside the SDR (specificity determining region, e.g., the non-ligand contacting sites) are substituted. For example, in any of the CDR sequences, at most one, two or three amino acids can be substituted.
  • the production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well known in the art. For example, humanized antibodies can be routinely produced.
  • the antibody or antibody fragment can be a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor monoclonal antibody that binds CD300b, and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks.
  • CDRs complementarity determining regions
  • the humanized immunoglobulin specifically binds to CD300b with an affinity constant of at least 10 7 M "1 , such as at least 10 8 M "1 at least 5 X 10 8 M "1 or at least 10 9 M 1 .
  • the antibody specifically binds CD300b with an affinity constant of at least 10 8 M 1 at least 5 X 10 8 M 1 or at least 10 9 M "1 .
  • Humanized monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from heavy and light variable chains of the donor mouse
  • the antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgGi, IgG2, IgG3 and IgG 4 .
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
  • Human framework regions, and mutations that can be made in humanized antibody framework regions, are known in the art (see, for example, in U.S. Patent No. 5,585,089, which is incorporated herein by reference).
  • Exemplary human antibodies are LEN and 21/28 CL.
  • the sequences of the heavy and light chain frameworks are known in the art.
  • Exemplary light chain frameworks of human MAb LEN have the following sequences: FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 1)
  • FR3 GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 3)
  • FR4 FGQGQTKLEIK (SEQ ID NO: 4)
  • Exemplary heavy chain frameworks of human MAb 21/28' CL have the following sequences:
  • RVTITRDTSASTAYMELSSLRSEDTAVYYCAR SEQ ID NO: 7
  • FR4 WGQGTLVTVSS (SEQ ID NO: 8).
  • Antibodies such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which include a heavy chain and light chain variable region and are capable of binding specific epitope determinants. These antibody fragments retain some ability to selectively bind with their antigen or receptor. These fragments include:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • Fv a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody such as scFv
  • scFv Single chain antibody
  • scFv single chain antibody
  • a suitable polypeptide linker as a genetically fused single chain molecule.
  • Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).
  • the variable region includes the variable region of the light chain and the variable region of the heavy chain expressed as individual polypeptides.
  • Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain.
  • the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. If the VH and the VL are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker.
  • the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
  • the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing scFvs are known in the art (see Whitlow et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991 ; Bird et al, Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al,
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No.
  • conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the VH and the VL regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
  • amino acid sequence of an antibody of interest locate one or more of the amino acids in the brief table above, identify a conservative substitution, and produce the conservative variant using well-known molecular techniques.
  • Effector molecules such as therapeutic, diagnostic, or detection moieties can be linked to an antibody that specifically binds CD300b, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used.
  • the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, IL.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • Nucleic acid sequences encoding the antibodies can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979; the
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • nucleic acids encoding sequences encoding an antibody that specifically binds
  • CD300b can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.
  • Nucleic acids can also be prepared by amplification methods.
  • Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustained sequence replication system
  • an antibody of use is prepared by inserting the cDNA, which encodes a variable region from an antibody that specifically binds CD300b, into a vector which comprises the cDNA encoding an effector molecule (EM). The insertion is made so that the variable region and the EM are read in frame so that one continuous polypeptide is produced.
  • the encoded polypeptide contains a functional Fv region and a functional EM region.
  • cDNA encoding a detectable marker (such as an enzyme) is ligated to a scFv so that the marker is located at the carboxyl terminus of the scFv.
  • a detectable marker is located at the amino terminus of the scFv.
  • cDNA encoding a detectable marker is ligated to a heavy chain variable region of an antibody that specifically binds CD300b, so that the marker is located at the carboxyl terminus of the heavy chain variable region.
  • the heavy chain- variable region can subsequently be ligated to a light chain variable region of the antibody that specifically binds CD300b using disulfide bonds.
  • cDNA encoding a marker is ligated to a light chain variable region of an antibody that binds CD300b, so that the marker is located at the carboxyl terminus of the light chain variable region.
  • the light chain- variable region can subsequently be ligated to a heavy chain variable region of the antibody that specifically binds CD300b using disulfide bonds.
  • the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells.
  • a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells.
  • One or more DNA sequences encoding the antibody or functional fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • Polynucleotide sequences encoding the antibody or functional fragment thereof can be operatively linked to expression control sequences.
  • An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
  • the expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • the polynucleotide sequences encoding the antibody or functional fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCh method using procedures well known in the art.
  • MgC can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody of functional fragment thereof and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Isolation and purification of a recombinantly expressed polypeptide can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the recombinant antibodies can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes.
  • the polypeptides should be substantially free of endotoxin.
  • Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies disclosed herein. See, Buchner et al., Anal.
  • a reducing agent must be present to separate disulfide bonds.
  • An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).
  • Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, incorporated by reference herein, and especially as described by Buchner et al., supra. Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer.
  • An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L- arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
  • the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution.
  • An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.
  • the antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis.
  • Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc.
  • Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicycylohexylcarbodimide) are well known in the art.
  • Inhibitory nucleic acids that decrease the expression and/or activity of CD300b can also be used in the methods disclosed herein.
  • One embodiment is a small inhibitory RNA (siRNA) for interference or inhibition of expression of a target.
  • siRNA small inhibitory RNA
  • Nucleic acid sequences encoding CD300b are disclosed in GENBANK® Accession No. NM_174892.3 and NM_199221.2 for human and mouse CD300b, respectively, which are both incorporated herein by reference.
  • siRNAs are generated by the cleavage of relatively long double-stranded RNA molecules by Dicer or DCL enzymes (Zamore, Science, 296: 1265-1269, 2002; Bernstein et al.,
  • siRNAs are assembled into RISC and guide the sequence specific ribonucleolytic activity of RISC, thereby resulting in the cleavage of mRNAs or other RNA target molecules in the cytoplasm.
  • siRNAs also guide heterochromatin- associated histone and DNA methylation, resulting in transcriptional silencing of individual genes or large chromatin domains.
  • CD300b siRNAs are commercially available, such as from Santa Cruz Biotechnology, Inc.
  • RNA suitable for interference or inhibition of expression of a target gene which RNA includes double stranded RNA of about 19 to about 40 nucleotides with the sequence that is substantially identical to a portion of an mRNA or transcript of a target gene, such as CD300b, for which interference or inhibition of expression is desired.
  • a sequence of the RNA "substantially identical" to a specific portion of the mRNA or transcript of the target gene for which interference or inhibition of expression is desired differs by no more than about 30 percent, and in some embodiments no more than about 10 percent, from the specific portion of the mRNA or transcript of the target gene.
  • the sequence of the RNA is exactly identical to a specific portion of the mRNA or transcript of the target gene.
  • siRNAs disclosed herein include double- stranded RNA of about 15 to about 40 nucleotides in length and a 3' or 5' overhang having a length of 0 to 5-nucleotides on each strand, wherein the sequence of the double stranded RNA is substantially identical to (see above) a portion of a mRNA or transcript of a nucleic acid encoding CD300b.
  • the double stranded RNA contains about 19 to about 25 nucleotides, for instance 20, 21, or 22 nucleotides substantially identical to a nucleic acid encoding CD300b.
  • the double stranded RNA contains about 19 to about 25 nucleotides 100% identical to a nucleic acid encoding CD300b. It should be not that in this context "about” refers to integer amounts only. In one example, "about” 20 nucleotides refers to a nucleotide of 19 to 21 nucleotides in length.
  • the length of the overhang is independent between the two strands, in that the length of one overhang is not dependent on the length of the overhang on other strand.
  • the length of the 3' or 5' overhang is 0-nucleotide on at least one strand, and in some cases it is 0-nucleotide on both strands (thus, a blunt dsRNA).
  • the length of the 3' or 5' overhang is 1 -nucleotide to 5- nucleotides on at least one strand.
  • the length of the 3 ' or 5 ' overhang is 2-nucleotides on at least one strand, or 2-nucleotides on both strands.
  • the dsRNA molecule has 3' overhangs of 2-nucleotides on both strands.
  • the double- stranded RNA contains 20, 21, or 22 nucleotides, and the length of the 3' overhang is 2-nucleotides on both strands.
  • the double-stranded RNA contains about 40-60% adenine+uracil (AU) and about 60-40% guanine+cytosine (GC). More particularly, in specific examples the double- stranded RNA contains about 50% AU and about 50% GC.
  • RNAs that further include at least one modified ribonucleotide, for instance in the sense strand of the double- stranded RNA.
  • the modified ribonucleotide is in the 3' overhang of at least one strand, or more particularly in the 3' overhang of the sense strand.
  • examples of modified ribonucleotides include ribonucleotides that include a detectable label (for instance, a fluorophore, such as rhodamine or
  • FITC a thiophosphate nucleotide analog
  • a deoxynucleotide (considered modified because the base molecule is ribonucleic acid), a 2'-fluorouracil, a 2'-aminouracil, a 2'-aminocytidine, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, an inosine, or a 2'0-Me-nucleotide analog.
  • Antisense and ribozyme molecules for CD300b are also of use in the method disclosed herein.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate an mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell producing CD300b.
  • the use of antisense methods to inhibit the in vitro translation of genes is well known in the art (see, for example, Marcus-Sakura, Anal. Biochem. 172:289, 1988).
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridin- e, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, amongst others.
  • triplex strategy Use of an oligonucleotide to stall transcription is known as the triplex strategy where an oligonucleotide winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al. , Antisense Res. and Dev. 1(3):227, 1991 ; Helene, C, Anticancer Drug Design 6(6):569), 1991. This type of inhibitory oligonucleotide is also of use in the methods disclosed herein.
  • Ribozymes which are RNA molecules possessing the ability to specifically cleave other single- stranded RNA in a manner analogous to DNA restriction endonucleases, are also of use. Through the modification of nucleotide sequences, which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, /. Amer. Med. Assn. 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • ribozymes There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature 334:585, 1988) and "hammerhead"-type. Tetrahymena-type, ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type, ribozymes for inactivating a specific mRNA species and 18-base recognition sequences are preferable to shorter recognition sequences.
  • RNA delivery systems are known and can be used to administer the siRNAs and other inhibitory nucleic acid molecules as therapeutics.
  • Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, nanoparticles, recombinant cells capable of expressing the therapeutic molecule(s) (see, e.g., Wu et al, J. Biol. Chem. 262, 4429, 1987), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like.
  • CD300b antagonists include molecules that are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art.
  • the screening methods that detect decreases in CD300b activity are useful for identifying compounds from a variety of sources for activity.
  • the initial screens may be performed using a diverse library of compounds, a variety of other compounds and compound libraries.
  • molecules that bind CD300b molecules that inhibit the expression of CD300b, and molecules that inhibit the activity of CD300b can be identified.
  • These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries.
  • CD300b antagonist can be identified as compounds from commercial sources, as well as commercially available analogs of identified inhibitors.
  • the small molecule can be, for examples, less than 900 daltons or less than 800 daltons.
  • CD300b antagonists can be identified from virtually any number of chemical extracts or compounds.
  • examples of such extracts or compounds that can be CD300b antagonists include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). CD300b antagonists can be identified from synthetic compound libraries that are commercially available from a number of companies including
  • CD300b antagonists can be identified from a rare chemical library, such as the library that is available from Aldrich
  • CD300b antagonists can be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
  • Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, such as less than about 750 or less than about 350 daltons can be utilized in the methods disclosed herein. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. In several embodiments, compounds of use has a Kd for CD300b of less than InM, less than lOnM, less than 1 ⁇ , less than 10 ⁇ , or less than lmM.
  • Interleukin (IL)-10 Interleukin (IL)-10
  • IL-10 is an immunosuppressive cytokine that suppresses release and function of
  • cytokines such as IL-1, IL-2, IL-6, TNF-a, and granulocyte macrophage colony stimulating factor (GM-CSF) (Williams et al. (2004) Immunology 113:281-92).
  • ⁇ -10 acts as a normal endogenous feedback system to control immune responses and inflammation.
  • IL-10 also acts as a chemotactic factor towards CD8+T cells, and is able to inhibit antigen-specific T cell proliferation.
  • Some of the activities of IL-10 require different portions of the protein sequence (e.g. C-terminus vs. N-terminus, Gesser et al. (1997) Proc Natl Acad Sci USA. 94:14620-5).
  • Th cells T helper cells
  • Th cells can be divided into different subsets that are distinguished by their cytokine production profiles.
  • Thl T cell clones produce IL-2 and IFNy whereas Th2 cell clones secrete IL-10, IL-4, and IL-5, generally following activation by antigens or mitogenic lectins.
  • Both classes of Th cell clones produce cytokines such as TNF-a, IL-3, and GM-CSF.
  • ThO A third category of Th cells (ThO) produces IL-2, IFNy, IL-4, IL-5, TNFa, IL-3, and GM-CSF simultaneously.
  • IL-10 suitable for use in the disclosed methods can be obtained from a number of sources. For example, it can be isolated from culture media of activated T-cells capable of secreting the protein. Additionally, the polypeptide or active fragments thereof can be chemically synthesized using standard techniques as known in the art. See, e.g., Merrifield, Science 233:341-47 (1986) and Atherton et al., Solid Phase Peptide Synthesis, a Practical Approach, I.R.L. Press, Oxford (1989).
  • IL-10 is obtained by recombinant techniques using isolated nucleic acids encoding for the IL-10 polypeptide.
  • the appropriate sequences can be obtained using standard techniques from either genomic or cDNA libraries. Libraries are constructed from nucleic acid extracted from the appropriate cells. See, e.g., PCT Publication No.
  • WO 91/00349 discloses recombinant methods to make IL-10.
  • Useful gene sequences can be found, e.g., in various sequence databases, e.g., GENBANK® and EMBL for nucleic acid, and PIR and Swiss-Prot for protein, c/o Intelligenetics, Mountain View, Calif.; or the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis.
  • Clones comprising sequences that encode human- IL-10 have been deposited by others with the American Type Culture Collection (ATCC), Rockville, Md. under the Accession Numbers 68191 and 68192. Identification of clones harboring the sequences encoding IL-10 is performed by either nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used. Oligonucleotide probes based on the deposited sequences disclosed in PCT Publication No. WO 91/00349 are particularly useful.
  • the molecule includes a phenylalanine at position 129 of the rat IL-10 precursor protein is replaced with a serine ("F129S”), see Published U.S. Patent Application No. 2009/0035256, incorporated herein by reference.
  • F129S serine
  • An exemplary amino acid sequence of human IL-10 is:
  • Addition IL-10 variants are of use, see for example, NCBI accession numbers NM012854, L02926, X60675 (rat) and NM000572, U63015, AF418271, AF247603, AF247604, AF247606, AF247605, AY029171, UL16720 (human), all as available on February 29, 2016, which are incorporated herein by reference.
  • IL-10 fusion proteins are also of use in the disclosed methods, see for example U.S.
  • the protein has a molecular weight of at least 10 kD; a net neutral charge at pH 6.8; a globular tertiary structure; human origin; and no ability to bind to surface receptors other than a receptor for the cytokine (e.g., the IL-10 receptor).
  • the enzymatically inactive polypeptide is IgG
  • the IgG portion can be glycosylated.
  • the enzymatically inactive polypeptide can include an IgG hinge region positioned such that the chimeric protein has IL-10 bonded to an IgG hinge region with the hinge region bonded to a longevity-increasing polypeptide.
  • the hinge region can serve as a spacer between the cytokine and the longevity- increasing polypeptide.
  • These molecules can be produced from a hybridoma (e.g., HB129) or other eukaryotic cells or baculovirus systems.
  • a flexible polypeptide spacer as defined herein, can be used. Using conventional molecular biology techniques, such a polypeptide can be inserted between the cytokine and the longevity-increasing polypeptide.
  • the Fc region can be mutated, if desired, to inhibit its ability to fix complement and bind the Fc receptor with high affinity.
  • substitution of Ala residues for Glu 318, Lys 320, and Lys 322 renders the protein unable to direct antibody dependent complement mediated cell lysis (ADCC).
  • Substitution of Glu for Leu 235 inhibits the ability of the protein to bind the Fc receptor with high affinity.
  • Appropriate mutations for human IgG also are known (see, e.g., Morrison et al., 1994, The Immunologist 2: 119-124 and Brekke et al., 1994, The Immunologist 2: 125).
  • Other mutations can also be used to inhibit these activities of the protein, and art-recognized methods can be used to assay for the ability of the protein to fix complement or bind the Fc receptor.
  • albumin e.g., human serum albumin
  • transferrin enzymes such as t-PA which have been inactivated by mutations
  • other proteins with a long circulating half-life and without enzymatic activity in humans.
  • the enzymatically inactive polypeptide used in the production of the fusion protein has, by itself, an in vivo circulating half-life greater than that of IL-10.
  • the half-life of the chimeric protein is at least 2 times that of IL-10 alone.
  • the half-life of the chimeric protein is at least 10 times that of IL-10 alone.
  • the circulating half-life of the fusion protein can be measured by an ELISA in a sample of serum obtained from a patient treated with the chimeric protein.
  • antibodies directed against the cytokine can be used as the capture antibodies, and antibodies directed against the enzymatically inactive protein can be used as the detection antibodies (or vice versa), allowing detection of only the chimeric protein in a sample.
  • Conventional methods for performing ELISAs can be used, and a detailed example of such an ELISA is provided herein.
  • a dosage of 0.01 mg/kg to 500 mg/kg body weight is sufficient for the treatment of sepsis or septic shock. In particular examples, the dosage is 10 ⁇ g/kg to 100 ⁇ g kg. Treatment is begun with the diagnosis or suspicion of septicemia or endotoxemia and is repeated at 12-hour intervals until stabilization of the subject's condition is achieved, on the basis of the observation that serum TNFa levels are undetectable by ELISA.
  • PEGylated IL-10 is also of use in the disclosed methods, see U.S. Published Patent
  • PEG Polyethylene glycol
  • pegylate means to attach at least one PEG molecule to another molecule, e.g. IL-10.
  • the attachment of polyethylene glycol has been shown to protect against proteolysis (see, e.g., Sada, et al., (1991) J. Fermentation Bioengineering 71:137-139).
  • PEG is a linear or branched polyether terminated with hydroxyl groups and having the general structure:
  • PEG polypeptides, polysaccharides, polynucleotides, and small organic molecules
  • PEG polypeptides, polysaccharides, polynucleotides, and small organic molecules
  • the most common route for PEG conjugation of proteins, such as IL-10 has been to activate the PEG with functional groups suitable for reaction with lysine and N-terminal amino acid groups.
  • the most common reactive groups involved in coupling of PEG to polypeptides are the alpha or epsilon amino groups of lysine.
  • the reaction of a PEGylation linker with a protein leads to the attachment of the PEG moiety predominantly at the following sites: the alpha amino group at the N- terminus of the protein, the epsilon amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. Since most recombinant proteins possess a single alpha and a number of epsilon amino and imidazloe groups, numerous positional isomers can be generated depending on the linker chemistry.
  • mPEGs Two widely used first generation activated monomethoxy PEGs (mPEGs) were succinimdyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15: 100-114; and Miron and Wilcheck (1993) Bioconjug. Chem. 4:568-569) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence, et al. U.S. Pat. No. 5,650,234), which react preferentially with lysine residues to form a carbamate linkage, but are also known to react with histidine and tyrosine residues.
  • the linkage to histidine residues on IFN-a has been shown to be a hydrolytically unstable imidazolecarbamate linkage (see, e.g., Lee and McNemar, U.S. Pat. No. 5,
  • Second generation PEGylation technology has been designed to avoid these unstable linkages as well as the lack of selectivity in residue reactivity.
  • Use of a PEG-aldehyde linker targets a single site on the N-terminus of a polypeptide through reductive animation.
  • IL-10 may be PEGylated using different types of linkers and pH to arrive at a various forms of a PEGylated molecule (see, e.g., U.S. Pat. No. 5,252,714, U.S. Pat. No. 5,643,575, U.S. Pat. No. 5,919,455, U.S. Pat. No. 5,932,462, U.S. Pat. No. 5,985,263, U.S. Pat. No. 7,052,686). These forms are of use in the disclosed methods.
  • Standard transfection methods can be used to produce prokaryotic, mammalian, yeast or insect cell lines, which express large quantities of any protein, including IL-10.
  • Exemplary E. coli strains suitable for both expression and cloning include W3110 (ATCC No. 27325), JA221, C600, ED767, DH1, LE392, HB 101, X1776 (ATCC No. 31244), X2282, RR1 (ATCC No. 31343).
  • Exemplary mammalian cell lines include COS-7 cells, mouse L cells and CHO cells.
  • Various expression vectors can be used to express the nucleic acid sequence encoding IL- 10, or a fragment, variant or fusion thereof.
  • Conventional vectors used for expression of recombinant proteins in prokaryotic or eukaryotic cells may be used.
  • Vectors include the pcD vectors described in Okayama et al., Mol. Cell. Biol., 3:280-289 (1983); and Takebe et al., Mol. Cell. Biol., 466-472 (1988).
  • Other SV40-based mammalian expression vectors include those disclosed in Kaufman et al., Mol. Cell. Biol., 2: 1304-1319 (1982) and U.S. Pat. No. 4,675,285, both of which are incorporated herein by reference.
  • SV40-based vectors are particularly useful in COS7 monkey cells (ATCC No. CRL 1651), as well as other mammalian cells such as mouse L cells. See also, Pouwels et al. (1989 and supplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.
  • Peptides can be expressed in soluble form such as a secreted product of a transformed yeast or mammalian cell.
  • the peptide can be purified according to standard procedures well known in the art. For example, purification steps could include ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and the like.
  • IL-10 may be expressed in insoluble form such as aggregates or inclusion bodies.
  • These proteins are purified as described herein, or by standard procedures known in the art. Examples of purification steps include separating the inclusion bodies from disrupted host cells by centrifugation, solubilizing the inclusion bodies with chaotropic agents and reducing agents, diluting the solubilized mixture, and lowering the concentration of chaotropic agent and reducing agent so that the protein assumes a biologically active conformation.
  • IL-10 polypeptides can be modified according to standard techniques to yield IL-10 polypeptides or fragments thereof, with a variety of desired properties.
  • IL-10 polypeptides can be readily designed and manufactured utilizing various recombinant DNA techniques, including these well known to those skilled in the art.
  • IL-10 is produced in E. coli as inclusion bodies which are isolated by lysing the E. coli cell and centrifuging the resultant supernatant at about 13,000 g. The resultant pellet is collected and washed by homogenizing in an appropriate buffer to remove contaminant proteins.
  • the inclusion bodies are solubilized in a suitable buffer containing 6 molar (M) guanidine hydrochloric acid (HC1) and 10 mM dithiothreitol (DTT) in the proportion of 10 ml buffer per gram of inclusion bodies. The mixture is incubated at 4°C for 3 hours.
  • solubilized inclusion bodies are diluted 100 fold with buffer containing 0.5M guanidine HC1, reduced glutathione, and oxidized glutathione in a ratio of 2:1 and protease inhibitors at pH 8.5, and allowed to refold for 18 hours at 4°C in the presence of a nitrogen atmosphere.
  • the refolded material is filtered and solid diammonium sulfate ((NH 4 )2S0 4 ) is added to make the final concentration 25%.
  • the material is loaded onto a hydrophilic interaction column using phenyl sepharose, butyl sepharose or toyo pearl.
  • the column is washed with 10 bed volumes of 25% (NH4)2S0 4 in buffer (TRIS 30 mM, (NH 4 ) 2 S0 4 at 25% saturation, and tetra sodium EDTA 10 mM at pH 8.5) and eluted with a buffer containing no diammonium sulfate (TRIS 30 mM, NaCl 30 mM, and tetra sodium EDTA 10 mM at pH 8.5).
  • the eluate peak fractions are collected, assayed, analyzed and pooled.
  • the pools are adjusted to pH 9.0 and conductivity 5.0 mhos (5.0 Siemens).
  • the pools are loaded onto a Q Sepharose column and the flow is collected. This flow-through contains the active fraction of IL-10.
  • the material that is bound to the column contains inactive IL-10 and is eluted with 1.0 M sodium chloride (NaCl).
  • the active fractions are pooled, analyzed, assayed and adjusted to pH 7.0 and conductivity 5.0-6.0 mhos (5.0-6.0 Siemens).
  • the material is loaded onto an S-Sepharose column.
  • the flow-through fractions are collected.
  • the column is washed with 10 bed volumes of 20mM HEPES (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) pH 7.0, which is the equilibration buffer.
  • the column is eluted with a NaCl gradient from 0-6M.
  • the peak fractions are pooled and analyzed, and contain active, 95% pure, IL-10.
  • the purified IL-10 is stored at 4°C under sterile aseptic conditions.
  • the final product has pyrogen levels of less than 0.1 endotoxin units (EU)/ml.
  • IL-10 can also be synthesized in solid or liquid phase as is known in the art.
  • Peptides can be synthesized at different substitution levels and the synthesis may follow a stepwise format or a coupling approach.
  • the stepwise method includes condensing amino acids to the terminal amino group sequentially and individually.
  • the coupling, or segment condensation, approach involves coupling fragments divided into several groups to the terminal amino acid.
  • Synthetic methods include azide, chloride, acid anhydride, mixed anhydride, active ester, Woodward reagent K, and carbodiimidazole processes as well as oxidation-reduction and other processes.
  • the synthetic peptides are usually purified by a method such as gel filtration chromatography or high performance liquid chromatography.
  • a CD 14 antagonist can also be used in the disclosed methods in combination with the CD300b antagonist.
  • the CD14 antagonist is an antagonistic antibody, such as a polyclonal, monoclonal, or antibody fragment.
  • therapeutic antibodies such as those specific for CD 14
  • the antibody can bind to any region of CD14, such as the N-terminus, C-terminus, or in between (such as an internal sequence comprising aa 71- 84 of human CD14; KRVDADADPRQYAD).
  • Exemplary nucleic acid sequences encoding human CD14 are provided in GENBANK® Accession Nos. CR457016.1 and M86511.1, and exemplary amino acid sequences of human CD 14 are provided in GENBANK® Accession Nos.
  • NP_001167576.1 and ADX31876.1 which are all incorporated by reference herein.
  • Such sequences can be used to generate CD14-specfic antibodies (such as polyclonal or monoclonal antibodies or fragments thereof), which can be used with the disclosed methods.
  • Antibodies that specifically bind CD14 are known in the art, see for example, U.S.
  • the CD14 antibody is the clone designated 1116 la6 described by Schimke et al, (PNAS 95: 13875-80, 1998, herein incorporated by reference). In one example, the CD14 antibody is the clone designated IC14 described by Reinhart et al., (Crit. Care Med 32: 1100-8, 2004, herein incorporated by reference).
  • Antibodies that specifically bind CD 14 and that can be used with the disclosed methods are commercially available.
  • antibodies to human CD 14 are available from Santa Cruz Biotechnology (such as catalog numbers sc-1182 (clone UCH-M1), sc-52457 (clone 61D3), sc- 7328 (clone BA-8) and sc58951 (clone 5A3B 11B5)), Abeam (such as catalog numbers ab45870, abl93322, abl33335, ab760, and ab91146), and Novus Biologicals (such as catalog numbers NB100-2807, NB100-77758 (clone M5E2), MAB3832 (clone 134620) and NP1-40683 (clone EPR3653), clone 18D11 from LifeSpan Biosciences, and MACS Milenyl Bioech (such as catalog number 130-098-063 (clone TUK4)).
  • the antibody is anti-CD14 (clone UCH-M
  • Humanized and chimeric forms of these antibodies can be utilized.
  • Antigen binding fragments of antibodies that specifically bind CD 14 are also of use. Methods of producing humanized antibodies, chimeric antibodies, and antigen binding fragments are disclosed above.
  • Inhibitory nucleic acids that decrease the expression and/or activity of CD 14 can also be used in the methods disclosed herein.
  • One embodiment is a small inhibitory RNA (siRNA) for interference or inhibition of expression of a target.
  • Another embodiment of an inhibitory nucleic acid are antisense and ribozyme molecules specific for CD 14.
  • Inhibitory nucleic acids specific for CD14 can be generated using routine methods and publicly available CD14 nucleic acid sequences, for example as described above.
  • TLR4 antagonists are available and can be used in the methods disclosed herein, for example in combination with a CD300b antagonist.
  • the TLR4 antagonist is a TLR4-specific antibody, such as a polyclonal, monoclonal, or antibody fragment.
  • therapeutic antibodies such as those specific for TLR4 can be humanized, or chimeric antibodies including the CDRs for these antibodies, can be used in the disclosed methods.
  • the antibody can bind to any region of TLR4, such as the N-terminus, C-terminus, or in between (such as an internal sequence comprising amino acids 242-321 of human TLR4).
  • the TLR4 antibody binds to a region of TLR4 that is found on the cell surface (such as an extracellular domain).
  • Exemplary nucleic acid sequences encoding human TLR4 are provided in GENBANK® Accession Nos. NM_003266.3, NM_138554.4, NM_138557.2 and AF177765.1, and exemplary amino acid sequences of human TLR4 are provided in GENBANK® Accession Nos.
  • NP 003257.1, NP_612564.1, NP_612567.1 and AAI17423.1 which are all incorporated by reference herein.
  • Such sequences can be used to generate TLR4-specfic antibodies (such as polyclonal or monoclonal antibodies or fragments thereof), which can be used with the disclosed methods.
  • TLR4 antibody is the clone designated NI-0101 by Novlmmune.
  • TLR4 antibody is the clone designated Ia6 by Novlmmune and described by Hennessy et al., (Nat Rev Drug Discovery 9:293-307, 2010, herein incorporated by reference).
  • Antibodies that specifically bind TLR4 and that can be used with the disclosed methods are commercially available.
  • antibodies to human TLR4 are available from Santa Cruz Biotechnology (such as catalog numbers sc-8694 (clone C-18), sc-10741 (clone H-80), sc-13593 (clone HTA125) and sc-529621 (clone 76B357.1)) and Abeam (such as catalog numbers ab22048 (clone 76B357.1), abl3556, ab47093, and abl50583)
  • Humanized and chimeric forms of these antibodies can be utilized.
  • Antigen binding fragments are also of use. Methods of producing humanized antibodies, chimeric antibodies, and antigen binding fragments are disclosed above.
  • the TLR4 antagonist is E5531 (6-0- ⁇ 2-deoxy-6-C ) -methyl-4-C ) -phosphono- 3-0-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-C ) -phosphono-d- glucopyranose tetrasodium salt).
  • the TLR4 antagonist is E5564 (EritoranTM).
  • TLR4 antagonists are provided in Leon et al., (Pharm. Res. 25: 1751-61, 2008, herein incorporated by reference), such as
  • the TLR4 antagonist is a small molecule phosphodiesterase inhibitor, such as AV411 (ibudilast), such as from Avigen (also see U.S. Patent No. 7,534,806, herein incorporated by reference).
  • AV411 ibudilast
  • Avigen also see U.S. Patent No. 7,534,806, herein incorporated by reference.
  • TLR4 antagonists that can be used in the disclosed methods also include specific mRNA- protein complexes (ribonucleoprotein complexes; mRNP), which mediate post- transcriptional regulation of mRNA stability and translation.
  • mRNP ribonucleoprotein complexes
  • mRNP K heterogeneous nuclear ribonucleoprotein K
  • hnRNP K was identified as a potential modulator of LPS-dependent translation of mRNA coding for key components of the TRL4 signaling pathway.
  • compounds that modulate the binding of hnRNP K to mRNA are of use in the disclosed methods. Such compounds are disclosed, for example, in U.S. Published patent application No. 20150152171, which is incorporated herein by reference.
  • Inhibitory nucleic acids that decrease the expression and/or activity of TLR4 can also be used in the methods disclosed herein.
  • One embodiment is a small inhibitory RNA (siRNA) for interference or inhibition of expression of a target.
  • siRNA small inhibitory RNA
  • Another embodiment of an inhibitory nucleic acid are antisense and ribozyme molecules specific for TLR4.
  • Inhibitory nucleic acids specific for TLR4 can be generated using routine methods and publicly available TLR4 nucleic acid sequences, for example as described above.
  • TLR4 antagonists that can be used with the disclosed methods are provided in U.S. Publication No. 20030077279, which is incorporated herein by reference.
  • PD-1, CTLA-4 and BTLA Antagonists are provided in U.S. Publication No. 20030077279, which is incorporated herein by reference.
  • CTLA-4 and BTLA Antagonists are provided in U.S. Publication No. 20030077279, which is incorporated herein by reference.
  • PD-1 antagonists, CTLA-4 antagonist, and/or BTLA antagonists are of use in the method disclosed herein, for example in combination with a CD300b antagonist.
  • the antagonist can be a chemical or biological compound.
  • the antagonist can be an antibody, including but not limited to a chimeric, humanized, or human antibody. Suitable antagonists also include antigen binding fragments of these antibodies (see above for a description of antigen binding fragments).
  • Methods of use for producing antibodies to CD300b are also of use to produce antibodies that specifically bind PD-1, PD-L1, PD-L2, CTLA-4 or BTLA.
  • the PD-1, CTLA4, or BTLA antagonist can be an inhibitory nucleic acid molecule.
  • Methods for preparing inhibitory nucleic acid molecules for CD300b can be used for producing inhibitory nucleic acids that specifically bind PD-1, PD-L1, PD-L2, CTLA-4 or BTLA.
  • the antagonist can be a small molecule, such as a molecule less than 900 daltons or less than 800 daltons.
  • the methods of use to select CD300b small molecule antagonist, disclosed above, can also be used to select small molecules to different targets, such as PD-1, PD-L1, PD-L2, CTLA-4 or BTLA.
  • the PD-1 antagonist is a PD-1 binding antagonist.
  • a PD-1 antagonist can be any chemical compound or biological molecule that blocks binding of PD-L1 or PD-L2 expressed on a cell to human PD-1 expressed on an immune cell (T cell, B cell or NKT cell)
  • Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1 ; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1 ; and PDCD1L2, PDL2, B7- DC, Btdc and CD273 for PD-L2.
  • Exemplary human PD-1 amino acid sequences can be found in
  • Exemplary human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Accession No.: NP 054862 and NP 079515, respectively, February 28,
  • the PD-1 binding antagonist is an antibody.
  • amino acid sequence of antibodies that bind PD-1 are disclosed, for example, in U.S.
  • Patent Publication No. 2006/0210567 which is incorporated herein by reference.
  • Antibodies that bind PD-1 are also disclosed in U.S. Patent Publication No. 2006/0034826, which is also incorporated herein by reference.
  • Antibodies that bind PD-1 are also disclosed in U.S. Patent No. 7,488,802, U.S. Patent No. 7,521,051, U.S. Patent No. 8,008,449, U.S. Patent No. 8,354,509, U.S. Patent No. 8,168,757, U.S. PCT Publication No. WO2004/004771, PCT Publication No.
  • the antibody can be KEYTRUDA® (pembrolizumab).
  • the antibody can be an anti-PD-1 antibody such as Nivolumab (ONO-4538/BMS-936558) or OPDIVO® from Ono Pharmaceuticals.
  • PD-L1 binding antagonists include YW243.55.S70, MPDL3280A, MDX-1105 and MEDI 4736, see U.S. Published Patent Application No. 2017/0044256. Examples of monoclonal antibodies that specifically bind to human PD-L1 , and are useful in the disclosed methods and compositions are disclosed in PCT Publication No. WO2013/019906, PCT
  • Antibodies that bind PD-1, PD-L2 and PD-1 are also disclosed in Patent No. 8,552, 154.
  • the antibody specifically binds PD-1 or a PD-L1 or PD-L2 with an affinity constant of at least 10 7 M "1 , such as at least 10 8 M 1 at least 5 X 10 8 M 1 or at least 10 9 M "1 .
  • Inhibitory nucleic acids that decrease the expression and/or activity of PD-1, PD-L1 or PD- L2 can also be used in the methods disclosed herein.
  • One embodiment is a small inhibitory RNA (siRNA) for interference or inhibition of expression of a target gene.
  • siRNA small inhibitory RNA
  • Nucleic acid sequences encoding PD-1, PD-L1 and PD-L2 are disclosed in GENBANK® Accession Nos. NM_005018, AF344424, NP_079515, and NP_054862, all incorporated by reference as available on February 28, 2017.
  • An immunoadhesin that specifically binds to human PD-1 or human PD-L1 can also be utilized.
  • An immunoadhesin is a fusion a fusion protein containing the extracellular or PD- 1 binding portion of PD-L1 or PD-L2 fused to a constant region such as an Fc region of an immunoglobulin molecule.
  • Examples of immunoadhesion molecules that specifically bind to PD-1 are disclosed in PCT Publication Nos. WO2010/027827 and WO2011/066342, both incorporated by reference. These immunoadhesion molecules include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein. Additional PD-1 antagonists that are fusion proteins are disclosed, for example, in U.S. Published Patent Application No. 2014/0227262, incorporated herein by reference. Dominant negative inhibitors are also of use in the disclosed methods.
  • a CTLA-4 antagonist is used in the methods disclosed herein.
  • CTLA-4 antagonist can be an antibody that specifically binds CTLA-4.
  • Antibodies that specifically bind CTLA-4 are disclosed in PCT Publication No. WO 2001/014424, PCT Publication No. WO 2004/035607, U.S. Publication No. 2005/0201994, European Patent No. EPl 141028, and European Patent No. EP 1212422 Bl. Additional CTLA-4 antibodies are disclosed in U.S. Patent No.
  • Antibodies that specifically bind CTLA-4 are also disclosed in Hurwitz et al., Proc. Natl. Acad. Sci.
  • CTLA-1 antagonist is Ipilmumab (also known as MDX-010 and MDX-101 and
  • CTLA-4 antagonist can be a dominant negative protein or an immunoadhesins, see for example U.S. Published Patent Application No. 2016/0264643, incorporated herein by reference.
  • Additional anti-CTLA4 antagonists include any inhibitor, including but not limited to a small molecule, that can inhibit the ability of CTLA4 to bind to its cognate ligand, disrupt the ability of
  • CTLA4 This includes small molecule inhibitors of CTLA4, antibodies that specifically bind
  • CTLA4 antisense molecules directed against CTLA4, adnectins directed against CTLA4, RNAi inhibitors (both single and double stranded) for CTLA4.
  • a BTLA antagonist is utilized in the methods disclosed herein.
  • Antibodies that specifically bind BTLA are disclosed, for example, in U.S. Published Patent
  • HVEM Herpesvirus entry mediator
  • Methods of Treatment or Prevention of Septic Shock and Pharmaceutical Compositions are provided herein for treating a subject with sepsis or septic shock, or for preventing or delaying septic shock or sepsis in a subject.
  • the subject can be any mammalian subject, including veterinary and human subjects. In specific non-limiting examples, the subject is a human. The subject can be an adult or a child.
  • the method includes administering any of the CD300b antagonists disclosed herein, in some examples in combination with one or more of IL-10 (or a fragment, variant, or fusion protein thereof), a CD14 antagonist (such as an antibody that specifically binds CD14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA), after the subject has been diagnosed with sepsis or septic shock, for example at least 1 hour, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours after the subject has been diagnosed with sepsis or septic shock (such as 1 hour to 1 week, 1 hour to 24 hours, 6 hours to 48 hours, or 24 hours to 96 hours after the subject has been diagnosed
  • the method includes administering any of the CD300b antagonists disclosed herein, in some examples in combination with one or more of IL-10 (or a fragment, variant, or fusion protein thereof), a CD14 antagonist (such as an antibody that specifically binds CD14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA), before the subject has been diagnosed with sepsis or septic shock, for example at least 1 hour, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours before the subject has been diagnosed with sepsis or septic shock (such as 1 hour to 1 week, 1 hour to 24 hours, 6 hours to 48 hours, or 24 hours to 96 hours before the subject has been diagnosed
  • the method can include administering to the subject a therapeutically effective amount of a
  • the method can include administering additional agents, such as, but not limited to, one or more of IL-10 (or a fragment, variant, or fusion protein thereof), a CD 14 antagonist (such as an antibody that specifically binds CD14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA).
  • additional agents such as, but not limited to, one or more of IL-10 (or a fragment, variant, or fusion protein thereof), a CD 14 antagonist (such as an antibody that specifically binds CD14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a
  • a pharmaceutical composition of the disclosure can optionally further include one or more of IL-10 (or a fragment, variant, or fusion protein thereof), a CD14 antagonist (such as an antibody that specifically binds CD 14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA).
  • the method can also include administering to the subject an effective amount of an anti-microbial agent.
  • a method for treating a subject with septic shock includes selecting a subject with septic shock; and administering to the subject a therapeutically effective amount of a CD300b antagonist, as disclosed herein.
  • a method for treating a subject with sepsis wherein the method includes selecting a subject with sepsis; and administering to the subject a therapeutically effective amount of a CD300b antagonist, as disclosed herein.
  • the CD300b antagonist is an antibody that specifically binds CD300b, such as a humanized or chimeric antibody. Other CD300b antagonist of use are disclosed above.
  • the method can also include selecting a subject with a bacterial infection, and administering to the subject a therapeutically effective amount of a CD300b antagonist, as disclosed herein.
  • the method can delay the onset of, or prevent, sepsis and/or septic shock.
  • any of these methods can also include administering a therapeutically effective amount of one or more of IL-10 (or a fragment, variant, or fusion protein thereof) a CD14 antagonist (such as an antibody that specifically binds CD14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA).
  • a CD14 antagonist such as an antibody that specifically binds CD14
  • TLR4 antagonist such as an antibody that specifically binds TLR4
  • a PD-1 antagonist such as an antibody that specifically binds PD-1
  • CTLA-4 antagonist such as an antibody that specifically binds CTLA-4
  • BTLA antagonist such as an antibody that specifically binds BTLA
  • Sepsis or septic shock can be caused by infection with a gram-negative or gram-positive bacteria.
  • the subject has septic shock, diagnosed by the presence of one or both of the following: (1) evidence of infection, through a positive blood culture (2) refractive hypotension (despite adequate fluid resuscitation) which in adults is diagnosed as a systolic blood pressure of less than about 90 mmHg, or a mean arterial pressure (MAP) of less than about 60 mmHg, or a reduction of 40 mmHg in the systolic blood pressure from baseline, while in children it is a blood pressure of less than two standard deviations (SD) of the normal blood pressure.
  • MAP mean arterial pressure
  • the subject can have two or more of the following: (a) heart rate of greater than about 90 beats per minute; (b) body temperature of less than about 36 or greater than about 38°C; (3) hyperventilation (high respiratory rate) greater than 20 breaths per minute or, on blood gas, a PaCC less than about 32 mmHg; (4) white blood cell count less than 4000 cells/mm 3 or greater than about 12000 cells/mm 3 ( ⁇ 4 x 10 9 or > 12 x 10 9 cells/L).
  • the use of the disclosed pharmaceutical composition provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer in at least one of the parameters described above.
  • the subject has an infection, such as with a gram-negative or a gram-positive bacteria, but does not have sepsis or septic shock.
  • the methods are utilize to delay or prevent the development of sepsis or septic shock.
  • the method includes selecting a subject with an infection with a gram-negative or a gram-positive bacteria, and administering to the subject one or more of the compositions disclosed herein.
  • the subject is at risk of sepsis or septic shock, but does not have the infection.
  • the methods are utilized to delay or prevent the development of sepsis or septic shock.
  • the method includes selecting a subject at risk for sepsis or septic shock caused by an infection with a gram-negative or a gram-positive bacteria, and administering to the subject one or more of the compositions disclosed herein.
  • the subject may be, for example, someone with a needle stick or exposure to a particular bacteria, such as a methicillin-resistant Staphylococcus aureus.
  • the gram-negative bacteria is Escherichia coli, Klebsiella pneumoniae, Proteus species, Pseudomonas aeruginosa or Salmonella typhimurium.
  • Methods are also provided herein for treating a subject with sepsis or septic shock resulting from an infection with gram- positive bacteria.
  • gram-positive bacteria is a species of Staphlococci, Streptococi or Pneumococci.
  • methods are provided herein for the treatment of sepsis or septic shock caused by either a gram-negative or gram-positive bacteria.
  • Methods are further provided for delaying or prevent sepsis or septic shock from a gram-negative or gram-positive bacteria.
  • a CD300b antagonist can optionally be administered with a therapeutically effective amount of IL-10 (or a fragment, variant, or fusion protein thereof), a CD 14 antagonist (such as an antibody that specifically binds CD 14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA).
  • a CD 14 antagonist such as an antibody that specifically binds CD 14
  • TLR4 antagonist such as an antibody that specifically binds TLR4
  • a PD-1 antagonist such as an antibody that specifically binds PD-1
  • CTLA-4 antagonist such as an antibody that specifically binds CTLA-4
  • BTLA antagonist such as an antibody that specifically binds BTLA
  • the CD300b antagonist can be administered by any means known to one of skill in the art (see Banga, A., "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995) either locally or systemically, such as by intramuscular, subcutaneous, intraperitoneal, intraarterial, or intravenous injection, but even oral, nasal, transdermal, vaginal, ocular, or anal administration is contemplated. In one embodiment, administration is by intravenous, subcutaneous or intramuscular injection.
  • a single administration of the CD300b antagonist can be administered to the subject, optionally with one or more of (such as 1, 2, 3, 4,5 or 6 of) IL-10 (or a fragment, variant, or fusion protein thereof), a CD 14 antagonist (such as an antibody that specifically binds CD 14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA).
  • multiple administrations can be utilized, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations.
  • administrations can be several (3, 4, 5, 6, etc.) times a day, twice a day, once a day, or once every other day.
  • the therapeutic agent(s) can be provided as an implant, an oily injection, or as a particulate system.
  • the particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle.
  • Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic protein as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 ⁇ are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ so that only nanoparticles are administered intravenously.
  • Microparticles are typically around 100 ⁇ in diameter and are administered subcutaneously or intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A.
  • polymers can be used for ion-controlled release.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537, 1993).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant IL-2 and urease (Johnston et al., Pharm. Res. 9:425, 1992; and Pec, /. Parent. Set Tech. 44(2):58, 1990).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993).
  • Numerous additional systems for controlled delivery of therapeutic proteins are known (e.g., U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No.
  • a pharmaceutical composition for intravenous administration would include about 0.1 ⁇ g to 10 mg of CD300b antagonist per patient per day. In some embodiments dosages from 0.1 up to about 100 mg per subject per day can be used. In other embodiments, suitable doses for antibodies include dose of from about 0.5 to about 100 mg/kg, such as about 1 to about 60, such as about 1 to about 50, such as about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 1 to about 3, about 0.5 to about 50 mg/kg, such as about 0.5 to about 40, about 0.5 to about 30, about 0.5 to about 20, about 0.5 to about 10, about 0.5 to about 5, about 0.5 to about 3, about 3 to about 7, about 8 to about 12, about 15 to about 25, about 18 to about 22, about 28 to about 32, about 10 to about 20, about 5 to about 15, or about 20 to about 450 mg/kg.
  • the doses described herein can be administered at any dosing frequency/frequency of administration,
  • a pharmaceutical composition for intravenous administration would include about 0.1 ⁇ g to 10 mg of one or more of (such as 1, 2, 3, 4,5 or 6 of) IL-10 (or a fragment, variant, or fusion protein thereof), a CD 14 antagonist (such as an antibody that specifically binds CD 14), a TLR4 antagonist (such as an antibody that specifically binds TLR4), a PD-1 antagonist, (such as an antibody that specifically binds PD-1), CTLA-4 antagonist (such as an antibody that specifically binds CTLA-4), and/or a BTLA antagonist (such as an antibody that specifically binds BTLA) per patient per day. Dosages from 0.1 up to about 100 mg per subject per day can be used, particularly if the agent is administered to a body cavity or into a lumen of an organ.
  • compositions Actual methods for preparing administrable compositions are known or apparent to those in the art and are described in more detail in such publications as Remingtons Pharmaceutical Sciences, 19 th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995. Antimicrobial agents can also be included in the composition.
  • compositions are administered depending on the dosage and frequency as required and tolerated by the subject.
  • the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result is achieved.
  • the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject.
  • Systemic or local administration can be utilized.
  • a pharmaceutical composition in another embodiment, includes a nucleic acid encoding one or more of the antagonists, such as siRNAs, disclosed herein.
  • a therapeutically effective amount of the polynucleotide can be administered to a subject, such as a subject with septic shock or sepsis.
  • the nucleic acid can be a siRNA.
  • polynucleotide is administered to a subject to treat sepsis or septic shock induced by a gram- negative bacteria.
  • a therapeutically effective amount of the polynucleotide is administered to a subject to treat sepsis or septic shock induced by a gram- positive bacteria.
  • a therapeutically effective amount of the polynucleotide is administered to a subject to delay or prevent sepsis or septic shock induced by a gram-negative bacteria.
  • a therapeutically effective amount of the polynucleotide is administered to a subject to delay or prevent sepsis or septic shock induced by a gram-positive bacteria.
  • nucleic acids are direct immunization with plasmid DNA, such as with a mammalian expression plasmid.
  • plasmid DNA such as with a mammalian expression plasmid.
  • the nucleotide sequence encoding a polypeptide can be placed under the control of a promoter to increase expression of the molecule.
  • nucleic acid constructs are well known in the art and taught, for example, in U.S. Patent No. 5,643,578; U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637; and U.S. Patent No. 5,880,103.
  • the methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves).
  • a polypeptide in another approach to using nucleic acids for immunization, can also be expressed by attenuated viral hosts or vectors or bacterial vectors.
  • Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, or other viral vectors can be used to express the peptide or protein.
  • vaccinia vectors and methods of administration are described in U.S. Patent No. 4,722,848.
  • BCG Bacillus Calmette Guerin provides another vector for expression of the peptides (see Stover, Nature 351 :456-460, 1991).
  • each recombinant virus in the composition in the range of from about 10 5 to about 10 10 plaque forming units/mg mammal, although a lower or higher dose can be administered.
  • the composition of recombinant viral vectors can be introduced into a subject with septic shock. Examples of methods for administering the composition into mammals include, but are not limited to, intravenous, subcutaneous, intradermal or intramuscular administration of the nucleic acid, such as virus or other vector including the nucleic acid encoding the disclosed polypeptides.
  • the quantity of recombinant viral vector, carrying the nucleic acid sequence of a polypeptide to be administered is based on the titer of virus particles.
  • An exemplary range of the virus to be administered is 10 5 to 10 10 virus particles per mammal, such as a human.
  • the subject is also administered an additional agent, such as antimicrobial agent or a corticosteroid.
  • an additional agent such as antimicrobial agent or a corticosteroid.
  • the subject is also administered activate protein C and/or intensive fluid resuscitation. In several examples, this administration is sequential. In other examples, this administration is simultaneous.
  • Suitable anti-microbial agents include any antibiotic, which includes any compound that decreases or abolishes the growth of a pathogen, such as a gram-negative bacteria, gram-positive bacterial, fungus or protozoa. These compounds include the following: amino glycosides such as amikacin, neomycin, streptomycin or gentamycin, ansamycins such as geldanamycin or
  • herbamycin a carbacephem such as loracarbef, a carbapenem such as meropenem, a cephalosporin (including first, second, third and fourth generation cepalosporins) such as cefazolin or cefepine, a glycopeptides such as vancomycina macrolide such as azithromycin, a penicillin such as ampicillin or amoxicillin, a polypeptide such as bacitracin, a quinolone such as ciprofloxacin, a sulfonamide such as mafenide, a tetracycline such as doxycycline.
  • a carbacephem such as loracarbef
  • carbapenem such as meropenem
  • cephalosporin including first, second, third and fourth generation cepalosporins
  • cefazolin or cefepine a glycopeptides
  • vancomycina macrolide such as azithromycin
  • CD300b was determined to be a novel LPS binding receptor. The mechanism underlying CD300b augmentation of septic shock was elicited. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting septic shock. It was demonstrated that CD300b/DAP12 associates with TLR4/CD14 upon LPS binding, promoting MyD88/TIRAP dissociation from the complex and the recruitment and activation of Syk and PI3K. This results in the activation of AKT, which subsequently leads to a reduced production of the anti- inflammatory cytokine IL-10 by macrophages, via a PI3K-AKT-dependent inhibition of the MEK1/2-ERK1/2-NFKB signaling pathway.
  • CD300b also enhanced TLR4/CD14- TRIF-IRF3 signaling responses, resulting in elevated IFN- ⁇ levels.
  • TLR4/CD14- TRIF-IRF3 signaling responses resulting in elevated IFN- ⁇ levels.
  • TLR4 signaling as regulated by CD300b, in myeloid cells in response to LPS was determined.
  • Fcy-chimeric or recombinant protein interactions with LPS was measured using the BIAcore T100 SPR instrument as disclosed below.
  • FITC-labeled TLR4 (LPS)-ligand competition and Ab blocking experiments were performed as disclosed below.
  • CLP and lethal endotoxemia were performed as disclosed below.
  • mice were i.v. injected with 200 ⁇ PBS-liposomes (control) or dichloromethylene biphosphate (CkMBPHiposomes (Encapsula NanoSciences) 24 h before the induction of lethal endotoxemia as described (Totsuka et al., 2014, Nat Commun 5, 4710).
  • mice were i.p. injected with 1 mg of anti-IL-10 (clone JES5-2A5, BioxCell) or control Ab (clone HRPN, BioxCell) 2 h before the induction of lethal endotoxemia.
  • anti-IL-10 clone JES5-2A5, BioxCell
  • control Ab clone HRPN, BioxCell
  • Bone marrow cells were isolated from WT, Cd300b , Cd300b A IL-10 ' Cd300f or TLR4 ' mice and differentiated into ⁇ or DC as disclosed below.
  • mice were i.v. injected with 2 x 10 6 of WT, Cd300b , or Cd300b IL-10 ⁇ 24 h before the induction of lethal endotoxemia. Extract preparation, immunoprecipitation and Western blot analysis
  • Efferocytosis of apoptotic thymocytes was performed as previously described (Tian et al., 2014) and is disclosed below.
  • RNA isolation and qRT-PCR were performed as disclosed below.
  • mice subjected to lethal endotoxemia or CLP were analyzed by using Kaplan-Meir survival curves and the log-rank test (GraphPad Prism Software, version 6.0). The statistical significance was assessed using ANOVA with Bonferroni post-test, or by the two-tailed unpaired Student i-test (GraphPad). Data are presented as mean + SEM, unless stated otherwise. Alpha level was set to 0.05.
  • CD300b binds LPS
  • CD300b can bind LPS.
  • SPR surface plasmon resonance
  • mCD300f-FcY mCD300a-Fcy
  • mCD300d-Fcy or the control protein, NITR- Fcy
  • pull-down assays demonstrated that mCD300b-Fcy protein, but not other related proteins, including those that contain the same Fcy-domain, co- immunoprecipitated with LPS (FIG. ID).
  • the kinetics was measured of LPS interaction with monomeric mCD300b protein, the well-known LPS-sensor mCD14, and a control protein, mLAIRl.
  • the dissociation constants (KD) for the binding of LPS to mCD300b and mCD14 were 6.39 x 10 "6 M and 8.5 x 10 "7 M, respectively (FIGS. 7A and 7B). No binding to mLAIRl was observed (FIG. 7C).
  • mCD300b/DAP12-, mCD300d/FcRy-, mCD300f-, and EV-expressing L929 cell lines were generated and incubated them with FITC-labeled LPS derived from E. coli or S. minnesota; DAP12 (Yamanishi et al., 2008, supra) and FcRy (Izawa et al., 2007, J Biol Chem 282, 17997-18008) were previously established as signaling adaptors for mCD300b and mCD300d, respectively. It was found that only cells expressing mCD300b or mCD300b/DAP12 bound LPS (0.5 - 1 h; FIGS. IE and IF).
  • the specificity of LPS recognition was assessed by testing additional TLR or NOD ligands. It was found that mCD300b did not recognize ligands for TLR2 (PAM3CSK4), TLR3 (Poly(I:Q), or NOD2 (MDP), further indicating that CD300b binding to LPS is specific (FIGS. 8D and 8E).
  • Competition experiments utilizing unlabeled LPS from either E. coli or S.
  • CD300b exacerbates the pathogenesis of septic shock
  • FIG. 2B Histopathologic analyses of the lungs from LPS-treated animals revealed that, in comparison to Cd300b ⁇ ' ⁇ mice, WT animals displayed a more severe inflammatory state associated with a greater influx of inflammatory cells, increased alveolar and interstitial edema, greater alveolar-capillary membrane thickening, and more hemorrhages (FIG. 2B). Notably, CD300b- deficiency did not completely protect mice from inflammatory damage, as shown in tissues from PBS-treated mice (FIGS. 2B and 8H).
  • CD300b- deficiency did not completely protect mice from inflammatory damage as shown in tissues from sham-treated mice (FIGS. 2E and 81).
  • the changes seen in lungs of WT and Cd300b ⁇ ' ⁇ mice with CLP were quite similar to those seen in LPS-treated mice of both genotypes (FIGS. 2B and 2E).
  • Assessment of the bacterial load in the blood, peritoneal cavity, lung, spleen and liver after CLP showed a dramatically higher level of bacterial burden in WT mice than in Cd300b ⁇ ' ⁇ animals (FIGS. 8J-8N).
  • Cd300b ⁇ ' ⁇ mice treated with LPS or CLP are characterized by having reduced mortality, less bacterial burden, reduced pro-inflammatory cytokine levels and higher IL-10 levels in comparison to Tmice (FIGS. 2 and 8).
  • CD300b amplifies LPS-induced septic shock by dampening IL-10 production
  • CD300b functions to amplify inflammatory responses triggered by bacterial infection via IL-10 inhibition.
  • anti-IL-10 or a control Ab was injected into WT and Cd300b ⁇ ' ⁇ mice 2 h before treatment with LPS.
  • IL-10 neutralization diminished the survival advantage of Cd300b ⁇ ' ⁇ over WT mice, while treatment with the control Ab had no effect on the survival of Cd300b ⁇ ' ⁇ mice (FIG. 3A).
  • CD300b is broadly expressed at the mRNA and protein levels among myeloid but not lymphoid cell populations (FIGS. 9A and 9B). It was hypothesized that CD300b-expressing ⁇ account for the increased mortality of septic animals. Thus, ⁇ from WT and Cd300b ⁇ ' ⁇ mice were depleted using dichloromethylene biphosphate (CkMBP -encapsulated liposomes (Totsuka et al., 2014, Nat Commun 5, 4710) before injection of LPS.
  • CkMBP -encapsulated liposomes Totsuka et al., 2014, Nat Commun 5, 4710
  • C MBP-liposomes selectively depleted ⁇ with no effect on neutrophil or DC numbers in the peritoneal cavity, lung or spleen of either WT or Cd300b ⁇ ' ⁇ mice (FIGS. 9C-9F).
  • TNFa levels were reduced following the ⁇ depletion in WT mice to levels similar to those observed in either ⁇ -depleted or non-depleted Cd300b ' mice, suggesting that ⁇ may be a primary source of TNFa.
  • levels of IL-10 in sera from ⁇ -depleted Cd300b ' mice were considerably lower than those in non-depleted Cd300b ' mice (FIG. 4C), indicating that ⁇ are the major source of IL-10 in Cd300b ' mice.
  • CD300b expression by macrophages shifts the balance toward inflammation in LPS-induced septic shock
  • cytokine secretion by LPS-treated ⁇ or BMDC was compared from WT, Cd300b A and Cd300f mice.
  • LPS-treated ⁇ from Cd300b mice produced significantly higher levels of IL-10, and markedly lower levels of TNFa, and IL-12 when compared to ⁇ from WT or Cd300f animals (FIG. 5 A).
  • IFNyproduction the amount of IFNy produced by ⁇ was very low, indicating that ⁇ are not a significant source of this cytokine (FIG. 5A).
  • CD300b expression by ⁇ but not DC altered the balance of cytokine production toward a more pro-inflammatory state supports the hypothesis that CD300b- expressing ⁇ amplify the effects of endotoxins.
  • This hypothesis was further tested by transferring ⁇ from WT or Cd300b ' mice into WT or Cd300b ' animals prior to LPS injection. Strikingly, transfer of Cd300b ' ⁇ , but not those from WT animals, improved the survival of WT mice from 0 to 65%, and Cd300b ' mice from 50 to 95%, in correlation with increased IL-10 serum levels and decreased TNFodevels (FIGS. 5B and 5C).
  • ⁇ -mediated IL-10 production contributes to the differential susceptibility to lethal peritonitis in WT versus Cd300b ' mice
  • ⁇ were transferred from Cd300b A IL-10 mice into WT or Cd300b animals prior to LPS injection.
  • Transfer of Cd300b ' IL-10 ' ⁇ significantly impaired the survival of WT and, importantly, Cd300b ' mice, and correlated with an enhanced serum level of pro-inflammatory cytokines (FIGS. 5B and 5C).
  • CD300b regulates TLR4/CD14-MyD88 complex assembly and IL-10 production
  • LPS is a well-defined inducer of TLR4 signaling.
  • LPS stimulation of ⁇ promotes the production of IL-10 as a feedback mechanism to inhibit the pro-inflammatory cytokine response (Siewe et al., 2006, Eur J Immunol 36, 3248-3255).
  • LPS binding to CD300b influences LPS-induced TLR4 signaling
  • the co-localization of CD300b and TLR4 was first assessed in unstimulated or LPS-treated ⁇ . It was found that while a small portion of CD300b co-localized with TLR4 in unstimulated cells, LPS treatment greatly enhanced the co- localization between CD300b and TLR4 (FIGS. IOC and 10D; the specificities of the anti-CD300b and anti-TLR4 Abs were validated using Cd300b A and TLR4 A ⁇ ).
  • TLR4/CD300b/DAP12 complex while the addition of anti-CD14 or anti-IgG isotype control Ab failed to block the complex formation (FIG. 6E), suggesting that the CD300b/TLR4 complex formation is mediated by LPS binding to CD300b.
  • Treatment with either anti-CD300b or anti- CD 14 Abs interfered with LPS binding to WT ⁇ , and had a cumulative effect when both Abs were used (FIGS. 10E and 10F), in line with a significant reduction of LPS binding to ⁇ from Cd300b '- or Cdl4 '- mice (FIGS. 10G and 10H).
  • Ab-mediated blocking of LPS binding to CD300b or CD14 inhibited pro-inflammatory cytokine production (FIG.
  • the LPS-induced association between CD300b and TLR4 was further validated by examining complex assembly in ⁇ treated with AC, a source of phosphatidylserine (PS), which previously were identified as a CD300b ligand (Murakami et al., 2014, supra).
  • PS phosphatidylserine
  • FIG. 11A The interaction between CD300b and TLR4 in the presence of AC alone was similar to that of unstimulated cells, indicating that AC do not trigger the association between CD300b and TLR4 in ⁇ (FIG. 11A), even though they stimulate the recruitment of DAP 12 to CD300b resulting in efferocytosis and an enhanced IL-10 production (FIGS. 11B and 11C).
  • MyD88 and TIRAP are primary adaptor proteins utilized for TLR4-LPS-induced signaling.
  • LPS treatment induced an early and transient recruitment of MyD88 and TIRAP reaching a maximum at 0.5 h ( ⁇ 3-fold over time 0 h) after LPS stimulation (FIGS. 6C and 6D).
  • Most of MyD88/TIRAP disassociated from the complex during prolonged stimulation with LPS (FIGS. 6C and 6D).
  • TLR4 pull-down experiments using Cd300b ' ⁇ demonstrated that CD300b was necessary for DAP12, pSyk, and pPDK recruitment, and the displacement of MyD88/TIRAP from the complex, as without CD300b no DAP12, and only small amounts of pSyk or pPDK were recruited to the TLR4 complex, while MyD88 as well as TIRAP remained associated with the receptor complex (FIG. 6D).
  • CD300b plays a role in regulating the TLR4/CD14-TRIF- IRF3 signaling pathway, thereby mediating IFN- ⁇ production, highlighting CD300b as a potential mediator influencing both the TLR4-MyD88 and TLR4/CD 14-TRIF signaling cascades.
  • CD300b/TLR4/DAP12-Syk-PI3K signaling complex limits the activation of the MEK1/2-ERK1/2-NFKB pathway in ⁇ , thereby dampening IL- 10 production, which likely potentiates lethal inflammation.
  • Anti-Cd300b Antibodies in a Mouse Model of Septic Shock Wild-type (WT) mice were injected with a toxic dose of LPS (37 mg/g) followed by a second injection, 1 h post-LPS administration, using either anti-CD300b (5 ⁇ g/g; R&D, Cat.-No: MAB2580, clone 339003) or anti-IgG control (5 ⁇ g/g, R&D, Cat.-No: MAB0061, clone 141945) antibody.
  • Mouse survival was monitored every 6 hours for 7 days and results of the endpoint study were graphed using a Kaplan-Meier survival plot.
  • an anti-CD300b antibody treatment according to a therapeutic protocol showed that administration of anti-CD300b significantly prolonged the survival of septic WT mice as compared to anti-IgG isotype control antibody treated mice.
  • Administration of anti-CD300b antibody 2 hours prior to LPS-injection resulted in a survival of about 85% of the anti-CD300b antibody-injected animals compared to mice that received an isotype control.
  • our data demonstrated that an administration of anti-CD300b antibody 6 hours after the LPS-injection resulted in a survival of about 75% of the anti-CD300b antibody-injected animals compared to the isotype control injected mice.
  • Reported KD values for mTLR4 and mMD-2 are 1.4 x 10 "5 M and 2.3 x 10 "6 M, respectively (Thomas et al., 2002, FEBS Lett 531, 184-188; Shin et al., 2007, Moll Cells 24, 119-124).
  • biochemical measurements like our SPR analysis, do not necessarily reflect the avidity of these receptors when interacting with LPS at the cells surface, due to the fact that receptors are often multimeric (e.g., CD300 receptors are most likely dimers; Martinez-Barriocanal et al., 2010, supra), differ in expression levels, and ligand binding often induces receptor clustering.
  • IL-10 is known to be protective in the LPS- and CLP-models of sepsis by decreasing pro-inflammatory cytokine levels (Howard et al., 1993, J Exp. Med. 177, 1205-1208; van der Poll et al., 1995, J. Immunol. 155, 5397-5401; Latifi et al., 2002, Infect.
  • IL-10 is produced by ⁇ , DC, B cells and T cells (Huhn et al., 1996, Blood 87, 699-705; Wang et al., 2012, N. Engl. J. Med. 366, 2122-2124), and to a lower extent by neutrophils and NK cells (Kasten et al., 2010, Biochem. Biophys. Res.
  • LPS-treated BMDC from WT mice produced higher levels of IL-10 than those from Cd300b ⁇ ' ⁇ mice. While DC theoretically could contribute to LPS-induced lethality, their involvement is likely less significant, at least in regards to CD300b function in regulating lethal inflammation. DC, unlike ⁇ , express relatively low levels of CD300b and TLR4 mRNA and protein, and they are found in significantly lower numbers in the peritoneal cavity and different tissues. In addition, there are reasons to believe that the same receptors may differentially regulate LPS-responses in ⁇ and DC, as CD1 lb has been shown to regulate the TLR4-LPS signaling response in DC but not in ⁇ (Ling et al., 2014, Nat. Commun. 5, 3039).
  • CD300b-mediated regulation of IL-10 production was defined, and the role of the CD300b/DAP12-Syk-PI3K complex was highlighted in TLR4 signaling.
  • DAP 12 was shown to play an important role in mediating bacterially-induced inflammation by functioning either as an activating (Turnbull et al., 2005, J. Exp. Med. 202, 363-369) or inhibitory molecule (Hamerman et al., 2005, Nat. Immunol. 6, 579-586.) depending on the severity of the disease.
  • TREM1 Bochon et al., 2000, J. Immunol. 164, 4991-4995
  • TREM2 Talnbull et al., 2006, J.
  • CD300b/DAP12/TLR4-Syk-PI3K signaling complex and the dissociation of MyD88/TIRAP from the complex.
  • the precise mechanism by which DAP12, Syk and PI3K regulate the displacement of MyD88/TIRAP and thereby the assembly of the receptor complex can be determined.
  • PI3K recruited to and activated in the complex could phosphorylate PtdIns(4,5)P2to PtdIns(3,4,5)P3, thereby reducing PtdIns(4,5)P2 levels and facilitating the dissociation of MyD88/TIRAP from the CD300b/TLR4 complex.
  • DAP12/FcYR-deficient ⁇ showed impaired internalization of TLR4, and consequently lacked signaling via the TRIF-IRF3 pathway, which is activated after endocytosis and leads to IFN- ⁇ production (Zanoni et al., 2011, supra). Therefore, it was examined if Cd300b ' ⁇ would display an impairment in the TLR4-TRIF signaling pathway, and it was found that LPS -treated ⁇ from WT mice produced significantly higher levels of IFN- ⁇ compared to Cd300b ' ⁇ .
  • CD14 is critical for the TLR4-MyD88-dependent TNFa response. Therefore, without being bound by theory, the role of CD300b as an LPS-sensor could be critical during severe bacterial infection, wherein the limitation of pro-inflammatory cytokine levels due to IL-10 production may be more detrimental than the potential harm that these cytokines may cause.
  • This relates to the observation that at high LPS concentrations the contributing role of CD14 (Zanoni et al., 2011, supra) may be dispensable (FIG. 12).
  • the findings disclosed herein show that CD300b/DAP12 regulates the TLR4-MyD88 as well as the TBKl-IKKs-IRF3 signaling cascade.
  • CD300f associates with IL-4Ra and regulates IL-4Ra- mediated responses by augmenting IL-4/IL-13- induced signaling, mediator release and priming (see also Moshkovits et al., 2015, Proc Natl Acad Sci U S A 112, 8708-8713).
  • CD300f associates with IL-4Ra and regulates IL-4Ra- mediated responses by augmenting IL-4/IL-13- induced signaling, mediator release and priming (see also Moshkovits et al., 2015, Proc Natl Acad Sci U S A 112, 8708-8713).
  • CD300f associates with IL-4Ra and regulates IL-4Ra- mediated responses by augmenting IL-4/IL-13- induced signaling, mediator release and priming (see also Moshkovits et al., 2015, Proc Natl Acad Sci U S A 112, 8708-8713).
  • CD300 family members have been shown to modulate LPS-induced
  • CD300b is a PS-binding phagocytic receptor promoting the engulfment of AC (efferocytosis) via DAP12 signaling (Murakami et al., 2014, supra), suggesting that the
  • CD300b/DAP12 complex plays a role in maintaining cellular homeostasis.
  • an important mechanism is provided that governs TLR4 signaling in ⁇ whereby CD300b/DAP12-Syk-PI3K association with TLR4 upon LPS binding results in lower IL-10 production.
  • CD300b may not form a robust complex with TLR4 and functions as a receptor that supports homeostasis through efferocytosis by ⁇ (FIG. 12).
  • the excess amount of endotoxin present shifts the function of CD300b to a receptor that responds to bacterial assault.
  • LPS suppresses CD300b-mediated efferocytosis and anti-inflammatory cytokine production, and stimulates the association of CD300b/DAP12-Syk-PI3K with TLR4; this leads to the displacement of MyD88/TIRAP, which likely results in the inhibition of its signaling, and blocks the production of IL-10 (FIG. 12). Furthermore, the data shows that CD300b activates the TLR4/CD 14-TRIF-IRF3 signaling pathway, resulting in enhanced IFN- ⁇ production.
  • LPS acts as a molecular switch to temporarily dispense CD300b-mediated efferocytosis, an anti-inflammatory function (Henson and Bratton, 2013, supra), to one that heightens the pro-inflammatory cytokine response (FIG. 12).
  • CD300b as a new LPS -recognizing receptor that regulates TLR4 signaling, thus controlling the balance between pro- and anti-inflammatory cytokine secretion during severe bacterial infection, and highlights CD300b as a potential therapeutic target for clinical intervention to manage septic shock in humans.
  • an anti-CD300b Antibody in Combination with an Anti-PDl, an Anti-CTLA-1 and/or an Anti-BTLA-4 Antibody
  • Wild-type mice are injected with a toxic dose of LPS (37 mg/kg) followed by a second injection, 6 h post-LPS administration, using anti-CD300b (5 ⁇ g/g; R&D, Cat.-No: MAB2580, clone 339003) in combination with either anti-PD-1 (200 ⁇ g/body, Bristol-Meyers Squibb, clone 4H2), anti-PDLl (200 ⁇ g/body, Bristol-Meyers Squibb, clone 14D8), anti-CTLA-4 (50 ⁇ g/body; R7D, Cat.-No: MAB434, clone 63828), anti-BTLA (400 ⁇ g/body; BioXCell, Cat-No: BE0132, clone 6A6) or anti-IgG control (5 ⁇ g/g, R&D, Cat.-No: MAB0061, clone 141945) antibody.
  • anti-CD300b 5 ⁇ g/
  • IL-10 ' C57BL/6 mice obtained from the Cancer and Inflammation Program, National Cancer Institute, MD, USA. Femurs and tibias of TLR4 ' mice were kindly provided University of Maryland, MD, USA, while Cdl4 'A ⁇ were provided by Harvard Medical School, MA, USA. All animals were bred and housed in a pathogen- free environment.
  • IgG Ab were from R&D, and were labeled with Alexa488 using the Alexa488 antibody labeling kit (Invitrogen), according to the manufacturer's instructions.
  • Syk (D3Z1E), pPI3K (#4228), PI3K (6G10), pAkt (D9E), Akt (#9272), pERKl/2 (3A7), ERK1/2 (#9102), pMEKl/2 (41G9), MEK1/2 (#9122), pJNK (G9), JNK (#9251), TIRAP (D6M9Z), pNFKB (93H1), NFKB (D14E12), pp38 (D3F9), p38 (D13E1), pTBKl (D52C2), TBK1 (D1B4), ⁇ , ⁇ , pIRF3 (4D4G), IRF3
  • D83B9 Abs were from Cell Signaling Technology. GAPDH (FL-355), DAP 12 (FL-113), TLR4 (25), and MyD88 (HFL-296) Abs, and HRP-conjugated secondary Abs (anti-mouse, anti-rabbit, anti-rat, and anti-goat) were from Santa Cruz Biotechnology.
  • the Alexa647 antibody labeling kit and the pSyk (F.724.5) and TLR4 (76B357.1) Abs were from Thermo Scientific.
  • Anti-CD14 (4C1) was from BD Biosciences and anti-human IgG-Fcy fragment specific Ab was from Jackson Immuno. Research.
  • the PI3K inhibitor, Wortmannin, the Syk inhibitor, Piceantannol, p38 inhibitor, SB203580, and the ERK1/2 inhibitor, PD98059 were obtained from Calbiochem, and dissolved in the diluent dimethyl sulfoxide (DMSO, Sigma).
  • Rhodamine-conjugated TLR2-TLR1 (PAM3CSK4)-, TLR3 (Poly(I:C))-, NOD2 (MDP)-specific ligands were from InvivoGen, and FITC-conjugated TLR4 (LPS) ligand or FITC-conjugated E. coli were obtained from Invitrogen.
  • LPS-E.coli (0127:B8) was obtained from Sigma.
  • LPS-E.coli (0111 :B4), LPS-E.coli (055:B5), LPS- E.coli (EH100 (Ra), LPS-E.coli (R515 (Re), Lipid A-E.coli (R515 (Re) and purified mTLR4-hFc were purchased from Enzo Life Sciences. Recombinant mCD14 was obtained from Sino Biological Inc, mLAIRl was purchased from R&D. DNA reagents
  • pCDH-EFl-T2A-puro vector System Biosciences
  • lentivirus expression constructs carrying mCD300b, mCD300f, FcRy, or DAP12 genes were previously described (Tian et al., 2014, supra; Murakami et al., 2014, supra; Yamanishi et al., 2012, supra).
  • constructs for the IgG-Fc portion fused to hCD300b hCD300b-Fcy
  • mCD300b mCD300b- Fcy
  • mCD300d mCD300d-Fcy
  • mCD300f mCD300f-Fcy
  • control protein NITR (NITR- Fcy)
  • extracellular domains in a pcDNA backbone were as described (Cannon et al., 2011, Immunogenetics 64, 39-47).
  • L929 and HEK293T cells were cultured in DMEM medium with 10% FBS.
  • HEK293T cells were transfected using PolyJet (Signagen).
  • Lentivirus particles were generated by co-transfection of HEK293T cells with pCDH-puro vector encoding mCD300b, mCD300d, mCD300f, mFcRy mDAP12, or psPAX2, and pMD2G helper plasmids.
  • L929 cells were infected with lentivirus particles for 24 h at 37°C, in the presence of 6 g/ml protamine sulfate. Selection with 20 ⁇ g/ml puromycin started 48 h after infection and clonal cell lines were obtained using the limiting dilution method as previously described (Murakami et al., 2014, supra). Chimeric proteins
  • HEK293T cells were transiently transfected with pcDNA3.0 plasmids encoding hCD300b- Fcy, mCD300b-Fcy, mCD300d-Fcy, mCD300f-FcY or NITR-Fcy constructs using PolyJet, and chimeric proteins were purified as previously described (Murakami et al., 2014, supra). Protein induction and purification
  • the Ig-domain of mCD300b was PCR-amplified from the pCDNA3-mCD300b-FcY plasmid (Cannon et al., 2011, supra), using primers 5'-
  • CD300b-Ig-domain was expressed in Rosetta 2 (DE3) E.coli using 1 mM IPTG (isopropyl l-thio- ⁇ - D-galactopyranoside) as inclusion bodies.
  • the protein was refolded by dilution into refolding buffer (0.4 M arginine HC1, 0.1 M Tris, pH 8, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione) for 7 days at 4°C; dialyzed against 150 mM NaCl and 25 mM MES, pH 6.5; concentrated with an Amicon stirred cell concentrator using an Ultracell 10-kDa ultrafiltration regenerated cellulose filter (Millipore); purified by gel filtration on Superdex HR 75 followed by ion exchange
  • the interaction with LPS was assessed by injection with various concentrations of hCD300b-Fcy, mCD300b-Fc, mCD300a-Fcy, mCD300d- Fcy, mCD300f-FcY, NITR-Fcy, or mCD300b, mCD14 and mLAIRl proteins, ranging from 0.125 to 5 ⁇ .
  • Dissociation constants (KD) were calculated using BIAevaluation software as described previously (Sgourakis et al., 2015, supra).
  • LPS blocking experiments purified mCD300b (0.5 ⁇ ) was pre- incubated for 0.5 h at 4°C with different concentrations of wild-type LPS: LPS-E.coli (0111 :B4), LPS-E.coli (0127:B8), LPS-E.coli (055:B5) or structural components of LPS: lipid A- E.coli (R515 (Re), LPS-E.coli (EH100 (Ra), and LPS-E.coli (R515 (Re), ranging from 1-100 ⁇ g/ml. Binding data were acquired with a flow rate of 20 ⁇ /min for 2 min. After 2 min dissociation, the bound analytes were removed by a 1 min regeneration phase with a washing buffer containing 2.5 M NaCl and 50 mM NaOH. Cell based TLR-ligand binding assays
  • Rhodamine-labeled TLR2/TLR1 PAM3CSK4-, TLR3 (Poly(I:C) , NOD2 (MDP)-specific ligands or FITC-labeled TLR4 (LPS)-ligand purified from either E. coli or S. minnesota (10 ⁇ g/ml) were incubated with mCD300b/DAP12-, mCD300d/FcRy-, mCD300f-, EV-expressing L929 cells or ⁇ from WT, Cd300b , and Cdl4 A mice, then incubated for up to 2 h at 37°C or 4°C. Binding was determined by flow cytometry and represented as mean fluorescence intensity (MFI) or percentage of LPS binding.
  • CLP Cecal Ligation Puncture
  • lethal endotoxemia CLP
  • CLP was performed as described previously (Leelahavanichkul et al., 2014, Am J Physiol Renal Physiol 307, F939-948). Briefly, the cecum was ligated and punctured twice with a 21-gauge needle, then gently squeezed to express a small amount of fecal material, and returned to the central abdominal cavity. Sham-control mice were subjected to a similar laparotomy without ligation and puncture. Pre-warmed normal saline (40 ml/kg) was immediately given intraperitoneally (i.p.) after surgery and slow release buprenorphrine (0.5 mg/kg) was given subcutaneously every 72 h for pain management.
  • Lethal endotoxemia was induced by i.p. injection of LPS (37 mg/kg, E.coli, serotype 0127:B8), dissolved in 0.1 ml PBS.
  • LPS 37 mg/kg, E.coli, serotype 0127:B8
  • serum cytokine levels were measured by flow cytometry.
  • Lung tissue specimens were collected after 12 h and fixed in 10% neutral-buffered formalin for histology.
  • TNFa, IFNy, IL-6, IL-10, IL-12, and MCP-1 concentrations were measured using the
  • CBA Cytometric Bead Array
  • Bacterial colony-forming units in blood, peritoneal cavity, lung, spleen and liver were assessed in CLP-treated WT and Cd300b ' mice. Bacterial burden was determined 24 h after CLP- treatment. The number of colony formation units (c.f.u.) was determined by plating 10-fold serial dilutions of blood. For assessing the bacterial load in organ tissues, equal amount (g) of tissue was homogenized and 10-fold serial dilutions were plated. A 100 ⁇ aliquot of each dilution was spread on brain heart infusion agar plates without antibiotics and incubated under aerobic conditions at 37°C for 24 h. Depletion of neutrophils and NK cells
  • mice were injected i.p. with 500 ⁇ g anti-Ly6G (clone 1A8, BioxCell) or control Ab (clone 2A3, BioxCell) 24 h before the induction of lethal endotoxemia.
  • mice were injected i.v. with 500 ⁇ g anti-NKl.l (clone PK136, BioxCell) or control Ab (clone CI.18.4, BioxCell) at 5, 3 and 1 day before the induction of lethal endotoxemia.
  • ⁇ were lysed for 1 h at 4°C in ice-cold lysis buffer A [20 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 1% NP40, supplemented with protease and phosphatase inhibitory cocktails (Sigma)].
  • Cellular debris was removed by centrifugation at 10,000 x g for 15 min at 4°C.
  • Equal amounts of protein, as determined by Bradford assay, were loaded for SDS-PAGE, then transferred onto nitrocellulose membranes.
  • Membranes were probed with Abs of interest, followed by enhanced chemiluminescence with secondary Abs conjugated to horseradish peroxidase.
  • mCD300b, mCD14 and mTLR4 proteins were mixed using equal molar ratios (1 : 1 : 1) in the presence or absence of 2 ⁇ g/ml LPS for 1 h at 4°C. Reactions were incubated with 2.5 mM of dithiobissuccinimidyl propionate (DSP) for 10 min at RT and the reaction was quenched using 0.5 M Tris, pH 7.5 for additional 15 min.
  • DSP dithiobissuccinimidyl propionate
  • LPS binding to mCD300b-, mCD300d-, mCD300f-, or NITR-Fcy chimeric proteins was determined using a streptavidin pulldown assay.
  • Biotin-conjugated LPS (InvivoGen) was mixed with different concentrations of Fcy-chimeric proteins. Reactions were incubated overnight at 4°C in lysis buffer C (20 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 0.5% NP40), followed by 1 h incubation with 5 ⁇ of streptavidin-magnetic beads (New England BioLabs). Beads were washed 3 x with lysis buffer C and reactions were analyzed by immunoblotting using an anti-human IgG-Fcy- specific Ab.
  • FITC-labeled LPS-E. coli (10 ⁇ g/ml) was incubated with mCD300b/DAP12-expressing L929 cells at 4°C in the presence of increasing concentrations of unlabeled LPS from either E. coli, or S. minnesota, ranging from 1 to 100 ⁇ g/ml.
  • v Zymosan A (a TLR2-ligand) was used as control.
  • vSamples were analyzed by flow cytometry and MFI values from reactions without unlabeled LPS were considered as the maximum binding level (0%
  • ⁇ from WT or Cd300b ' mice were pretreated for 12 h with anti-IgG isotype control Ab (5 ⁇ g), anti-CD300b Ab (5 ⁇ g), anti-CD 14 Ab (5 ⁇ g) or both anti- CD300b Ab (2.5 ⁇ g), anti-CD14 Ab (2.5 ⁇ g) before the addition of 10 ug/ml FITC-labeled LPS for 2 h at 37°C or 4°C. Binding was analyzed by flow cytometry.
  • mice ⁇ from WT, Cd300b ' , and TLR4 'A mice were plated for 12 h prior on number 1.5 glass dishes (MatTek Corporation). Then, cells were either treated with LPS (2 ⁇ g/ml) for 20 min at 37°C or left unstimulated (NT), followed by staining with Alexa647 -conjugated anti-TLR4 Ab or an anti-IgG isotype control Ab for 2 h at 4°C. Next, cells were washed twice with PBS and fixed with methanohacetic acid (95%:5%) for 10 min at -20°C.
  • CD300b and TLR4 expression profiles on ⁇ from the bone marrow, peritoneal cavity, lung or spleen cells were stained with Alexa488-conjugated anti-CD300b and APC- conjugated anti-TLR4 Abs.
  • the expression of CD300b and TLR4 was determined on ⁇ (F4/80 + , CDl lb hi , CDl lc " Ly6G " SSC l0 ) by using cell type specific Abs, as indicated in the text.
  • lung or splenic cells were first isolated by homogenization and then passed through nylon mesh strainers (70 ⁇ , Fisher Scientific). Cells were treated with an anti-CD16/32 Ab (2.4G2, BD Biosciences) to block Fc receptor binding, and then stained with the indicated Abs in PBS containing 0.2% FBS. All Abs were obtained from BioLegend and included the following molecules: CD16/CD32 (93), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CDl lb (Ml/70), CDl lc (N418), CD19 (MB 19-1), CD21 (eBio8D9), CD23 (B3B4), B220 (RA3-6B2),
  • NK1.1 PK136
  • PDCA-1 eBio927
  • F4/80 BM8
  • TLR4 SA15-21
  • Ly6G Ly6G
  • Dead cells were excluded using ZOMBIE NIRTM (BioLegend) staining following the manufactures recommendations. Stained cells were sorted with a FACS Aria- Red (BD Bioscience) and analyzed with FlowJo software (v.10, Tree Star).
  • Bone marrow cells were isolated from femurs and tibias of WT, Cd300b ' , Cd300b ⁇ ' ⁇ IL-10 ' , Cd300f or TLR4 ' mice. Differentiation of ⁇ was induced by culturing bone marrow cells in RPMI 1640 medium supplemented with 10% FBS and 30% L929-conditioned medium (a source of macrophage colony-stimulating factor), while differentiation of BMDC was induced by culturing cells in RPMI 1640 medium supplemented with 10% FBS and 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF). ⁇ or BMDC were cultured for 7 days with one renewal of the culture medium. Phagocytosis of apoptotic cells
  • Thymocytes from C57BL/6 mice were incubated with 10 ⁇ dexamethasone in RPMI medium with 1% FBS for 6 h for the generation of apoptotic cells (AC) (Tian et al., 2014, Nat Commun 5, 3146; Yamanishi et al., 2012, supra).
  • the AC were labeled with pHrodo succinimidyl ester (Invitrogen) according to the manufacturer's instruction.
  • ⁇ (2 x 10 5 ) were incubated with pHrodo-labeled AC at a ratio of 1:2 for various lengths of time at 37°C and stained using an anti- mouse F4/80 Ab.
  • Cells were washed and suspended in basic buffer (pH 8.8) to quench the fluorescence of non-engulfed pHrodo-labeled AC before the flow cytometry analysis.
  • RNA was isolated using the RNAquous-4PCR kit (Ambion) following the
  • cDNA was synthesized with Qscript cDNA synthesis kits (Quanta Biosciences) and quantitative real-time PCR (qRT-PCR) was performed as previously described (Murakami et al., 2014, Cell Death Differ 21, 1746-1757).
  • Oligonucleotide primers for amplifying murine Cd300b, TLR4 and GAPDH were purchased from Qiagen.

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Abstract

L'invention concerne une composition pharmaceutique comprenant un antagoniste de CD300b destinée à être utilisée dans le traitement ou la prévention du développement d'un sepsis ou d'un choc septique chez un sujet. La composition pharmacologique peut éventuellement comprendre de l'IL-10, un antagoniste de TLR4 ou un antagoniste de CD14. L'invention concerne également des procédés pour traiter ou prévenir le développement d'un sepsis ou d'un choc septique chez un sujet. Les procédés comprennent l'administration, au sujet, d'une quantité thérapeutiquement efficace d'un antagoniste de CD300b. Les procédés peuvent éventuellement comprendre l'administration d'une quantité thérapeutiquement efficace d'IL-10, d'un antagoniste de TLR4, d'un antagoniste de CD14, d'un antagoniste de PD-1, d'un antagoniste de CTLA-4 et/ou d'un antagoniste de BTLA. L'antagoniste de CD300b, l'antagoniste de TLR4, l'antagoniste de CD14, l'antagoniste de PD-1, l'antagoniste de CTLA 4 et/ou l'agoniste de BTLA peuvent être un anticorps ou un fragment de liaison à l'antigène correspondant.
PCT/US2017/021654 2016-03-14 2017-03-09 Utilisation d'antagonistes de cd300b pour traiter un sepsis et un choc septique WO2017160599A1 (fr)

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WO2021097685A1 (fr) * 2019-11-19 2021-05-27 深圳迈瑞生物医疗电子股份有限公司 Kit et procédé de détection mixte de pct et de présepsine et leur utilisation
WO2021155097A1 (fr) * 2020-01-29 2021-08-05 The Wistar Institute Of Anatomy And Biology Blocage de cd40-l pour améliorer une thérapie par anticorps synthétiques
WO2023069919A1 (fr) * 2021-10-19 2023-04-27 Alector Llc Anticorps anti-cd300lb et leurs procédés d'utilisation

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