WO2015038538A1 - Compositions and methods for treating sepsis - Google Patents

Compositions and methods for treating sepsis Download PDF

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Publication number
WO2015038538A1
WO2015038538A1 PCT/US2014/054773 US2014054773W WO2015038538A1 WO 2015038538 A1 WO2015038538 A1 WO 2015038538A1 US 2014054773 W US2014054773 W US 2014054773W WO 2015038538 A1 WO2015038538 A1 WO 2015038538A1
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subject
antibody
levels
sepsis
responsive
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PCT/US2014/054773
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French (fr)
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Subramaniam Krishnan
Wade Blair
Andriani PATERA
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Medimmune, Llc
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Publication of WO2015038538A1 publication Critical patent/WO2015038538A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Sepsis is a potentially life-threatening complication of a severe infection. Sepsis occurs when chemicals released into the bloodstream to fight the infection trigger inflammation throughout the body. This systemic inflammation can trigger a cascade of changes that can damage multiple organ systems, causing them to fail. The systemic response can lead to septic shock, which is characterized by a precipitous drop in blood pressure, cardiovascular collapse, and/or multiple organ failure.
  • the present invention features compositions and methods for identifying a subject responsive to PD-1 pathway blockade or treating a patient preselected as responsive to PD-1 pathway blockade.
  • the invention provides methods for treating sepsis, septic shock, systemic inflammatory response syndrome, and compensatory anti-inflammatory response syndrome, as well as methods for reducing sepsis- induced lymphocyte apoptosis, reducing T cell proliferation, increasing IFN- ⁇ or IL-2 levels, or otherwise reducing immune dysfunction in a subject, for example, in a subject pre-selected as responsive to PD- 1 pathway blockade.
  • the invention generally provides a method of identifying a subject (e.g., human) responsive to PD- 1 pathway blockade, the method involving detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels (e.g., by immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
  • immunoprecipitation in a biological sample (e.g., blood) of the subject relative to a reference, thereby identifying the subject as responsive to PD-1 pathway blockade.
  • a biological sample e.g., blood
  • the invention generally provides a method of identifying a subject responsive to PD- 1 pathway blockade, the method involving detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels in a biological sample of the subject relative to a reference, where the detection is by any one or more of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
  • the invention provides a method of reducing T cell proliferation in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 and/or anti-PD-Ll antibody, where the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby reducing T cell proliferation in the subject.
  • the invention provides a method of reducing sepsis-induced lymphocyte apoptosis in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 or anti-PD-Ll antibody, where the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby reducing sepsis-induced lymphocyte apoptosis in the subject.
  • the invention provides a method of increasing IFN- ⁇ or IL-2 levels in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 or anti-PD-Ll antibody, where the subject is preselected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby increasing IFN- ⁇ or IL-2 levels in the subject.
  • the invention provides a method of reducing immune dysfunction in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 or anti-PD-Ll antibody, where the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-L1 levels relative to a reference, thereby reducing immune dysfunction in the subject.
  • the detection is by any one or more of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
  • the invention provides a method of treating sepsis in a subject identified as responsive to PD-1 pathway blockade, the method involving detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels in a biological sample of the subject relative to a reference, thereby identifying the subject as responsive to PD-1 pathway blockade; and administering to the subject an anti-PD-1 or anti-PD-Ll antibody, thereby treating sepsis in the subject.
  • the invention provides a method of identifying a subject responsive to PD- 1 pathway blockade, the method involving detecting immune dysfunction where CD8+ PD-1 >36 and PD-Ll ⁇ 5 expression; decreased HLA-DR expression; and decreased TNF-a levels in LPS -stimulated whole blood, where the detection identifies the patient as responsive to anti-PD-1 and/or anti-PD-Ll antibody therapy.
  • the invention provides a method of identifying a subject responsive to PD- 1 pathway blockade, the method involving detecting immune dysfunction where CD8+ PD-1 >36 and PD-Ll ⁇ 5 expression; decreased HLA-DR expression; and decreased TNF-a levels in LPS -stimulated whole blood, where the detection is by a method is any one or more of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and immunoprecipitation, where the detection identifies the patient as responsive to anti-PD-1 and/or anti-PD-Ll antibody therapy.
  • the invention provides a method of treating sepsis in a subject identified as responsive to PD-1 pathway blockade, the method involving detecting immune dysfunction where CD8+ PD-1 >36 and PD-Ll ⁇ 5 expression; decreased HLA-DR expression; and decreased TNF-a levels in LPS -stimulated whole blood, where the detection identifies the patient as responsive to PD-1 pathway blockade; and administering to the subject an anti-PD-1 or anti-PD-Ll antibody, thereby treating sepsis in the subject.
  • the invention provides a kit for treating sepsis, the kit containing an effective amount of an antibody that specifically binds PD-1 and/or PD-Ll, and instructions for using the kit to treat sepsis.
  • the invention provides a kit for identifying a subject responsive to PD-1 pathway blockade, the kit containing a capture reagent that binds PD-1 and a capture reagent that binds PD-Ll .
  • the method identifies the subject as in need of treatment with an anti-PD-1 or anti-PD-Ll antibody. In other embodiments, the method characterizes the level or stage of immune dysfunction in the subject. In other embodiments of any of the above aspects or any other aspect of the invention delineated herein CD8+ PD-1 levels are increased (e.g., by at least about 25, 30, 35, 40, 45, 50, 55, 60, 65 percent or more). In various embodiments of the above aspects or any other aspect of the invention delineated herein, CD8+ PD-1 >36% and PD-Ll ⁇ 5% expression.
  • CD8+ PD-1 levels are greater than at least about 36% relative to a reference and PD-Ll levels are less than about 5% relative to the level in a reference.
  • the method further involves detecting a decrease in HLA-DR expression levels relative to a reference.
  • the method further involves detecting a decrease in TNF-a levels in LPS -stimulated whole blood.
  • the method characterizes the immunosuppressive phase of sepsis in the subject.
  • the administration of an anti-PD-1 and/or anti-PD-Ll antibody restores cytokine production
  • the antibody decreases sepsis-induced lymphocyte apoptosis, or restores immunity in the subject.
  • sepsis is bacterial or fungal sepsis.
  • the antibody neutralizes PD-1 and/or PD-Ll .
  • the antibody is LOPD180 or the antibody contains one or more variable regions of a LOPD180 antibody.
  • the anti-PD-1 or anti- PD-Ll antibody is administered at about 10 mg/kg, 30 mg/kg, or 60 mg/kg.
  • the method increases an immune response in the subject, increases T-cell proliferation, IFN- ⁇ production, and/or IL-2 production in the subject.
  • the administering is by intravenous injection.
  • anti-PD-1 antibody an antibody that selectively binds a PD-1 polypeptide.
  • LOPD 180 is an exemplary PD-1 antibody.
  • anti-PD-Ll antibody an antibody that selectively binds a PD-Ll polypeptide.
  • exemplary anti-PD-Ll antibodies are described for example at WO
  • PD-1 polypeptide is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_005009 and having PD-Ll and/or PD-L2 binding activity or other PD- 1 biological activity.
  • PD-1 nucleic acid molecule is meant a polynucleotide encoding a PD-1 polypeptide.
  • An exemplary PD-1 nucleic acid molecule sequence is provided at NCBI Accession No. NM_005018.
  • PD-Ll polypeptide is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001254635 and having PD-1 and CD80 binding activity or other PD-Ll biological activity.
  • PD-Ll nucleic acid molecule is meant a polynucleotide encoding a PD-Ll polypeptide.
  • An exemplary PD-Ll nucleic acid molecule sequence is provided at NCBI Accession No. NM_001267706.
  • responsive in the context of therapy is meant susceptible to treatment.
  • PD-1 pathway blockage is meant treatment with an agent that negatively regulates PD-1 signalling.
  • Treatment with an anti-PDl antibody or anti-PDLl antibody is an exemplary PD- 1 pathway blockade.
  • responsive to PD-1 pathway blockade is meant having an immune dysfunction characterized by increased PD-1 and decreased PD-Ll on a CD8+ T cell.
  • a septic patient is characterized as having (i) immune dysfunction where CD8+ PD-1 >36% and PD-Ll ⁇ 5% expression; (ii) decreased HLA-DR expression relative to a reference; and/or (iii) decreased TNF-a levels in LPS -stimulated whole blood relative to a reference.
  • CD8+ PD-1 expression is greater than at least about 25%, 30%, 35%, 40%, 50%, 60%, 75%, 85% or more relative to a reference.
  • PD-Ll is less than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% expression relative to a reference.
  • a “biomarker” or “marker” as used herein generally refers to a protein, nucleic acid molecule, clinical indicator, or other analyte that is associated with a disease.
  • a marker of sepsis is differentially present in a biological sample obtained from a subject having or at risk of developing sepsis relative to a reference.
  • a marker can be used to characterize the stage of immune and/or inflammatory dysfunction in a subject diagnosed as having sepsis, septic shock, systemic inflammatory response syndrome, compensatory anti-inflammatory response syndrome or related disorders.
  • a marker is differentially present if the mean or median level of the marker present in the sample is statistically different from the level present in a reference.
  • a reference level may be, for example, the level present in a sample obtained from a healthy control subject the level obtained from the subject at an earlier timepoint, i.e., prior to treatment, or the level obtained from a subject at a defined stage of septic illness.
  • Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney and odds ratio.
  • Biomarkers alone or in combination, provide measures of relative likelihood that a subject belongs to a phenotypic status of interest.
  • the differential presence of a marker of the invention in a subject sample can be useful in characterizing the immune dysfunction present in a subject identified as having or at risk of developing sepsis, for determining the prognosis of the subject, for evaluating therapeutic efficacy, or for selecting a treatment regimen.
  • active or “activity” in regard to a PD-1 and/or PD-Ll polypeptide refers to a portion of a PD-1 and/or PD-Ll polypeptide that has a biological, inhibitory, and/or immunosuppressive activity of a native PD-1 and/or PD-Ll polypeptide.
  • PD-1 has one or more immunoregulatory activities associated with PD-1.
  • PD-1 is a negative regulator of the TcR/CD28 -mediated immune response.
  • PD-1 and/or PD-L1 biological activity when used herein refers to a biological function that results from the activity of the native PD-1 and/or PD-L1 polypeptide.
  • a 5 particular PD-1 and/or PD-L1 biological activity includes, for example, negative regulation of immune responses, induction of lymphocyte apoptosis, reduced T cell proliferation, and reduced IFN- ⁇ production, and reduced IL-2 production.
  • antibody refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigenic) binding site, regardless whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies.
  • antibody also includes antibody fragments such as Fab, F(ab')2, Fv, 15 scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e. , the ability to bind PD- 1 specifically. Typically, such fragments would comprise an antigen- binding domain.
  • antigen-binding domain refers to a part of an antibody molecule that comprises amino acids responsible for 0 the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as “epitope” or "antigenic determinant.”
  • An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable 5 region (VH), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain, but still retains some antigen-binding function of the intact antibody.
  • Binding fragments of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab',
  • bifunctional antibody is understood to have each of its binding sites identical. Digestion of antibodies with the enzyme, papain, results in two identical antigen-binding fragments, known also as “Fab” fragments, and a "Fc” fragment, having no antigen-binding activity but having the ability to crystallize. Digestion of antibodies with the enzyme, pepsin, results in the a F(ab')2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab')2 fragment has the ability to crosslink antigen. “Fv” when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. "Fab” when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
  • mAb refers to monoclonal antibody.
  • Antibodies of the invention comprise without limitation whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.
  • biological sample any tissue, cell, fluid, or other material derived from an organism.
  • kits for identifying a subject as responsive to PD-1 pathway blockade using a capture reagent that binds a PD-1 or PD-L1 polypeptide e.g., anti-PD-1 or anti-PD-Ll antibody.
  • capture reagent is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
  • detecting refer to both quantitative and qualitative determinations, and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase “determining a level" of an analyte or “detecting” an analyte is used.
  • the term "effective amount” refers to a dosage or amount that is sufficient to reduce the activity of PD-1 to result in amelioration of symptoms in a patient or to achieve a desired biological outcome, e.g., increased cytolytic activity of T cells, reinduction of immune tolerance, reduction or increase of the PD- 1 activity associated with the negative regulation of T-cell mediated immune response, etc.
  • subject refers to an animal which is the object of treatment, observation, or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non- human primate, murine, bovine, equine, canine, ovine, or feline.
  • the term "decrease” or “increase” is meant to alter negatively or positively, respectively.
  • An alteration may be by 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even by 100%.
  • the PD- 1 or PD-L1 polypeptide or polynucleotide level present in a patient sample may be compared to the level of said polypeptide or polynucleotide present in a corresponding healthy cell or tissue.
  • the standard of comparison is the level of PD-1 or PD-L1 polypeptide or polynucleotide level present in serum of a subject that does not have sepsis.
  • the standard of comparison is the level of PD-1 or PD-L1 polypeptide or polynucleotide level present in a biological sample of a subject that has an early stage of sepsis (e.g.
  • the PD-1 or PD- Ll polypeptide or polynucleotide level polypeptide level present in a patient sample may be compared to the level of said polypeptide present in a corresponding sample obtained at an earlier time point. If desired, such levels are monitored periodically.
  • Periodic patient monitoring includes, for example, a schedule of tests that are administered daily, bi-weekly, bi-monthly, monthly, bi- annually, or annually.
  • sepsis refers to a medical condition characterized by a whole -body inflammatory state (called a systemic inflammatory response syndrome or SIRS) associated with a severe infection. Sepsis is commonly caused by the immune system's response to a serious infection, (e.g. , from bacteria, fungi, viruses, and parasites in the blood, urinary tract, lungs, skin, or other tissues). Indications of sepsis can range from infection to multiple organ dysfunction syndrome. Common symptoms of sepsis include those related to a specific infection, but usually accompanied by high fevers, hot, flushed skin, elevated heart rate, hyperventilation, altered mental status, swelling, and low blood pressure. In the very young and elderly, or in people with weakened immune systems, the pattern of symptoms may be atypical, with hypothermia and without an easily localizable infection.
  • nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringency See, e.g. , Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
  • stringent salt concentration will ordinarily be less than about 750 mM
  • NaCl and 75 mM trisodium citrate preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g. , formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • ssDNA denatured salmon sperm DNA
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • the invention is not limited to the specific antibodies described herein as binding a
  • PD-1 or PD-L1 polypeptide encompasses other anti-PD-1 and PD-L1 antibodies, including antibodies that are substantially identical to the antibodies delineated herein (e.g., LOPD180).
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
  • BLAST program may be used, with a probability score between e "3 and e "100 indicating a closely related sequence.
  • sample includes a biologic sample such as any tissue, cell, fluid, or other material derived from an organism.
  • telomere binding binds is meant a compound (e.g., antibody) that recognizes and binds a molecule (e.g., polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.
  • a compound e.g., antibody
  • molecule e.g., polypeptide
  • two molecules that specifically bind form a complex that is relatively stable under physiologic conditions.
  • Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity.
  • binding is considered specific when the affinity constant KA is higher than 10 6 M _1 , or more preferably higher than 10 8 M _1 .
  • non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions.
  • the appropriate binding conditions such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g. , serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • the terms "comprises,” “comprising,” “containing,” “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • Figure 1 provides four graphs depicting PD-1, PD-L1, and HLA-DR expression in septic and non-septic patients.
  • Septic and non-septic patients were identified, and heparinized blood samples were obtained at a maximum of 4 time points during their septic course.
  • Peripheral blood mononuclear cells were stained for lymphocyte (CD4, upper left panel; CD8, upper right panel) and monocyte markers (CD14, lower panels).
  • Immunostaining was also performed for PD-1 (upper panels), PD-L1 (lower left panel), and HLA-DR (lower right panel).
  • Flow cytometry revealed an increase in PD-1 and PD-L1 expression in CD8 T cells and monocytes from septic versus non-septic patients.
  • HLA-DR expression was decreased in monocytes from septic versus non-septic patients as well.
  • Data are from 43 septic (70 data points) and 16 non-septic patients (16 data points).
  • Figures 2A-2B show that markers of immune exhaustion increased with protracted sepsis. Flow cytometry revealed an increase in expression of immune exhaustion markers over time in ICU.
  • Figure 2A is a graph showing that PD-1 expression increased as PD-L1 expression decreased on CD8 T cells in samples from septic patients over the course of stay in ICU.
  • Figure 2B is a graph showing separation of a CD8 subset into an exhausted phenotype.
  • Figure 3 is a graph showing that septic patients with an immune exhausted phenotype had a higher incidence of secondary infections.
  • CD8+ PD-l high PD-Ll low is defined as CD8+ PD-1 >36 and CD8+ PD-Ll ⁇ 5 expression
  • CD8+ PD-l low PD- Ll high is defined as CD8+ PD-1 ⁇ 36 and CD8+ PD-Ll >5 expression.
  • Groups were analyzed for presence of more than two pathogens, secondary infections, type and route of infection (VAP or peritonitis).
  • Figure 4 depicts graphs showing that blockade of PD-1 or PD-Ll decreased sepsis- induced lymphocyte apoptosis.
  • PBMCs from septic patients (top panels) or non-septic patients (bottom panels) were incubated overnight in media containing inactive isotype control antibody, anti-PD-1 antibody (left panels), or anti-PD-Ll antibody (right panels). The next morning, cells were washed and immunostained for lymphocyte markers. TUNEL assay was performed to detect apoptosis. Apoptosis was quantitated in cells located in the lymphocyte gate (determined by forward and side scatter properties - see, e.g.
  • FIG. 5 A for a representative flow histogram consisting of but not limited to CD4 T cells, CD8 T cells, NK cells, ⁇ T cells, and NKT cells.
  • Apoptosis was increased in lymphocytes that had been incubated in inactive isotype control antibody from septic versus non-septic patients, 10.4 ⁇ 1.5% and 6.1 ⁇ 1.0% respectively (p ⁇ 0.01).
  • overnight incubation with anti-PD-1 and anti-PD-Ll reduced apoptosis in lymphocytes from septic but not non-septic patients 40.7% and 29.9% respectively for anti- PD-1 and anti-PD-Ll.
  • Figures 5A and 5B depicts representative flow cytometric histograms of lymphocyte gating strategy and detection of lymphocyte apoptosis via TUNEL assay.
  • Blood from a septic patient was obtained and PBMCs isolated by ficol gradient.
  • Figure 5A depicts representative flow cytometric histograms of lymphocyte gating strategy. The lymphocyte fraction in the PBMCs was identified by characteristic forward and side scatter properties.
  • Figure 5B depicts representative flow cytometric histograms of detection of lymphocyte apoptosis via TUNEL assay.
  • CD4 (left panels) and CD8 T cells (right panels) were identified by cell specific antibodies.
  • PBMCs were plated overnight with inactive, isotype control antibody (top panels), anti-PD-1 antibody (middle panels), or anti-PD-Ll antibody (bottom panels).
  • Apoptosis in CD4 T cells incubated in inactive isotype control antibody was 7.5%.
  • Anti-PD-1 and anti-PD-Ll decreased CD4 apoptosis to 1.25 and 2.0% respectively.
  • a similar protective effect was seen in CD8 T cells.
  • Figure 6 depicts four graphs showing quantitation of apoptosis in CD4 T cells via TUNEL in septic and non-septic patients.
  • Peripheral blood mononuclear cells (PBMCs) from septic patients (top panels) or non-septic patients (bottom panels) were incubated overnight in media containing anti-PD-1 antibody (left panels), or anti-PD-Ll antibody (right panels).
  • PBMCs were incubated overnight with inactive isotype control antibody The following morning, cells were washed and underwent immunostaining followed by detection of apoptosis via TUNEL assay. Flow cytometry was used to quantitate apoptosis in different lymphocyte subsets including CD4 T cells.
  • Figure 7 depicts four graphs showing that sepsis impaired lymphocyte IFN- ⁇ production.
  • Peripheral blood mononuclear cells (PBMCs) from septic or non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN- ⁇ .
  • Flow cytometric analysis revealed a decrease in the percentage of total lymphocytes and NKT cells that were IFN- ⁇ positive in septic compared to non-septic patients; p ⁇ 0.02. Data are from 15 septic (21 data points) and 7 non-septic patients (7 data points) obtained during their illness.
  • Figure 8 includes four graphs showing that sepsis impaired lymphocyte IL-2 production.
  • Peripheral blood mononuclear cells (PBMCs) from septic or non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IL-2.
  • Flow cytometric analysis revealed a decrease in the percentage of total lymphocytes, CD3 T cells, and NKT cells that were IL-2 positive in septic versus non-septic patients, p ⁇ 0.03, p ⁇ 0.02, and p ⁇ 0.002 respectively.
  • Data are from 15 septic (21 data points) and 7 non-septic patients (7 data points) obtained throughout their illness - all blood draws.
  • Figure 9 includes graphs showing that anti-PD-1 and anti-PD-Ll antibodies increased IFN- ⁇ production in sepsis.
  • Peripheral blood mononuclear cells (PBMCs) from septic patients were incubated overnight in media containing anti-PD-1 antibody (top panels) or anti-PD-Ll antibody (bottom panels).
  • PBMCs from septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN- ⁇ .
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs from septic patients were incubated overnight in media containing inactive isotype control antibody.
  • cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN- ⁇ .
  • the lymphocyte gate was identified by characteristic forward and side scatter properties.
  • NK cells were identified as CD3 " , CD56 + .
  • the percentage of NK cells that were positive for IFN- ⁇ that were incubated in inactive isotype control antibody was 9.9%.
  • Treatment with anti-PD-1 and anti-PD-Ll increased the percentage of NK cells that were IFN- ⁇ positive to 34.8 and 21.8% respectively.
  • Figure 11 includes six graphs showing that anti-PD-1 and anti-PD-Ll improved IL-2 production in sepsis.
  • Peripheral blood mononuclear cells (PBMCs) from septic patients were incubated overnight in media containing anti-PD-1 antibody (top panels) or anti-PD-Ll antibody (bottom panels).
  • PBMCs from septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IL-2.
  • Figure 12 provides six graphs showing that anti-PD-1 and anti-PD-Ll antibodies did not significantly increase IFN- ⁇ in non-septic patients.
  • Peripheral blood mononuclear cells (PBMCs) from non-septic patients were incubated overnight in media containing anti-PD- 1 antibody (top panels) or anti-PD-Ll antibody (bottom panels).
  • PBMCs from non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN- ⁇ .
  • Figure 13 provides six graphs showing that anti-PD-1 and anti-PD-Ll did not significantly increase IL-2 in non-septic patients.
  • Peripheral blood mononuclear cells (PBMCs) from non-septic patients were incubated overnight in media containing anti-PD- 1 antibody (top panels) or anti-PD-Ll antibody (bottom panels).
  • PBMCs from non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IL-2.
  • Flow cytometric analysis revealed that neither anti-PD-1 nor anti-PD-Ll antibody caused a significant increase in the percentage of cells that were positive for IL-2. Data are from blood draws from 6-7 non-septic patients (6-7 data points throughout their illness).
  • Figure 15 provides sequences of anti-PD 1 antibody (LOPD180) polypeptides.
  • Figure 16 provides exemplary sequences of human PD-1, PD-L1, and anti-PD 1 antibody (LOPD180) polypeptides and nucleic acid molecules.
  • the present invention features compositions and methods for identifying a subject responsive to PD-1 pathway blockade or treating a patient preselected as responsive to PD-1 pathway blockade.
  • the invention provides methods for treating sepsis, septic shock, systemic inflammatory response syndrome, and compensatory anti-inflammatory response syndrome, as well as methods for reducing sepsis- induced lymphocyte apoptosis, reducing T cell proliferation, increasing IFN- ⁇ or IL-2 levels, or otherwise reducing immune dysfunction in a subject, for example, in a subject pre-selected as responsive to PD- 1 pathway blockade.
  • the present invention is based, at least in part, on the discovery that subjects suffering from sepsis are a heterogeneous population with differing levels of immune and/or inflammatory dysfunction; that as they develop progressively increasing levels of immune exhaustion with protracted sepsis, they can successfully be treated with anti-PD-1 and anti- PD-Ll antibodies to reduce lymphocyte apoptosis and increase immunity.
  • a major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections.
  • many immunosuppressive mechanisms exist increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-Ll) are thought to play key roles in sepsis.
  • PD-1 inhibitory receptor programmed cell death 1
  • PD-Ll inhibitory receptor programmed cell death 1
  • the newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-Ll antibodies to prevent apoptosis and improve lymphocyte function in septic patients.
  • lymphocytes from septic patients produced decreased IFN- ⁇ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-Ll expression compared to non-septic patients (p ⁇ 0.05).
  • Monocytes from septic patients had increased PD-Ll and decreased HLA-DR expression compared to non-septic patients (p ⁇ 0.01).
  • CD8 T cell expression of PD-1 increased over time in ICU as PD-Ll decreased.
  • donors with the highest CD8 PD-1 expression and lowest PD-Ll expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype.
  • the invention provides for the identification of septic patients having higher PD-1 and lower PD-Ll expression on CD8 cells, such that CD8+ PD-1 >36 and PD-Ll ⁇ 5 expression. These patients also displayed reduced levels of HLA-DR expression on monocytes compared with septic patients where CD8+ PD- 1 ⁇ 36 and PD-Ll >5% expression. Interestingly, these subjects had an increased rate of secondary infections, VAP and peritonitis.
  • septic patients characterized as having (i) immune dysfunction where CD8+ PD-1 >36 and PD-Ll ⁇ 5% expression; (ii) decreased LHA-DR expression; and/or (iii) decreased TNF-a levels in LPS -stimulated whole blood, are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
  • Agents that inhibit or decrease the interaction between PD- 1 and its ligands PD-Ll and/or PD-L2 in selected subjects are useful in modulating the immune response in subjects having CD8+ PD-l hlgh PD-Ll low T cells associated with sepsis.
  • PD- 1 (Programmed Death 1) is an inhibitory receptor and a counterpart of CTLA-4. This disclosure relates to modulation of immune responses mediated by the PD-1 receptor.
  • 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-Ll (B7-H1) and PD-L2 (B7-DC).
  • PD- 1 is a member of the immunoglobulin (Ig) superfamily that contains a single Ig V- like domain in its extracellular region.
  • the PD- 1 cytoplasmic domain contains two tyrosines, with the most membrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM (immuno-receptor tyrosine-based inhibitory motif).
  • ITIM immunoglobulin
  • Human and murine PD-1 proteins share about 60% amino acid identity with conservation of four potential N-glycosylation sites, and residues that define the Ig-V domain.
  • the ITIM in the cytoplasmic region and the ITIM-like motif surrounding the carboxy-terminal tyrosine (TEYATI in human and mouse) are also conserved between human and murine orthologues.
  • PD- 1 is expressed on activated T cells, B cells, and monocytes.
  • Experimental data implicates the interactions of PD-1 with its ligands in downregulation 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.
  • PD-1 deficiency in the C57BL/6 mice results in chronic progressive lupus-like glomerulonephritis and arthritis.
  • PD-1 deficiency leads to severe cardiomyopathy due to the presence of heart-tissue-specific self -reacting antibodies.
  • Programmed cell death 1 ligands 1 (PD-Ll) PD-Ll
  • PD-Ll molecules Two forms of human PD-Ll molecules have been identified (Freeman et al. J. Exp. Med. 2000. 192: 1027; Dong et al. 1999. Nature Medicine. 5: 1365).
  • One form is a naturally occurring PD-Ll soluble polypeptide, i.e. , having a short hydrophilic domain and no transmembrane domain, and is referred to herein as soluble PD-Ll .
  • the second form is a cell- associated polypeptide, i.e., having a transmembrane and cytoplasmic domain, referred to herein as PD-Ll.
  • PD-L2 molecules have also been identified. (Genbank Accession no.
  • Sepsis, SIRS, and septic shock are associated with activation of the innate immunity and coagulation systems. Sepsis and septic shock are characterized clinically by systemic inflammation, coagulopathy, hypotension and multiple organ dysfunction (J.-L. Vincent et al. , Annuals of Medicine 34 (2002) 606-613). During severe sepsis, a network of specific proteases activates clotting, fibrinolytic and complement factors. These proteases can also trigger tissue and organ damage and enhance non-specific proteolysis of clotting and complement factors in plasma (J. Wite et al, Intensive Care Medicine 8 (1982) 215-222; S. J. Weiss, New England Journal of Medicine 320 (1989) 365-376).
  • cytokine storm cytokine storm
  • immunosuppressive state that occurs after the initial stage of the disorder [1-4]. Numerous interacting mechanisms of immunosuppression occur in sepsis including increased T regulatory cells, increased myeloid derived suppressor cells, apoptotic depletion of immune effector cells, and a shift from a TH1 to an anergic or TH2 immune phenotype [5-8].
  • T cell exhaustion Another recently recognized mechanism of immunosuppression in sepsis is T cell exhaustion [3]. T cell exhaustion was first described in states of chronic viral infection with persistent high levels of antigen exposure [9-11]. It is typified by the presence of T cells which have lost effector function, i.e., they fail to proliferate, produce cytokines, or induce cytotoxic cell death in targeted cells [10]. Exhausted T cells also have an increased tendency to undergo apoptosis because of changes in the ratio of pro-and anti-apoptotic Bcl-2 family members.
  • CD279 negative co- stimulatory molecule PD-1
  • CD274 a member of the B7-CD28 super family
  • PD-L1 a member of the B7-CD28 super family
  • CD273 a member of the B7-CD28 super family
  • PD-1 is promptly induced and subsequently expressed on the surface of CD4 and CD8 T cells whereupon it interacts with PD-L1 and PD-L2.
  • PD-L1 is broadly expressed on both hematopoietic and non-hematopoietic cells and its expression is significantly upregulated during states of inflammation such as sepsis [11].
  • anti-PD- 1 antibody therapy has been in the field of oncology, in which it has been demonstrated to be highly effective in inducing remissions in patients with a variety of malignancies [14, 15], anti-PD- 1 has also shown significant success in infectious disease.
  • Another goal of the study was to determine if a correlation existed between lymphocyte apoptosis and putative mediators of apoptosis including lymphocyte PD- 1 and PD-L1 expression and monocyte PD-L1 expression to gain insight into possible mechanisms for apoptotic cell death and the lymphocytopenia that typically accompany sepsis.
  • a biomarker e.g. , PDl and/or PD-L1
  • a biomarker is an analyte or clinical indicator whose presence, absence, or level relative to a reference is indicative of a disease state.
  • a marker is differentially present in a sample taken from a subject of one phenotypic status (e.g. , CD8+ PD-l high PD-Ll low T cells) as compared with another phenotypic status (e.g. , CD8+ PD- l low PD-Ll high T cells).
  • a biomarker is differentially present between different phenotypic statuses if the mean or median expression level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t- test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. Therefore, they are useful as markers for characterizing a disease. In one embodiment, levels of PDl and/or PD-Llare typically increased in immune cells of a subject having sepsis. In other embodiments, the relative levels of expression are characterized.
  • CD8+ PD- l high PD-Ll low T cells having higher PD-1 together with lower PD-L1 expression on CD8 cells (CD8+ PD- l high PD-Ll low T cells), where CD8+ PD-1 >36 and PD-L1 ⁇ 5%.
  • these subjects were further characterized as having reduced levels of HLA-DR expression on monocytes compared with CD8+ PD-l low PD-Ll hlgh septic patients (CD8+ PD- 1 ⁇ 36 and PD-L1 >5 expression).
  • the subject is further characterized as having decreased TNF-a levels in LPS -stimulated whole blood.
  • the biologic sample is a tissue sample or biologic fluid sample.
  • Biological fluid samples include blood, blood serum, plasma, cerebrospinal fluid, urine, and saliva, or any other biological fluid useful in the methods of the invention.
  • the biological fluid is blood, serum, or plasma.
  • the biological sample is a blood sample comprising a peripheral blood mononuclear cells, lymphocytes, monocytes, T cells, and/or NK Cells.
  • the sample comprises T cell identified by CD3+, CD4+, or CD8+
  • the present invention provides a number of diagnostic assays that are useful for the identification or characterization of the stage of immune and/or inflammatory dysfunction in a subject diagnosed as having any one or more of sepsis, septic shock, systemic inflammatory response syndrome, and/or a related disorder.
  • diagnostic assays of the invention identify a subject responsive to PD-1 pathway blockade.
  • sepsis is characterized by quantifying the level of one or more of the following markers: PD1 and/or PD-L1, for example, on a CD8+ T cell. While the examples provided below describe specific methods of detecting levels of these markers, the skilled artisan appreciates that the invention is not limited to such methods. Marker levels are quantifiable by any standard method, such methods include, but are not limited to real-time PCR, Southern blot, PCR, mass spectroscopy, and/or antibody binding.
  • septic patients characterized as having (i) immune dysfunction where CD8+ PD-1 >36 and PD-L1 ⁇ 5 expression; (ii) decreased HLA-DR expression; and/or (iii) decreased TNF-a levels in LPS-stimulated whole blood. Patients meeting one or more of these critera (e.g. , all) are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
  • the level of a marker is compared to a reference.
  • the reference is the level of marker present in a control sample obtained from a patient that does not have sepsis.
  • the reference is a baseline level of marker present in a biologic sample derived from a patient prior to, during, or after treatment for sepsis.
  • the reference is a standardized curve. The level of any one or more of the markers described herein (e.g. , PDl and/or PD-Ll) is used, alone or in combination with other standard methods, to identify a subject as having sepsis.
  • biomarkers of this invention can be detected by any suitable method.
  • the methods described herein can be used individually or in combination for a more accurate detection of the biomarkers (e.g. , immunoassay, mass spectrometry, and the like).
  • the biomarkers of the invention are measured by immunoassay.
  • Immunoassay typically utilizes an antibody (or other agent that specifically binds the marker) to detect the presence or level of a biomarker in a sample.
  • Antibodies can be produced by methods well known in the art, e.g. , by immunizing animals with the biomarkers.
  • Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.
  • This invention contemplates traditional immunoassays including, for example, Western blot, sandwich immunoassays including ELISA and other enzyme immunoassays, fluorescence -based immunoassays, chemiluminescence,.
  • Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance, which is measured.
  • Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR).
  • Immunoassays can be carried out on solid substrates (e.g. , chips, beads, microfluidic platforms, membranes) or on any other forms that supports binding of the antibody to the marker and subsequent detection.
  • a single marker may be detected at a time or a multiplex format may be used.
  • Multiplex immunoanalysis may involve planar microarrays (protein chips) and bead-based microarrays (suspension arrays).
  • Antibodies that selectively bind PD-1 and/or PD-Ll and inhibit the binding or activation of PD-1 and/or PD-Ll are useful in the methods of the invention.
  • Anti-PD-1 antibodies and their antigen-binding fragments have been described (see e.g., U.S. Patent No. 7,488,802, which is herein incorporated by reference in its entirety).
  • LOPD180 is an exemplary PD-1 antibody, further described in U.S. Prov. Appl. entitled “Antibodies against PD-1 and Uses Thereof," to Buchanan, A. et al., filed herewith, and herein incorporated by reference in its entirety.
  • Anti- PD-Ll (B7-H1) antibodies are known in the art and described, for example, in WO 2011/066389 and in U.S. Publ. No. 2013/0034559, which is herein incorporated by reference in its entirety.
  • antibodies can be made, for example, using traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256: 495-499), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display performed with antibody, libraries (Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597).
  • Antibodies A Laboratory Manual, eds. Harlow et al, Cold Spring Harbor Laboratory, 1988.
  • the invention is not limited to any particular source, species of origin, method of production.
  • Intact antibodies also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, designated as the ⁇ chain and the ⁇ chain, are found in antibodies.
  • immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • each light chain is composed of an N-terminal variable domain (VL) and a constant domain (CL).
  • Each heavy chain is composed of an N-terminal variable domain (VH), three or four constant domains (CH), and a hinge region.
  • the CH domain most proximal to VH is designated as CHI.
  • the VH and VL domains consist of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence called complementarity determining regions (CDRs).
  • the CDRs contain most of the residues responsible for specific interactions with the antigen.
  • the three CDRs are referred to as CDR1, CDR2, and CDR3.
  • CDR constituents on the heavy chain are referred to as HI, H2, and H3, while CDR constituents on the light chain are referred to as LI, L2, and L3, accordingly.
  • CDR3 and, particularly H3, are the greatest source of molecular diversity within the antigen-binding domain.
  • H3, for example, can be as short as two amino acid residues or greater than 26.
  • the Fab fragment (Fragment antigen-binding) consists of the VH-CH1 and VL-CL domains covalently linked by a disulfide bond between the constant regions.
  • a so-called single chain (sc) Fv fragment (scFv) can be constructed.
  • a scFv a flexible and adequately long polypeptide links either the C-terminus of the VH to the N-terminus of the VL or the C-terminus of the VL to the N-terminus of the VH.
  • a 15-residue (Gly4Ser)3 peptide is used as a linker but other linkers are also known in the art.
  • Antibody diversity is a result of combinatorial assembly of multiple germline genes encoding variable regions and a variety of somatic events.
  • the somatic events include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete VH region and the recombination of variable and joining gene segments to make a complete VL region.
  • D diversity
  • J joining
  • the recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V(D) J junctions.
  • the disclosure provides anti-PD-Ll and/or anti-PDl CDRs derived from human immunoglobulin gene libraries.
  • the structure for carrying a CDR will generally be an antibody heavy or light chain or a portion thereof, in which the CDR is located at a location corresponding to the CDR of naturally occurring VH and VL.
  • the structures and locations of immunoglobulin variable domains may be determined, for example, as described in Kabat et al , Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of
  • Antibodies of the invention may optionally comprise antibody constant regions or parts thereof.
  • a VL domain may have attached, at its C terminus, antibody light chain constant domains including human CK or Ck chains.
  • a specific antigen-binding domain based on a VH domain may have attached all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g. , IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgGl and IgG4.
  • antibodies of this invention may be used to detect, measure, and inhibit proteins that differ somewhat from PD-L1 and PD1.
  • the antibodies are expected to retain the specificity of binding so long as the target protein comprises a sequence which is at least about 60%, 70%, 80%, 90%, 95%, or more identical to any sequence of at least 100, 80, 60, 40, or 20 of contiguous amino acids described herein.
  • the percent identity is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215:
  • BLAST Basic Local Alignment Tool
  • Antibodies of the invention may include variants of these sequences that retain the ability to specifically bind their targets. Such variants may be derived from the sequence of these antibodies by a skilled artisan using techniques well known in the art. For example, amino acid substitutions, deletions, or additions, can be made in the FRs and/or in the CDRs. While changes in the FRs are usually designed to improve stability and immunogenicity of the antibody, changes in the CDRs are typically designed to increase affinity of the antibody for its target. Variants of FRs also include naturally occurring immunoglobulin allotypes.
  • affinity-increasing changes may be determined empirically by routine techniques that involve altering the CDR and testing the affinity antibody for its target. For example, conservative amino acid substitutions can be made within any one of the disclosed CDRs. Various alterations can be made according to the methods described in Antibody Engineering, 2nd ed., Oxford University Press, ed.
  • the nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Derivatives and analogs of antibodies of the invention can be produced by various techniques well known in the art, including recombinant and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany).
  • a method for making a VH domain which is an amino acid sequence variant of a VH domain of the invention comprises a step of adding, deleting, substituting, or inserting one or more amino acids in the amino acid sequence of the presently disclosed VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations for a specific binding to PD-lor PD-L1 and, optionally, testing the ability of such antigen-binding domain to modulate PD-lor PD-L1 activity.
  • An analogous method can be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.
  • a further aspect of the disclosure provides a method of preparing antigen-binding fragment that specifically binds with PD-lor PD-L1.
  • One such technique, error-prone PCR is described by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).
  • Another method that may be used is to direct mutagenesis to CDRs of VH or VL genes.
  • Such techniques are disclosed by Barbas et al. (Proc. Nat. Acad. Sci. U.S.A. (1994) 91 : 3809-3813) and Schier ei a/. (J. Mol. Biol. (1996) 263: 551-567).
  • one or more, or all three CDRs may be grafted into a repertoire of VH or
  • VL domains which are then screened for an antigen-binding fragment specific for PD-1 or PD-L1.
  • a portion of an immunoglobulin variable domain will comprise at least one of the CDRs substantially as set out herein and, optionally, intervening framework regions from the scFv fragments as set out herein.
  • the portion may include at least about 50% of either or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions.
  • construction of antibodies by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps.
  • Other manipulation steps include the introduction of linkers to join variable domains to further protein sequences including immunoglobulin heavy chain constant regions, other variable domains (for example, in the production of diabodies), or proteinaceous labels as discussed in further detail below.
  • antibodies of the invention may comprise antigen- binding fragments containing only a single CDR from either VL or VH domain.
  • Either one of the single chain specific binding domains can be used to screen for complementary domains capable of forming a two-domain specific antigen-binding fragment capable of, for example, binding to PD-L1 and PD1.
  • the screening may be accomplished by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO92/01047, in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two- chain specific binding domain is selected in accordance with phage display techniques as described.
  • Antibodies of the invention can be linked to another functional molecule, e.g. , another peptide or protein (albumin, another antibody, etc.).
  • another functional molecule e.g. , another peptide or protein (albumin, another antibody, etc.).
  • the antibodies can be linked by chemical cross-linking or by recombinant methods.
  • the antibodies may also be linked to one of a variety of
  • nonproteinaceous polymers e.g. , polyethylene glycol, polypropylene glycol, or
  • poly oxy alky lenes in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791, 192; or 4,179,337.
  • the antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life.
  • Exemplary polymers and methods to attach them are also shown in U.S. Pat. Nos. 4,766, 106; 4,179,337; 4,495,285, and 4,609,546.
  • the disclosed antibodies may also be altered to have a glycosylation pattern that differs from the native pattern.
  • one or more carbohydrate moieties can be deleted and/or one or more glycosylation sites added to the original antibody.
  • Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences known in the art.
  • Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. Such methods are described in WO 87/05330 and in Aplin et al. (1981) CRC Crit. Rev.
  • Detectable labels also include enzyme labels such as horseradish peroxidase or alkaline phosphatase. Detectable labels further include chemical moieties such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.
  • Antibodies in which CDR sequences differ only insubstantially from those set forth herein are encompassed within the scope of this invention.
  • an amino acid is substituted by a related amino acid having similar charge, hydrophobic, or stereochemical characteristics. Such substitutions would be within the ordinary skills of an artisan.
  • Changes to FRs include, but are not limited to, humanizing a non- human derived or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g. , changing the class or subclass of the constant region, changing specific amino acid residues which might alter the effector function such as Fc receptor binding, e.g.
  • the present disclosure further provides isolated nucleic acids encoding the disclosed antibodies.
  • the nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant.
  • Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.
  • the nucleic acids provided herein comprise a coding sequence for a CDR, a VH domain, and/or a VL domain disclosed herein.
  • the invention also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes which comprise at least one nucleic acid encoding a CDR, a VH domain, and/or a VL domain disclosed here.
  • the disclosure further provides a host cell which comprises one or more constructs as above.
  • nucleic acids encoding any CDR (HI, H2, H3, LI, L2, or L3), VH or VL domain, as well as methods of making of the encoded products.
  • the method comprises expressing the encoded product from the encoding nucleic acid. Expression may be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a VH or VL domain, or specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.
  • Antigen-binding fragments, VH and/or VL domains and encoding nucleic acid molecules and vectors may be isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function.
  • suitable host cells include bacteria, plant cells, mammalian cells, and yeast and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma cells, and many others.
  • a common bacterial host is E. coli. Any protein expression system compatible with the invention may be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al , 1999.
  • Suitable vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids or viral, e.g., phage, or phagemid, as appropriate.
  • phage e.g., phagemid
  • a further aspect of the disclosure provides a host cell comprising a nucleic acid as disclosed here.
  • a still further aspect provides a method comprising introducing such nucleic acid into a host cell.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. , vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • the introduction of the nucleic acid into the cells may be followed by causing or allowing expression from the nucleic acid, e.g. , by culturing host cells under conditions for expression of the gene.
  • the antibodies of the invention can be used to prevent, diagnose, or treat medical disorders in mammals, especially, in humans, including patients diagnosed as having sepsis, septic shock, systemic inflammatory response syndrome, compensatory anti-inflammatory response syndrome and related disorders.
  • Antibodies of the invention can also be used for isolating PD-1 , or PD-L1 , as well as PD-1 and/or PD-L1 - expressing cells.
  • the antibodies can be used to treat a subject at risk of or susceptible to a disorder or having a disorder associated with aberrant PD-1 and/or PD-L1 expression or function.
  • the antibodies or antibody compositions of the present invention are administered in therapeutically effective amounts.
  • a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition of the subject.
  • a therapeutically effective amount of antibody ranges from about 0.001 to about 30 mg/kg body weight, preferably from about 0.01 to about 25 mg/kg body weight, from about 0.1 to about 20 mg/kg body weight, or from about 1 to about 10 mg/kg.
  • an anti-PD-1 or anti-PD-Ll antibody is administered at about 10 mg/kg, 30 mg/kg, or 60 mg/kg.
  • the dosage may be adjusted, as necessary, to suit observed effects of the treatment.
  • the appropriate dose is chosen based on clinical indications by a treating physician.
  • the antibodies may be given as a bolus dose, to maximize the circulating levels of antibodies for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
  • the antibodies of the invention may also be used to detect the presence of PD- 1 and/or PD-L1 in biological samples. Detection methods that employ antibodies are well known in the art and include, for example, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, immunoprecipitation.
  • the antibodies may be provided in a diagnostic kit that incorporates one or more of these techniques to detect PD-1 and/or PD- LI. Such a kit may contain other components, packaging, instructions, or other material to aid the detection of the protein.
  • the antibodies are intended for diagnostic purposes, it may be desirable to modify them, for example, with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope or an enzyme).
  • a detectable label such as a fluorescent group, a radioisotope or an enzyme.
  • the antibodies of the invention may be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase can be detected by its ability to convert tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
  • TMB tetramethylbenzidine
  • binding partners include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
  • the antibody binds to PD-L1 and is a monoclonal, human antibody comprising comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence provided in the Figures.
  • the antibody is LOPD180 having the variable region sequences provided at Figures 15 and 16.
  • the invention provides for the use of a PD-1 and/or PD-L1 antibody as a therapeutic agent.
  • Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed.
  • the duration of the therapy depends on the stage of sepsis being treated, the age and condition of the patient, the stage and type of an infectious disease, and how the patient's body responds to the treatment.
  • Drug administration may be performed at different intervals (e.g. , daily, weekly, or monthly). Therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to build healthy new cells and regain its strength.
  • Disclosed herein is a therapeutic method for administration of anti-PD-1 and/or anti-
  • PD-L1 antibody to a subject to treat sepsis, septic shock, compensatory anti-inflammatory response syndrome, and related disorders.
  • the invention may be used for the treatment of virtually any disease associated with sepsis in a subject.
  • the methods of the invention involve the administration of anti-PD-1 and/or anti-PD-Ll antibody in a therapeutically effective dose.
  • antibody of the invention e.g. , an antibody that binds PD- 1/PD-Ll
  • administration of antibody of the invention may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in preventing, ameliorating, or reducing sepsis.
  • An antibody of the invention, or other negative regulator of PD1 and/or PD-L1 may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by sepsis.
  • the compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for parenteral (e.g. , subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route.
  • parenteral e.g. , subcutaneously, intravenously, intramuscularly, or intraperitoneally
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g. , Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
  • Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration.
  • parenteral intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration.
  • therapeutic agents for example, therapeutic
  • formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration.
  • controlled release formulations which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in a sarcoma (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target proliferating neoplastic cells by using
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings.
  • the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
  • a composition of the invention may be administered within a pharmaceutically- acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by excessive cell proliferation. Administration may begin before the patient is symptomatic.
  • administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration.
  • therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the compounds.
  • PD1 and/or PD-L1 polypeptides or polynucleotides include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • the formulations can be administered to human patients in therapeutically effective amounts (e.g. , amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition.
  • therapeutically effective amounts e.g. , amounts which prevent, eliminate, or reduce a pathological condition
  • the preferred dosage of a nucleobase oligomer of the invention is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.
  • subjects suffering from sepsis comprise a heterogeneous population having differing levels of immune and/or inflammatory dysfunction.
  • septic subjects display increased levels of immune exhaustion.
  • Such subjects are characterized as having higher PD-1 and lower PD-L1 expression on CD8 cells, such that CD8+ PD-1 >36 and PD-L1 ⁇ 5 expression.
  • Such patients display an 'CD8+ pD 1 high PD .
  • L1 iow T cells These patients also haye reduced levels of HLA-DR expression on monocytes compared with CD8+ PD-l low PD-Ll high septic patients where CD8+ PD-1 ⁇ 36 and PD-L1 >5 expression.
  • these patients show decreased TNF-a levels in LPS -stimulated whole blood. Patients characterized as meeting one or more of these criteria are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
  • a number of standard treatment regimens are available for the selected patients. These treatments can be used in combination with the methods of the invention.
  • the therapy of sepsis rests on intravenous fluids, antibiotics, surgical drainage of infected fluid collections, and appropriate support for organ dysfunction. This may include hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition— preferably by enteral feeding, but if necessary by parenteral nutrition— is important during prolonged illness. In those with high blood sugar levels, insulin to bring it down to 7.8-10 mmol/L (140-180 mg/dL) is recommended with lower levels potentially worsening outcomes. Medication to prevent deep vein thrombosis and gastric ulcers may also be used.
  • fluids are titrated in response to heart rate, blood pressure, and urine output; restoring large fluid deficits can require 6 to 10L of crystalloids.
  • fluids should be
  • the mixed venous oxygen saturation (Sv02), i.e., the oxygen saturation of venous blood as it returns to the heart as measured at the vena cava, is optimized. If the Sv02 is less than 70%, blood is given to reach a hemoglobin of 10 g/dl and then inotropes are added until the Sv02 is optimized.
  • norepinephrine noradrenaline
  • dopamine the former appears safer. If a single pressor is not sufficient in improving the blood pressure, epinephrine (adrenaline) may be added in.
  • Elective tracheal intubation and mechanical ventilation may be performed to reduce oxygen demand if the Sv02 remains low despite optimization of hemodynamics.
  • Etomidate is not recommended as a medication to help with intubation in this situation due to concerns of adrenal insufficiency and increased mortality.
  • the diagnostic methods of the invention are also useful for monitoring the course of a sepsis in a patient and/or for assessing the efficacy of a therapeutic regimen.
  • the diagnostic methods of the invention are used periodically to monitor the polynucleotide or polypeptide levels of one or more of PD-1 and/or PD-L1.
  • sepsis is characterized using a diagnostic assay of the invention prior to administering therapy. This assay provides a baseline that describes the level of one or more markers of sepsis prior to treatment. Additional diagnostic assays are administered during the course of therapy to monitor the efficacy of a selected therapeutic regimen.
  • a therapy is identified as efficacious when a diagnostic assay of the invention detects a decrease in marker levels relative to the baseline level of marker prior to treatment.
  • the level of immune dysfunction is characterized by measuring PD- 1 and/or PD-Ll on CD8+ T cells. Where CD8+ PD-1 >36 and PD-Ll ⁇ 5 expression; HLA-DR expression is decreased; and/or TNF-a levels are decreased in LPS -stimulated whole blood, such patients are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
  • the disease state or treatment of a subject having sepsis, or a propensity to develop such a condition can be monitored using the methods and compositions of the invention.
  • the expression of markers present in a bodily fluid such as blood, blood serum, or plasma, is monitored.
  • Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a subject or in assessing disease progression.
  • Therapeutics that decrease the expression of a marker of the invention e.g. , PD-1 and/or PD-Ll are taken as particularly useful in the invention.
  • kits for the treatment or prevention of sepsis, septic shock, systemic inflammatory response syndrome, and and compensatory anti-inflammatory response syndrome are provided.
  • the invention further provides kits for identifying a subject as responsive to PD- 1 pathway blockade.
  • the kit includes a therapeutic or prophylactic composition containing an effective amount of an inhibitory antibody that disrupts the biological activity of a PD1 and/or PD-Ll polypeptide in unit dosage form. In another embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of an anti- PD1 and/or anti-PD-Ll antibody in unit dosage form.
  • a diagnostic kit of the invention provides a capture reagent (e.g., an anti-PDl and/or anti-PD-Ll antibody) for measuring increased CD8+ PD-1 and decreased PD-Ll (e.g., where CD8+ PD-1 >36 and PD-Ll ⁇ 5 expression).
  • a capture reagent e.g., an anti-PDl and/or anti-PD-Ll antibody
  • the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • an antibody or agent of the invention is provided together with instructions for administering the antibody or agent to a subject having or at risk of developing sepsis.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of sepsis.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology;
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • CD8 T cell PD-1 increased as PD-L1 decreased during stay in ICU ( Figure 2A).
  • the subset of donors with increased PD-1 together with decreased PD-L1 expression on CD8 cells (CD8+ PD-l high PD-Ll low ' , CD8+ PD-1 >36% and PD-L1 ⁇ 5% expression) also had reduced levels of HLA-DR expression on monocytes compared with CD8+ PD-l low PD- Ll high septic patients (CD8 + PD-1 ⁇ 36% and PD-L1 >5% expression) and critically ill non- septic patients ( Figure 2B).
  • Apoptosis was quantified in patient lymphocytes after overnight incubation with either isotype control antibody, anti-PD-1 antibody, or anti-PD-Ll antibody ( Figure 4).
  • apoptosis Quantitation of apoptosis in total lymphocytes, i.e. , all lymphocytes present in the lymphocyte gate identified by forward and side scatter on flow cytometry (see Figure 5A) and consisting primarily of CD4 + , CD8 + , NKT cells, and NK cells was examined.
  • Total lymphocyte apoptosis was increased by -70% in septic patients when compared to non-septic patients after overnight incubation in isotype (inactive) control antibody, i.e., 10.4 + 1.5% in septic patients versus 6.1 ⁇ 1.0% in non-septic patients (p ⁇ 0.01) ( Figure 4).
  • lymphocytes incubated in media containing anti-PD-1 or anti-PD-Ll antibody had a highly significant decrease in apoptosis, p ⁇ 0.002, ( Figures 4 and 5B).
  • No effect of anti-PD-1 or anti-PD-Ll antibody on lymphocyte apoptosis was observed in samples from non-septic patients, possibly due to their lower level of baseline apoptosis which was often less than 5% ( Figure 4).
  • a highly similar effect of anti-PD-1 and anti-PD-Ll antibody on sepsis-induced apoptosis was observed in CD4 T cells from septic and non-septic patients ( Figure 6).
  • PBMCs Peripheral blood mononuclear cells
  • IL-2 production was decreased in total lymphocytes, CD3 T cells, and NKT cells in septic versus non-septic patients (Figure 8). Overnight incubation of cells showed a significant effect of anti-PD-1 and anti-PD-Ll antibodies in increasing IFN- ⁇ production in total lymphocytes and NKT cells ( Figures 9 and 10). Examination showed that only a subset of patients' samples responded to anti-PD-1 or anti-PD-Ll. Anti-PD-1 and anti-PD-Ll had similar effects of increasing IL-2 production in specific lymphocyte subsets ( Figure 11). There was no significant effect of anti-PD-1 or anti-PD-Ll to increase IFN- ⁇ or IL-2 in lymphocytes from non-septic patients ( Figures 12 and 13).
  • a characteristic hematologic finding in patients with sepsis is an apoptosis-induced reduction in their absolute lymphocyte count, often to values that are less than 20-30% of that for healthy controls (Munford et al , Am J Respir Crit Care Med 2001, 163(2):316-321 ; Hotchkiss et al, Crit Care Med 1999, 27(7): 1230-1251; Venet et al, J Immunol 2012, 189(10):5073-5081).
  • persistent lymphopenia in sepsis is associated with increased mortality. Lymphocyte depletion (as reflected by lymphopenia) may contribute to morbidity and mortality by impairing host immunity (Kasten et al. , Infect Immun 2010,
  • T cell exhaustion was first reported in animal models of chronic viral infection and was thought to be due to persistent exposure to high levels of antigen [9-11]. Patients with sepsis often have a protracted course with primary and secondary infections, a scenario that likely includes persistent high circulating antigens thereby facilitating development of T cell exhaustion [3,33,34].
  • Zhang et al. reported that anti-PD- 1 was increased on monocytes from septic patients and that anti-PD- 1 antibody decreased T cell apoptosis and improved immune effector function [36].
  • a recent important study by Singh et al. showed that in vitro blockade of PD-1 improved T cell IFN- ⁇ production and decreased apoptosis in patients with active infections due to M. tuberculosis [37].
  • a second major finding of these investigators was that when patients with active tuberculosis were treated with effective medication to eradicate M.
  • tuberculosis the number of PD-1 -expressing T cells decreased and inversely correlated with IFN- ⁇ T-cell response against M. tuberculosis. We believe that this work has major implications for the broader field of sepsis because of the similarities of active tuberculosis with protracted sepsis.
  • the effect of anti-PD- 1 and anti-PD-Ll to improve IFN- ⁇ production by T cells may be a particularly beneficial in sepsis given its ability to improve monocyte/macrophage function which is impaired in sepsis [4, 38,39].
  • a clinical trial of IFN- ⁇ in sepsis is currently underway and is being targeted to those patients whose circulating monocytes have low HLA-DR expression, (see clinicaltrials.gov).
  • Anti-PD- 1 antibody has been highly successful in a subset of patients with various types of malignancies [14,15]. In general, those patients whose tumors expressed PD-L1 on immunohistochemical analysis have responded to therapy with anti-PD-1 antibody. As PD-1 and PD-L1 can also be early activation markers, it is inadvisable to use these markers alone to diagnose an immunosuppressive state.
  • PD-1 and PD-L1 can also be early activation markers, it is inadvisable to use these markers alone to diagnose an immunosuppressive state.
  • patients with sepsis whose monocytes have decreased HLA-DR expression and/or patients whose LPS-stimulated whole blood response shows decreased TNF-a production are considered good candidates for immuno- stimulatory therapy [4].
  • Anti-PD-1 and anti-PD-Ll antibodies have had extraordinary success in cancer trials and are considered to represent a major breakthrough in the field [40].
  • Anti-PD-1 antibody induced remission in approximately 20-25% of patients with a diversity of tumors including malignant melanoma, renal cell cancer, and non-small cell lung cancer.
  • a remarkable feature of anti-PD-1 and anti-PD-1 therapy is the fact that some patients have durable cancer remissions that last for many months in the absence of continued therapy [41]. Cancer and sepsis share many of the same immunosuppressive mechanisms including increased T regulatory cells, increased myeloid derived suppressor cells, and T cell exhaustion [4-8,42].
  • anti-PD-1 and anti-PD-Ll antibodies ameliorated key immune defects consistent with reversal of T cell exhaustion in PBMCs from septic patients. Both antibodies appeared equally effective in their capabilities. Thus, lymphocyte PD-1 expression, in conjunction with other cellular markers and clinical and laboratory findings, may contribute to identifying septic patients in which anti-PD-1 or anti-PD-Ll antibody therapy may be beneficial.
  • lymphocyte PD-1 expression in conjunction with other cellular markers and clinical and laboratory findings, may contribute to identifying septic patients in which anti-PD-1 or anti-PD-Ll antibody therapy may be beneficial.
  • the present findings indicate that T cell exhaustion is a major etiology of immune dysfunction in sepsis and that reversal of putative T cell exhaustion using anti-PD-1 or anti-PD-Ll offers promise in the therapy of this highly lethal disorder.
  • Septic patients Patients at Barnes Jewish Hospital who were older than 18 years of age and who fulfilled a consensus panel definition of sepsis (Levy et al. , Crit Care Med 2003, 31 (4): 1250-1256) were included in the study (Table 1). Sepsis was defined as the presence of systemic inflammatory response syndrome (SIRS) and a known or suspected source of infection. Patients with HIV infection, viral hepatitis, or who were receiving SIRS.
  • immunosuppressive medications except corticosteroids at a dose of ⁇ 10 mg prednisone or equivalent per day) were excluded. Consent for blood draws was obtained from the patient or a legally authorized representative.
  • BAL bronchial alveolar lavage
  • BLD blood
  • C. diff. Clostridia difficile, CT, CT scan
  • CSR chest x-ray
  • GPC Gram positive cocci
  • MRSA methicillin resistant Straph, aureus
  • pressors vaso pressors
  • VAP ventilator associated pneumonia
  • WCC white cell count
  • Control subjects consisted of critically-ill non-septic patients admitted to the ICU for care following major surgery, trauma, or myocardial ischemia (Table 1). Exclusion criteria were identical to that for patients with sepsis. Consent for blood draws was obtained from the patient or a legally authorized representative.
  • PBMCs Peripheral blood mononuclear cells
  • PD-1 and PD-L1 were identified by forward scatter (FSC) and side scatter (SSC) properties as described previously (Boomer et al , JAMA 2011, 306(23) :2594- 2605). Monocytes were identified by FSC and SSC properties and by CD14+
  • T cell subsets were further identified by CD3+, CD4+, or CD8+ immunostaining.
  • NK cells were identified as CD3-/CD56+ while natural killer T (NKT) cells were identified as CD3+/CD56+.
  • PBMCs that had undergone overnight incubation with either isotype-control antibody, anti-PD-1 antibody, or anti-PD-Ll antibody were stimulated with PMA/ionomycin plus brefeldin for 5 hrs as previously described (Unsinger et al. , J Immunol 2010, 184(7):3768- 3779; Unsinger et al. ,. J Infect Dis 2012, 206(4):606-616). Following stimulation, cells were washed, stained with anti-CD3 and anti-CD56 antibodies, fixed with 1% paraformaldehyde, permeabilized with IX perm/wash (Biolegend) and stained with fluorescently labeled anti- IFN- ⁇ or anti-IL-2 antibodies. Patient hematologic values
  • ICU patients had daily complete blood count analysis performed as part of the standard of care.
  • Patient clinical laboratory values that were recorded in this study included absolute lymphocyte, absolute monocyte, and absolute granulocyte cell counts and were quantitated in the clinical laboratories at Barnes Jewish Hospital (Table 2).
  • the PD-l/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum. Proc Natl Acad Sci U S A 2008, 105(7):2658-2663.
  • Hotchkiss RS Swanson PE, Freeman BD, Tinsley KW, Cobb JP, Matuschak GM, Buchman TG, Karl IE: Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 1999, 27(7): 1230-1251.
  • Venet F Venet F, Foray AP, Villars-Mechin A, Malcus C, Poitevin- Later F, Lepape A, Monneret G: IL-7 Restores Lymphocyte Functions in Septic Patients. J Immunol 2012, 189(10):5073-5081. 26. Kasten KR, Prakash PS, Unsinger J, Goetzman HS, England LG, Cave CM, Seitz AP, Mazuski CN, Zhou TT, Morre M et at Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gamma delta T-cell IL-17 production in a murine model of sepsis. Infect Immun 2010, 78(l l):4714-4722.
  • Pollack A Promising new cancer drugs empower the body's own defense system.
  • Cheever MA Twelve immunotherapy drugs that could cure cancers. Immunol Rev 2008, 222:357-368.

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Abstract

The present invention provides compositions and methods for the diagnosis and treatment of sepsis. The present invention features compositions and methods for identifying a subject responsive to PD-1 pathway blockade or treating a patient preselected as responsive to PD-1 pathway blockade. In particular embodiments, the invention provides methods for treating sepsis, septic shock, systemic inflammatory response syndrome, and compensatory anti-inflammatory response syndrome, as well as methods for reducin sepsisinduced lymphocyte apoptosis, reducing T cell proliferation, increasing IFN-γ or IL-2 levels, or otherwise reducing immune dysfunction in a subject, for example, in a subject pre-selected as responsive to PD-1 pathway blockade.

Description

COMPOSITIONS AND METHODS FOR TREATING SEPSIS
BACKGROUND OF THE INVENTION
Sepsis is a potentially life-threatening complication of a severe infection. Sepsis occurs when chemicals released into the bloodstream to fight the infection trigger inflammation throughout the body. This systemic inflammation can trigger a cascade of changes that can damage multiple organ systems, causing them to fail. The systemic response can lead to septic shock, which is characterized by a precipitous drop in blood pressure, cardiovascular collapse, and/or multiple organ failure.
There are approximately 800,000 sepsis cases per year in the U.S. at a cost of $17 billion, with an equal number in the rest of the world. Despite the introduction of antibiotics over fifty years ago, the mortality rate among subjects diagnosed with septic shock is 30- 50%. One possible explanation for this high mortality rate is that septic patients represent a heterogeneous population who should not be treated as if they all suffer from the same disorder. Rather, there may be important biological differences that characterize the illness of patients in the early stages of sepsis versus those that succumb to secondary infections during late stage sepsis. Accordingly, improved methods for characterizing sepsis, septic shock, systemic inflammatory response syndrome, and related disorders in a subject and selecting an effective therapy for that subject are urgently required.
SUMMARY OF THE INVENTION
As described below, the present invention features compositions and methods for identifying a subject responsive to PD-1 pathway blockade or treating a patient preselected as responsive to PD-1 pathway blockade. In particular embodiments, the invention provides methods for treating sepsis, septic shock, systemic inflammatory response syndrome, and compensatory anti-inflammatory response syndrome, as well as methods for reducing sepsis- induced lymphocyte apoptosis, reducing T cell proliferation, increasing IFN-γ or IL-2 levels, or otherwise reducing immune dysfunction in a subject, for example, in a subject pre-selected as responsive to PD- 1 pathway blockade.
In one aspect, the invention generally provides a method of identifying a subject (e.g., human) responsive to PD- 1 pathway blockade, the method involving detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels (e.g., by immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
immunoprecipitation) in a biological sample (e.g., blood) of the subject relative to a reference, thereby identifying the subject as responsive to PD-1 pathway blockade.
In another aspect, the invention generally provides a method of identifying a subject responsive to PD- 1 pathway blockade, the method involving detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels in a biological sample of the subject relative to a reference, where the detection is by any one or more of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
immunoprecipitation, thereby identifying the subject as responsive to PD-1 pathway blockade.
In yet another aspect, the invention provides a method of reducing T cell proliferation in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 and/or anti-PD-Ll antibody, where the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby reducing T cell proliferation in the subject.
In still another aspect, the invention provides a method of reducing sepsis-induced lymphocyte apoptosis in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 or anti-PD-Ll antibody, where the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby reducing sepsis-induced lymphocyte apoptosis in the subject.
In a related aspect, the invention provides a method of increasing IFN-γ or IL-2 levels in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 or anti-PD-Ll antibody, where the subject is preselected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby increasing IFN-γ or IL-2 levels in the subject.
In another related aspect, the invention provides a method of reducing immune dysfunction in a subject pre-selected as responsive to PD-1 pathway blockade, the method involving administering to the subject an anti-PD-1 or anti-PD-Ll antibody, where the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-L1 levels relative to a reference, thereby reducing immune dysfunction in the subject. In one embodiment, the detection is by any one or more of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
immunoprecipitation
In another aspect, the invention provides a method of treating sepsis in a subject identified as responsive to PD-1 pathway blockade, the method involving detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels in a biological sample of the subject relative to a reference, thereby identifying the subject as responsive to PD-1 pathway blockade; and administering to the subject an anti-PD-1 or anti-PD-Ll antibody, thereby treating sepsis in the subject.
In yet another aspect, the invention provides a method of identifying a subject responsive to PD- 1 pathway blockade, the method involving detecting immune dysfunction where CD8+ PD-1 >36 and PD-Ll <5 expression; decreased HLA-DR expression; and decreased TNF-a levels in LPS -stimulated whole blood, where the detection identifies the patient as responsive to anti-PD-1 and/or anti-PD-Ll antibody therapy.
In still another aspect, the invention provides a method of identifying a subject responsive to PD- 1 pathway blockade, the method involving detecting immune dysfunction where CD8+ PD-1 >36 and PD-Ll <5 expression; decreased HLA-DR expression; and decreased TNF-a levels in LPS -stimulated whole blood, where the detection is by a method is any one or more of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and immunoprecipitation, where the detection identifies the patient as responsive to anti-PD-1 and/or anti-PD-Ll antibody therapy.
In a related aspect, the invention provides a method of treating sepsis in a subject identified as responsive to PD-1 pathway blockade, the method involving detecting immune dysfunction where CD8+ PD-1 >36 and PD-Ll <5 expression; decreased HLA-DR expression; and decreased TNF-a levels in LPS -stimulated whole blood, where the detection identifies the patient as responsive to PD-1 pathway blockade; and administering to the subject an anti-PD-1 or anti-PD-Ll antibody, thereby treating sepsis in the subject.
In another aspect, the invention provides a kit for treating sepsis, the kit containing an effective amount of an antibody that specifically binds PD-1 and/or PD-Ll, and instructions for using the kit to treat sepsis. In another aspect, the invention provides a kit for identifying a subject responsive to PD-1 pathway blockade, the kit containing a capture reagent that binds PD-1 and a capture reagent that binds PD-Ll .
In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the method identifies the subject as in need of treatment with an anti-PD-1 or anti-PD-Ll antibody. In other embodiments, the method characterizes the level or stage of immune dysfunction in the subject. In other embodiments of any of the above aspects or any other aspect of the invention delineated herein CD8+ PD-1 levels are increased (e.g., by at least about 25, 30, 35, 40, 45, 50, 55, 60, 65 percent or more). In various embodiments of the above aspects or any other aspect of the invention delineated herein, CD8+ PD-1 >36% and PD-Ll <5% expression. In particular embodiments, CD8+ PD-1 levels are greater than at least about 36% relative to a reference and PD-Ll levels are less than about 5% relative to the level in a reference. In other embodiments, the method further involves detecting a decrease in HLA-DR expression levels relative to a reference. In other embodiments, the method further involves detecting a decrease in TNF-a levels in LPS -stimulated whole blood. In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the method characterizes the immunosuppressive phase of sepsis in the subject. In other embodiments, the administration of an anti-PD-1 and/or anti-PD-Ll antibody restores cytokine production,
decreases sepsis-induced lymphocyte apoptosis, or restores immunity in the subject. In particular embodiments, sepsis is bacterial or fungal sepsis. In other embodiments, the antibody neutralizes PD-1 and/or PD-Ll . In other embodiments of the above aspects or any other aspect of the invention, the antibody is LOPD180 or the antibody contains one or more variable regions of a LOPD180 antibody. In particular embodiments, the anti-PD-1 or anti- PD-Ll antibody is administered at about 10 mg/kg, 30 mg/kg, or 60 mg/kg. In other embodiments of the above aspects or any other aspect of the invention, the method increases an immune response in the subject, increases T-cell proliferation, IFN-γ production, and/or IL-2 production in the subject. In still other embodiments, the administering is by intravenous injection. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al. , Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By "anti-PD-1 antibody is meant" an antibody that selectively binds a PD-1 polypeptide. LOPD 180 is an exemplary PD-1 antibody.
By "anti-PD-Ll antibody" is meant an antibody that selectively binds a PD-Ll polypeptide. Exemplary anti-PD-Ll antibodies are described for example at WO
2011/066389, which is herein incorporated by reference.
By "PD-1 polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_005009 and having PD-Ll and/or PD-L2 binding activity or other PD- 1 biological activity.
By "PD-1 nucleic acid molecule" is meant a polynucleotide encoding a PD-1 polypeptide. An exemplary PD-1 nucleic acid molecule sequence is provided at NCBI Accession No. NM_005018.
By "PD-Ll polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001254635 and having PD-1 and CD80 binding activity or other PD-Ll biological activity.
By "PD-Ll nucleic acid molecule" is meant a polynucleotide encoding a PD-Ll polypeptide. An exemplary PD-Ll nucleic acid molecule sequence is provided at NCBI Accession No. NM_001267706.
Select exemplary sequences delineated herein are shown at Figures 15 and 16.
By "responsive" in the context of therapy is meant susceptible to treatment.
By "PD-1 pathway blockage" is meant treatment with an agent that negatively regulates PD-1 signalling. Treatment with an anti-PDl antibody or anti-PDLl antibody is an exemplary PD- 1 pathway blockade. By "responsive to PD-1 pathway blockade" is meant having an immune dysfunction characterized by increased PD-1 and decreased PD-Ll on a CD8+ T cell. In particular embodiments, a septic patient is characterized as having (i) immune dysfunction where CD8+ PD-1 >36% and PD-Ll <5% expression; (ii) decreased HLA-DR expression relative to a reference; and/or (iii) decreased TNF-a levels in LPS -stimulated whole blood relative to a reference. Such subjects are selected as in need of anti-PD-1 and/or anti-PD-Ll antibody therapy. In other embodiments, CD8+ PD-1 expression is greater than at least about 25%, 30%, 35%, 40%, 50%, 60%, 75%, 85% or more relative to a reference. In still other embodiments, PD-Ll is less than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% expression relative to a reference.
A "biomarker" or "marker" as used herein generally refers to a protein, nucleic acid molecule, clinical indicator, or other analyte that is associated with a disease. In one embodiment, a marker of sepsis is differentially present in a biological sample obtained from a subject having or at risk of developing sepsis relative to a reference. In another
embodiment, a marker can be used to characterize the stage of immune and/or inflammatory dysfunction in a subject diagnosed as having sepsis, septic shock, systemic inflammatory response syndrome, compensatory anti-inflammatory response syndrome or related disorders. A marker is differentially present if the mean or median level of the marker present in the sample is statistically different from the level present in a reference. A reference level may be, for example, the level present in a sample obtained from a healthy control subject the level obtained from the subject at an earlier timepoint, i.e., prior to treatment, or the level obtained from a subject at a defined stage of septic illness. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative likelihood that a subject belongs to a phenotypic status of interest. The differential presence of a marker of the invention in a subject sample can be useful in characterizing the immune dysfunction present in a subject identified as having or at risk of developing sepsis, for determining the prognosis of the subject, for evaluating therapeutic efficacy, or for selecting a treatment regimen.
As used herein, "active" or "activity" in regard to a PD-1 and/or PD-Ll polypeptide refers to a portion of a PD-1 and/or PD-Ll polypeptide that has a biological, inhibitory, and/or immunosuppressive activity of a native PD-1 and/or PD-Ll polypeptide. PD-1 has one or more immunoregulatory activities associated with PD-1. For example, PD-1 is a negative regulator of the TcR/CD28 -mediated immune response.
"PD-1 and/or PD-L1 biological activity" when used herein refers to a biological function that results from the activity of the native PD-1 and/or PD-L1 polypeptide. A 5 particular PD-1 and/or PD-L1 biological activity includes, for example, negative regulation of immune responses, induction of lymphocyte apoptosis, reduced T cell proliferation, and reduced IFN-γ production, and reduced IL-2 production.
The term "antibody," as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigenic) binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies.
Unless otherwise modified by the term "intact," as in "intact antibodies," for the purposes of this disclosure, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, 15 scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e. , the ability to bind PD- 1 specifically. Typically, such fragments would comprise an antigen- binding domain.
The terms "antigen-binding domain," "antigen-binding fragment," and "binding fragment" refer to a part of an antibody molecule that comprises amino acids responsible for 0 the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as "epitope" or "antigenic determinant." An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable 5 region (VH), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain, but still retains some antigen-binding function of the intact antibody.
Binding fragments of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab',
30 F(ab')2, Fv, and single-chain antibodies. An antibody other than a "bispecific" or
"bifunctional" antibody is understood to have each of its binding sites identical. Digestion of antibodies with the enzyme, papain, results in two identical antigen-binding fragments, known also as "Fab" fragments, and a "Fc" fragment, having no antigen-binding activity but having the ability to crystallize. Digestion of antibodies with the enzyme, pepsin, results in the a F(ab')2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab')2 fragment has the ability to crosslink antigen. "Fv" when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. "Fab" when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
The term "mAb" refers to monoclonal antibody. Antibodies of the invention comprise without limitation whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.
By "biologic sample" is meant any tissue, cell, fluid, or other material derived from an organism.
The invention provides kits for identifying a subject as responsive to PD-1 pathway blockade using a capture reagent that binds a PD-1 or PD-L1 polypeptide (e.g., anti-PD-1 or anti-PD-Ll antibody). By "capture reagent" is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
As used herein, the terms "determining", "assessing", "assaying", "measuring" and
"detecting" refer to both quantitative and qualitative determinations, and as such, the term "determining" is used interchangeably herein with "assaying," "measuring," and the like. Where a quantitative determination is intended, the phrase "determining an amount" of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase "determining a level" of an analyte or "detecting" an analyte is used.
The term "effective amount" refers to a dosage or amount that is sufficient to reduce the activity of PD-1 to result in amelioration of symptoms in a patient or to achieve a desired biological outcome, e.g., increased cytolytic activity of T cells, reinduction of immune tolerance, reduction or increase of the PD- 1 activity associated with the negative regulation of T-cell mediated immune response, etc.
The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non- human primate, murine, bovine, equine, canine, ovine, or feline.
The term "decrease" or "increase" is meant to alter negatively or positively, respectively. An alteration may be by 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even by 100%.
By "reference" is meant a standard of comparison. For example, the PD- 1 or PD-L1 polypeptide or polynucleotide level present in a patient sample may be compared to the level of said polypeptide or polynucleotide present in a corresponding healthy cell or tissue. In one embodiment, the standard of comparison is the level of PD-1 or PD-L1 polypeptide or polynucleotide level present in serum of a subject that does not have sepsis. In another embodiment, the standard of comparison is the level of PD-1 or PD-L1 polypeptide or polynucleotide level present in a biological sample of a subject that has an early stage of sepsis (e.g. , with a day of admission to the ICU). In particular embodiments, the PD-1 or PD- Ll polypeptide or polynucleotide level polypeptide level present in a patient sample may be compared to the level of said polypeptide present in a corresponding sample obtained at an earlier time point. If desired, such levels are monitored periodically.
By "periodic" is meant at regular intervals. Periodic patient monitoring includes, for example, a schedule of tests that are administered daily, bi-weekly, bi-monthly, monthly, bi- annually, or annually.
The term "sepsis" refers to a medical condition characterized by a whole -body inflammatory state (called a systemic inflammatory response syndrome or SIRS) associated with a severe infection. Sepsis is commonly caused by the immune system's response to a serious infection, (e.g. , from bacteria, fungi, viruses, and parasites in the blood, urinary tract, lungs, skin, or other tissues). Indications of sepsis can range from infection to multiple organ dysfunction syndrome. Common symptoms of sepsis include those related to a specific infection, but usually accompanied by high fevers, hot, flushed skin, elevated heart rate, hyperventilation, altered mental status, swelling, and low blood pressure. In the very young and elderly, or in people with weakened immune systems, the pattern of symptoms may be atypical, with hypothermia and without an easily localizable infection.
The invention provides antibodies that bind a PD-1 or PD-L1 polypeptide, as well as nucleic acid molecules encoding such antibodies. LOPD180 is an exemplary antibody useful in the methods of the invention. Accordingly, nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g. , Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM
NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g. , formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g. , sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
The invention is not limited to the specific antibodies described herein as binding a
PD-1 or PD-L1 polypeptide, but encompasses other anti-PD-1 and PD-L1 antibodies, including antibodies that are substantially identical to the antibodies delineated herein (e.g., LOPD180). By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e"3 and e"100 indicating a closely related sequence.
As used herein, the term "sample" includes a biologic sample such as any tissue, cell, fluid, or other material derived from an organism.
By "specifically binds" is meant a compound (e.g., antibody) that recognizes and binds a molecule (e.g., polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample. For example, two molecules that specifically bind form a complex that is relatively stable under physiologic conditions.
Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than 106 M_1, or more preferably higher than 108 M_1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g. , serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.
Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01 % of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Any compounds, compositions, or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
As used herein, the singular forms "a", "an", and "the" include plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes reference to more than one biomarker. Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.
The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."
As used herein, the terms "comprises," "comprising," "containing," "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides four graphs depicting PD-1, PD-L1, and HLA-DR expression in septic and non-septic patients. Septic and non-septic patients were identified, and heparinized blood samples were obtained at a maximum of 4 time points during their septic course. Peripheral blood mononuclear cells were stained for lymphocyte (CD4, upper left panel; CD8, upper right panel) and monocyte markers (CD14, lower panels).
Immunostaining was also performed for PD-1 (upper panels), PD-L1 (lower left panel), and HLA-DR (lower right panel). Flow cytometry revealed an increase in PD-1 and PD-L1 expression in CD8 T cells and monocytes from septic versus non-septic patients. HLA-DR expression was decreased in monocytes from septic versus non-septic patients as well. Data are from 43 septic (70 data points) and 16 non-septic patients (16 data points). Septic and non-septic patients had up to four serial blood samples obtained depending upon the duration of their illness and/or discharge from the ICU; - first draw = days 1-3 after admission to the ICU, days 4-7 (second blood draw), days 8-12 (third blood draw), and days 12-21 (fourth blood draw) after sepsis onset. Most non-septic patients were discharged from the ICU within 4-5 days and therefore, they had fewer serial blood samples obtained compared to septic patients.
Figures 2A-2B show that markers of immune exhaustion increased with protracted sepsis. Flow cytometry revealed an increase in expression of immune exhaustion markers over time in ICU. Figure 2A is a graph showing that PD-1 expression increased as PD-L1 expression decreased on CD8 T cells in samples from septic patients over the course of stay in ICU. Figure 2B is a graph showing separation of a CD8 subset into an exhausted phenotype. As PD-1 and PD-Ll can also be activation markers, data were further separated into a CD8+ PD-lhigh PD-Lllow subset defined as CD8+ PD-1 >36 and CD8+ PD-Ll <5 expression (n= 22 samples), and a CD8+ PD-llow PD-Llhigh subset defined as CD8+ PD-1 <36 and CD8+ PD-Ll >5 expression (n= 47 samples), based on levels above or below the mean CD8+ PD-1 and CD8+ PD-Ll expression for non-septic controls. Selection of sepsis donor samples expressing high PD-1 and low PD-Ll on CD8+ T cells (CD8+ PD-lhigh PD- Ll10", shown in boxed region) revealed a significantly lower level of % HLA-DR+ CD14+ monocytes compared with the CD8+ PD-llow PD-Ll hlgh subset, indicative of a generally more immune suppressed state. Mean per group is indicated by the horizontal bars.
Figure 3 is a graph showing that septic patients with an immune exhausted phenotype had a higher incidence of secondary infections.
Septic patient samples were separated into CD8+ PD-lhigh PD-Lllow and CD8+ PD-llow PD- Llhigh subsets based on PD-1 and PD-Ll immunostaining where CD8+ PD-lhigh PD-Lllow is defined as CD8+ PD-1 >36 and CD8+ PD-Ll <5 expression, and CD8+ PD-llow PD- Llhigh is defined as CD8+ PD-1 <36 and CD8+ PD-Ll >5 expression. Groups were analyzed for presence of more than two pathogens, secondary infections, type and route of infection (VAP or peritonitis). The percentage of patients positive for each parameter tested are shown for the CD8+ PD-lhigh PD-Lllow and CD8+ PD-llow PD-Llhigh subsets. This data analysis revealed an increased number of secondary infections, VAP and peritonitis in septic patients with a CD8+ PD-lhigh PD-Lllow phenotype (n=14 patients) compared with a CD8+ pD 1 iow PD.L1 high phenotype (n=2i patients).
Figure 4 depicts graphs showing that blockade of PD-1 or PD-Ll decreased sepsis- induced lymphocyte apoptosis. PBMCs from septic patients (top panels) or non-septic patients (bottom panels) were incubated overnight in media containing inactive isotype control antibody, anti-PD-1 antibody (left panels), or anti-PD-Ll antibody (right panels). The next morning, cells were washed and immunostained for lymphocyte markers. TUNEL assay was performed to detect apoptosis. Apoptosis was quantitated in cells located in the lymphocyte gate (determined by forward and side scatter properties - see, e.g. , Figure 5 A for a representative flow histogram) consisting of but not limited to CD4 T cells, CD8 T cells, NK cells, γδ T cells, and NKT cells. Apoptosis was increased in lymphocytes that had been incubated in inactive isotype control antibody from septic versus non-septic patients, 10.4 ±1.5% and 6.1± 1.0% respectively (p< 0.01). Compared to incubation in isotype control antibodies, overnight incubation with anti-PD-1 and anti-PD-Ll reduced apoptosis in lymphocytes from septic but not non-septic patients 40.7% and 29.9% respectively for anti- PD-1 and anti-PD-Ll. The failure of anti-PD-1 and anti-PD-Ll antibodies to decrease apoptosis in non-septic patients may have been due to the fact that baseline apoptosis in non- septic patients is low, frequently less than 5%. Data are the mean values for all time points for 24 septic and 7 non-septic patients.
Figures 5A and 5B depicts representative flow cytometric histograms of lymphocyte gating strategy and detection of lymphocyte apoptosis via TUNEL assay. Blood from a septic patient was obtained and PBMCs isolated by ficol gradient. Figure 5A depicts representative flow cytometric histograms of lymphocyte gating strategy. The lymphocyte fraction in the PBMCs was identified by characteristic forward and side scatter properties. Figure 5B depicts representative flow cytometric histograms of detection of lymphocyte apoptosis via TUNEL assay. CD4 (left panels) and CD8 T cells (right panels) were identified by cell specific antibodies. PBMCs were plated overnight with inactive, isotype control antibody (top panels), anti-PD-1 antibody (middle panels), or anti-PD-Ll antibody (bottom panels). Apoptosis in CD4 T cells incubated in inactive isotype control antibody was 7.5%. Anti-PD-1 and anti-PD-Ll decreased CD4 apoptosis to 1.25 and 2.0% respectively. A similar protective effect was seen in CD8 T cells.
Figure 6 depicts four graphs showing quantitation of apoptosis in CD4 T cells via TUNEL in septic and non-septic patients. Peripheral blood mononuclear cells (PBMCs) from septic patients (top panels) or non-septic patients (bottom panels) were incubated overnight in media containing anti-PD-1 antibody (left panels), or anti-PD-Ll antibody (right panels). As a control, PBMCs were incubated overnight with inactive isotype control antibody The following morning, cells were washed and underwent immunostaining followed by detection of apoptosis via TUNEL assay. Flow cytometry was used to quantitate apoptosis in different lymphocyte subsets including CD4 T cells. Compared to treatment with inactive isotype control antibody, incubation with anti-PD-1 and anti-PD-Ll antibody decreased apoptosis in CD4 T cells from septic patients; p < 0.01 and p < 0.05 respectively. There was no effect of anti-PD-1 or anti-PD-Ll in non-septic patients, possibly due to the fact that baseline apoptosis in the non-septic patients was so low. Data are the mean values for all time points for 19 septic and 7 non-septic patients.
Figure 7 depicts four graphs showing that sepsis impaired lymphocyte IFN-γ production. Peripheral blood mononuclear cells (PBMCs) from septic or non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN-γ. Flow cytometric analysis revealed a decrease in the percentage of total lymphocytes and NKT cells that were IFN-γ positive in septic compared to non-septic patients; p < 0.02. Data are from 15 septic (21 data points) and 7 non-septic patients (7 data points) obtained during their illness.
Figure 8 includes four graphs showing that sepsis impaired lymphocyte IL-2 production. Peripheral blood mononuclear cells (PBMCs) from septic or non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IL-2. Flow cytometric analysis revealed a decrease in the percentage of total lymphocytes, CD3 T cells, and NKT cells that were IL-2 positive in septic versus non-septic patients, p < 0.03, p < 0.02, and p < 0.002 respectively. Data are from 15 septic (21 data points) and 7 non-septic patients (7 data points) obtained throughout their illness - all blood draws.
Figure 9 includes graphs showing that anti-PD-1 and anti-PD-Ll antibodies increased IFN-γ production in sepsis. Peripheral blood mononuclear cells (PBMCs) from septic patients were incubated overnight in media containing anti-PD-1 antibody (top panels) or anti-PD-Ll antibody (bottom panels). As a control, PBMCs from septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN-γ. Flow cytometric analysis revealed that, compared to inactive isotype control antibody, both anti-PD-1 and anti-PD-Ll antibody caused an increase in the percentage of total lymphocytes and NKT cells that were IFN-γ positive. Data are from 15 septic patients (21 data points) throughout their illness - all blood draws. Figure 10 depicts representative flow cytometric histograms showing that anti-PD-1 and anti-PD-Ll antibodies increased IFN-γ in NK cells from septic patients. Peripheral blood mononuclear cells (PBMCs) from septic patients were incubated overnight in media containing anti-PD-1 antibody (middle, right panels) or anti-PD-Ll antibody (bottom panels). As a control, PBMCs from septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN-γ. The lymphocyte gate was identified by characteristic forward and side scatter properties. NK cells were identified as CD3", CD56+. The percentage of NK cells that were positive for IFN-γ that were incubated in inactive isotype control antibody was 9.9%. Treatment with anti-PD-1 and anti-PD-Ll increased the percentage of NK cells that were IFN-γ positive to 34.8 and 21.8% respectively.
Figure 11 includes six graphs showing that anti-PD-1 and anti-PD-Ll improved IL-2 production in sepsis. Peripheral blood mononuclear cells (PBMCs) from septic patients were incubated overnight in media containing anti-PD-1 antibody (top panels) or anti-PD-Ll antibody (bottom panels). As a control, PBMCs from septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IL-2. Flow cytometric analysis revealed that, compared to inactive isotype control antibody, both anti- PD-1 and anti-PD-Ll antibody caused an increase in the percentage of CD3 T and NKT cells that were IL-2 positive. Data are from blood draws from 15 septic patients (21 data points throughout their illness).
Figure 12 provides six graphs showing that anti-PD-1 and anti-PD-Ll antibodies did not significantly increase IFN-γ in non-septic patients. Peripheral blood mononuclear cells (PBMCs) from non-septic patients were incubated overnight in media containing anti-PD- 1 antibody (top panels) or anti-PD-Ll antibody (bottom panels). As a control, PBMCs from non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IFN-γ. Flow cytometric analysis showed no significant effect of anti- PD-1 or anti-PD-Ll antibodies to increase IFN-γ compared to inactive isotype control antibody in any cell subtype. Data are from blood draws from 6-7 non-septic patients (6-7 data points throughout their illness).
Figure 13 provides six graphs showing that anti-PD-1 and anti-PD-Ll did not significantly increase IL-2 in non-septic patients. Peripheral blood mononuclear cells (PBMCs) from non-septic patients were incubated overnight in media containing anti-PD- 1 antibody (top panels) or anti-PD-Ll antibody (bottom panels). As a control, PBMCs from non-septic patients were incubated overnight in media containing inactive isotype control antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 hrs, washed, immunostained with phenotypic markers to CD3 and CD56, fixed, and stained for intracellular IL-2. Flow cytometric analysis revealed that neither anti-PD-1 nor anti-PD-Ll antibody caused a significant increase in the percentage of cells that were positive for IL-2. Data are from blood draws from 6-7 non-septic patients (6-7 data points throughout their illness).
Figure 14 is a graph showing that PD-1 expression and lymphopenia are correlated. Additional indirect evidence for a causal role of the PD-1 :PD-L1 pathway in sepsis-induced lymphocyte apoptosis is provided by data which show a correlation between the % of PD-1 positivity on CD4 T cells and the percentage of CD4 T cells that are TUNEL positive, p < 0.01, R2 = 0.35. Data are for 21 samples from 21 patients.
Figure 15 provides sequences of anti-PD 1 antibody (LOPD180) polypeptides.
Figure 16 provides exemplary sequences of human PD-1, PD-L1, and anti-PD 1 antibody (LOPD180) polypeptides and nucleic acid molecules.
DETAILED DESCRIPTION OF THE INVENTION
As described below, the present invention features compositions and methods for identifying a subject responsive to PD-1 pathway blockade or treating a patient preselected as responsive to PD-1 pathway blockade. In particular embodiments, the invention provides methods for treating sepsis, septic shock, systemic inflammatory response syndrome, and compensatory anti-inflammatory response syndrome, as well as methods for reducing sepsis- induced lymphocyte apoptosis, reducing T cell proliferation, increasing IFN-γ or IL-2 levels, or otherwise reducing immune dysfunction in a subject, for example, in a subject pre-selected as responsive to PD- 1 pathway blockade. The present invention is based, at least in part, on the discovery that subjects suffering from sepsis are a heterogeneous population with differing levels of immune and/or inflammatory dysfunction; that as they develop progressively increasing levels of immune exhaustion with protracted sepsis, they can successfully be treated with anti-PD-1 and anti- PD-Ll antibodies to reduce lymphocyte apoptosis and increase immunity.
A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-Ll) are thought to play key roles in sepsis. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-Ll antibodies to prevent apoptosis and improve lymphocyte function in septic patients.
As reported herein below, lymphocytes from septic patients produced decreased IFN- γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-Ll expression compared to non-septic patients (p <0.05). Monocytes from septic patients had increased PD-Ll and decreased HLA-DR expression compared to non-septic patients (p< 0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-Ll decreased. In addition, donors with the highest CD8 PD-1 expression and lowest PD-Ll expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-Ll antibody decreased apoptosis and increased IFN-γ and IL-2 production in some donors; (p<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (p< 0.01). In vitro blockade of the PD-LPD-Ll pathway decreased apoptosis and improved immune cell function blood samples from septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-Ll in animal models of bacterial and fungal infections and the relative safety of anti-PD-1 /anti-PD-Ll in human oncology trials to date strongly support the use of these antibodies for the treatment of sepsis, a disorder with a high mortality.
In one preferred embodiment, the invention provides for the identification of septic patients having higher PD-1 and lower PD-Ll expression on CD8 cells, such that CD8+ PD-1 >36 and PD-Ll <5 expression. These patients also displayed reduced levels of HLA-DR expression on monocytes compared with septic patients where CD8+ PD- 1 <36 and PD-Ll >5% expression. Interestingly, these subjects had an increased rate of secondary infections, VAP and peritonitis.
In sum, septic patients characterized as having (i) immune dysfunction where CD8+ PD-1 >36 and PD-Ll <5% expression; (ii) decreased LHA-DR expression; and/or (iii) decreased TNF-a levels in LPS -stimulated whole blood, are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
Agents (e.g. , anti-PD-1 antibody, anti-PD-Ll antibody) that inhibit or decrease the interaction between PD- 1 and its ligands PD-Ll and/or PD-L2 in selected subjects are useful in modulating the immune response in subjects having CD8+ PD-lhlgh PD-Ll low T cells associated with sepsis.
PD-1 (Programmed Death 1)
PD- 1 (Programmed Death 1) is an inhibitory receptor and a counterpart of CTLA-4. This disclosure relates to modulation of immune responses mediated by the PD-1 receptor. 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-Ll (B7-H1) and PD-L2 (B7-DC).
PD- 1 is a member of the immunoglobulin (Ig) superfamily that contains a single Ig V- like domain in its extracellular region. The PD- 1 cytoplasmic domain contains two tyrosines, with the most membrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM (immuno-receptor tyrosine-based inhibitory motif). The presence of an ITIM on PD-1 indicates that this molecule functions to attenuate antigen receptor signaling by recruitment of cytoplasmic phosphatases. Human and murine PD-1 proteins share about 60% amino acid identity with conservation of four potential N-glycosylation sites, and residues that define the Ig-V domain. The ITIM in the cytoplasmic region and the ITIM-like motif surrounding the carboxy-terminal tyrosine (TEYATI in human and mouse) are also conserved between human and murine orthologues.
PD- 1 is expressed on activated T cells, B cells, and monocytes. Experimental data implicates the interactions of PD-1 with its ligands in downregulation of central and peripheral immune responses. In particular, proliferation in wild-type T cells but not in PD-1 - deficient T cells is inhibited in the presence of PD-Ll. Additionally, PD-1 -deficient mice exhibit an autoimmune phenotype. PD-1 deficiency in the C57BL/6 mice results in chronic progressive lupus-like glomerulonephritis and arthritis. In Balb/c mice, PD-1 deficiency leads to severe cardiomyopathy due to the presence of heart-tissue-specific self -reacting antibodies.
In general, a need exists to provide safe and effective therapeutic methods for immune disorders such as, for example, sepsis, septic shock, systemic inflammatory response syndrome, and related disorders in selected subjects. Modulation of the immune responses involved in these disorders can be accomplished by manipulation of the PD-1 pathway. Programmed cell death 1 ligands 1 (PD-Ll)
Two forms of human PD-Ll molecules have been identified (Freeman et al. J. Exp. Med. 2000. 192: 1027; Dong et al. 1999. Nature Medicine. 5: 1365). One form is a naturally occurring PD-Ll soluble polypeptide, i.e. , having a short hydrophilic domain and no transmembrane domain, and is referred to herein as soluble PD-Ll . The second form is a cell- associated polypeptide, i.e., having a transmembrane and cytoplasmic domain, referred to herein as PD-Ll. PD-L2 molecules have also been identified. (Genbank Accession no.
AF344424; Latchman et al. 2001. Nature Immunology. 2: 1).
Sepsis
Sepsis, SIRS, and septic shock are associated with activation of the innate immunity and coagulation systems. Sepsis and septic shock are characterized clinically by systemic inflammation, coagulopathy, hypotension and multiple organ dysfunction (J.-L. Vincent et al. , Annuals of Medicine 34 (2002) 606-613). During severe sepsis, a network of specific proteases activates clotting, fibrinolytic and complement factors. These proteases can also trigger tissue and organ damage and enhance non-specific proteolysis of clotting and complement factors in plasma (J. Wite et al, Intensive Care Medicine 8 (1982) 215-222; S. J. Weiss, New England Journal of Medicine 320 (1989) 365-376). "Sepsis-like" symptoms are observed in the exposed individuals mainly due to the overwhelming systemic inflammatory response of the body. The overreaction typically includes excessive production of cytokines ("cytokine storm") and destructive proteases and disturbances in metabolic, oxygenation, coagulation, and vascular functions leading to multi-organ dysfunction.
Although most new therapeutic approaches to sepsis have focused on blocking the early hyper-inflammatory phase, recent studies have highlighted the profound
immunosuppressive state that occurs after the initial stage of the disorder [1-4]. Numerous interacting mechanisms of immunosuppression occur in sepsis including increased T regulatory cells, increased myeloid derived suppressor cells, apoptotic depletion of immune effector cells, and a shift from a TH1 to an anergic or TH2 immune phenotype [5-8].
Another recently recognized mechanism of immunosuppression in sepsis is T cell exhaustion [3]. T cell exhaustion was first described in states of chronic viral infection with persistent high levels of antigen exposure [9-11]. It is typified by the presence of T cells which have lost effector function, i.e., they fail to proliferate, produce cytokines, or induce cytotoxic cell death in targeted cells [10]. Exhausted T cells also have an increased tendency to undergo apoptosis because of changes in the ratio of pro-and anti-apoptotic Bcl-2 family members. One of the contributing factors for development of T cell exhaustion is signaling by the negative co- stimulatory molecule PD-1 (CD279), a member of the B7-CD28 super family, following interaction with its ligands PD-L1 (CD274) and PD-L2 (CD273) [9,11-13].
Following T cell activation, PD-1 is promptly induced and subsequently expressed on the surface of CD4 and CD8 T cells whereupon it interacts with PD-L1 and PD-L2. PD-L1 is broadly expressed on both hematopoietic and non-hematopoietic cells and its expression is significantly upregulated during states of inflammation such as sepsis [11]. Although much of the focus and excitement of anti-PD- 1 antibody therapy has been in the field of oncology, in which it has been demonstrated to be highly effective in inducing remissions in patients with a variety of malignancies [14, 15], anti-PD- 1 has also shown significant success in infectious disease. Multiple independent investigators have reported that blockade of the PD- 1 :PD-L1 pathway restores T cell effector function, increases IFN-γ production, prevents apoptosis, and improves survival in various pathologic mouse models of sepsis [16-20]. The present study compared and contrasted the ability of anti-PD- 1 and anti- PD-L1 antibodies to decrease apoptosis and improve effector function in leukocytes from patients with sepsis. Another goal of the study was to determine if a correlation existed between lymphocyte apoptosis and putative mediators of apoptosis including lymphocyte PD- 1 and PD-L1 expression and monocyte PD-L1 expression to gain insight into possible mechanisms for apoptotic cell death and the lymphocytopenia that typically accompany sepsis. Biomarkers
As reported herein below, the invention provides for the use of markers to characterize the degree, stage, or severity of immune and/or inflammatory dysfunction in a septic subject. In particular embodiments, a biomarker (e.g. , PDl and/or PD-L1) is an analyte or clinical indicator whose presence, absence, or level relative to a reference is indicative of a disease state. In one embodiment, a marker is differentially present in a sample taken from a subject of one phenotypic status (e.g. , CD8+ PD-lhigh PD-Ll low T cells) as compared with another phenotypic status (e.g. , CD8+ PD- llow PD-Llhigh T cells). A biomarker is differentially present between different phenotypic statuses if the mean or median expression level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t- test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. Therefore, they are useful as markers for characterizing a disease. In one embodiment, levels of PDl and/or PD-Llare typically increased in immune cells of a subject having sepsis. In other embodiments, the relative levels of expression are characterized. For example, having higher PD-1 together with lower PD-L1 expression on CD8 cells (CD8+ PD- lhigh PD-Lllow T cells), where CD8+ PD-1 >36 and PD-L1 <5%. In other embodiments, these subjects were further characterized as having reduced levels of HLA-DR expression on monocytes compared with CD8+ PD-llow PD-Llhlgh septic patients (CD8+ PD- 1 <36 and PD-L1 >5 expression). In still other embodiments, the subject is further characterized as having decreased TNF-a levels in LPS -stimulated whole blood.
Types of biological samples
The level of PD1 and/or PD-L1 protein or polynucleotide is measured in different types of biologic samples. In one embodiment, the biologic sample is a tissue sample or biologic fluid sample. Biological fluid samples include blood, blood serum, plasma, cerebrospinal fluid, urine, and saliva, or any other biological fluid useful in the methods of the invention. In particular embodiments, the biological fluid is blood, serum, or plasma. In one preferred embodiment, the biological sample is a blood sample comprising a peripheral blood mononuclear cells, lymphocytes, monocytes, T cells, and/or NK Cells. In other embodiments, the sample comprises T cell identified by CD3+, CD4+, or CD8+
immunostaining, as described herein below.
Diagnostic assays
The present invention provides a number of diagnostic assays that are useful for the identification or characterization of the stage of immune and/or inflammatory dysfunction in a subject diagnosed as having any one or more of sepsis, septic shock, systemic inflammatory response syndrome, and/or a related disorder. In particular embodiments, diagnostic assays of the invention identify a subject responsive to PD-1 pathway blockade. In one
embodiment, sepsis is characterized by quantifying the level of one or more of the following markers: PD1 and/or PD-L1, for example, on a CD8+ T cell. While the examples provided below describe specific methods of detecting levels of these markers, the skilled artisan appreciates that the invention is not limited to such methods. Marker levels are quantifiable by any standard method, such methods include, but are not limited to real-time PCR, Southern blot, PCR, mass spectroscopy, and/or antibody binding.
In particular embodiments, septic patients characterized as having (i) immune dysfunction where CD8+ PD-1 >36 and PD-L1 <5 expression; (ii) decreased HLA-DR expression; and/or (iii) decreased TNF-a levels in LPS-stimulated whole blood. Patients meeting one or more of these critera (e.g. , all) are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
In particular embodiments, the level of a marker is compared to a reference. In one embodiment, the reference is the level of marker present in a control sample obtained from a patient that does not have sepsis. In another embodiment, the reference is a baseline level of marker present in a biologic sample derived from a patient prior to, during, or after treatment for sepsis. In yet another embodiment, the reference is a standardized curve. The level of any one or more of the markers described herein (e.g. , PDl and/or PD-Ll) is used, alone or in combination with other standard methods, to identify a subject as having sepsis.
Detection of Biomarkers
The biomarkers of this invention can be detected by any suitable method. The methods described herein can be used individually or in combination for a more accurate detection of the biomarkers (e.g. , immunoassay, mass spectrometry, and the like).
In particular embodiments, the biomarkers of the invention (e.g. , PDl and/or PD-Ll) are measured by immunoassay. Immunoassay typically utilizes an antibody (or other agent that specifically binds the marker) to detect the presence or level of a biomarker in a sample. Antibodies can be produced by methods well known in the art, e.g. , by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.
This invention contemplates traditional immunoassays including, for example, Western blot, sandwich immunoassays including ELISA and other enzyme immunoassays, fluorescence -based immunoassays, chemiluminescence,. Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance, which is measured. Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR).
Immunoassays can be carried out on solid substrates (e.g. , chips, beads, microfluidic platforms, membranes) or on any other forms that supports binding of the antibody to the marker and subsequent detection. A single marker may be detected at a time or a multiplex format may be used. Multiplex immunoanalysis may involve planar microarrays (protein chips) and bead-based microarrays (suspension arrays).
Antibodies
Antibodies that selectively bind PD-1 and/or PD-Ll and inhibit the binding or activation of PD-1 and/or PD-Ll are useful in the methods of the invention. Anti-PD-1 antibodies and their antigen-binding fragments have been described (see e.g., U.S. Patent No. 7,488,802, which is herein incorporated by reference in its entirety). LOPD180 is an exemplary PD-1 antibody, further described in U.S. Prov. Appl. entitled "Antibodies Against PD-1 and Uses Thereof," to Buchanan, A. et al., filed herewith, and herein incorporated by reference in its entirety. Anti- PD-Ll (B7-H1) antibodies are known in the art and described, for example, in WO 2011/066389 and in U.S. Publ. No. 2013/0034559, which is herein incorporated by reference in its entirety.
In general, antibodies can be made, for example, using traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256: 495-499), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display performed with antibody, libraries (Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597). For other antibody production techniques, see also Antibodies: A Laboratory Manual, eds. Harlow et al, Cold Spring Harbor Laboratory, 1988. The invention is not limited to any particular source, species of origin, method of production.
Intact antibodies, also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, designated as the λ chain and the κ chain, are found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of antibody structure, see Harlow et al, supra. Briefly, each light chain is composed of an N-terminal variable domain (VL) and a constant domain (CL). Each heavy chain is composed of an N-terminal variable domain (VH), three or four constant domains (CH), and a hinge region. The CH domain most proximal to VH is designated as CHI. The VH and VL domains consist of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence called complementarity determining regions (CDRs). The CDRs contain most of the residues responsible for specific interactions with the antigen. The three CDRs are referred to as CDR1, CDR2, and CDR3. CDR constituents on the heavy chain are referred to as HI, H2, and H3, while CDR constituents on the light chain are referred to as LI, L2, and L3, accordingly. CDR3 and, particularly H3, are the greatest source of molecular diversity within the antigen-binding domain. H3, for example, can be as short as two amino acid residues or greater than 26.
The Fab fragment (Fragment antigen-binding) consists of the VH-CH1 and VL-CL domains covalently linked by a disulfide bond between the constant regions. To overcome the tendency of non-covalently linked VH and VL domains in the Fv to dissociate when co- expressed in a host cell, a so-called single chain (sc) Fv fragment (scFv) can be constructed. In a scFv, a flexible and adequately long polypeptide links either the C-terminus of the VH to the N-terminus of the VL or the C-terminus of the VL to the N-terminus of the VH. Most commonly, a 15-residue (Gly4Ser)3 peptide is used as a linker but other linkers are also known in the art.
Antibody diversity is a result of combinatorial assembly of multiple germline genes encoding variable regions and a variety of somatic events. The somatic events include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete VH region and the recombination of variable and joining gene segments to make a complete VL region. The recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V(D) J junctions. These mechanisms of diversity occur in the developing B cell prior to antigen exposure. After antigenic stimulation, the expressed antibody genes in B cells undergo somatic mutation.
Based on the estimated number of germline gene segments, the random recombination of these segments, and random VH-VL pairing, up to 1.6x107 different antibodies could be produced (Fundamental Immunology, 3rd ed., ed. Paul, Raven Press, New York, N.Y., 1993). When other processes which contribute to antibody diversity (such as somatic mutation) are taken into account, it is thought that upwards of 1x1010 different antibodies could be potentially generated (Immunoglobulin Genes, 2nd ed., eds. Jonio et al, Academic Press, San Diego, Calif., 1995). Because of the many processes involved in antibody diversity, it is highly unlikely that independently generated antibodies will have identical or even substantially similar amino acid sequences in the CDRs.
The disclosure provides anti-PD-Ll and/or anti-PDl CDRs derived from human immunoglobulin gene libraries. The structure for carrying a CDR will generally be an antibody heavy or light chain or a portion thereof, in which the CDR is located at a location corresponding to the CDR of naturally occurring VH and VL. The structures and locations of immunoglobulin variable domains may be determined, for example, as described in Kabat et al , Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of
Health Publications, Bethesda, Md., 1991.
DNA and amino acid sequences of anti-PD-Ll and anti-PDl antibodies and fragments thereof are set forth in the Figures.
Antibodies of the invention {e.g., anti-PD-Ll and/or anti-PDl) may optionally comprise antibody constant regions or parts thereof. For example, a VL domain may have attached, at its C terminus, antibody light chain constant domains including human CK or Ck chains. Similarly, a specific antigen-binding domain based on a VH domain may have attached all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g. , IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgGl and IgG4.
One of ordinary skill in the art will recognize that the antibodies of this invention may be used to detect, measure, and inhibit proteins that differ somewhat from PD-L1 and PD1.
The antibodies are expected to retain the specificity of binding so long as the target protein comprises a sequence which is at least about 60%, 70%, 80%, 90%, 95%, or more identical to any sequence of at least 100, 80, 60, 40, or 20 of contiguous amino acids described herein.
The percent identity is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215:
403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48: 444-453, or the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4: 11-17.
In addition to the sequence homology analyses, epitope mapping (see, e.g. , Epitope
Mapping Protocols, ed. Morris, Humana Press, 1996) and secondary and tertiary structure analyses can be carried out to identify specific 3D structures assumed by the disclosed antibodies and their complexes with antigens. Such methods include, but are not limited to,
X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11 :7-13) and computer modeling of virtual representations of the presently disclosed antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Derivatives
Antibodies of the invention {e.g., anti-PD-Ll and/or anti-PDl) may include variants of these sequences that retain the ability to specifically bind their targets. Such variants may be derived from the sequence of these antibodies by a skilled artisan using techniques well known in the art. For example, amino acid substitutions, deletions, or additions, can be made in the FRs and/or in the CDRs. While changes in the FRs are usually designed to improve stability and immunogenicity of the antibody, changes in the CDRs are typically designed to increase affinity of the antibody for its target. Variants of FRs also include naturally occurring immunoglobulin allotypes. Such affinity-increasing changes may be determined empirically by routine techniques that involve altering the CDR and testing the affinity antibody for its target. For example, conservative amino acid substitutions can be made within any one of the disclosed CDRs. Various alterations can be made according to the methods described in Antibody Engineering, 2nd ed., Oxford University Press, ed.
Borrebaeck, 1995. These include but are not limited to nucleotide sequences that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a "silent" change. For example, the nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
Derivatives and analogs of antibodies of the invention can be produced by various techniques well known in the art, including recombinant and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany).
In one embodiment, a method for making a VH domain which is an amino acid sequence variant of a VH domain of the invention comprises a step of adding, deleting, substituting, or inserting one or more amino acids in the amino acid sequence of the presently disclosed VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations for a specific binding to PD-lor PD-L1 and, optionally, testing the ability of such antigen-binding domain to modulate PD-lor PD-L1 activity. An analogous method can be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.
A further aspect of the disclosure provides a method of preparing antigen-binding fragment that specifically binds with PD-lor PD-L1.
Analogous shuffling or combinatorial techniques are also disclosed by Stemmer
(Nature (1994) 370: 389-391), who describes the technique in relation to a β-lactamase gene but observes that the approach may be used for the generation of antibodies.
In further embodiments, one may generate novel VH or VL regions carrying one or more sequences derived from the sequences disclosed herein using random mutagenesis of one or more selected VH and/or VL genes. One such technique, error-prone PCR, is described by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).
Another method that may be used is to direct mutagenesis to CDRs of VH or VL genes. Such techniques are disclosed by Barbas et al. (Proc. Nat. Acad. Sci. U.S.A. (1994) 91 : 3809-3813) and Schier ei a/. (J. Mol. Biol. (1996) 263: 551-567).
Similarly, one or more, or all three CDRs may be grafted into a repertoire of VH or
VL domains, which are then screened for an antigen-binding fragment specific for PD-1 or PD-L1.
A portion of an immunoglobulin variable domain will comprise at least one of the CDRs substantially as set out herein and, optionally, intervening framework regions from the scFv fragments as set out herein. The portion may include at least about 50% of either or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of antibodies by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains to further protein sequences including immunoglobulin heavy chain constant regions, other variable domains (for example, in the production of diabodies), or proteinaceous labels as discussed in further detail below.
A skilled artisan will recognize that antibodies of the invention may comprise antigen- binding fragments containing only a single CDR from either VL or VH domain. Either one of the single chain specific binding domains can be used to screen for complementary domains capable of forming a two-domain specific antigen-binding fragment capable of, for example, binding to PD-L1 and PD1. The screening may be accomplished by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO92/01047, in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two- chain specific binding domain is selected in accordance with phage display techniques as described.
Antibodies of the invention (e.g., anti-PD-Ll and/or anti-PDl) described herein can be linked to another functional molecule, e.g. , another peptide or protein (albumin, another antibody, etc.). For example, the antibodies can be linked by chemical cross-linking or by recombinant methods. The antibodies may also be linked to one of a variety of
nonproteinaceous polymers, e.g. , polyethylene glycol, polypropylene glycol, or
poly oxy alky lenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791, 192; or 4,179,337. The antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods to attach them are also shown in U.S. Pat. Nos. 4,766, 106; 4,179,337; 4,495,285, and 4,609,546.
The disclosed antibodies may also be altered to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate moieties can be deleted and/or one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences known in the art. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. Such methods are described in WO 87/05330 and in Aplin et al. (1981) CRC Crit. Rev.
Biochem., 22: 259-306. Removal of any carbohydrate moieties from the antibodies may be accomplished chemically or enzymatically, for example, as described by Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259: 52; and Edge et al. (1981) Anal. Biochem., 118: 131 and by Thotakura et al. (1987) Meth. Enzymol., 138: 350. The antibodies may also be tagged with a detectable, or functional, label. Detectable labels include radiolabels such as 1311 or 99Tc, which may also be attached to antibodies using conventional chemistry. Detectable labels also include enzyme labels such as horseradish peroxidase or alkaline phosphatase. Detectable labels further include chemical moieties such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.
Antibodies, in which CDR sequences differ only insubstantially from those set forth herein are encompassed within the scope of this invention. Typically, an amino acid is substituted by a related amino acid having similar charge, hydrophobic, or stereochemical characteristics. Such substitutions would be within the ordinary skills of an artisan. Unlike in CDRs, more substantial changes can be made in FRs without adversely affecting the binding properties of an antibody. Changes to FRs include, but are not limited to, humanizing a non- human derived or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g. , changing the class or subclass of the constant region, changing specific amino acid residues which might alter the effector function such as Fc receptor binding, e.g. , as described in U.S. Pat. Nos. 5,624,821 and 5,648,260 and Lund et al. (1991) J. Immun. 147: 2657-2662 and Morgan et al. (1995) Immunology 86: 319-324, or changing the species from which the constant region is derived.
One of skill in the art will appreciate that the modifications described above are not all-exhaustive, and that many other modifications would obvious to a skilled artisan in light of the teachings of the present disclosure.
Nucleic Acids, Cloning and Expression Systems
The present disclosure further provides isolated nucleic acids encoding the disclosed antibodies. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.
The nucleic acids provided herein comprise a coding sequence for a CDR, a VH domain, and/or a VL domain disclosed herein.
The invention also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes which comprise at least one nucleic acid encoding a CDR, a VH domain, and/or a VL domain disclosed here.
The disclosure further provides a host cell which comprises one or more constructs as above.
Also provided are nucleic acids encoding any CDR (HI, H2, H3, LI, L2, or L3), VH or VL domain, as well as methods of making of the encoded products. The method comprises expressing the encoded product from the encoding nucleic acid. Expression may be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a VH or VL domain, or specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.
Antigen-binding fragments, VH and/or VL domains and encoding nucleic acid molecules and vectors may be isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function.
Systems for cloning and expression of a polypeptide in a variety of different host cells are well known in the art. For cells suitable for producing antibodies, see Gene Expression Systems, Academic Press, eds. Fernandez et al, 1999. Briefly, suitable host cells include bacteria, plant cells, mammalian cells, and yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma cells, and many others. A common bacterial host is E. coli. Any protein expression system compatible with the invention may be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al , 1999.
Suitable vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids or viral, e.g., phage, or phagemid, as appropriate. For further details see, for example, Sambrook et al , Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for manipulation of nucleic acid, for example, in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, 2nd Edition, eds. Ausubel et al , John Wiley & Sons, 1992.
A further aspect of the disclosure provides a host cell comprising a nucleic acid as disclosed here. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. , vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction of the nucleic acid into the cells may be followed by causing or allowing expression from the nucleic acid, e.g. , by culturing host cells under conditions for expression of the gene. Methods of Use
The antibodies of the invention (e.g. , anti-PD-Ll and/or anti-PDl) can be used to prevent, diagnose, or treat medical disorders in mammals, especially, in humans, including patients diagnosed as having sepsis, septic shock, systemic inflammatory response syndrome, compensatory anti-inflammatory response syndrome and related disorders. Antibodies of the invention can also be used for isolating PD-1 , or PD-L1 , as well as PD-1 and/or PD-L1 - expressing cells. Furthermore, the antibodies can be used to treat a subject at risk of or susceptible to a disorder or having a disorder associated with aberrant PD-1 and/or PD-L1 expression or function.
The antibodies or antibody compositions of the present invention are administered in therapeutically effective amounts. Generally, a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition of the subject. A therapeutically effective amount of antibody ranges from about 0.001 to about 30 mg/kg body weight, preferably from about 0.01 to about 25 mg/kg body weight, from about 0.1 to about 20 mg/kg body weight, or from about 1 to about 10 mg/kg. In one particular embodiment, an anti-PD-1 or anti-PD-Ll antibody is administered at about 10 mg/kg, 30 mg/kg, or 60 mg/kg. The dosage may be adjusted, as necessary, to suit observed effects of the treatment. The appropriate dose is chosen based on clinical indications by a treating physician.
The antibodies may be given as a bolus dose, to maximize the circulating levels of antibodies for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
The antibodies of the invention may also be used to detect the presence of PD- 1 and/or PD-L1 in biological samples. Detection methods that employ antibodies are well known in the art and include, for example, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, immunoprecipitation. The antibodies may be provided in a diagnostic kit that incorporates one or more of these techniques to detect PD-1 and/or PD- LI. Such a kit may contain other components, packaging, instructions, or other material to aid the detection of the protein.
Where the antibodies are intended for diagnostic purposes, it may be desirable to modify them, for example, with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope or an enzyme). If desired, the antibodies of the invention may be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase can be detected by its ability to convert tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. For detection, suitable binding partners include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
In yet another embodiment, the antibody binds to PD-L1 and is a monoclonal, human antibody comprising comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence provided in the Figures. In a preferred embodiment, the antibody is LOPD180 having the variable region sequences provided at Figures 15 and 16.
Therapy
Therapy may be provided wherever therapy for sepsis is needed: a hospital, an emergency room, intensive care unit, the doctor's office, a clinic, a hospital's outpatient department, or anywhere therapy for sepsis is required or desired. In one embodiment, the invention provides for the use of a PD-1 and/or PD-L1 antibody as a therapeutic agent.
Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed. The duration of the therapy depends on the stage of sepsis being treated, the age and condition of the patient, the stage and type of an infectious disease, and how the patient's body responds to the treatment. Drug administration may be performed at different intervals (e.g. , daily, weekly, or monthly). Therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to build healthy new cells and regain its strength.
Disclosed herein is a therapeutic method for administration of anti-PD-1 and/or anti-
PD-L1 antibody to a subject to treat sepsis, septic shock, compensatory anti-inflammatory response syndrome, and related disorders. The invention may be used for the treatment of virtually any disease associated with sepsis in a subject. The methods of the invention involve the administration of anti-PD-1 and/or anti-PD-Ll antibody in a therapeutically effective dose.
Formulation of Pharmaceutical Compositions
The administration of antibody of the invention (e.g. , an antibody that binds PD- 1/PD-Ll) for the treatment of sepsis may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in preventing, ameliorating, or reducing sepsis. An antibody of the invention, or other negative regulator of PD1 and/or PD-L1 , may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by sepsis. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g. , subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g. , Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic
formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in a sarcoma (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target proliferating neoplastic cells by using carriers or chemical derivatives to deliver the therapeutic agent to a sarcoma cell. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
A composition of the invention, may be administered within a pharmaceutically- acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by excessive cell proliferation. Administration may begin before the patient is symptomatic.
Any appropriate route of administration may be employed, for example,
administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for delivering an agent that disrupts the activity of PD1 and/or PD-L1 polypeptides or polynucleotides include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
The formulations can be administered to human patients in therapeutically effective amounts (e.g. , amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition. The preferred dosage of a nucleobase oligomer of the invention is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.
Selection of a treatment method
As reported herein below, subjects suffering from sepsis comprise a heterogeneous population having differing levels of immune and/or inflammatory dysfunction. As their disease progresses, septic subjects display increased levels of immune exhaustion. Such subjects are characterized as having higher PD-1 and lower PD-L1 expression on CD8 cells, such that CD8+ PD-1 >36 and PD-L1 <5 expression. Such patients display an 'CD8+ pD 1high PD.L1iow T cells , These patients also haye reduced levels of HLA-DR expression on monocytes compared with CD8+ PD-llow PD-Llhigh septic patients where CD8+ PD-1 <36 and PD-L1 >5 expression. Finally, these patients show decreased TNF-a levels in LPS -stimulated whole blood. Patients characterized as meeting one or more of these criteria are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
A number of standard treatment regimens are available for the selected patients. These treatments can be used in combination with the methods of the invention. The therapy of sepsis rests on intravenous fluids, antibiotics, surgical drainage of infected fluid collections, and appropriate support for organ dysfunction. This may include hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition— preferably by enteral feeding, but if necessary by parenteral nutrition— is important during prolonged illness. In those with high blood sugar levels, insulin to bring it down to 7.8-10 mmol/L (140-180 mg/dL) is recommended with lower levels potentially worsening outcomes. Medication to prevent deep vein thrombosis and gastric ulcers may also be used.
In severe sepsis, broad spectrum antibiotics are recommended within 1 hour of making the diagnosis. For every hour delay in the administration there is an associated 6% rise in mortality. Antibiotic regimens should be reassessed daily and narrowed if appropriate. Duration of treatment is typically 7-10 days with the type of antibiotic used directed by the results of cultures. Early goal directed therapy (EGDT) is an approach to the management of severe sepsis during the initial 6 hours after diagnosis. A step-wise approach should be used, with the physiologic goal of optimizing cardiac preload, afterload, and contractility. It has been found to reduce mortality in those with sepsis.
In EGDT, fluids are titrated in response to heart rate, blood pressure, and urine output; restoring large fluid deficits can require 6 to 10L of crystalloids. In cases where a central venous catheter is used to measure blood pressures dynamically, fluids should be
administered until the central venous pressure (CVP) reaches 8-12 cm of water (or 10-15 cm of water in mechanically ventilated patients). Once these goals are met, the mixed venous oxygen saturation (Sv02), i.e., the oxygen saturation of venous blood as it returns to the heart as measured at the vena cava, is optimized. If the Sv02 is less than 70%, blood is given to reach a hemoglobin of 10 g/dl and then inotropes are added until the Sv02 is optimized.
Once the subject has been sufficiently fluid resuscitated but the mean arterial pressure is not greater than 65 mmHg vasopressors are recommended. While current
recommendations suggest either norepinephrine (noradrenaline) or dopamine, the former appears safer. If a single pressor is not sufficient in improving the blood pressure, epinephrine (adrenaline) may be added in.
Elective tracheal intubation and mechanical ventilation may be performed to reduce oxygen demand if the Sv02 remains low despite optimization of hemodynamics. Etomidate is not recommended as a medication to help with intubation in this situation due to concerns of adrenal insufficiency and increased mortality.
Patient monitoring
The diagnostic methods of the invention are also useful for monitoring the course of a sepsis in a patient and/or for assessing the efficacy of a therapeutic regimen. In one embodiment, the diagnostic methods of the invention are used periodically to monitor the polynucleotide or polypeptide levels of one or more of PD-1 and/or PD-L1. In one example, sepsis is characterized using a diagnostic assay of the invention prior to administering therapy. This assay provides a baseline that describes the level of one or more markers of sepsis prior to treatment. Additional diagnostic assays are administered during the course of therapy to monitor the efficacy of a selected therapeutic regimen. A therapy is identified as efficacious when a diagnostic assay of the invention detects a decrease in marker levels relative to the baseline level of marker prior to treatment. In particular, the level of immune dysfunction is characterized by measuring PD- 1 and/or PD-Ll on CD8+ T cells. Where CD8+ PD-1 >36 and PD-Ll <5 expression; HLA-DR expression is decreased; and/or TNF-a levels are decreased in LPS -stimulated whole blood, such patients are selected for anti-PD-1 and/or anti-PD-Ll antibody therapy.
The disease state or treatment of a subject having sepsis, or a propensity to develop such a condition can be monitored using the methods and compositions of the invention. In one embodiment, the expression of markers present in a bodily fluid, such as blood, blood serum, or plasma, is monitored. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a subject or in assessing disease progression. Therapeutics that decrease the expression of a marker of the invention (e.g. , PD-1 and/or PD-Ll) are taken as particularly useful in the invention.
Kits
The invention provides kits for the treatment or prevention of sepsis, septic shock, systemic inflammatory response syndrome, and and compensatory anti-inflammatory response syndrome. The invention further provides kits for identifying a subject as responsive to PD- 1 pathway blockade.
In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of an inhibitory antibody that disrupts the biological activity of a PD1 and/or PD-Ll polypeptide in unit dosage form. In another embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of an anti- PD1 and/or anti-PD-Ll antibody in unit dosage form.
A diagnostic kit of the invention provides a capture reagent (e.g., an anti-PDl and/or anti-PD-Ll antibody) for measuring increased CD8+ PD-1 and decreased PD-Ll (e.g., where CD8+ PD-1 >36 and PD-Ll <5 expression).
In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. If desired an antibody or agent of the invention is provided together with instructions for administering the antibody or agent to a subject having or at risk of developing sepsis. The instructions will generally include information about the use of the composition for the treatment or prevention of sepsis. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology;
clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);
"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);
"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES Example 1. Sepsis increased CD8 PD-1 and monocyte PD-L1 expression. Examination of PD-1 expression on CD4 and CD8 T cells showed that sepsis caused an increase in CD8, but not CD4 PD-1 expression compared to non-septic patients (Figure 1). The percentage of monocytes that expressed PD-L1 was increased over two-fold in septic versus non-septic patients (Figure 1). As is characteristically described in patients with sepsis (Monneret et al. , Mol Med 2008, 14(l-2):64-78), monocyte HLA-DR expression was significantly decreased in septic versus non-septic patients, p < 0.001, (Figure 1). In addition, analysis of expression over time of patients in the intensive care unit (ICU) revealed that CD8 T cell PD-1 increased as PD-L1 decreased during stay in ICU (Figure 2A). Furthermore, the subset of donors with increased PD-1 together with decreased PD-L1 expression on CD8 cells (CD8+ PD-lhigh PD-Lllow' , CD8+ PD-1 >36% and PD-L1 <5% expression) also had reduced levels of HLA-DR expression on monocytes compared with CD8+ PD-llow PD- Llhigh septic patients (CD8+ PD-1 <36% and PD-L1 >5% expression) and critically ill non- septic patients (Figure 2B). This finding is further validated upon reciprocal analysis of HLA-DR expression on CD14 monocytes, where selection of donor samples with less than 50% HLA-DR+ monocytes had a higher proportion of CD8+ PD- lhigh PD-L1 low T cells than those with greater than 50% HLA-DR+ monocytes (data not shown). CD8+ PD-lhigh PD- Lllow patient samples were detected at all time points tested during stay in ICU, but increasing over time (26.7%, 37.5%, 40% and 100% of samples per blood Draw A to D respectively). Similarly, CD14 HLA-DR low, PD-lhigh PD-Lllow patient samples were detected at all time points tested during stay in ICU, and also increased over time (42.8%, 50%, 75% and 100% of samples per blood Draw A to D respectively). Interestingly, this same subgroup of septic patients had an increased rate of secondary infections, VAP and peritonitis, compared to other septic patients (p<0.05, Figure 3). These data support the idea that septic patients are a heterogeneous population at different stages of disease and with differing immunologic status upon presentation to the ICU, but develop progressively increasing levels of immune exhaustion with protracted sepsis.
Example 2. Anti-PD-1 and anti-PD-Ll decreased sepsis-induced apoptosis in lymphocytes.
Apoptosis was quantified in patient lymphocytes after overnight incubation with either isotype control antibody, anti-PD-1 antibody, or anti-PD-Ll antibody (Figure 4).
Quantitation of apoptosis in total lymphocytes, i.e. , all lymphocytes present in the lymphocyte gate identified by forward and side scatter on flow cytometry (see Figure 5A) and consisting primarily of CD4+, CD8+, NKT cells, and NK cells was examined. Total lymphocyte apoptosis was increased by -70% in septic patients when compared to non-septic patients after overnight incubation in isotype (inactive) control antibody, i.e., 10.4 + 1.5% in septic patients versus 6.1± 1.0% in non-septic patients (p< 0.01) (Figure 4). Compared to lymphocytes incubated with isotype control antibody, lymphocytes incubated in media containing anti-PD-1 or anti-PD-Ll antibody had a highly significant decrease in apoptosis, p< 0.002, (Figures 4 and 5B). No effect of anti-PD-1 or anti-PD-Ll antibody on lymphocyte apoptosis was observed in samples from non-septic patients, possibly due to their lower level of baseline apoptosis which was often less than 5% (Figure 4). A highly similar effect of anti-PD-1 and anti-PD-Ll antibody on sepsis-induced apoptosis was observed in CD4 T cells from septic and non-septic patients (Figure 6).
Example 3. Anti-PD-l/anti-PD-Ll ameliorated sepsis-induced impairment in production of IFN-γ and IL-2.
Peripheral blood mononuclear cells (PBMCs) from septic or non-septic patients were divided equally into wells and incubated overnight with isotype control antibody, anti-PD-1 antibody, or anti-PD-Ll antibody. The next morning, cells were washed, stained for various lymphocyte subsets and stimulated (see Methods). Comparison of the intracellular production of IFN-γ (expressed as the % IFN-γ " cells) demonstrated impaired IFN-γ production in total lymphocytes and NKT cells from septic versus non-septic patients, when incubated with inactive isotype control antibody (Figure 7). Likewise, IL-2 production was decreased in total lymphocytes, CD3 T cells, and NKT cells in septic versus non-septic patients (Figure 8). Overnight incubation of cells showed a significant effect of anti-PD-1 and anti-PD-Ll antibodies in increasing IFN-γ production in total lymphocytes and NKT cells (Figures 9 and 10). Examination showed that only a subset of patients' samples responded to anti-PD-1 or anti-PD-Ll. Anti-PD-1 and anti-PD-Ll had similar effects of increasing IL-2 production in specific lymphocyte subsets (Figure 11). There was no significant effect of anti-PD-1 or anti-PD-Ll to increase IFN-γ or IL-2 in lymphocytes from non-septic patients (Figures 12 and 13).
A characteristic hematologic finding in patients with sepsis is an apoptosis-induced reduction in their absolute lymphocyte count, often to values that are less than 20-30% of that for healthy controls (Munford et al , Am J Respir Crit Care Med 2001, 163(2):316-321 ; Hotchkiss et al, Crit Care Med 1999, 27(7): 1230-1251; Venet et al, J Immunol 2012, 189(10):5073-5081). Importantly, persistent lymphopenia in sepsis is associated with increased mortality. Lymphocyte depletion (as reflected by lymphopenia) may contribute to morbidity and mortality by impairing host immunity (Kasten et al. , Infect Immun 2010,
78(l l):4714-4722; Hotchkiss et al, J Immunol 1999, 162(7):4148-4156). To confirm a role for PD-1 :PD-L1 interaction in lymphocyte apoptosis, and thereby, lymphopenia in sepsis, the correlation between PD- 1 expression on CD4 T cells and apoptosis was examined. At the first blood draw for septic patients (time point A), there was a positive correlation between the percentage of CD4 T cells that were PD-1 positive and the degree of CD4 apoptosis reflected in TUNEL positivity (Figure 14).
The present results show that blockade of either PD-1 or its ligand PD-L1 reverses two pathophysiologic hallmarks of sepsis. Anti-PD-1 and anti-PD-Ll antibodies markedly decreased sepsis-induce lymphocyte apoptosis and restored the ability of immune effector cells to produce cytokines that are essential for host immunity. These in vitro findings in patient leukocytes strengthen the concept that blockade of the PD-1 :PD-L1 pathway offers a promising new approach in the treatment of sepsis [17,28]. Although most previous therapeutic trials in sepsis have focused on blockade of the initial hyper-inflammatory phase, there is increased recognition that if patients survive this initial stage of the disorder, they progress to an immunosuppressive state [4,28-32]. New treatment protocols have resulted in the fact that the majority of deaths in sepsis now occur after the first four days of sepsis (the hyper- inflammatory phase) and during the immunosuppressive phase [33]. Furthermore, microbiologic studies of patients dying of sepsis showed that over 50% of the infecting organisms were classified as opportunistic pathogens (opportunistic bacteria and fungae), a finding which is highly compatible with impaired immunity [33]. In this setting, use of immuno-adjuvant agents including anti-PD-1 or anti-PD-Ll antibodies is a logical approach to restore host immunity and potentially improve survival.
Research into the mechanistic basis of immunosuppression in sepsis has determined that multiple overlapping etiologies exist including increased T regulatory and myeloid derived suppressor cells and apoptotic depletion of T and B cells [5-8]. A relatively newly recognized etiology of immunosuppression in sepsis is T cell exhaustion. T cell exhaustion was first reported in animal models of chronic viral infection and was thought to be due to persistent exposure to high levels of antigen [9-11]. Patients with sepsis often have a protracted course with primary and secondary infections, a scenario that likely includes persistent high circulating antigens thereby facilitating development of T cell exhaustion [3,33,34]. A recent postmortem study of spleens and lungs obtained from patients dying of sepsis demonstrated findings highly consistent with T cell exhaustion [3,10]. These findings included severely depressed splenocyte cytokine production, decreased T cell IL-7 receptor (CD 127) expression, and increased PD-1 and PD-L1 expression on T cells and macrophages respectively. These postmortem studies also demonstrated that PD-L1 was highly expressed on tissue parenchymal cells, i.e. , on splenic endothelial and bronchial epithelial cells, thereby providing opportunity for PD-1 activation [3]. Guignant and colleagues documented a correlation between PD- 1 expression on circulating immune cells of septic patients and decreased T cell proliferative capacity, increased nosocomial infections, and mortality [35].
Zhang et al. reported that anti-PD- 1 was increased on monocytes from septic patients and that anti-PD- 1 antibody decreased T cell apoptosis and improved immune effector function [36]. A recent important study by Singh et al. showed that in vitro blockade of PD-1 improved T cell IFN-γ production and decreased apoptosis in patients with active infections due to M. tuberculosis [37]. A second major finding of these investigators was that when patients with active tuberculosis were treated with effective medication to eradicate M.
tuberculosis, the number of PD-1 -expressing T cells decreased and inversely correlated with IFN-γ T-cell response against M. tuberculosis. We believe that this work has major implications for the broader field of sepsis because of the similarities of active tuberculosis with protracted sepsis.
In addition to data that T cell exhaustion exists in patients with chronic viral infections and sepsis, there is evidence from animal studies that treatment with anti-PD- 1 and anti-PD-Ll antibodies can reverse T cell dysfunction, increase pathogen clearance, and improve survival. Four different investigative teams reported that blockade of the PD-1 pathway prevents apoptotic cell death, restores host immunity, and decreases mortality in clinically-relevant models of bacterial and fungal sepsis [16-20]. The present results showing that anti-PD- 1 and anti-PD-Ll antibodies restore cytokine production and prevent apoptosis in immune cells from patients with sepsis are highly consistent with these animal studies and underscore their potential efficacy in clinical sepsis. The effect of anti-PD- 1 and anti-PD-Ll to improve IFN- γ production by T cells may be a particularly beneficial in sepsis given its ability to improve monocyte/macrophage function which is impaired in sepsis [4, 38,39]. A clinical trial of IFN- γ in sepsis is currently underway and is being targeted to those patients whose circulating monocytes have low HLA-DR expression, (see clinicaltrials.gov).
An important factor in the potential clinical utility of anti-PD-1 or anti-PD-Ll antibodies in sepsis is identifying which patients would be optimal candidates for blocking therapy. Anti-PD- 1 antibody has been highly successful in a subset of patients with various types of malignancies [14,15]. In general, those patients whose tumors expressed PD-L1 on immunohistochemical analysis have responded to therapy with anti-PD-1 antibody. As PD-1 and PD-L1 can also be early activation markers, it is inadvisable to use these markers alone to diagnose an immunosuppressive state. Currently, patients with sepsis whose monocytes have decreased HLA-DR expression and/or patients whose LPS-stimulated whole blood response shows decreased TNF-a production are considered good candidates for immuno- stimulatory therapy [4]. Increased CD8 T cell PD-1 expression in conjunction with these two criteria might identify patients who are good candidates for anti-PD-1 antibody in sepsis. Recent studies as well as work from our own investigations have shown that patients with sepsis who have a persistently low absolute lymphocyte counts have a greatly increased risk of dying of sepsis. We postulate that these patients would be ideal candidates for anti-PD-1 antibody. The positive correlation between PD-1 expression on CD4 T cells and apoptosis, (Fig. 14), as well as the potent anti-apoptotic effect of anti-PD-1 suggests that anti-PD-1 would be highly advantageous in this setting by acting to increase lymphocyte numbers and function.
Anti-PD-1 and anti-PD-Ll antibodies have had extraordinary success in cancer trials and are considered to represent a major breakthrough in the field [40]. Anti-PD-1 antibody induced remission in approximately 20-25% of patients with a diversity of tumors including malignant melanoma, renal cell cancer, and non-small cell lung cancer. A remarkable feature of anti-PD-1 and anti-PD-1 therapy is the fact that some patients have durable cancer remissions that last for many months in the absence of continued therapy [41]. Cancer and sepsis share many of the same immunosuppressive mechanisms including increased T regulatory cells, increased myeloid derived suppressor cells, and T cell exhaustion [4-8,42]. This commonality in immune pathology in cancer and sepsis could be due to the fact that both cancer and sepsis may evolve into states of chronic low grade inflammation and persistent antigen exposure. Therefore, immunotherapy that is effective in reversing immune dysfunction in cancer might have similar effects in sepsis. This finding could explain why anti-PD-1 and anti-PD-Ll are effective in these two seemingly disparate disorders. Both anti-PD-1 and anti-PD-Ll antibodies have been well tolerated in clinical trials to date
[14,15,43]. Although serious autoimmune reactions can occur in patients treated with anti- PD-1 or anti-PD-Ll antibodies, these reactions are uncommon. Patients with sepsis typically may not require as prolonged therapy with anti-PD-l/anti-PD-Ll therapy as patients with cancer. Therefore, severe autoimmune reactions will likely be less of a problem in patients with sepsis.
In conclusion, anti-PD-1 and anti-PD-Ll antibodies ameliorated key immune defects consistent with reversal of T cell exhaustion in PBMCs from septic patients. Both antibodies appeared equally effective in their capabilities. Thus, lymphocyte PD-1 expression, in conjunction with other cellular markers and clinical and laboratory findings, may contribute to identifying septic patients in which anti-PD-1 or anti-PD-Ll antibody therapy may be beneficial. Collectively, the present findings indicate that T cell exhaustion is a major etiology of immune dysfunction in sepsis and that reversal of putative T cell exhaustion using anti-PD-1 or anti-PD-Ll offers promise in the therapy of this highly lethal disorder.
The results described herein were obtained using the following materials and methods.
Patient selection and demographics
A total of 43 septic patients were included in the study. Relevant clinical and laboratory values for septic and critically-ill non-septic patients regarding median age, gender, sites of infection, severity of illness scores, mortality, length of ICU stay, etc. are provided at Table 1.
Tab!e 1.
PATIENT CHARACTERISTICS
Septic
# Patients 43
Age
median 64
range 21-90
Gender
m.aie 21.
female 22
A ache
median 16
range 7-33
SOFA, (se uential organ failure assessment)
median 8
range 2 -IS
Length of 1CU stay
median 1.0 5 range 0-98 2-15
Mortality (%
survived 34 (79) 13 (87) expired 9 21} 2 (13)
Vasopressor ~ dependent shock 29 1 Admission ICU Diagnosis'
Community-acquired neumonia 14
Ventilator-associated neumonia 6
Peritonitis 15
Wound infection 2
Line infection 6
Trauma 10
Post-op (major surgery! 3
Intra-cranlal hemorrhage 2 Co-morbidities
Diabetes 17 5
Heart Disease 35 14
Morbid Obesity 2 0
Neurologic 6 3
Rena Disease 6 0
Respiratory 14 2
Liver 1 0
Additional patient data are presented at Tables 2 and 3. Thirty nine of the 43 septic patients were located in ICUs; 3 septic patients were located in lesser acuity treatment areas including observation units. Fifteen critically-ill non- septic patients were included in the study. Two non-septic critically-ill patients became septic with ventilator-associated pneumonia during their initial ICU admission. Data from these two patients is included in both septic and non-septic columns based upon their particular phase of illness, i.e. , non-septic or septic phase. Most non-septic patients did not remain in the ICU past 4 days and therefore, only one blood draw was obtained in these patients.
Septic patients: Patients at Barnes Jewish Hospital who were older than 18 years of age and who fulfilled a consensus panel definition of sepsis (Levy et al. , Crit Care Med 2003, 31 (4): 1250-1256) were included in the study (Table 1). Sepsis was defined as the presence of systemic inflammatory response syndrome (SIRS) and a known or suspected source of infection. Patients with HIV infection, viral hepatitis, or who were receiving
immunosuppressive medications (except corticosteroids at a dose of < 10 mg prednisone or equivalent per day) were excluded. Consent for blood draws was obtained from the patient or a legally authorized representative.
Table 2. Septic Patients
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
* Admitted as non-septic but developed sepsis
BAL, bronchial alveolar lavage, BLD, blood, C. diff. Clostridia difficile, CT, CT scan; CSR, chest x-ray, GPC, Gram positive cocci; MRSA, methicillin resistant Straph, aureus; pressors, vaso pressors; VAP, ventilator associated pneumonia; WCC, white cell count
Critically-ill non-septic patients: Control subjects consisted of critically-ill non-septic patients admitted to the ICU for care following major surgery, trauma, or myocardial ischemia (Table 1). Exclusion criteria were identical to that for patients with sepsis. Consent for blood draws was obtained from the patient or a legally authorized representative.
Table 3. Critically III Non-Septic Patients
Figure imgf000057_0001
Blood collection and processing
Patients provided consent for a maximum of 4 blood samples (5ml/sample) obtained serially at days 1-3 after admission to the ICU, days 4-7 (second blood draw), days 8-12 (third blood draw), and days 12-21 (fourth blood draw) after sepsis onset. The same serial blood draw protocol was used in non-septic patients. Heparinized blood was collected through an indwelling central venous or arterial catheter or by peripheral venipuncture. The blood was immediately transported and processed in the laboratory. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient separation. Plasma was collected and stored at -80°C for subsequent analysis. The cells were washed and resuspended in RPMI 1640 and processed for immunostaining or overnight incubation as previously described.
Flow Cytometry
Antibodies for flow cytometric determinations were purchased from Biolegend (San
Diego, CA, USA), BD Biosciences (San Diego, CA, USA), or eBiosciences (San Diego, CA, USA). Cellular expression of PD-1 and PD-L1 on acutely isolated PBMCs was performed on the day of blood draw. Lymphocytes were identified by forward scatter (FSC) and side scatter (SSC) properties as described previously (Boomer et al , JAMA 2011, 306(23) :2594- 2605). Monocytes were identified by FSC and SSC properties and by CD14+
immunostaining. T cell subsets were further identified by CD3+, CD4+, or CD8+ immunostaining. NK cells were identified as CD3-/CD56+ while natural killer T (NKT) cells were identified as CD3+/CD56+. Effects of anti-PD-1 and anti-PD-Ll on lymphocyte apoptosis
Cells (~1 X 107) were incubated overnight. Cells were treated with either isotype- control antibody, anti-PD-1 antibody, or anti-PD-Ll antibody. Anti-PD-1 antibody and anti- PD-Ll antibody were provided by Medimmune and were all human IgGl . The effect of anti- PD-1 and anti-PD-Ll antibody on lymphocyte apoptosis following overnight incubation was quantitated via the TUNEL assay as previously described (Brahmamdam et al. , J Leukoc Biol 2010, 88(2):233-240).
Effects of anti-PD-1 and anti-PD-Ll on stimulated IFN-y and IL-2 production
PBMCs that had undergone overnight incubation with either isotype-control antibody, anti-PD-1 antibody, or anti-PD-Ll antibody were stimulated with PMA/ionomycin plus brefeldin for 5 hrs as previously described (Unsinger et al. , J Immunol 2010, 184(7):3768- 3779; Unsinger et al. ,. J Infect Dis 2012, 206(4):606-616). Following stimulation, cells were washed, stained with anti-CD3 and anti-CD56 antibodies, fixed with 1% paraformaldehyde, permeabilized with IX perm/wash (Biolegend) and stained with fluorescently labeled anti- IFN-γ or anti-IL-2 antibodies. Patient hematologic values
Depending upon severity of illness, ICU patients had daily complete blood count analysis performed as part of the standard of care. Patient clinical laboratory values that were recorded in this study included absolute lymphocyte, absolute monocyte, and absolute granulocyte cell counts and were quantitated in the clinical laboratories at Barnes Jewish Hospital (Table 2).
Secondary infections - Definition of hospital-acquired secondary infections
Data on nosocomial infections occurring while patients were in the ICU were abstracted from medical records using standard Center for Disease Control case definitions (www.cdc.gov/hai/) .
Statistical analysis
Data were analysed with the statistical software Prism (GraphPad, San Diego, CA, USA). Data are reported as the mean + SEM. For comparison of two groups, the Student's t- test was employed. A paired i-test was used when comparing samples from the same patient which were treated identically except for incubation with either anti-PD-1 or anti-PD-Ll antibodies. One-way ANOVA with Tukey's multiple comparison tests was used to analyse data in which there were more than two groups. Significance was reported at p <0.05.
References
1. Cohen J, Opal S, Calandra T: Sepsis studies need new direction. Lancet Infect Dis 2012, 12(7):503-505.
2. Ward PA: Immunosuppression in sepsis. JAMA 2011 , 306(23):2618-2619.
3. Boomer JS, To K, Chang KC, Takasu O, Osborne DF, Walton AH, Bricker TL,
Jarman SD, 2nd, Kreisel D, Krupnick AS et at Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA 2011 , 306(23):2594-2605.
4. Monneret G, Venet F, Pachot A, Lepape A: Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony. Mol Med 2008, 14(l-2):64-78. 5. Munford RS, Pugin J: Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am J Respir Crit Care Med 2001, 163(2):316-321. 6. Felmet KA, Hall MW, Clark RS, Jaffe R, Carcillo JA: Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure. J Immunol 2005, 174(6):3765-3772.
7. Venet F, Chung CS, Monneret G, Huang X, Horner B, Garber M, Ayala A:
Regulatory T cell populations in sepsis and trauma. J Leukoc Biol 2008,
83(3):523-535.
8. Delano MJ, Scumpia PO, Weinstein JS, Coco D, Nagaraj S, Kelly-Scumpia KM, O'Malley KA, Wynn JL, Antonenko S, Al-Quran SZ et al: MyD88-dependent expansion of an immature GR-l(+)CDllb(+) population induces T cell suppression and Th2 polarization in sepsis. J Exp Med 2007, 204(6): 1463-1474. 9. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C et al: PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 2006,
443(7109):350-354.
10. Wherry EJ: T cell exhaustion. Nat Immunol 2011, 12(6):492-499.
11. Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ: The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol 2007, 8(3):239-245.
12. Keir ME, Butte MJ, Freeman GJ, Sharpe AH: PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008, 26:677-704.
13. Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N et al: Autoimmune dilated cardiomyopathy in PD- 1 receptor-deficient mice. Science 2001, 291(5502):319-322.
14. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF,
Powderly JD, Carvajal RD, Sosman JA, Atkins MB et al: Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012,
366(26) :2443-2454.
15. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG,
Camacho LH, Kauh J, Odunsi K et al: Safety and activity of anti-PD-Ll antibody in patients with advanced cancer. N Engl J Med 2012, 366(26):2455-2465.
16. Lazar-Molnar E, Gacser A, Freeman GJ, Almo SC, Nathenson SG, Nosanchuk JD:
The PD-l/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum. Proc Natl Acad Sci U S A 2008, 105(7):2658-2663.
Huang X, Venet F, Wang YL, Lepape A, Yuan Z, Chen Y, Swan R, Kherouf H, Monneret G, Chung CS et at PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis. Proc Natl Acad Sci U S A 2009, 106(15):6303-6308.
Brahmamdam P, Inoue S, Unsinger J, Chang KC, McDunn JE, Hotchkiss RS:
Delayed administration of anti-PD-1 antibody reverses immune dysfunction and improves survival during sepsis. J Leukoc Biol 2010, 88(2):233-240.
Zhang Y, Zhou Y, Lou J, Li J, Bo L, Zhu K, Wan X, Deng X, Cai Z: PD-L1 blockade improves survival in experimental sepsis by inhibiting lymphocyte apoptosis and reversing monocyte dysfunction. Crit Care 2010, 14(6):R220.
Chang KC, Burnham CA, Compton SM, Rasche DP, Mazuski R, Smcdonough J, Unsinger J, Korman AJ, Green JM, Hotchkiss RS: Blockade ofthe negative co- stimulatory molecules PD-1 and CTLA-4 improves survival in primary and secondary fungal sepsis. Crit Care 2013, 17(3):R85.
Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G et at 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med 2003, 31(4): 1250-1256.
Unsinger J, McGlynn M, Kasten KR, Hoekzema AS, Watanabe E, Muenzer JT, McDonough JS, Tschoep J, Ferguson TA, McDunn JE et al: IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis. /
Immunol 2010, 184(7):3768-3779.
Unsinger J, Burnham CA, McDonough J, Morre M, Prakash PS, Caldwell CC, Dunne WM, Jr., Hotchkiss RS: Interleukin-7 ameliorates immune dysfunction and improves survival in a 2-hit model of fungal sepsis. / Infect Dis 2012, 206(4):606- 616.
Hotchkiss RS, Swanson PE, Freeman BD, Tinsley KW, Cobb JP, Matuschak GM, Buchman TG, Karl IE: Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 1999, 27(7): 1230-1251.
Venet F, Foray AP, Villars-Mechin A, Malcus C, Poitevin- Later F, Lepape A, Monneret G: IL-7 Restores Lymphocyte Functions in Septic Patients. J Immunol 2012, 189(10):5073-5081. 26. Kasten KR, Prakash PS, Unsinger J, Goetzman HS, England LG, Cave CM, Seitz AP, Mazuski CN, Zhou TT, Morre M et at Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gamma delta T-cell IL-17 production in a murine model of sepsis. Infect Immun 2010, 78(l l):4714-4722.
27. Hotchkiss RS, Swanson PE, Knudson CM, Chang KC, Cobb JP, Osborne DF, Zollner KM, Buchman TG, Korsmeyer SJ, Karl IE: Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis. J Immunol 1999,
162(7):4148-4156.
28. Hotchkiss RS, Monneret G, Payen D: Immunosuppression in sepsis: a novel
understanding of the disorder and a new therapeutic approach. Lancet Infect Dis
2013, 13(3):260-268.
29. Christaki, E., Anyfanti P, Opal SM: Immunomodulatory therapy for sepsis: an update.
Expert Rev Anti Infect Ther 2011, 9: 1013-1033.
30. Hotchkiss RS, Opal S: Immunotherapy for sepsis-a new approach against an ancient foe. N Engl J Med 2010, 363(l):87-89.
31. Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med
2003, 348(2): 138-150.
32. Hall MW, Knatz NL, Vetterly C, Tomarello S, Wewers MD, Volk HD, Carcillo JA:
Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome. Intensive Care Med 2011, 37(3):525-532.
33. Otto GP, Sossdorf M, Claus RA, Rodel J, Menge K, Reinhart K, Bauer M,
Riedemann NC: The late phase of sepsis is characterized by an increased microbiological burden and death rate. Crit Care 2011, 15(4):R183.
34. Torgersen C, Moser P, Luckner G, Mayr V, Jochberger S, Hasibeder WR, Dunser MW: Macroscopic postmortem findings in 235 surgical intensive care patients with sepsis. Anesth Analg 2009, 108(6): 1841-1847.
35. Guignant C, Lepape A, Huang X, Kherouf H, Denis L, Poitevin F, Malcus C, Cheron A, Allaouchiche B, Gueyffier F et al: Programmed death-1 levels correlate with increased mortality, nosocomial infection and immune dysfunctions in septic shock patients. Crit Care 2011, 15(2):R99.
36. Zhang Y, Li j, Zhou Y, Bo L, Zhu J, Zhu K, Wan X, Cai Z, Deng X. Upregulation of programmed death- 1 on T cells and programmed death ligand- 1 on monocytes in septic shock patients. Crit Care 2011, 15(1):R70
37. Singh A, Mohan A, Dey AB, Mitra DK: Inhibiting the programmed Death 1
pathway rescues Mycobacterium tuberculosis-specific interferon γ- producing T cells from apoptosis in patients with pulmonary tuberculosis. J. Infectious Dis. 2013, 208:603-615
38. Docke WD, Randow F, Syrbe U, Krausch D, Asadullah K, Reinke P, Volk HD, Kox W: Monocyte deactivation in septic patients: restoration by IFN-gamma treatment. Nat Med 1997, 3(6):678-681.
39. Munoz C, Carlet J, Fitting C, Misset B, Bleriot JP, Cavaillon JM: Dysregulation of in vitro cytokine production by monocytes during sepsis. J Clin Invest 1991 ,
88(5):1747-1754.
340. Pollack A: Promising new cancer drugs empower the body's own defense system.
In: The New York Times(Health section). June 3, 2013.
41. Lipson EJ, Sharfman WH, Drake CG, Wollner I, Taube JM, Anders RA, Xu H, Yao S, Pons A, Chen L et at Durable cancer regression off- treatment and effective reinduction therapy with an anti-PD-1 antibody. Clin Cancer Res 2013, 19(2): 462- 468.
42. Cheever MA: Twelve immunotherapy drugs that could cure cancers. Immunol Rev 2008, 222:357-368.
43. Hamid O, Robert C, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P, Joseph RW, Weber JS, Dronca R, et al. Safety and tumor responses with lambrolizumab in melanoma. N Engl J. Med. 2013, epub (ahead of print)
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents, publications, and accession numbers mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent, publication, and accession number was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:
1. A method of identifying a subject responsive to PD-1 pathway blockade, the method comprising detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels in a biological sample of the subject relative to a reference, thereby identifying said subject as responsive to PD- 1 pathway blockade.
2. The method of claim 1, wherein said detection is by a method selected from the group consisting of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and immunoprecipitation.
3. A method of identifying a subject responsive to PD-1 pathway blockade, the method comprising detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels in a biological sample of the subject relative to a reference, wherein said detection is by a method selected from the group consisting of immunostaining, ELISA, FACS,
radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
immunoprecipitation, thereby identifying said subject as responsive to PD-1 pathway blockade.
4. A method of increasing T cell proliferation in a subject pre-selected as responsive to PD-1 pathway blockade, the method comprising administering to said subject an anti-PD-1 and/or anti-PD-Ll antibody, wherein the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby reducing T cell proliferation in said subject.
5. A method of reducing sepsis-induced lymphocyte apoptosis in a subject pre-selected as responsive to PD-1 pathway blockade, the method comprising administering to said subject an anti-PD-1 or anti-PD-Ll antibody, wherein the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby reducing sepsis-induced lymphocyte apoptosis in said subject.
6. A method of increasing IFN-γ or IL-2 levels in a subject pre-selected as responsive to PD- 1 pathway blockade, the method comprising administering to said subject an anti-PD-1 or anti-PD-Ll antibody, wherein the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby increasing IFN-γ or IL-2 levels in said subject.
7. A method of reducing immune dysfunction in a subject pre-selected as responsive to PD-1 pathway blockade, the method comprising administering to said subject an anti-PD-1 or anti- PD-Ll antibody, wherein the subject is pre-selected by detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels relative to a reference, thereby reducing immune dysfunction in said subject.
8. The method of claim 7, wherein said detection is by a method selected from the group consisting of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and immunoprecipitation
9. A method of treating sepsis in a subject identified as responsive to PD-1 pathway blockade, the method comprising:
(a) detecting an increase in CD8+ PD-1 levels and a decrease in CD8+ PD-Ll levels in a biological sample of the subject relative to a reference, thereby identifying said subject as responsive to PD-1 pathway blockade; and
(b) administering to said subject an anti-PD-1 or anti-PD-Ll antibody, thereby treating sepsis in the subject.
10. The method of any of claims 1-9, wherein the method identifies the subject as in need of treatment with an anti-PD-1 or anti-PD-Ll antibody.
11. The method of claim 1 or 3, wherein said method characterizes the level or stage of immune dysfunction in the subject.
12. The method of any of claims 1-9, wherein CD8+ PD-1 levels are greater than at least about 36% and PD-Ll levels are less than about 5% relative to said levels in a reference.
13. The method of any of claims 1-9, wherein said method further comprises detecting a decrease in HLA-DR expression levels relative to a reference.
14. The method of any of claims 1-9, wherein said method further comprises detecting a decrease in TNF-a levels in LPS-stimulated whole blood.
15. A method of identifying a subject responsive to PD-1 pathway blockade, the method comprising detecting
(i) immune dysfunction where CD8+ PD-1 >36 and PD-L1 <5 expression; (ii) decreased HLA-DR expression; and
(iii) decreased TNF-a levels in LPS-stimulated whole blood, wherein said detection identifies the patient as responsive to anti-PD- 1 and/or anti-PD-Ll antibody therapy.
16. A method of identifying a subject responsive to PD-1 pathway blockade, the method comprising detecting
(i) immune dysfunction where CD8+ PD-1 >36 and PD-L1 <5 expression;
(ii) decreased HLA-DR expression; and
(iii) decreased TNF-a levels in LPS-stimulated whole blood, wherein said detection is by a method selected from the group consisting of immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and
immunoprecipitation, wherein said detection identifies the patient as responsive to anti-PD- 1 and/or anti-PD-Ll antibody therapy.
17. A method of treating sepsis in a subject identified as responsive to PD-1 pathway blockade, the method comprising detecting
(i) immune dysfunction where CD8+ PD-1 >36 and PD-L1 <5 expression;
(ii) decreased HLA-DR expression; and
(iii) decreased TNF-a levels in LPS-stimulated whole blood, wherein said detection identifies the patient as responsive to PD-1 pathway blockade; and
administering to said subject an anti-PD- 1 or anti-PD-Ll antibody, thereby treating sepsis in the subject.
18. The method of any of claims 1-17, wherein said method characterizes the immunosuppressive phase of sepsis in the subject.
19. The method of any of claims 3-17, wherein said administration restores cytokine production in the subject.
20. The method of any of claims 9-17, wherein said administration decreases sepsis- induced lymphocyte apoptosis in said subject.
21. The method of any of claims 2-17, wherein said administration restores host immunity.
22. The method of any of claims 2-17, wherein sepsis is bacterial or fungal sepsis.
23. The method of any of claims 2-17, wherein the antibody neutralizes PD-1 and/or PD- Ll.
24. The method of any of claims 2-17, wherein the antibody comprises one or more variable regions of a LOPD180 antibody.
25. The method of any of claims 2-17, wherein the antibody is LOPD180.
26. The method of any of claims 2-17, wherein the anti-PD-1 or anti-PD-Ll antibody is administered at about 10 mg/kg, 30 mg/kg, or 60 mg/kg.
27. The method of any of claims 2-17, wherein the method increases an immune response in the subject.
28. The method of any of claims 27, wherein the method increases T-cell proliferation, IFN-γ production, and/or IL-2 production in the subject.
29. The method of any of claims 2-17, wherein the administering is by intravenous injection.
30. The method of any of claims 1-17, wherein the biological sample is a blood sample.
31. A kit for treating sepsis comprising an effective amount of an antibody that specifically binds PD-1 and/or PD-Ll, and instructions for using the kit to treat sepsis.
32. A kit for identifying a subject responsive to PD-1 pathway blockade, the kit comprising a capture reagent that binds PD-1 and a capture reagent that binds PD-Ll.
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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016164799A1 (en) 2015-04-10 2016-10-13 The Regents Of The University Of California Methods of determining patient populations amenable to immunomodulatory treatment of cancer
US9724390B2 (en) 2015-02-03 2017-08-08 Oncomed Pharmaceuticals, Inc. Tumor necrosis factor receptor soluble factor binding (TNFRSF-binding) agents
WO2017144681A1 (en) 2016-02-25 2017-08-31 Cell Medica Switzerland Ag Binding members to pd-l1
US9885721B2 (en) 2014-05-29 2018-02-06 Spring Bioscience Corporation PD-L1 antibodies and uses thereof
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US10336824B2 (en) 2015-03-13 2019-07-02 Cytomx Therapeutics, Inc. Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of thereof
US10344090B2 (en) 2013-12-12 2019-07-09 Shanghai Hangrui Pharmaceutical Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US10428145B2 (en) 2015-09-29 2019-10-01 Celgene Corporation PD-1 binding proteins and methods of use thereof
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
US10620211B2 (en) 2015-02-03 2020-04-14 Ventana Medical Systems, Inc. Histochemical assay for evaluating expression of programmed death ligand 1 (PD-L1)
US10751414B2 (en) 2016-09-19 2020-08-25 Celgene Corporation Methods of treating psoriasis using PD-1 binding antibodies
US10766958B2 (en) 2016-09-19 2020-09-08 Celgene Corporation Methods of treating vitiligo using PD-1 binding antibodies
US11168144B2 (en) 2017-06-01 2021-11-09 Cytomx Therapeutics, Inc. Activatable anti-PDL1 antibodies, and methods of use thereof
US11299544B2 (en) 2013-03-15 2022-04-12 Genentech, Inc. Biomarkers and methods of treating PD-1 and PD-L1 related conditions
US11530269B2 (en) 2014-07-11 2022-12-20 Ventana Medical Systems, Inc. Anti-PD-L1 antibodies and diagnostic uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070122378A1 (en) * 2005-06-08 2007-05-31 Gordon Freeman Methods and compositions for the treatment of persistent infections
US20120301892A1 (en) * 2011-05-27 2012-11-29 Xenotech Llc In vitro test system to evaluate xenobiotics as immune-modulators of drug transport and metabolism in human hepatocytes
US20130045202A1 (en) * 2008-12-09 2013-02-21 Genentech, Inc. Anti-pd-l1 antibodies and articles of manufacture

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070122378A1 (en) * 2005-06-08 2007-05-31 Gordon Freeman Methods and compositions for the treatment of persistent infections
US20130045202A1 (en) * 2008-12-09 2013-02-21 Genentech, Inc. Anti-pd-l1 antibodies and articles of manufacture
US20120301892A1 (en) * 2011-05-27 2012-11-29 Xenotech Llc In vitro test system to evaluate xenobiotics as immune-modulators of drug transport and metabolism in human hepatocytes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DURAISWAMY ET AL.: "Therapeutic PD-1 Pathway Blockade Augments with other Modalities of Immunotherapy to Prevent Immune Decline in Ovarian Cancer", CANCER RES, vol. 1, no. 73, 1 December 2013 (2013-12-01), pages 1 - 21 *
ZHANG ET AL.: "PD-L1 blockade improves survival in experimental sepsis by inhibiting lymphocyte apoptosis and reversing monocyte dysfunction", CRITICAL CARE, vol. 14, no. R220, 2010, pages 1 - 9 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US11299544B2 (en) 2013-03-15 2022-04-12 Genentech, Inc. Biomarkers and methods of treating PD-1 and PD-L1 related conditions
US11365255B2 (en) 2013-12-12 2022-06-21 Suzhou Suncadia Biopharmaceuticals Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US10344090B2 (en) 2013-12-12 2019-07-09 Shanghai Hangrui Pharmaceutical Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US10775383B2 (en) 2014-05-29 2020-09-15 Ventana Medical Systems, Inc. PD-L1 antibodies and uses thereof
US9885721B2 (en) 2014-05-29 2018-02-06 Spring Bioscience Corporation PD-L1 antibodies and uses thereof
US11530269B2 (en) 2014-07-11 2022-12-20 Ventana Medical Systems, Inc. Anti-PD-L1 antibodies and diagnostic uses thereof
US9724390B2 (en) 2015-02-03 2017-08-08 Oncomed Pharmaceuticals, Inc. Tumor necrosis factor receptor soluble factor binding (TNFRSF-binding) agents
US10620211B2 (en) 2015-02-03 2020-04-14 Ventana Medical Systems, Inc. Histochemical assay for evaluating expression of programmed death ligand 1 (PD-L1)
US10336824B2 (en) 2015-03-13 2019-07-02 Cytomx Therapeutics, Inc. Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of thereof
US11174316B2 (en) 2015-03-13 2021-11-16 Cytomx Therapeutics, Inc. Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of use thereof
US10669339B2 (en) 2015-03-13 2020-06-02 Cytomx Therapeutics, Inc. Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of use thereof
EP3281010A4 (en) * 2015-04-10 2018-08-01 The Regents of The University of California Methods of determining patient populations amenable to immunomodulatory treatment of cancer
US11215616B2 (en) 2015-04-10 2022-01-04 National Institutes Of Health (Nih), (Dhhs), U.S. Government Methods of determining patient populations amenable to immunomodulatory treatment of cancer
WO2016164799A1 (en) 2015-04-10 2016-10-13 The Regents Of The University Of California Methods of determining patient populations amenable to immunomodulatory treatment of cancer
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
US10428145B2 (en) 2015-09-29 2019-10-01 Celgene Corporation PD-1 binding proteins and methods of use thereof
US11434294B2 (en) 2016-02-25 2022-09-06 Cell Medica, Inc. Binding members to PD-L1
EP4086285A1 (en) 2016-02-25 2022-11-09 Cell Medica Switzerland AG Binding members to pd-l1
WO2017144681A1 (en) 2016-02-25 2017-08-31 Cell Medica Switzerland Ag Binding members to pd-l1
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US10751414B2 (en) 2016-09-19 2020-08-25 Celgene Corporation Methods of treating psoriasis using PD-1 binding antibodies
US11168144B2 (en) 2017-06-01 2021-11-09 Cytomx Therapeutics, Inc. Activatable anti-PDL1 antibodies, and methods of use thereof

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