WO2020172391A1 - Methods and compositions relating to the treatment of gvhd - Google Patents

Methods and compositions relating to the treatment of gvhd Download PDF

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Publication number
WO2020172391A1
WO2020172391A1 PCT/US2020/019012 US2020019012W WO2020172391A1 WO 2020172391 A1 WO2020172391 A1 WO 2020172391A1 US 2020019012 W US2020019012 W US 2020019012W WO 2020172391 A1 WO2020172391 A1 WO 2020172391A1
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Prior art keywords
inhibitor
cell
subject
versus
disease
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PCT/US2020/019012
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French (fr)
Inventor
Leslie S. KEAN
Benjamin Watkins
John Horan
Muna QAYED
Amelia LANGSTON
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The Children's Medical Center
Emory University
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Publication of WO2020172391A1 publication Critical patent/WO2020172391A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • GVHD Graft versus host disease
  • GVHD graft-versus-host disease
  • GVHD chronic GVHD
  • patients with chronic GVHD commonly demonstrate skin involvement resembling lichen planus or the cutaneous manifestations of scleroderma; dry oral mucosa with ulcerations and sclerosis of the gastrointestinal tract; and a rising serum bilirubin concentration, amongst other clinical manifestations.
  • Various immunosuppressive agents have been employed to protect patients from such damage.
  • GVHD is controlled using immunosuppressive agents including cyclosporin A, corticosteroids including prednisone, and methylprednisolone, cyclophosphamide, and FK506, amongst others.
  • immunosuppressive agents including cyclosporin A, corticosteroids including prednisone, and methylprednisolone, cyclophosphamide, and FK506, amongst others.
  • these drugs are associated with a high incidence of side effects when used at therapeutic doses.
  • Such side effects include neurotoxicity, nephrotoxicity, increased risk of relapse of the underlying malignancy, increased risk of infections and/or hepatotoxicity.
  • GvHD results from over activation of the immune system, specifically over active transplanted T cells.
  • the CD80/86 inhibitor abatacept is demonstrated herein to suppress this immune activity to a surprising degree, particularly in combination with a calcineurin inhibitor or methotrexate.
  • administration of an immune suppressing CD80/86 inhibitor alters the biology of T cells in a way that increases their anti -cancer activity (i.e. by downregulating immune checkpoint molecules), preventing cancer relapses in transplant patients to a striking degree.
  • the methods described herein therefore provide both i) a suprising degree of efficacy in suppressing the immune response underlying GvHD and ii) an unexpected result as far as increasing the expression of checkpoint molecules which may increase the effectiveness of the anti-cancer aspects of the immune system.
  • a method for treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction comprising:
  • a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor
  • the condition caused by or associated with T cell dysfunction is selected from Graft-versus-host disease (GvHD), leukemia or lymphoma relapse.
  • GvHD Graft-versus-host disease
  • a method for treating or preventing GvHD comprising administering to a subject in need thereof, a therapeutically effective amount of a
  • composition comprising a CD80/86 inhibitor.
  • the administration is prophylactic.
  • the subject is a subject who is or has received a bone marrow transplant.
  • a method of treating a hematopoietic disease in a subject in need thereof comprising: a. administering a bone marrow transplant to the subject; and
  • the hematopoietic disease is a hematopoietic cancer.
  • the hematopoietic disease is leukemia.
  • the CD80/86 inhibitor is an antibody or antigen binding fragment thereof, an aptamer, or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86.
  • the CD80/86 inhibitor binds specifically to CD80 and/or 86.
  • the CD80/86 inhibitor is abatacept.
  • the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
  • the receptor is CTLA-4 and/or CD28.
  • the immune checkpoint polypeptides are selected from CTLA4, Lag-3, HAVCR2 (TIM-3) and/orPDCDl.
  • the immune checkpoint polypeptides are selected from Lag-3, HAVCR2 (TIM-3) and/orPDCDl.
  • the T cell population comprises CD3+, CD4+ and/or CD8+ T cells.
  • the T cell population comprises Regulatory T cells (TregS ) .
  • the inhibitor is targeted to T-cells.
  • the inhibitor comprises a T cell targeting moiety.
  • the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide.
  • the T cell-specific cell surface polypeptide is selected from the group consisting of CD3, CD4 and/or CD8.
  • the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof, an aptamer, or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
  • the method further comprises administering a therapeutically effective amount of an inhibitor of the expression or activity of one or more immune checkpoint polypeptides.
  • the condition caused by or associated with T cell dysfunction is selected from Graft-versus-host disease (GvHD) leukemia or lymphoma relapse.
  • GvHD Graft-versus-host disease
  • the method comprises administering a therapeutically effective amount of a Calcineurin inhibitor.
  • the Calcineurin inhibitor binds specifically to Calcineurin.
  • the Calcineurin inhibitor is selected from cyclosporine and tacrolimus.
  • the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
  • the method comprises administering a therapeutically effective amount of a dihydrofolate reductase (DHFR) inhibitor.
  • DHFR dihydrofolate reductase
  • the dihydrofolate reductase (DHFR) inhibitor is an antibody or antigen-binding fragment thereof, an aptamer, or a dihydrofolate reductase (DHFR) inhibitor fragment of a natural receptor or ligand of dihydrofolate reductase (DHFR).
  • the dihydrofolate reductase (DHFR) inhibitor binds specifically to dihydrofolate reductase (DHFR).
  • the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
  • the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
  • the CD80/86 inhibitor inhibits the expression or activity of one or more polypeptides in T cells selected from the polypeptides listed in table 1.
  • the Graft-versus-host disease is selected from acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
  • the condition caused by or associated with T cell dysfunction is selected from non-relapse mortality (NRM) and infection.
  • NEM non-relapse mortality
  • the subject is a subject who is or has received a Hematopoietic cell transplant (HCT).
  • HCT Hematopoietic cell transplant
  • UTD unrelated donor
  • composition comprising:
  • b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a
  • DHFR dihydrofolate reductase
  • the Calcineurin inhibitor is selected from cyclosporine or tacrolimus.
  • the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
  • the CD80/86 inhibitor is abatacept.
  • Described herein is a method of treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction in a subject in need thereof, the method comprising administering to the subject a composition of any of the preceding claims.
  • the condition caused by or associated with T cell dysfunction is selected from Graft-versus-host disease (GvHD) or leukemia.
  • GvHD Graft-versus-host disease
  • leukemia leukemia
  • the Graft-versus-host disease is selected from acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD) and chronic Graft-versus-host disease (cGvHD).
  • a method of treating or preventing a hematopoietic disease in a subject in need thereof comprising administering to the subject a composition of any of the preceding claims.
  • NRM non-relapse mortality
  • HCT Hematopoietic cell transplant
  • the Hematopoietic cell transplant originates from an unrelated donor (URD).
  • hematopoietic disease in a subject in need thereof, the method comprising:
  • DHFR dihydrofolate reductase inhibitor
  • the hematopoietic disease is a hematopoietic cancer.
  • the hematopoietic disease is leukemia.
  • a pharmaceutical composition formulated for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction comprising: a. a CD80/86 inhibitor; and b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor; and c. a pharmaceutically acceptable carrier.
  • a CD80/86 inhibitor comprising: a. a CD80/86 inhibitor; and b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor; and c. a pharmaceutically acceptable carrier.
  • DHFR dihydrofolate reductase
  • a pharmaceutical composition for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), severe acute Graft-versus-host disease
  • aAGVHD chronic Graft-versus-host disease
  • cGvHD chronic Graft-versus-host disease
  • a pharmaceutical composition for the treatment or prevention of a hematopoietic disease in a subject in need thereof is a pharmaceutical composition for the treatment or prevention of a hematopoietic disease in a subject in need thereof.
  • a pharmaceutical composition for the treatment of a hematopoietic disease wherein the hematopoietic disease is a hematopeitic cancer.
  • a pharmaceutical composition for the treatment or prevention of a non-relapse mortality (NRM) in a subject who is need of or who has received a Hematopoietic Cell Transplant (HCT) comprising administering to the subject a composition of any of the preceding claims.
  • NEM non-relapse mortality
  • HCT Hematopoietic Cell Transplant
  • FIGs. 1A-1D depict the results of a Transcriptomic Analysis.
  • FIG. 1A Weighted Gene Correlation Network Analysis (WGCNA) heatmap. Heatmap demonstrates the correlation (using a blue- red false color scale) of WGCNA gene modules (colored turquoise, blue, green and red) in transcriptomic amples grouped by clinical metadata. Row/Column order is determined by hierarchical clustering or the module eigenmen with clinical metadata for each sample.
  • FIG. IB Gene Set Enrichment Analysis (GSEA) of the turquoise WGCNA module in a ranked list of differentially expressed genes between patients receiving Abatacept (left) and patients receiving Standard prophylaxis (right).
  • GSEA Gene Set Enrichment Analysis
  • FIG. 1C Reactome database terms exhibiting enrichment in the turquoise module genes using a Fisher’s exact test. Threshold at a significance value of 0.00 latter Bonferroni correlation for multiple hypotheses. Node size indicates number of genes in each term (range 10-390). Edges of graph indicate shared genes between terms (range 1-301).
  • FIG. ID Visualization of cytoscape pathway analysis demonstrating network connections of transcripts identified as being positively correlated with grade of acute GVHD.
  • FIG. 2 shows a table listing the Baseline Characteristics of the Participants.
  • the 7/8 P value represents the comparison of the 7/8 abatacept cohort and the CIBMTR control cohort.
  • FIG. 3 shows a table listing Rates of Acute and Chronic GVHD, SGFS, Relapse, RFS, NRM, and OS.
  • the 7/8 P value represents the comparison of the 7/8 abatacept cohort and the CIBMTR control cohort.
  • FIG. 4 shows a table listing of antibodies used for flow cytometry and the gating strategy of immune populations.
  • FIG. 5 shows a table listing the rates of neutrophil and platelet engraftment, CMV and EBV reactivation, CMV End-organ Disease, and PTLD.
  • FIG. 6 shows a table listing serious adverse events.
  • FIG. 7 shows a table listing baseline characteristics of the Participants.
  • FIG. 8 shows a table listing the rates of neutrophil and platelet engraftment, acute and chonic
  • GVHD severe GVHD-free survival, relapse, RFS, NRM and OS.
  • FIG. 9 shows a table listing the subanalysis of acute GVHD II-IV at day 180.
  • FIGs. 10A-10J depict key GVHD Outcomes in accordance with the Mount Sinai Acute GVHD International Consortium (MAGIC) algorithm prediction of 6-month NRM
  • FIGa. 1 lA-1 IB show the Key Transplant Outcomes.
  • FIG.l 1A 7/8 Cohort, Cumulative incidence of non-relapse mortality.
  • FIG. l IB 8/8 Cohort, Cumulative incidence of non-relapse mortality.
  • 7/8 Cohort Cumulative incidence of relapse-free survival.
  • 8/8 Cohort Cumulative incidence of relapse- free survival.
  • 7/8 Cohort Cumulative incidence of overall survival.
  • 8/8 Cohort Cumulative incidence of overall survival.
  • 7/8 Cohort Blue: CIBMTR control cohort. Solid Red: 7/8 abatacept cohort, matched to the CIBMTR controls. Dashed Red: 7/8 Intention-to-treat (ITT) cohort. P values represent comparison of 7/8 abatacept cohort and CIBMTR control cohort.
  • 8/8 Cohort Blue: Placebo. Red: Abatacept.
  • FIGs. 12A-12D depict the results of a Transcriptomic Analysis.
  • Fig. 12A Scehmata of the Transcriptomic analysis pipeline.
  • FIG. 12B Weighted Gene Coexpression Network Analysis (WGCNA) heatmap. Heatmap demonstrates the correlation (using a blue-red false color scale) of WGCNA gene modules (colored Turquoise, Blue, Green and Red) in transcriptomic samples grouped by clinical metadata. Row/Column order is determined by hierarchical clustering of the module eigengene with clinical metadata for each sample.
  • FIG. 12A Scehmata of the Transcriptomic analysis pipeline.
  • FIG. 12B Weighted Gene Coexpression Network Analysis (WGCNA) heatmap. Heatmap demonstrates the correlation (using a blue-red false color scale) of WGCNA gene modules (colored Turquoise, Blue, Green and Red) in transcriptomic samples grouped by clinical metadata. Row/Column order is determined by hierarchical clustering of the module eigengene with clinical metadata for each
  • GSEA Gene Set Enrichment Analysis of the Turquoise WGCNA module in a ranked list of differentially expressed genes between patients receiving abatacept (left) and patients receiving standard prophylaxis (right). The gene list was generated with a cut-off of FDR ⁇ 0.001 by gene-set permutation testing.
  • FIG. 12D Ingenuity Pathway Analysis of the 93-gene subset of the Turquoise module. Yellow: Proliferation genes; Black: Apoptosis genes; Blue: Checkpoint genes; Green: Metabolism genes; Red: T cell activation genes. Solid lines: Direct interactions; Dashed lines: Indirect interactions.
  • FIGs. 13A-13B show a consort Diagram.
  • FIG. 13A 7/8 stratum.
  • FIG. 13B 8/8 stratum.
  • Patients were randomized between 3/1/2013 and 12/9/14, following a protocol amendment, subjects in the 7/8 cohort were non-randomly assigned to receive abatacept.
  • FIGs. 14A-14L show the Post-transplant Immune Monitoring.
  • FIG.14A White blood cell count.
  • FIG.14B Absolute granulocyte count.
  • FIG. 14C Absolute lymphocyte count.
  • FIG. 14D Absolute B cell count.
  • FIG. 14E Absolute NK cell count.
  • FIG. 14F Absolute T cell count.
  • FIG. 14G Absolute CD4+ T cell count.H: Absolute CD8+ T cell count.
  • SEM standard error of the mean
  • FIG. 14K Percentage of patients achieving >95% donor CD3 engraftment at one year post-transplant.
  • FIG. 14L Percentage of patients achieving >95% donor CD33 engraftment at one year post-transplant.
  • FIGs. 15A-15F show the clinical outcomes of the 7/8 cohort compared to a CIBMTR cohort who received CNI/MTX+ATG.
  • FIG.15A Cumulative incidence of Grade 2-4 AGVHD.
  • FIG.15B Cumulative incidence of Grade 3-4 AGVHD.
  • FIG.15C Cumulative incidence of Severe Acute GVHD- Free Survival (SGFS).
  • FIG.15D Cumulative incidence of RFS.
  • FIG.15E Cumulative incidence of NRM.
  • FIG.15F Cumulative incidence of OS.
  • Blue CIBMTR control cohort, CNI/MTX+ATG. Solid Red: 7/8 abatacept cohort, matched to the CIBMTR ATG controls. Dashed Red: 7/8 Intention-to-treat (ITT) cohort.
  • P values represent comparison of 7/8 abatacept cohort and CIBMTR control cohort.
  • FIGs. 16A-16E show purified CD4+ T cells from patients of the ABA2 trial at Day 21 and Day 28. RNASeq was performed on these cells. An unsupervised multiparameter analysis was performed to determine what transcripts, and cellular pathways were correlated with key outcomes including:
  • FIG. 16A Module and Metamodule Analysis using the technique of‘Weighted Gene Correlation Network Analysis’ to identify which transcriptomic modules closely correlate with clinical outcomes.
  • FIG. 16B Image of the Turquoise Module, that correlates with standard GVHD prophylaxis and anti-correlates with exposure to abatacept.
  • FIG. 16C A Gene Set enrichment analysis which confirms that the Turquoise module correlates with patients prophylaxed with standard agents and Not with patients given abatacept.
  • FIG. 16D Pathway analysis of the Turquoise module identifying multiple cell proliferation pathways that correlate with this Module.
  • FIG. 16E GSEA analysis confirming that a CellCycle gene set correlates with standard versus abatacept samples.
  • FIGs. 17A-17B show Ki-67 expression as it correlates with GVHD for standard and abatacept-treated patients separately.
  • FIG. 17A Measurements of the xpression of a major proliferation marker, Ki-67. It was determined whether this marker correlated with GVHD in patients prophylaxed with either Standard (FIG.17A), or Abatacept (FIG. 17B). In the bar graph shown in Fig. 17A, the series are, from left to right: 0, 1, 2, 3 and 4.
  • FIGs. 17A-17B The box-whisker plot shows that the level of Ki- 67 expression increases with increasing severity of GVHD in patients prophylaxed with a standard GVHD regimen.
  • This box-whisker plot shows that the level of Ki-67 expression DOES NOT INCREASE with increasing severity of GVHD in patients prophylaxed with an abatacept-containing GVHD regimen.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”. This result is in agreement with the pathway analysis shown in FIG. 16, and suggests that patients prophylaxed with abatacept control proliferation post-transplant, whether or not they get GVHD.
  • FIGs. 18A-18C show that patients prophylaxed with Abatacept demonstrate less Ki-67, CD28 and CTLA4 expression compared to standard.
  • CTLA4 CTLA4 is expressed at a lower level in patients who received abatacept.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None, Abatacept, and Standard.
  • Abatacept controls GVHD by limiting proliferation (see, e.g. FIGs. 16A-E, 17A-B and 18A-C). but also decreases the expression of check-point molecules like CTLA4 (FIG. 18C).
  • FIGs. 19A-19B show CTLA4 expression data as it correlates with GVHD for standard and abatacept-treated patients separately.
  • FIG. 19A This graph shows that patients that are not given abataceptincrease expression of CTLA4 when they develop GVHD.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • FIG. 19B Patients given Abatacept as part of their prophylaxis do not upregulate CTLA4 when they get GVHD.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • FIGs. 20A-20C show the successful reconstitution of T cells in patients prophylaxed with Abatacept.
  • Patients prophylaxed with Abatacept successfully reconstitute total T cells (FIG. 20A), CD4+ T cells (FIG. 20B) and CD8+ T cells (FIG. 20C) post-transplant.
  • the series are, ABA (as depicted in the line with the squares) and Placebo (as depicted in the line with the triangles).
  • the series are, ABA (as depicted in the line with the squares) and Placebo (as depicted in the line with the triangles).
  • the series are, ABA (as depicted in the line with the squares) and Placebo (as depicted in the line with the triangles).
  • FIGs. 21A-21E show a Weighted Correlation Network Analysis (WGCNA) Analysis.
  • FIG. 21 B Identification of the Turquoise Module.
  • FIGs. 21C-21E shows the GSEA and Pathway Analysis of the Turquoise Module.
  • FIGs. 22A-22C show the successful T Cell Reconstitution in Abatacept treated Patients.
  • the series are, ABA (line with square boxes) and Placebo (ine with triangles).
  • the series are, ABA (line with square boxes) and Placebo (line with triangles).
  • the series are, ABA (line with square boxes) and Placebo (line with triangles).
  • FIGs. 23A-23I show graphs depicting the expression data of proliferation markers: (FIGs. 23A-C) Ki-67; (FIGs. 23D-23F) AURKA (Aurora Kinase A); and (FIGs. 23G-23I) EZH2 (Enhancer of zeste homolog 2).
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and “3 and 4”.
  • the bar graph shown in Fig. 23C the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • FIGs. 24A-24I show graphs depicting the expression data of following T cell activation markers: (FIGs. 24A- 24C) CD28; (FIGs. 24D-24F) CD38; and (FIGs. 24G-24I) CD59.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the bar graph shown in Fig. 24C the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • FIGs. 25A-250 show graphs depicting the expression data of the following Cytokines and Receptors activation markers: (FIGs. 25A-25C) IL7; (FIGs. 25D-25F) IL12RB2; (FIGs. 25G-25I)
  • IL18R1 (FIG. 25J-25L) IL21; and (FIGs. 25M-250) IL32.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • Fig. 25A the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, “1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, “1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and “3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • FIGS. 26A-26I show graphs depicting the expression data of the following Integrins and Adhesion Molecule marker: (FIGs. 26A-26C) CCR3; (FIGs. 26D-26F) CCR5; (FIGs. 26G-26I) CXXL13.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the bar graph shown in Fig. 26C the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • Fig. 26C the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • Fig. 26C the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • FIGs. 27A-27I show graphs depicting the expression data of the following Checkpoint Molecule markers: (FIGs. 27A-27C) CTLA4; (FIGs. 27D-27F) PD-1; FIGs. 27G-27I) TIM-3.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the bar graph shown in Fig. 27C the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • Fig. 27A-27C the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None, Abatacept, and Standard.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • the series are, from left to right: None,“1 and 2”, and“3 and 4”.
  • This invention relates to compositions and methods for the treatment of graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • HCT hematopoietic stem cell transplantation
  • GVHD There are two forms of GVHD: the acute and chronic GVHD.
  • Acute GVHD usually occurs within the first three months following a transplant. T-cells present in the donor's bone marrow at the time of transplant attack the patient's skin, liver, stomach, and/or intestines. The earliest signs of acute GVHD are usually a skin rash that appears on the hand, feet and face. Other than blistering skin, patients with severe GVHD also develop large amounts of watery or bloody diarrhea with cramping due to the donor's T-cells' attack on the stomach and intestines. Jaundice (yellowing of the skin and eyes) is the usual indication that GVHD disease involves the liver. The more organs involved and the worse the symptoms, the worse the GVHD disease.
  • the technology described herein is directed to the treatment of acute Graft versus host disease (GVHD) with CD80/86 inhibitors.
  • CD80/86 inhibition suppresses the immune activity of T cells that causes GvHD while potentially increasing the immune activity of T cells that prevents cancer relapse.
  • the latter is accomplished by decoupling T cell costimulation from T cell checkpoint expression, leading to downregulation of checkpoint molecules including CTLA4, Lag-3, TIM-3, and PD-1, thereby simultaneously controlling GVHD and preventing increased relapse of the underlying malignancy.
  • the data provided herein demonstrate a general control of proliferation, activation markers, cytokines and their receptors, integrin and adhesion molecules, as well as checkpoint molecules.
  • a method of treating or preventing a hematopoietic disease, Graft-versus-host disease (GvHD), T cell dysfunction, and/or a condition caused by or associated with T cell dysfunction comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising: (a) a CD80/86 inhibitor.
  • a pharmaceutical composition comprising: (a) a CD80/86 inhibitor.
  • administration of such reagents inhibits the expression or activity of one or more immune checkpoint polypeptides in T cells and promotes reconstituting a functional T cell population.
  • a method of treating hematopoietic disease comprising administering to a subject i) a bone marrow or HCT transplant and ii) a therapeutically effective amount of a pharmaceutical composition comprising: (a) a CD80/86 inhibitor.
  • the steps i) and ii) can be conducted sequentially or concurrently. If conducted sequentially, step i) can be performed first, followed by step ii). Or step ii) can be performed first, followed by step i). Or step ii) can be performed first, followed by step i), followed by one or more reptitions of step ii).
  • the term“inhibitor” refers to an agent which can decrease the expression and/or activity of the target molecule or activity or process, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more.
  • the efficacy of an inhibitor of, for example, CD80/86 e.g. its ability to decrease the level and/or activity of CD80/86 can be determined, e.g. by measuring the level of interaction of CD80/86 with one of its interaction partners e.g. CD28 and/or by measuring the level of CD80/86 protein (or its mRNA).
  • Methods for measuring the level of interaction of polypeptides are known to one of skill in the art, e.g. Immunoprecipitation with antibodies directed against CD80/86, followed by Western Blotting against the interacting polypeptide e.g. CD28 can be used to determine the level of interaction between two polypeptides.
  • Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RT-PCR with primers can be used to determine the level of RNA, and Western blotting with an antibody can be used to determine the level of a polypeptide.
  • an inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule.
  • Exemplary inhibitors, e.g., of CD80/86 can include a small molecule, nucleic acid (e.g., inhibitory nucleic acid), polypeptide, antibody reagent, or genome editing system.
  • nucleic acid e.g., inhibitory nucleic acid
  • polypeptide e.g., antibody reagent
  • genome editing system e.g., the terms“compound” or“agent” are used interchangeably and refer to molecules and/or compositions.
  • the compounds/agents include, but are not limited to, drugs, chemical compounds, and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; peptides; aptamers; and antibodies and intrabodies, or fragments thereof.
  • “drug” as used herein refers to an agent approved for medical use, e.g., by the FDA.
  • an inhibitor of can be an antibody or antigen binding fragment thereof, an aptamer, or an inhibitory fragment of a natural receptor or ligand of the target. In some embodiments of any of the aspects, the inhibitor binds specifically to the target. In some embodiments of any of the aspects, the inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. In some embodiments of any of the aspects, the inhibitor binds specifically to the target. In some embodiments of any of the aspects, the inhibitor does not bind to CTLA-4 and/or CD28 and/or the subject is not administered a therapy that binds to CTLA-4 and/or CD28.
  • CD80 or“cluster of differentiation 80” refers to a Ig superfamily membrane protein found on certain immune cells and which binds to CD28 or CTLA-4 on T cells.
  • the sequences of CD80 expression products are known for a number of species, e.g., human CD80 (NCBI Gene ID No: 941) mRNA (NCBI Ref Seq: NM_005191.4) and polypeptide (NCBI Ref Seq:
  • “CD86” or“cluster of differentiation 86” refers to a closely realted Ig superfamily membrane protein found on certain immune cells and which binds to CD28 or CTLA-4 on T cells.
  • the sequences of CD86 expression products are known for a number of species, e.g., human CD86 (NCBI Gene ID No: 942) mRNA (NCBI Ref Seqs: NM)001206924.1, NM OO 1206925.1, NM_175862.5, NM_176892.
  • a CD80/86 inhibitor inhibits both CD80 and CD86. In some embodiments of any of the aspects, a CD80/86 inhibitor inhibits only CD80. In some embodiments of any of the aspects, a CD80/86 inhibitor inhibits only CD86. In some embodiments of any of the aspects, a CD80/86 inhibitor binds specifically to both CD80 and CD86. In some embodiments of any of the aspects,
  • a CD80/86 inhibitor binds specifically to only CD80. In some embodiments of any of the aspects, a CD80/86 inhibitor binds specifically to only CD86.
  • An exemplary but non-limiting CD80/86 inhibitor is abatacept or a reagent comprising the six CDRs of abatacept.
  • the CD80/86 inhibitor is belatacept.
  • the inhibitor is targeted to T cells, e.g., it comprises a T cell targeting moiety.
  • the inhibitor is administered or provided in a composition which further comprises a T cell targeting molecule or moiety.
  • a moiety can be, e.g., an antibody or antigen-binding fragment thereof, an aptamer, or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
  • Targeting can be achieved, e.g. by conjugating the inhibitor to a targeting group or including the inhibitor in a composition comprising a targeting group (e.g. a nanoparticule).
  • Targeting groups can include, e.g., a T cell targeting agent, e.g., an antibody, that binds to T cells, or a cell permeation agent.
  • T cell targeting groups can include antibodies to T cell surface markers (e.g., CD3, CD4, CD8, CD25, CD127, and/or CD196).
  • the CD80/86 inhibitor inhibits the expression or activity in T cells of one or more genes or polypeptides selected from Table 1.
  • the subject can be further administered an immune checkpoint inhibitor (e.g., an inhibitor of the expression or activity of one or more immune checkpoint polypeptides), a calcineurin inhibitor, or a dihydrofolate reductase (DHFR) inhibitor.
  • an immune checkpoint inhibitor e.g., an inhibitor of the expression or activity of one or more immune checkpoint polypeptides
  • a calcineurin inhibitor e.g., calcineurin inhibitor
  • DHFR dihydrofolate reductase
  • the calcineurin inhibitor is an antibody or antigen-binding fragment thereof, an aptamer, or a calcineurin inhibitor fragment of a natural receptor or ligand of calcineurin.
  • Calcineeurin refers to a serine/threonine protein phosphtase which is calcium and calmodulin dependent. Calcineurin positively resgulates T cells. Calcineurin is a homodimer of Calcinuerin A (NCBI Gene ID: 5530, 5532, or 5533) and Calcineurin B (NCBI Gene ID: 5534 or 5535). Exemplary but non-limiting calcineurin inhibitors include cyclosporine and tacrolimus.
  • “dihydrofolate reductase” or“DHFR” refers to an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid as part of nucleic acid synthesis.
  • the sequences of DHFR expression products are known for a number of species, e.g., human DHFR (NCBI Gene ID No: 1719) mRNA (NCBI Ref Seqs: NM 00791.4, NM_001290354.2, and NM_001290357.2) and polypeptide (NCBI Ref Seqs: NP 001277286.1, NP 001277283.1, and NP_000782.1).
  • Exemplary but non-limiting DHFR inhibitors include methotrexate, trimethoprim, pyrimethamine, diaminopteridines,
  • diaminotriazines diaminopyrroloquinazolines, stilbenes, chalcones, deoxybenzoins, diaminoquinazolines, diaminopyrimidines, and diaminotriazines.
  • the immune system has multiple inhibitory pathways that are critical for maintaining self tolerance and modulating immune responses.
  • T-cells the amplitude and quality of response isinitiated through antigen recognition by the T-cell receptor and is regulated by immune checkpoint proteins that balance co-stimulatory and inhibitory signals.
  • a subject or patient is treated with at least one inhibitor of an immune checkpoint protein.
  • immune checkpoint protein refers to a protein which, when active, exhibits an inhibitory effect on immune activity, e.g., T cell activity.
  • Exemplary immune checkpoint proteins can include PD-1 (e.g., NCBI Gene ID: 5133); PD-L1 (e.g, NCBI Gene ID: 29126); PD-L2 (e.g., NCBI Gene ID: 80380); TIM-3 (e.g, NCBI Gene ID: 84868); CTLA4 (e.g., NCBI Gene ID: 1493); TIGIT (e.g, NCBI Gene ID: 201633); KIR (e.g., NCBI Gene ID: 3811); LAG3 (e.g., NCBI Gene ID: 3902); DDl-a (e.g., NCBI Gene ID: 64115); A2AR (e.g, NCBI Gene ID: 135); B7-H3 (e.g, NCBI Gene ID: 80381); B7-H4 (e.g, NCBI Gene ID: 79679); BTLA (e.g, NCBI Gene ID: 151888); IDO (e.g, NCBI Gene ID: 3620); TDO (e
  • B7 family ligands include, but are not limited to, B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7.
  • Non-limiting examples of immune checkpoint inhibitors can include :MGA271 (B7-H3: MacroGenics); ipilimumab (CTLA-4; Bristol Meyers Squibb); pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb) ; atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3: Immuntep); BMS-986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137; Bristol-Meyers Squibb); PF-05082566 (CD137; Pfizer); IPH2101 (KIR; Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX-1127 (CD27; CellDex); MEDI-6769 (0x40; Medlmm
  • the methods described herein can result in inhibition of one or more immune checkpoint polypeptides.
  • the immune checkpoint polypeptides are selected from CTLA4, Lag-3, HAVCR2 (TIM-3), PDCD1, Ki-67, CD-28.
  • the immune checkpoint polypeptides are selected from CTLA4, Lag-3, HAVCR2 (TIM-3), PDCD1, Ki-67, CD-28.
  • the methods described herein can result in reconstitution of a T cell population.
  • the T cell population comprises CD3+, CD4+ and/or CD8+ T cells.
  • the T cell population comprises Regulatory T cells (TregS ) .
  • Regulatory T cell or “Treg” refers to those T cells (lymphocytes) that have immunoregulatory properties and the ability to suppress the proliferation and/or effector function of other T cell populations.
  • a number of cell surface molecules are used to characterize and define Treg cells as described below herein.
  • Regulatory T cells or Treg cells play an important role for the maintenance of immunological tolerance by suppressing the action of autoreactive effector cells and have been shown to be critically involved in preventing the development of autoimmune reactions.
  • a Treg cell can be a T cell expressing one or markers selected from the group consisting of CTLA4; PDL1; LAP; GARP; CD25; and CD27.
  • a Treg cell can be a CD8- (e.g., NCBI Gene ID: 925) CD4+ (e.g., NCBI Gene ID: 920) CD3+ cell.
  • X+ wherein "X” is a cell surface marker, indicates the marker is present in the indicated cell, while “X-” indicates the marker is not present.
  • X is a cell surface marker
  • X- indicates the marker is not present.
  • designators are often used when sorting or identifying cells by FACS, in which gates can be established to divide cells based on the level of expression of the marker.
  • the methods described herein relate to treating a subject having or diagnosed as having a hematopoietic disease with one or more of the compositions or agents described herein.
  • Subjects having a hematopoietic disease, e.g., cancer can be identified by a physician using current methods of diagnosing cancer.
  • hematopoietic disease refers to any blood disorder including but not limited to hematopoietic malignancy, hematopoietic cancer, hemoglobinopathy, and immunodeficiency.
  • the hematopoietic disease can be hematopoietic cancer or leukemia.
  • T cell lymphopenia refers to a temporary or continuous decrease in lymphocytes (including but not limited to T cells, B cells and natural killer cells) in a subject.
  • the T cells are Tregs.
  • Exemplary conditions caused by or associated with T cell lymphopenia include but are not limited to Graft-versus-host disease (GvHD), non-relapse mortality (NRM), infection, and leukemia.
  • GvHD Graft-versus-host disease
  • NRM non-relapse mortality
  • infection infection
  • leukemia leukemia
  • the subject is a subject who will or has received a bone marrow transplant.
  • the subject is a subject who will or has received a hematopoietic cell transplant (HCT), e.g, from an unrelated donor (URD).
  • HCT hematopoietic cell transplant
  • T cell dysfuction refers to a temporary or continuous decrease in T cell function in a subject.
  • a temporary or continues descrease in T cell function includes but is not limited reduced proliferative capacity, decreased effector function, and overexpression of multiple inhibitory receptors on T cells.
  • the T cells are Tregs.
  • Exemplary conditions caused by or associated with T cell dysfunction include but are not limited to Graft-versus-host disease (GvHD), non-relapse mortality (NRM), infection, and leukemia.
  • GvHD Graft-versus-host disease
  • NRM non-relapse mortality
  • infection and leukemia.
  • the subject is a subject who will or has received a bone marrow transplant.
  • the subject is a subject who will or has received a hematopoietic cell transplant (HCT), e.g, from an unrelated donor (URD).
  • HCT hematopoietic cell transplant
  • GvHD can be acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), or chronic Graft-versus-host disease (cGvHD).
  • AGvHD acute Graft-versus-host disease
  • aAGVHD severe acute Graft-versus-host disease
  • cGvHD chronic Graft-versus-host disease
  • the administration described herein can be prophylactic.
  • described herein is a prophylactic method of treatment.
  • prophylactic refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom.
  • Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to organ rejection or immune activity of a graft against the host.
  • the subject can be a subject who will or has received a bone marrow transplant but who has not exhibited a sign or symptom of GvHD at the time administration is commenced.
  • prophylactic treatment is not prevention of all symptoms or signs of a disease.
  • compositions and methods described herein can be administered to a subject having or diagnosed as having, e.g., a hematopoetic disease.
  • the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of e.g., a hematopoetic disease.
  • "alleviating a symptom” is ameliorating any condition or symptom associated with a disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • compositions described herein can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
  • the term“effective amount” as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the term "therapeutically effective amount” therefore refers to an amount of the composition that is sufficient to provide a particular effect when administered to atypical subject.
  • An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact“effective amount”. However, for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e..
  • composition comprising a CD80/86 inhibitor; and at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor.
  • DHFR dihydrofolate reductase
  • compositions comprising all possible pairwise, 3-way, and 4- way combinations are contemplated herein.
  • the composition comprises a CD80/86 inhibitor and an immune checkpoint inhibitor.
  • the composition comprises a CD80/86 inhibitor and a Calcineurin inhibitor.
  • the composition comprises a CD80/86 inhibitor and a dihydrofolate reductase (DHFR) inhibitor.
  • DHFR dihydrofolate reductase
  • a combination comprising a CD80/86 inhibitor; and at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor, e.g., for use in a method described herein.
  • an immune checkpoint inhibitor e.g., a Calcineurin inhibitor
  • DHFR dihydrofolate reductase
  • the combination comprises a CD80/86 inhibitor and an immune checkpoint inhibitor.
  • the combination comprises a CD80/86 inhibitor and a Calcineurin inhibitor.
  • the combination comprises a CD80/86 inhibitor and a dihydrofolate reductase (DHFR) inhibitor.
  • the two or more reagents of the combination can be present in the same solution, formulation, mixture, vial, or container.
  • the two or more reagents of the combination can be present in separate solutions, formulations, mixtures, vials, or containers.
  • they can be provided in separate solutions in a single kit with instructions to administer them to the same patient, e.g., concurrently or sequentially.
  • the technology described herein relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a CD80/86 inhibitor and one or more additional agents as described herein, and optionally a pharmaceutically acceptable carrier.
  • the active ingredients of the pharmaceutical composition comprise a CD80/86 inhibitor as described herein.
  • the active ingredients of the pharmaceutical composition consist essentially of a CD80/86 inhibitor as described herein.
  • the active ingredients of the pharmaceutical composition consist of a CD80/86 inhibitor as described herein.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent as described herein.
  • the pharmaceutical composition comprising a CD80/86 inhibitor as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS ® -type dosage forms and dose-dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms of a CD80/86 inhibitor as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a CD 80/86 inhibitor as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure
  • compositions comprising a CD80/86 inhibitor can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water- in-oil emulsion.
  • Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • the a CD80/86 inhibitor can be administered in a sustained release formulation.
  • Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled- release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each of which is incorporated herein by reference.
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza
  • composition or combiantion described herein is administered as a monotherapy, e.g., another treatment for the disease or condition is not administered to the subject.
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
  • a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemel
  • bisphosphonates such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophore s), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C
  • elformithine elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
  • lonidainine lonidainine
  • maytansinoids such as maytansine and ansamitocins
  • mitoguazone mitoxantrone
  • mopidanmol mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
  • TAXOL® paclitaxel Bristol-Myers Squibb Oncology, Princeton, N.J.
  • ABRAXANE® Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel
  • TAXOTERE® doxetaxel Rhone -Poulenc Rorer, Antony, France
  • chloranbucil GEMZAR® gemcitabine
  • 6- thioguanine mercaptopurine
  • methotrexate platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;
  • the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
  • an effective dose of a composition comprising a CD80/86 inhibitor as described herein can be administered to a patient once.
  • an effective dose of a composition comprising a CD80/86 inhibitor can be administered to a patient repeatedly.
  • subjects can be administered a therapeutic amount of a composition comprising a CD80/86 inhibitor, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
  • Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active ingredient.
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
  • dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
  • a composition can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the dosage ranges for the administration of a CD80/86 inhibitor, according to the methods described herein depend upon, for example, the form of the active ingredient, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for immune system activity, inflammation, or the extent to which, for example, Tregs are desired to be induced.
  • the dosage should not be so large as to cause adverse side effects, such as pathological immune system suppression.
  • the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • Efficacy of a CD80/86 inhibitor in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered“effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. Treg activity or checkpoint protein activity. Efficacy can also be measured by a failure of an individual to worsen as assessed by
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. Treg activity or levels). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of acute and chronic GvHD. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a GVHD marker is observed, e.g.
  • a dose of a CD80/86 inhibitor can be assessed by measuringthe expression or activity of one or more inflammatory and immunomodulatory polypeptides (including but not limited to TNFa, IL-6, IL-Ib, IL-8, sIL-2R, and IL- lRa, BAFF, anti-dsDNA, sIL-2Ra, and sCD13).
  • one or more inflammatory and immunomodulatory polypeptides including but not limited to TNFa, IL-6, IL-Ib, IL-8, sIL-2R, and IL- lRa, BAFF, anti-dsDNA, sIL-2Ra, and sCD13.
  • a non-limiting example of a protocol for such an assay is as follows assessing mRNA or protein levels of TNFa via, e.g., PCR-based assays, an enzyme-linked immunosorbent assay and/or western blotting, respectively.
  • the efficacy of a given dosage combination can also be assessed in an animal model, e.g. mouse models of acute and chronic GvHD.
  • an animal model e.g. mouse models of acute and chronic GvHD.
  • sclerodermatous (pro-fibrotic) cGvHD models that are characterized by fibrotic changes in the dermis, which can involve the lung, liver and salivary glands. Fibrosis in the mouse models begins within 30 days of transplantation.
  • the pathology is dependent on CD4+ T cells that release Th2 cytokines, which can stimulate other cells to release fibrosing cytokines (including but not limited to IL-13 and T ⁇ Rb) resulting in the sclerodermatous changes.
  • cytokines including but not limited to IL-13 and T ⁇ Rb
  • the effects of a dose of a CD80/86 inhibitor can be assessed by measuring the fibrotic changes using immunohistochemical methods and by measuring the expression or activity of fribrosing cytokines including but not limited to IL-13 and TGFD e.g. using PCR-based assays, an enzyme-linked immunosorbent assay and/or western blotting, respectively.
  • the effects of a dose of a CD80/86 inhibitor can be assessed by measuring the expression or activity of one or more marker including CD4+cells and/or CD8+T cells.
  • a marker including CD4+cells and/or CD8+T cells For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
  • “decrease”,“reduced”,“reduction”, or“inhibit” are all used herein to mean a decrease by a statistically significant amount.
  • “reduce,”“reduction” or“decrease” or“inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g.
  • “reduction” or“inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms“increased”,“increase”,“enhance”, or“activate” are all used herein to mean an increase by a statically significant amount.
  • the terms“increased”,“increase”, “enhance”, or“activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a“increase” is a statistically significant increase
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms,“individual,”“patient” and“subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • A“subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems.
  • Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.
  • Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
  • Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
  • the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e.. division beyond normal limits), invasion (i.e.. intrusion on and destruction of adjacent tissues), and metastasis (i.e. , spread to other locations in the body via lymph or blood).
  • metastasize refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a“metastatic tumor” or a“metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • the term“benign” or“non- malignanf’ refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • A“cancer cell” or“tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g. , leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • neoplasm refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues.
  • a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,
  • lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma;
  • rhabdomyosarcoma rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s
  • NHL low grade/follicular non-Hodgkin’s lymphoma
  • SL small lymphocytic
  • NHL intermediate grade/follicular NHL
  • intermediate grade diffuse NHL high grade immunoblast
  • Macroglobulinemia chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome
  • A“cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material.
  • transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.
  • Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
  • expression refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing.
  • Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
  • the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder.
  • the term“treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a condition. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • the term“pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term“specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • the inhibitor is an inhibitory nucleic acid.
  • inhibitors of the expression of a given gene can be an inhibitory nucleic acid.
  • inhibitory nucleic acid refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like.
  • the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA).
  • RNA interference Double -stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi).
  • the inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript.
  • the use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
  • iRNA refers to an agent that contains RNA (or modified nucleic acids as described below herein) and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • an iRNA as described herein effects inhibition of the expression and/or activity of a target, e.g. CD80/86.
  • contacting a cell with the inhibitor e.g.
  • an iRNA results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA.
  • administering an inhibitor e.g.
  • an iRNA to a subject results in a decrease in the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the subject without the presence of the iRNA.
  • the iRNA can be a dsRNA.
  • a dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used.
  • One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence.
  • the target sequence can be derived from the sequence of an mRNA formed during the expression of the target, e.g., it can span one or more intron boundaries.
  • the other strand includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
  • the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive.
  • the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive.
  • the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive.
  • the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule.
  • a“part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).
  • dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
  • Exemplary embodiments of types of inhibitory nucleic acids can include, e.g,. siRNA, shRNA,miRNA, and/or amiRNA, which are well known in the art.
  • siRNA, shRNA,miRNA, and/or amiRNA are well known in the art.
  • One skilled in the art would be able to design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of, e.g., CD80 or CD86, e.g., using publically available design tools.
  • siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).
  • the RNA of an iRNA is chemically modified to enhance stability or other beneficial characteristics.
  • the nucleic acids described herein may be synthesized and/or modified by methods well established in the art, such as those described in“Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.
  • base modifications e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners
  • RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages.
  • RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • the modified RNA will have a phosphorus atom in its intemucleoside backbone.
  • Modified RNA backbones can include, for example, phosphorothioates, chiral
  • phosphorothioates phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
  • thionophosphoramidates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • methyleneimino and methylenehydrazino backbones morpholino linkages
  • sulfonate and sulfonamide backbones are amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular— CH2— NH— CH2— , —CH2—N(CH3)—0—CH2— [known as a methylene (methylimino) or MMI backbone], --CH2--0-- N(CH3)— CH2— ,— CH2— N(CH3)— N(CH3)— CH2— and -N(CH3)-CH2-CH2-[wherein the native phosphodiester backbone is represented as— O— P— O— CH2— ] .
  • RNA mimetics suitable or contemplated for use in iRNAs both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This stmcture effectively "locks" the ribose in the 3'-endo structural conformation.
  • the addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
  • Modified RNAs can also contain one or more substituted sugar moieties.
  • the iRNAs, e.g., dsRNAs, described herein can include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, orN-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl.
  • Exemplary suitable modifications include 0[(CH2)nO] mCH3, 0(CH2).n0CH3, 0(CH2)nNH2, 0(CH2) nCH3,
  • dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br,
  • CN CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties.
  • the modification includes a 2' methoxyethoxy (2'-0— CH2CH20CH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below
  • 2'- dimethylaminoethoxyethoxy also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE
  • 2'-0— CH2— O— CH2— N(CH2)2 also described in examples herein below.
  • modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • An inhibitory nucleic acid can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6- azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5- trifluoromethyl
  • nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the invention.
  • These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
  • Another modification of an inhibitory nucleic acid featured in the invention involves chemically linking to the inhibitory nucleic acid to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et ah, Biorg. Med. Chem.
  • athioether e.g., beryl-S-tritylthiol (Manoharan et ah, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et ah, Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol
  • Acids Res., 1990, 18:3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino- carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
  • ligand refers to a molecule which can bind, e.g., specifically bind, to a second molecule or receptor.
  • a ligand can be, e.g., an polypeptide, an antibody, antibody fragment, antibody portion, and/or affibody.
  • polypeptide is intended to encompass a singular "polypeptide” as well as plural “polypeptides,” and includes any chain or chains of two or more amino acids.
  • amino acid chain and “contiguous amino acid sequence” are all encompassed within the definition of a “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with, any of these terms.
  • the term further includes polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • post-translational modification(s) including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.
  • amino acids Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; lie), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys).
  • Amino acid residues provided herein are preferred to be in the "L” isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue provided the desired
  • homologous refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue ( e.g similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is "unrelated" or “non- homologous" shares less than 40% identity. Determination of homologs of the genes or peptides described herein may be easily ascertained by the skilled artisan.
  • sequences provided here can be modified, comprise conservative amino acid
  • the first polypeptide has an amino acid sequence with at least 99% homology to the second polypeptide.
  • the third polypeptide has an amino acid sequence with at least 99% homology to the fourth polypeptide.
  • the first polypeptide has an amino acid sequence that is non-homologous to the second polypeptide.
  • the third polypeptide has an amino acid sequence that is non-homologous to the fourth polypeptide.
  • the first or second polypeptide has an amino acid sequence that is non-homologous to the third and/or fourth polypeptides.
  • conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties.
  • Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • a “conservative substitution” of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not substantially alter activity.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H.
  • Conservative substitutions that permit the formation of the tetrameric coiled coil structure described herein can be used.
  • directed evolution can be used to subject the polypeptides described herein to random mutagenesis and the resulting polypeptides are screened for desired qualities (e.g, using circular dichroism or binding assays). These methods are known in the art. See Wang et al. Cell, Volume 160, Issue 4, 2015, Pages 785-797; or Daugherty et al. Protein Engineering, Design and Selection, Volume 11, Issue 9, 1998, Pages 825-832.
  • nucleic acid aptamers refers to single-stranded nucleic acids that are capable of binding to cells and target molecules.
  • Nucleic acid aptamers include RNA, DNA, and/or synthetic nucleic acid analogs (e.g., PNA) capable of specifically binding target molecules. Aptamers are an attractive alternative to antibodies for cell selection because of their high level of specificity and affinity for cell surface markers.
  • nucleic acid includes one or more types of:
  • polydeoxyribonucleotides containing 2-deoxy-D-ribose
  • polyribonucleotides containing D-ribose
  • nucleic acid also includes polymers of ribomicleosides or deoxyribomicleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like.
  • Nucleic acids include single- and double -stranded DNA, as well as single- and double -stranded RNA.
  • nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.
  • antibody or“antibody reagent” broadly refers to any immunoglobulin (Ig) molecule or compositions of Igs and/or immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen) comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.
  • immunoglobulin immunoglobulin
  • immunologically active portions of immunoglobulin molecules i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen
  • immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.
  • Such mutant, variant, or derivative antibody formats are known in the art.
  • an immunoglobulin molecule e.g ., Fanzavecchia et al.,
  • Antibodies also refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen or target binding sites or“antigen-binding fragments.”
  • the antibody or immunoglobulin molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2,
  • the light chain can be a kappa chain or a lambda chain.
  • each heavy chain is comprised of a heavy chain variable domain (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains: CHI, CH2, and CH3.
  • Each light chain is comprised of a light chain variable domain (abbreviated herein LCVR as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well-known to those skilled in the art. The chains are usually linked to one another via disulfide bonds.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody.
  • the Fc region may be a native sequence Fc region or a variant Fc region.
  • the Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain, and a CH3 domain, and optionally comprises a CH4 domain.
  • the Fc portion of an antibody mediates several important effector functions, for example, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives.
  • Neonatal Fc receptors are the critical components determining the circulating half- life of antibodies.
  • at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered.
  • antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD80 or CD86).
  • Antigen-binding functions of an antibody can be performed by fragments of a full-length antibody.
  • Such antibody fragment embodiments may also be incorporated in bispecific, dual specific, or multi-specific formats such as a dual variable domain (DVD-Ig) format; specifically binding to two or more different antigens.
  • DVD-Ig dual variable domain
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature, 341: 544-546; PCT Publication No.
  • WO 90/05144 which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).
  • scFv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123); Kontermann and Dubel eds., Antibody Engineering, Springer- Verlag, N.Y. (2001), p. 790 (ISBN 3-540-41354-5).
  • a“bispecific antibody” refers to an antibody that can simultaneously bind to two different types of antigen.
  • single chain antibodies also include "linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. (1995) Protein Eng. 8(10): 1057-1062; and U.S. Pat. No. 5,641,870).
  • An immunoglobulin constant (C) domain refers to a heavy (CH) or light (CL) chain constant domain.
  • Murine and human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.
  • the antibody reagent is a humanized antibody.
  • humanized antibody refers to antibodies that comprise heavy and light chain variable domain sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. Accordingly, “humanized” antibodies are a form of a chimeric antibody, that are engineered or designed to comprise minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a“composite human antibody” or“deimmunized antibody” are specific types of engineered or humanized antibodies designed to reduce or eliminate T cell epitopes from the variable domains.
  • humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • humanized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody.
  • FR framework
  • CDR complementary determining region
  • the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab'). sub.2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanized antibody only contains a humanized light chain.
  • a humanized antibody only contains a humanized heavy chain.
  • a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.
  • a humanized antibody may be selected from any class of
  • immunoglobulins including IgM, IgG, IgD, IgA and IgE, and any isotype including without limitation IgGl, IgG2, IgG3, and IgG4.
  • the humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In an exemplary embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences.
  • the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence.
  • the term "consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.
  • a "human antibody,”“non-engineered human antibody,” or“fully human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art.
  • the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al. Proc. Natl. Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
  • Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous mouse immunoglobulin genes have been partially or completely inactivated.
  • the human antibody can be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (such B lymphocytes can be recovered from an individual or can have been immunized in vitro). See, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol., 147 (l):86-95 (1991); and U.S. Pat. No. 5,750,373.
  • an "antigen” is a molecule that is bound by a binding site on a polypeptide agent, such as a binding protein, an antibody or antibody fragment, or antigen-binding fragment thereof.
  • a polypeptide agent such as a binding protein, an antibody or antibody fragment, or antigen-binding fragment thereof.
  • antigens are bound by antibody ligands and are capable of raising an antibody response in vivo.
  • An antigen can be a polypeptide, protein, nucleic acid or other molecule.
  • the antibody binding site as defined by the variable loops (LI, L2, L3 and HI, H2, H3) is capable of binding to the antigen.
  • the term "antigenic determinant" refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.
  • epitope includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by a binding protein.
  • An epitope may be determined by obtaining an X-ray crystal structure of an antibody: antigen complex and determining which residues on the antigen are within a specified distance of residues on the antibody of interest, wherein the specified distance is, 5 ⁇ or less, e.g., 5 A, 4A, 3 A, 2A, lA or any distance in between.
  • an“epitope” can be formed on a polypeptide both from contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein.
  • Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • An "epitope" includes the unit of structure conventionally bound by an immunoglobulin VH VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • an epitope comprises of 8 or more contiguous or non-contiguous amino acid residues in the target sequence in which at least 50%, 70% or 85% of the residues are within the specified distance of the antibody or binding protein in the X-ray crystal structure.
  • antibody fragment refers to a protein fragment that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.
  • antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain;
  • Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • the method comprising administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor abatacept, thereby inhibiting the expression or activity of one or more immune checkpoint polypeptides in T cells and reconstituting the T cell population.
  • T cell lymphopenia is selected from Graft-versus-host disease (GvHD) or leukemia.
  • a method for treating or preventing GvHD comprising administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor.
  • a method of treating a hematopoietic disease in a subject in need thereof comprising: a. administering a bone marrow transplant to the subject; and
  • the hematopoietic disease is leukemia.
  • the CD80/86 inhibitor is an antibody or antigen-binding fragment thereof, an aptamer, or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86.
  • the method of any of paragraphs 1-9, wherein the CD80/86 inhibitor binds specifically to CD80 and/or 86.
  • the method of any of paragraphs 1-10, wherein the CD80/86 inhibitor is abatacept.
  • the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
  • the receptor is CTLA-4 and/or CD28
  • the immune checkpoint polypeptides are selected from CTLA4, Ki-67, CD-28.
  • the immune checkpoint polypeptides are selected from CTLA4, Ki-67, CD-28, PD-1, TIM-3, and LAG-3.
  • the T cell population comprises CD3+, CD4+ and/or CD8+ T cells.
  • the T cell population comprises CD3+, CD4+ and/or CD8+ T cells.
  • TregS Regulatory T cells
  • the inhibitor is targeted to T-cells.
  • the inhibitor comprises a T cell targeting moiety.
  • the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide.
  • CD3, CD4, CD8, CD25, CD127, and/or CD196 selected from the group consisting of CD3, CD4, CD8, CD25, CD127, and/or CD196.
  • the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof, an aptamer, or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
  • a method for treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction comprising:
  • GvHD Graft-versus-host disease
  • leukemia leukemia
  • lymphoma relapse A method for treating or preventing GvHD, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CD 80/86 inhibitor.
  • the method of paragraph 3 wherein the administration is prophylactic.
  • the subject is a subject who is in need of or has received a bone marrow transplant.
  • a method of treating a hematopoietic disease in a subject in need thereof the method comprising: a. administering a bone marrow transplant to the subject; and
  • the hematopoietic disease is a hematopoietic cancer.
  • the hematopoietic disease is leukemia.
  • the CD80/86 inhibitor is an antibody or antigen binding fragment thereof; an aptamer; or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86.
  • the method of any of paragraphs 1-9, wherein the CD80/86 inhibitor binds specifically to CD80 and/or 86.
  • any of paragraphs 1-11 wherein the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
  • the method of paragraph 12, wherein the receptor is CTLA-4 and/or CD28.
  • the immune checkpoint polypeptides are selected from the group selected from Lag-3, HAVCR2 (TIM-3), and/or PDCD1.
  • the T cell population comprises CD3+, CD4+, and/or CD8+ T cells.
  • the inhibitor is targeted to T-cells.
  • the method of paragraph 18, wherein the inhibitor comprises a T cell targeting moiety.
  • the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide.
  • the T cell-specific cell surface polypeptide is selected from the group consisting of CD3, CD4, and/or CD8.
  • the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof; an aptamer; or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
  • the method of any of paragraphs 1-23, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia, and lymphoma relapse.
  • GvHD Graft-versus-host disease
  • leukemia leukemia
  • lymphoma relapse The method of any of the preceding paragraphs, further comprising administering a
  • DHFR dihydrofolate reductase
  • the dihydrofolate reductase (DHFR) inhibitor is an antibody or antigen-binding fragment thereof; an aptamer; or a dihydrofolate reductase (DHFR) inhibitor fragment of a natural receptor or ligand of dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
  • the CD80/86 inhibitor inhibits the expression or activity of one or more T cell polypeptides selected from the polypeptides listed in Table 1.
  • the Graft-versus-host disease is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus- host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
  • T cell dysfunction is selected from the group consisting of non-relapse mortality (NRM) and infection.
  • NRM non-relapse mortality
  • HCT Hematopoietic Cell Transplant
  • UTD Unrelated Donor
  • b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a
  • DHFR dihydrofolate reductase
  • the composition of paragraph 39 wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus.
  • the method of paragraph 43 wherein the condition caused by or associated with T cell dysfunction is selected from the group of Graft-versus-host disease (GvHD) and leukemia.
  • GvHD Graft-versus-host disease
  • the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
  • NEM non-relapse mortality
  • HCT Hematopoietic Cell Transplant
  • HCT Hematopoietic Cell Transplant
  • UTD Unrelated Donor
  • a method of treating a hematopoietic disease in a subject in need thereof comprising: a. administering a hematopoietic stem transplant to the subject;
  • c. further administering a therapeutically effective amount of a Calcineurin inhibitor and/or a therapeutically effective amount of a dihydrofolate reductase inhibitor (DHFR).
  • DHFR dihydrofolate reductase inhibitor
  • a method for treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction comprising:
  • the hematopoietic disease is a hematopoietic cancer.
  • the hematopoietic disease is leukemia.
  • the CD80/86 inhibitor is an antibody or antigen binding fragment thereof; an aptamer; or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86.
  • the method of any of paragraphs 1-9, wherein the CD80/86 inhibitor binds specifically to CD80 and/or 86.
  • any of paragraphs 1-11 wherein the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
  • the method of paragraph 12, wherein the receptor is CTLA-4 and/or CD28.
  • the immune checkpoint polypeptides are selected from the group selected from Lag-3, HAVCR2 (TIM-3), and/or PDCD1.
  • the T cell population comprises CD3+, CD4+, and/or CD8+ T cells.
  • the inhibitor is targeted to T-cells.
  • the method of paragraph 18, wherein the inhibitor comprises a T cell targeting moiety.
  • the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide.
  • the T cell-specific cell surface polypeptide is selected from the group consisting of CD3, CD4, and/or CD8.
  • the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof; an aptamer; or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
  • the method of any of paragraphs 1-23, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia, and lymphoma relapse.
  • GvHD Graft-versus-host disease
  • leukemia leukemia
  • lymphoma relapse The method of any of the preceding paragraphs, further comprising administering a
  • DHFR dihydrofolate reductase
  • the dihydrofolate reductase (DHFR) inhibitor is an antibody or antigen-binding fragment thereof; an aptamer; or a dihydrofolate reductase (DHFR) inhibitor fragment of a natural receptor or ligand of dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
  • the CD80/86 inhibitor inhibits the expression or activity of one or more T cell polypeptides selected from the polypeptides listed in Table 1.
  • the Graft-versus-host disease is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus- host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
  • T cell dysfunction is selected from the group consisting of non-relapse mortality (NRM) and infection.
  • NRM non-relapse mortality
  • HCT Hematopoietic Cell Transplant
  • UTD Unrelated Donor
  • b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a
  • DHFR dihydrofolate reductase
  • the composition of paragraph 39 wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus.
  • the method of paragraph 43 wherein the condition caused by or associated with T cell dysfunction is selected from the group of Graft-versus-host disease (GvHD) and leukemia.
  • the method of paragraph 44 wherein the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
  • hematopoietic disease is a hematopoietic cancer.
  • hematopoietic disease is leukemia.
  • b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor; and
  • GvHD Graft- versus-host disease
  • aAGVHD severe acute Graft-versus-host disease
  • cGvHD chronic Graft-versus-host disease
  • leukemia and lymphoma relapse The pharmaceutical composition of paragraph 52 for the treatment or prevention of a
  • Transplant comprising administering to the subject a composition of any of the preceding paragraphs.
  • Example 1 Control of relapse when abatacept is combined with calcineurin inhibition and methotrexate: an unexpected discovery made with the ABA2 trial.
  • the ABA2 trial was designed by the inventors to improve survival after unrelated-donor hematopoietic stem cell transplantation (HCT) by adding the T cell costimulation blockade agent, abatacept, to background immunosuppression with a standard regimen containing a calcineurin inhibitor and methotrexate.
  • Abatacept is an immunosuppressive drug, marketed by Bristol Myers Squibb, which is approved for Rheumatoid Arthritis.
  • the hoped-for effect in the ABA2 trial was that the addition of abatacept would decrease the most deadly complication of HCT, which is a syndrome called“Acute Graft-versus-Host Disease” (AGVHD). Severe AGVHD is caused by over-activation of the immune system after transplant, which leads to organ damage in patients that is caused by the transplanted T cells.
  • results The ABA2 had the hoped for outcome with respect to AGVHD: Patients prophylaxed with abatacept demonstrated significantly less AGVHD than patients who received standard calcineurin inhibition + methotrexate alone. The magnitude of this effect has been significant, and has led to the FDA granting abatacept a Breakthrough Therapy Designation for prevention of GVHD after unrelated donor HCT. While improvement in AGVHD was the anticipated outcome of ABA2, the inventors have made an additional discovery in the course of analyzing the data for ABA2 that was totally unexpected given the previously -understood mechanism of action of abatacept. The inventors found that the addition of abatacept for AGVHD prophylaxis did not increase relapse after transplant. In one cohort of ABA2 patients (called the“7/8 cohort”), the one-year relapse rates were 9.3% in the
  • CNI/MTX+abatacept patients compared to 15.4% in CNI/MTX patients.
  • the second cohort of ABA2 patients (called the“8/8 cohort”) the one-year relapse rates were 13.8% for patients receiving abatacept and 20.5% for patients receiving placebo.
  • the inventors analyzed the transcriptomes of CD4+ T cells reconstituting after transplant in patients on this study.
  • Example 2 Transcriptomic Analysis of CD4+ T Cell Dysfunction during GVHD: Evidence for Profound Reprograming of T Cell Signaling during Acute Gvhd That Is Controlled during CD28:CD80/86 Costimulation Blockade with Abatacept
  • AGVHD acute graft-versus-host-disease
  • HCT hematopoietic stem cell transplant
  • Methotrexate compared to controls receiving CNI/MTX alone.
  • MTX Methotrexate
  • W GCNA Weighted Gene Correlation Network Analysis
  • This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from
  • Example 3 Costimulation Blockade with Abatacept for severe acute GVHD
  • Allogeneic HCT is an effective treatment for aggressive hematologic malignancies, often representing the only option for cure.
  • URD unrelated donors
  • the major disadvantage of URD HCT is an increased risk for non-relapse mortality (NRM) mediated by severe acute GVHD (AGVHD), chronic GVHD (CGVHD) and infection 1 7 .
  • NRM non-relapse mortality
  • AGVHD severe acute GVHD
  • CGVHD chronic GVHD
  • infection 1 7 The use of an HFA mismatched URD accentuates these risks, and carries a higher incidence of both severe AGVHD (up to 37% 3 ) and NRM (up to 45% 3 ).
  • AGVHD severe acute GVHD
  • CGVHD chronic GVHD
  • NRM up to 45% 3
  • the majority of patients in ethnic minorities will not have an HFA-matched URD, but will have a 7/8 URD donor available 8 , underscoring the importance of improving the safety of these high-risk transplants.
  • T cell-mediated immune activation that causes AGVHD mirrors that occurring during organ rejection and autoimmunity.
  • Studies in these diseases have led to the development of T cell costimulation blockade agents that inhibit T cell activation.
  • T cell costimulation blockade agents that inhibit T cell activation. 9
  • One of the most potent approaches occurs through blockade of CD28:CD80/CD86 signaling 10 , with the first costimulation blockade agent,
  • CTLA4Ig or‘abatacept,’ approved for use in rheumatoid arthritis 11 14 and the related compound, belatacept, approved for renal transplantation 15 16 .
  • ABA2 is a Phase 2 study of abatacept in addition to standard calcineurin inhibition (‘CNT, using either cyclosporine or tacrolimus) and methotrexate (‘MTX’, 4 doses, 15 mg/m2 on Day +1 and 10 mg/m2 on Days +3, +6 and +11 relative to transplant). CNI was continued through day 100, and weaned between days 100-180 post-transplant as tolerated. For patients receiving abatacept, four doses were delivered, 10 mg/kg/dose, on Days -1, +5, +14, +28.
  • CNT standard calcineurin inhibition
  • MTX methotrexate
  • Pre-transplant conditioning used one of four regimens: busulfan/fludarabine, busulfan/cyclophosphamide, total body irradiation/cyclophosphamide, fludarabine/melphalan (details in the ABA2 protocol (see, e.g. Example 4).
  • This trial had two strata: For 8/8 patients, a randomized double-blind placebo-controlled design was utilized, with patients randomized 1: 1 to abatacept or placebo. For 7/8 patients, a single arm open label design was used, with a pre-specified matched analysis comparing outcomes with patients from the CIBMTR registry, transplanted between 2008-2015. Details of control selection are provided in the ABA2 protocol (see also Example 4). It is important to note that the 7/8 stratum was initially designed as a randomized, double-blind placebo-controlled trial. However, after more than a year of open enrollment, the 7/8 stratum was experiencing very slow enrollment (FIGs.
  • SGFS AGVHD-Free Survival
  • the decision rule was to reject the null hypothesis if p8/8 > 0.8 for the 8/8 HLA-matched URD and p7/8 > 0.8 for the 7/8 HLA-mismatched URD.
  • a sample size of 70 per arm was calculated to achieve 80% Bayesian power to detect a reduction in severe AGVHD from 20% to 10% (Bayesian Type I probability ⁇ 0.2).
  • Patients in the 8/8 cohort were randomized at a 1: 1 ratio using a non- adaptive randomization with a block size of 8 stratified by patient age (patients ⁇ 21 years of age versus patients > 21 years).
  • a sample size of 40 patients was calculated to achieve 80% Bayesian power to detect a reduction in severe AGVHD from 30% to 10% (Bayesian Type I probability ⁇ 0.2).
  • the control arm was constructed using a cohort from the CIBMTR. Up to 4 controls per patient were selected, with matching on disease type, disease status, age within 10 years, or 50% of age (whichever was smaller), and preference for a match on performance score if more than 4 controls were available.
  • the comparative data presented shows outcomes for 38 or 39 abatacept patients (compared to CNI/MTX or CNI/MTX+ATG, respectively) as well as the ITT data. As shown throughout the results, the outcomes for the comparator groups of patients were indistinguishable from the outcomes for the ITT population.
  • Acute GVHD ABA2 demonstrated improvement in AGVHD in both 7/8 and 8/8 URD HCT patients receiving abatacept.
  • the primary endpoint was the Cl of severe AGVHD at Day +100.
  • FIG. 2 and FIG.10A in the 7/8 stratum, patients receiving abatacept demonstrated a substantial decrease in the Day +100 Cl of severe AGVHD, with 2.2% severe AGVHD in patients receiving CNI/MTX+abatacept versus 30.2% in CNI/MTX CIBMTR controls, (p ⁇ 0.001), greatly exceeding the expectations of the trial’s statistical design.
  • SGFS Severe AGVHD-Free Survival
  • SGFS was 97.7% (abatacept ITT) versus 58.7% (CNI/MTX) (p ⁇ 0.001), and 62.2% (CNI/MTX/ATG) (p ⁇ 0.001).
  • FIG. 10E The first figure.
  • both the 7/8 and 8/8 strata demonstrated successful neutrophil and platelet reconstitution, donor engraftment, and leukocyte reconstitution post transplant (as measured by multi -parameter flow cytometry).
  • FIG. 12B The most pertinent SAM, color- coded Turquoise, encapsulates a 476-gene module (FIG. 12B) that strongly correlated with patients receiving CNI/MTX prophylaxis and away from patients receiving CNI/MTX+abatacept prophylaxis (FIG. 12C), providing the first list of genes controlled by costimulation blockade during HCT.
  • haploidentical transplant 34 39 The ABA2 study was designed to test whether targeted in vivo CD80/86 costimulation blockade with abatacept could improve outcomes for both 7/8 and 8/8 URD HCT.
  • CNI/MTX+placebo (FIG. 3). This indicates that a regimen containing CNI/MTX+ abatacept can‘level the playing field’ for those patients (often ethnic minorities) for whom the only URD option is HLA- mismatched. Moreover, while not a controlled analysis, the survival results for the 7/8 ABA2 patients also compare favorably to a retrospective study of post-transplant cyclophosphamide and ATG in HLA- mismatched URD transplants recently published by the European Bone Marrow Transplant Consortium 40 .
  • abatacept may have activity in treating CGVHD and that extending the dosing of abatacept could provide protection against chronic as well as AGVHD 45,46 .
  • extending the dosing of abatacept can improve chronic as well as acute GVHD outcomes, while preserving the beneficial graft-versus-leukemia effect observed in the ABA2 trial.
  • the ABA2 trial demonstrated the safety and efficacy of in vivo costimulation blockade with abatacept in preventing severe AGVHD after 7/8 URD HCT, with a significant impact on moderate-severe AGVHD and steroid-refractory AGVHD in 8/8 patients.
  • abatacept has been granted a Breakthrough Therapy Designation by the FDA for the prevention of AGVHD after URD HCT.
  • the substantial improvement in one- and two-year survival indicators in the 7/8 cohort demonstrate that the addition of abatacept is practice-changing for these otherwise high-risk transplants.
  • CTLA4Ig prevents both acute and chronic GVHD
  • Ciurea SO Zhang MJ
  • Bacigalupo AA et al. Haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia. Blood
  • Table 1 Gene symbols and NCBI Gene ID number of human Transcripts Correlated with AGVHD Grade. The genes listed in this table correlate with the severity of Acute GVHD in patients undergoing a hematopoietic stem cell transplant.
  • nucleic acid sequences of the genes described herein have been assigned NCBI and ENSBL accession numbers for different species such as human, mouse and rat.
  • sequences for any of the genes described herein can be readily retrieved from either database by one of ordinary skill in the art.
  • sequence of a gene, transcript, or polypeptide described herein is the sequence available in the NCBI or ENSMBL database as of the filing date of this application. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
  • Control Selection For the 7/8 stratum, pre-specified controls from the CIBMTR were selected from a cohort of patients who received 7/8 URD HCT for hematologic malignancy, and who received CNI/MTX for GVHD prophylaxis, without the use of either anti-thymocyte globulin (ATG) or alemtuzumab. A secondary analysis using CIBMTR controls who had received CNI/MTX+ATG was also performed. Matching was based on age (within 50% or 10 years, whichever was smaller), diagnosis, CIBMTR disease status, and performance score when >4 matches were available, with all controls having a performance score >80%.
  • a secondary analysis using CIBMTR controls who had received CNI/MTX+ATG was also performed. Matching was based on age (within 50% or 10 years, whichever was smaller), diagnosis, CIBMTR disease status, and performance score when >4 matches were available, with all controls having a performance score >80%.
  • GVHD grading, chimerism analysis and adverse event grading The NIH consensus criteria were used for the grading of acute 1 and chronic GVHD2. Lineage specific (CD33+ and CD3+) donor chimerism analysis was performed to monitor donor engraftment. The National Cancer Institute Common Terminology Criteria for Adverse Events version 4 was used to classify adverse events. Review and adjudication of the grading of AGVHD and CGVHD was performed centrally by blinded members of the protocol team, using the Blood and Marrow Transplant Clinical Trial Network (BMTCTN) Manual of Procedures, as described in Section 3.10 of the protocol.
  • BMTCTN Blood and Marrow Transplant Clinical Trial Network
  • Transcriptomic analysis Flow cytometric CD4+ T cell sorting was performed on patients enrolled at two laboratory sites (Emory University and Seattle Children’s Research Institute) at Days 21 and 28 post-transplant, using either a FACSJazz or FACSAria flow cytometric cell sorter. For transcriptomic analysis, only samples that were sorted immediately after the blood draw (without shipping) from patients at the two laboratory sites were included, since shipping of samples from other sites resulted in a significant impact on the transcriptomic signature. This resulted in 65 analyzed samples from patients receiving CNI/MTX+abatacept and 41 analyzed samples from patients receiving
  • parameters for signed-hybrid network construction included a soft-thresholding power of 6, mean connectivity of 10, and minimum module size of 50. Enrichment analysis was performed using the piano4 package using Reactome8 pathway terms from the MSigDB v6.17,9.
  • ABA2 dataset we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0- 4), relapse, non-relapse mortality, and all-cause mortality.
  • SAMs Self-Assembling Modules
  • Transcriptional Network Visualization Genes in the Turquoise SAM for which expression level was positively correlated with the grade of AGVHD through linear regression were then analyzed using ingenuity pathway analysis (IPA, http://www.ingenuity.com) to identify direct and indirect interactions between genes, supported by a meta-analysis of scientific literature contained in the Ingenuity Knowledge Base. Default search parameters were used, including all direct and indirect gene-gene interactions supported by human studies with highest confidence.
  • IPA ingenuity pathway analysis
  • GVHD biomarker analysis using the Mount Sinai Acute GVHD International Consortium (MAGIC) biomarker algorithm In patients who developed AGVHD, the prognostic biomarkers ST2 and REG3a were analyzed by enzyme-linked immunoabsorbent assay, as previously described 11 13 .
  • MAGIC Mount Sinai Acute GVHD International Consortium
  • MAGIC analyses For MAGIC analyses, if AGVHD was treated with systemic steroids, a sample collected at the time of treatment (within a window of 7 days prior to treatment to 3 days after start of treatment) was used for analysis. If lower-grade AGVHD was diagnosed but never systemically treated, a sample drawn at the time of GVHD diagnosis (+/- 7 days) was used for analysis. The MAGIC algorithm probability (MAP) of NRM was calculated using the previously published model 14 and the median MAP was compared between the groups.
  • MAP MAGIC algorithm probability
  • Demographic, disease, and transplant-related factors were compared between treatment groups using c2 for categorical variables and Wilcoxon rank-sum tests for continuous variables.
  • Time- dependent outcomes without the presence of competing events including OS, Severe AGVHD-free survival (SGFS)15, and RFS, were examined using Kaplan-Meier survival curves, and resulting survival distributions were compared using a log-rank test.
  • cumulative incidence functions CIF
  • Gray Gray’s test was used to compare CIF between treatment groups.
  • Viral Reactivation and Disease [00274] The inventors prospectively measured CMV reactivation and end-organ disease, as well as EBV reactivation and PTLD (FIGs. 14I-14J, FIG. 5). CMV and EBV reactivation rates are described for >300 IU/ml for CMV and >1000 IU/ml for EBV because all centers were able to quantify values that exceeded these cut-offs.
  • SAEs Severe Adverse Events
  • CD80 refers to all naturally occuring variants or isoforms of CD80.
  • CD80 polypeptide sequence is presented in SEQ ID NO: 1.
  • the CD80 polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 1.
  • CD86 refers to all naturally occuring variants or isoforms of CD86.
  • the CD86 polypeptide sequence is presented in SEQ ID NO: 2.
  • the CD86 polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 2.
  • Calcineurin refers to all naturally occuring variants or isoforms of Calcineurin.
  • the Calcineurin polypeptide sequence is presented in SEQ ID NO: 3.
  • the Calcineurin polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 3.
  • dihydrofolate reductase sequences for dihydrofolate reductase are known for a number of species, e.g., human dihydrofolate reductase (NCBI Gene ID: 1719), mRNA (NCBI Ref Seq: NM_000791.4), and polypeptide (NCBI Ref Seq: NP_000782.1).
  • Dihydrofolate reductase refers to all naturally occuring variants or isoforms of dihydrofolate reductase.
  • the dihydrofolate reductase polypeptide sequence is presented in SEQ ID NO: 4.
  • the dihydrofolate reductase polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 4.

Abstract

.Provided herein are novel methods and compositions for the treatment of chronic and acute Graft-versus-host disease (GVHD). In another aspect, described herein is a method for treating or preventing GvHD, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor.

Description

METHODS AND COMPOSITIONS RELATING TO THE TREATMENT OF GVHD
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 62/809,010 filed February 22, 2019 and 62/956,707 filed January 3, 2020 the content of each of which is incorporated herein by reference in its entirety.
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant No. FD004099, awarded by the National Institutes of Health and Grant No. IND# 111738 awarded by the U.S. Food & Drug
Administration. The government has certain rights in the invention.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 12, 2020, is named 701039-094490WOPT_SL.txt and is 10,928 bytes in size.
TECHNICAL FIELD
[0004] The technology described herein relates to novel methods and compositions for the treatment of chronic and acute Graft versus host disease (GVHD).
BACKGROUND
[0005] Acute and chronic Graft-versus-host disease (GVHD) are life-threatening complications that can occur after certain stem cell or bone marrow transplants. GVHD occurs when immune cells transplanted from a non-identical donor (the graft) recognize the transplant recipient (the host) as foreign, thereby initiating an immune reaction that causes disease in the transplant recipient. Clinical
manifestations of acute GVHD include a classic maculopapular rash; persistent nausea and/or emesis; abdominal cramps with diarrhea; and a rising serum bilirubin concentration. In contrast, patients with chronic GVHD commonly demonstrate skin involvement resembling lichen planus or the cutaneous manifestations of scleroderma; dry oral mucosa with ulcerations and sclerosis of the gastrointestinal tract; and a rising serum bilirubin concentration, amongst other clinical manifestations.
[0006] Various immunosuppressive agents have been employed to protect patients from such damage. Currently, GVHD is controlled using immunosuppressive agents including cyclosporin A, corticosteroids including prednisone, and methylprednisolone, cyclophosphamide, and FK506, amongst others. However, these drugs are associated with a high incidence of side effects when used at therapeutic doses. Such side effects include neurotoxicity, nephrotoxicity, increased risk of relapse of the underlying malignancy, increased risk of infections and/or hepatotoxicity. Thus, urgent need exists for improved treatment and prophylaxis for chronic and acute GVHD with acceptable toxicity profiles.
SUMMARY
[0007] GvHD results from over activation of the immune system, specifically over active transplanted T cells. The CD80/86 inhibitor abatacept is demonstrated herein to suppress this immune activity to a surprising degree, particularly in combination with a calcineurin inhibitor or methotrexate. Even more surprisingly, administration of an immune suppressing CD80/86 inhibitor (e.g., abatacept) alters the biology of T cells in a way that increases their anti -cancer activity (i.e. by downregulating immune checkpoint molecules), preventing cancer relapses in transplant patients to a striking degree. The methods described herein therefore provide both i) a suprising degree of efficacy in suppressing the immune response underlying GvHD and ii) an unexpected result as far as increasing the expression of checkpoint molecules which may increase the effectiveness of the anti-cancer aspects of the immune system.
[0008] In one aspect, described herein is a method for treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction, the method comprising:
administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor,
thereby inhibiting the expression or activity of one or more immune checkpoint polypeptides in T cells and reconstituting the T cell population.
[0009] In some embodiments of any of the aspects, the condition caused by or associated with T cell dysfunction is selected from Graft-versus-host disease (GvHD), leukemia or lymphoma relapse.
[0010] In another aspect, described herein is a method for treating or preventing GvHD, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a
pharmaceutical composition comprising a CD80/86 inhibitor.
[0011] In some embodiments of any of the aspects, the administration is prophylactic.
[0012] In some embodiments of any of the aspects, the subject is a subject who is or has received a bone marrow transplant.
[0013] In another aspect, described herein is a method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a bone marrow transplant to the subject; and
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor.
[0014] In some embodiments of any of the aspects, the hematopoietic disease is a hematopoietic cancer.
[0015] In some embodiments of any of the aspects, the hematopoietic disease is leukemia.
[0016] In some embodiments of any of the aspects, the CD80/86 inhibitor is an antibody or antigen binding fragment thereof, an aptamer, or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86.
[0017] In some embodiments of any of the aspects, the CD80/86 inhibitor binds specifically to CD80 and/or 86.
[0018] In some embodiments of any of the aspects, the CD80/86 inhibitor is abatacept.
[0019] In some embodiments of any of the aspects, the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
[0020] In some embodiments of any of the aspects, the receptor is CTLA-4 and/or CD28.
[0021] In some embodiments of any of the aspects, the immune checkpoint polypeptides are selected from CTLA4, Lag-3, HAVCR2 (TIM-3) and/orPDCDl.
[0022] In some embodiments of any of the aspects, the immune checkpoint polypeptides are selected from Lag-3, HAVCR2 (TIM-3) and/orPDCDl.
[0023] In some embodiments of any of the aspects, the T cell population comprises CD3+, CD4+ and/or CD8+ T cells.
[0024] In some embodiments of any of the aspects, the T cell population comprises Regulatory T cells (TregS).
[0025] In some embodiments of any of the aspects, the inhibitor is targeted to T-cells.
[0026] In some embodiments of any of the aspects, the inhibitor comprises a T cell targeting moiety.
[0027] In some embodiments of any of the aspects, the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide.
[0028] In some embodiments of any of the aspects, the T cell-specific cell surface polypeptide is selected from the group consisting of CD3, CD4 and/or CD8.
[0029] In some embodiments of any of the aspects, the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof, an aptamer, or a natural ligand that specifically binds the T cell-specific cell surface polypeptide. [0030] In some embodiments of any of the aspects, the method further comprises administering a therapeutically effective amount of an inhibitor of the expression or activity of one or more immune checkpoint polypeptides.
[0031] In some embodiments of any of the aspects, the condition caused by or associated with T cell dysfunction is selected from Graft-versus-host disease (GvHD) leukemia or lymphoma relapse.
[0032] In some embodiments of any of the aspects, the method comprises administering a therapeutically effective amount of a Calcineurin inhibitor.
[0033] In some embodiments of any of the aspects, the Calcineurin inhibitor binds specifically to Calcineurin.
[0034] In some embodiments of any of the aspects, the Calcineurin inhibitor is selected from cyclosporine and tacrolimus.
[0035] In some embodiments of any of the aspects, the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
[0036] In some embodiments of any of the aspects, the method comprises administering a therapeutically effective amount of a dihydrofolate reductase (DHFR) inhibitor.
[0037] In some embodiments of any of the aspects, the dihydrofolate reductase (DHFR) inhibitor is an antibody or antigen-binding fragment thereof, an aptamer, or a dihydrofolate reductase (DHFR) inhibitor fragment of a natural receptor or ligand of dihydrofolate reductase (DHFR).
[0038] In some embodiments of any of the aspects, the dihydrofolate reductase (DHFR) inhibitor binds specifically to dihydrofolate reductase (DHFR).
[0039] In some embodiments of any of the aspects, the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
[0040] In some embodiments of any of the aspects, the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
[0041] In some embodiments of any of the aspects, the CD80/86 inhibitor inhibits the expression or activity of one or more polypeptides in T cells selected from the polypeptides listed in table 1.
[0042] In some embodiments of any of the aspects, the Graft-versus-host disease (GvHD) is selected from acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
[0043] In some embodiments of any of the aspects, the condition caused by or associated with T cell dysfunction is selected from non-relapse mortality (NRM) and infection.
[0044] In some embodiments of any of the aspects, the subject is a subject who is or has received a Hematopoietic cell transplant (HCT). [0045] In some embodiments of any of the aspects, the Hematopoietic cell transplant (HCT) originates from an unrelated donor (URD).
[0046] In another aspect, described herein is acomposition comprising:
a. a CD 80/86 inhibitor; and
b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a
dihydrofolate reductase (DHFR) inhibitor.
[0047] In some embodiments of any of the aspects, the Calcineurin inhibitor is selected from cyclosporine or tacrolimus.
[0048] In some embodiments of any of the aspects, the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
[0049] In some embodiments of any of the aspects, the CD80/86 inhibitor is abatacept.
[0050] In another aspect, described herein is a method of treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction in a subject in need thereof, the method comprising administering to the subject a composition of any of the preceding claims.
[0051] In some embodiments of any of the aspects, the condition caused by or associated with T cell dysfunction is selected from Graft-versus-host disease (GvHD) or leukemia.
[0052] In some embodiments of any of the aspects, the Graft-versus-host disease (GvHD) is selected from acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD) and chronic Graft-versus-host disease (cGvHD).
[0053] In another aspect, described herein is a method of treating or preventing a hematopoietic disease in a subject in need thereof, the method comprising administering to the subject a composition of any of the preceding claims.
[0054] In another aspect, described herein is a method of treating or preventing a non-relapse mortality (NRM) in a subject who is or has received a Hematopoietic cell transplant (HCT), the method comprising administering to the subject a composition of any of the preceding claims.
[0055] In some embodiments of any of the aspects, the Hematopoietic cell transplant (HCT) originates from an unrelated donor (URD).
[0056] In another aspect, described herein is a method of treating a hematopoietic disease in a subject in need thereof, the method comprising:
a. administering a Hematopoietic stem transplant to the subject; and
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor; and c. further administering a therapeutically effective amount of a Calcineurin inhibitor and/or a therapeutically effective amount of a dihydrofolate reductase inhibitor (DHFR).
[0057] In some embodiments of any of the aspects, the hematopoietic disease is a hematopoietic cancer.
[0058] In some embodiments of any of the aspects, the hematopoietic disease is leukemia.
[0059] In another aspect, described herein is a pharmaceutical composition formulated for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction, comprising: a. a CD80/86 inhibitor; and b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor; and c. a pharmaceutically acceptable carrier.
[0060] In some embodiments of any of the aspects, described herein is a pharmaceutical composition for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), severe acute Graft-versus-host disease
(aAGVHD), and chronic Graft-versus-host disease (cGvHD), leukemia and lymphoma relapse.
[0061] In some embodiments of any of the aspects, described herein is a pharmaceutical composition for the treatment or prevention of a hematopoietic disease in a subject in need thereof.
[0062] In some embodiments of any of the aspects, described herein is a pharmaceutical composition for the treatment of a hematopoietic disease, wherein the hematopoietic disease is a hematopeitic cancer.
[0063] In some embodiments of any of the aspects, described herein is a pharmaceutical composition for the treatment or prevention of a non-relapse mortality (NRM) in a subject who is need of or who has received a Hematopoietic Cell Transplant (HCT) comprising administering to the subject a composition of any of the preceding claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] FIGs. 1A-1D depict the results of a Transcriptomic Analysis. (FIG. 1A) Weighted Gene Correlation Network Analysis (WGCNA) heatmap. Heatmap demonstrates the correlation (using a blue- red false color scale) of WGCNA gene modules (colored turquoise, blue, green and red) in transcriptomic amples grouped by clinical metadata. Row/Column order is determined by hierarchical clustering or the module eigengegen with clinical metadata for each sample. (FIG. IB) Gene Set Enrichment Analysis (GSEA) of the turquoise WGCNA module in a ranked list of differentially expressed genes between patients receiving Abatacept (left) and patients receiving Standard prophylaxis (right). Gene list generated with a cut-off of FDR < 0.001 by gene-set permutation test. (FIG. 1C) Reactome database terms exhibiting enrichment in the turquoise module genes using a Fisher’s exact test. Threshold at a significance value of 0.00 latter Bonferroni correlation for multiple hypotheses. Node size indicates number of genes in each term (range 10-390). Edges of graph indicate shared genes between terms (range 1-301). (FIG. ID) Visualization of cytoscape pathway analysis demonstrating network connections of transcripts identified as being positively correlated with grade of acute GVHD.
[0065] FIG. 2 shows a table listing the Baseline Characteristics of the Participants. The 7/8 P value represents the comparison of the 7/8 abatacept cohort and the CIBMTR control cohort.
[0066] FIG. 3 shows a table listing Rates of Acute and Chronic GVHD, SGFS, Relapse, RFS, NRM, and OS. The 7/8 P value represents the comparison of the 7/8 abatacept cohort and the CIBMTR control cohort.
[0067] FIG. 4 shows a table listing of antibodies used for flow cytometry and the gating strategy of immune populations.
[0068] FIG. 5 shows a table listing the rates of neutrophil and platelet engraftment, CMV and EBV reactivation, CMV End-organ Disease, and PTLD.
[0069] FIG. 6 shows a table listing serious adverse events.
[0070] FIG. 7 shows a table listing baseline characteristics of the Participants.
[0071] FIG. 8 shows a table listing the rates of neutrophil and platelet engraftment, acute and chonic
GVHD, severe GVHD-free survival, relapse, RFS, NRM and OS.
[0072] FIG. 9 shows a table listing the subanalysis of acute GVHD II-IV at day 180.
[0073] FIGs. 10A-10J depict key GVHD Outcomes in accordance with the Mount Sinai Acute GVHD International Consortium (MAGIC) algorithm prediction of 6-month NRM
[0074] FIGa. 1 lA-1 IB show the Key Transplant Outcomes. (FIG.l 1A) 7/8 Cohort, Cumulative incidence of non-relapse mortality. (FIG. l IB) 8/8 Cohort, Cumulative incidence of non-relapse mortality. 7/8 Cohort, Cumulative incidence of relapse-free survival. 8/8 Cohort, Cumulative incidence of relapse- free survival. 7/8 Cohort, Cumulative incidence of overall survival. 8/8 Cohort, Cumulative incidence of overall survival. 7/8 Cohort: Blue: CIBMTR control cohort. Solid Red: 7/8 abatacept cohort, matched to the CIBMTR controls. Dashed Red: 7/8 Intention-to-treat (ITT) cohort. P values represent comparison of 7/8 abatacept cohort and CIBMTR control cohort. 8/8 Cohort: Blue: Placebo. Red: Abatacept.
[0075] FIGs. 12A-12D depict the results of a Transcriptomic Analysis. (Fig. 12A) Scehmata of the Transcriptomic analysis pipeline. (FIG. 12B) Weighted Gene Coexpression Network Analysis (WGCNA) heatmap. Heatmap demonstrates the correlation (using a blue-red false color scale) of WGCNA gene modules (colored Turquoise, Blue, Green and Red) in transcriptomic samples grouped by clinical metadata. Row/Column order is determined by hierarchical clustering of the module eigengene with clinical metadata for each sample. (FIG. 12C) Gene Set Enrichment Analysis (GSEA) of the Turquoise WGCNA module in a ranked list of differentially expressed genes between patients receiving abatacept (left) and patients receiving standard prophylaxis (right). The gene list was generated with a cut-off of FDR<0.001 by gene-set permutation testing. (FIG. 12D) Ingenuity Pathway Analysis of the 93-gene subset of the Turquoise module. Yellow: Proliferation genes; Black: Apoptosis genes; Blue: Checkpoint genes; Green: Metabolism genes; Red: T cell activation genes. Solid lines: Direct interactions; Dashed lines: Indirect interactions.
[0076] FIGs. 13A-13B show a consort Diagram. (FIG. 13A) 7/8 stratum. (FIG. 13B) 8/8 stratum. Patients were randomized between 3/1/2013 and 12/9/14, following a protocol amendment, subjects in the 7/8 cohort were non-randomly assigned to receive abatacept.
[0077] FIGs. 14A-14L show the Post-transplant Immune Monitoring. (FIG.14A) White blood cell count. (FIG.14B) Absolute granulocyte count. (FIG. 14C) Absolute lymphocyte count. (FIG. 14D) Absolute B cell count. (FIG. 14E) Absolute NK cell count. (FIG. 14F) Absolute T cell count. (FIG. 14G) Absolute CD4+ T cell count.H: Absolute CD8+ T cell count. In FIGs. 14A-14H, data are shown as the mean cells/microliter + standard error of the mean (SEM). Dashed red = 7/8 ABA ITT; Solid red = 8/8 ABA; Solid Blue = 8/8 Placebo; Green Diamond=Healthy Controls.I: Cumulative incidence of CMV reactivation to >300 IU/ml. (FIG. 14J) Cumulative incidence of EBV reactivation to >1000 IU/ml.
(FIG. 14K) Percentage of patients achieving >95% donor CD3 engraftment at one year post-transplant. (FIG. 14L) Percentage of patients achieving >95% donor CD33 engraftment at one year post-transplant.
[0078] FIGs. 15A-15F show the clinical outcomes of the 7/8 cohort compared to a CIBMTR cohort who received CNI/MTX+ATG. (FIG.15A) Cumulative incidence of Grade 2-4 AGVHD. (FIG.15B) Cumulative incidence of Grade 3-4 AGVHD. (FIG.15C) Cumulative incidence of Severe Acute GVHD- Free Survival (SGFS). (FIG.15D) Cumulative incidence of RFS. (FIG.15E) Cumulative incidence of NRM. (FIG.15F) Cumulative incidence of OS. Blue: CIBMTR control cohort, CNI/MTX+ATG. Solid Red: 7/8 abatacept cohort, matched to the CIBMTR ATG controls. Dashed Red: 7/8 Intention-to-treat (ITT) cohort. P values represent comparison of 7/8 abatacept cohort and CIBMTR control cohort.
[0079] FIGs. 16A-16E show purified CD4+ T cells from patients of the ABA2 trial at Day 21 and Day 28. RNASeq was performed on these cells. An unsupervised multiparameter analysis was performed to determine what transcripts, and cellular pathways were correlated with key outcomes including:
exposure to abatacept or not. Relapse or not. GVHD or not. CMV reactivation or not. (FIG. 16A) Module and Metamodule Analysis using the technique of‘Weighted Gene Correlation Network Analysis’ to identify which transcriptomic modules closely correlate with clinical outcomes. (FIG. 16B) Image of the Turquoise Module, that correlates with standard GVHD prophylaxis and anti-correlates with exposure to abatacept. (FIG. 16C) A Gene Set enrichment analysis which confirms that the Turquoise module correlates with patients prophylaxed with standard agents and Not with patients given abatacept. (FIG. 16D) Pathway analysis of the Turquoise module identifying multiple cell proliferation pathways that correlate with this Module. (FIG. 16E) GSEA analysis confirming that a CellCycle gene set correlates with standard versus abatacept samples.
[0080] FIGs. 17A-17B show Ki-67 expression as it correlates with GVHD for standard and abatacept-treated patients separately. (FIG. 17A) Measurements of the xpression of a major proliferation marker, Ki-67. It was determined whether this marker correlated with GVHD in patients prophylaxed with either Standard (FIG.17A), or Abatacept (FIG. 17B). In the bar graph shown in Fig. 17A, the series are, from left to right: 0, 1, 2, 3 and 4. (FIGs. 17A-17B) The box-whisker plot shows that the level of Ki- 67 expression increases with increasing severity of GVHD in patients prophylaxed with a standard GVHD regimen. This box-whisker plot shows that the level of Ki-67 expression DOES NOT INCREASE with increasing severity of GVHD in patients prophylaxed with an abatacept-containing GVHD regimen. In the bar graph shown in Fig. 17B, the series are, from left to right: None,“1 and 2”, and“3 and 4”. This result is in agreement with the pathway analysis shown in FIG. 16, and suggests that patients prophylaxed with abatacept control proliferation post-transplant, whether or not they get GVHD.
[0081] FIGs. 18A-18C show that patients prophylaxed with Abatacept demonstrate less Ki-67, CD28 and CTLA4 expression compared to standard. (FIG. 18A-18C) CTLA4: CTLA4 is expressed at a lower level in patients who received abatacept. In the bar graph shown in Fig. 18 A, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 18B, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 18C, the series are, from left to right: None, Abatacept, and Standard. Abatacept controls GVHD by limiting proliferation (see, e.g. FIGs. 16A-E, 17A-B and 18A-C). but also decreases the expression of check-point molecules like CTLA4 (FIG. 18C).
[0082] FIGs. 19A-19B show CTLA4 expression data as it correlates with GVHD for standard and abatacept-treated patients separately. (FIG. 19A) This graph shows that patients that are not given abataceptincrease expression of CTLA4 when they develop GVHD. In the bar graph shown in Fig. 19A, the series are, from left to right: None,“1 and 2”, and“3 and 4”. (FIG. 19B) Patients given Abatacept as part of their prophylaxis do not upregulate CTLA4 when they get GVHD. In the bar graph shown in Fig. 19B, the series are, from left to right: None,“1 and 2”, and“3 and 4”.
[0083] FIGs. 20A-20C show the successful reconstitution of T cells in patients prophylaxed with Abatacept. Patients prophylaxed with Abatacept successfully reconstitute total T cells (FIG. 20A), CD4+ T cells (FIG. 20B) and CD8+ T cells (FIG. 20C) post-transplant. In the graph shown in Fig. 20A, the series are, ABA (as depicted in the line with the squares) and Placebo (as depicted in the line with the triangles). In the graph shown in Fig. 20B, the series are, ABA (as depicted in the line with the squares) and Placebo (as depicted in the line with the triangles). In the graph shown in Fig. 20C, the series are, ABA (as depicted in the line with the squares) and Placebo (as depicted in the line with the triangles).
[0084] FIGs. 21A-21E show a Weighted Correlation Network Analysis (WGCNA) Analysis. (FIG. 21 B) Identification of the Turquoise Module. (FIGs. 21C-21E) shows the GSEA and Pathway Analysis of the Turquoise Module.
[0085] FIGs. 22A-22C show the successful T Cell Reconstitution in Abatacept treated Patients. In the graph shown in Fig. 22A, the series are, ABA (line with square boxes) and Placebo (ine with triangles). In the graph shown in Fig. 22B, the series are, ABA (line with square boxes) and Placebo (line with triangles). In the graph shown in Fig. 22C, the series are, ABA (line with square boxes) and Placebo (line with triangles).
[0086] FIGs. 23A-23I show graphs depicting the expression data of proliferation markers: (FIGs. 23A-C) Ki-67; (FIGs. 23D-23F) AURKA (Aurora Kinase A); and (FIGs. 23G-23I) EZH2 (Enhancer of zeste homolog 2). In the bar graph shown in Fig. 23A, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 23B, the series are, from left to right: None,“1 and 2”, and “3 and 4”. In the bar graph shown in Fig. 23C, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 23D, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 23E, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 23F, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 23G, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 23H, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 231, the series are, from left to right: None,“1 and 2”, and“3 and 4”. FIGs. 24A-24I show graphs depicting the expression data of following T cell activation markers: (FIGs. 24A- 24C) CD28; (FIGs. 24D-24F) CD38; and (FIGs. 24G-24I) CD59. In the bar graph shown in Fig. 24A, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 24B, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 24C, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 24D, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 24E, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 24F, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 24G, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 24H, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 241, the series are, from left to right: None,“1 and 2”, and“3 and 4”.
[0087] FIGs. 25A-250 show graphs depicting the expression data of the following Cytokines and Receptors activation markers: (FIGs. 25A-25C) IL7; (FIGs. 25D-25F) IL12RB2; (FIGs. 25G-25I)
IL18R1; (FIG. 25J-25L) IL21; and (FIGs. 25M-250) IL32. In the bar graph shown in Fig. 25A, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 25B, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 25C, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 25D, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 25E, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 25F, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 25G, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 25H, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 251, the series are, from left to right: None, “1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 25J, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 25K, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 25L, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 25M, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 25N, the series are, from left to right: None,“1 and 2”, and “3 and 4”. In the bar graph shown in Fig. 250, the series are, from left to right: None,“1 and 2”, and“3 and 4”.FIGs. 26A-26I show graphs depicting the expression data of the following Integrins and Adhesion Molecule marker: (FIGs. 26A-26C) CCR3; (FIGs. 26D-26F) CCR5; (FIGs. 26G-26I) CXXL13. In the bar graph shown in Fig. 26A, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 26B, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 26C, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 26D, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 26E, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 26F, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 26G, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 26H, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 261, the series are, from left to right: None,“1 and 2”, and“3 and 4”.
[0088] FIGs. 27A-27I show graphs depicting the expression data of the following Checkpoint Molecule markers: (FIGs. 27A-27C) CTLA4; (FIGs. 27D-27F) PD-1; FIGs. 27G-27I) TIM-3. In the bar graph shown in Fig. 27A, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 27B, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 27C, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 27D, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 27E, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 27F, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 27G, the series are, from left to right: None, Abatacept, and Standard. In the bar graph shown in Fig. 27H, the series are, from left to right: None,“1 and 2”, and“3 and 4”. In the bar graph shown in Fig. 271, the series are, from left to right: None,“1 and 2”, and“3 and 4”.
DETAILED DESCRIPTION
[0089] This invention relates to compositions and methods for the treatment of graft-versus-host disease (GVHD). For patients with malignant and non-malignant hematologic diseases, hematopoietic stem cell transplantation (HCT) is sometimes the last resort in the treatment of the patient. HCT is nowadays routinely performed. However, HCT is one of the most challenging and complex areas of modem medicine. Some of the key areas for medical management are the problems of GVHD during which the T cells in the donor graft has an immune response to the patient’s organs, possibly leading to organ damage and death of the HCT patient.
[0090] There are two forms of GVHD: the acute and chronic GVHD. Acute GVHD usually occurs within the first three months following a transplant. T-cells present in the donor's bone marrow at the time of transplant attack the patient's skin, liver, stomach, and/or intestines. The earliest signs of acute GVHD are usually a skin rash that appears on the hand, feet and face. Other than blistering skin, patients with severe GVHD also develop large amounts of watery or bloody diarrhea with cramping due to the donor's T-cells' attack on the stomach and intestines. Jaundice (yellowing of the skin and eyes) is the usual indication that GVHD disease involves the liver. The more organs involved and the worse the symptoms, the worse the GVHD disease.
[0091] The technology described herein is directed to the treatment of acute Graft versus host disease (GVHD) with CD80/86 inhibitors. CD80/86 inhibition suppresses the immune activity of T cells that causes GvHD while potentially increasing the immune activity of T cells that prevents cancer relapse. The latter is accomplished by decoupling T cell costimulation from T cell checkpoint expression, leading to downregulation of checkpoint molecules including CTLA4, Lag-3, TIM-3, and PD-1, thereby simultaneously controlling GVHD and preventing increased relapse of the underlying malignancy. The data provided herein demonstrate a general control of proliferation, activation markers, cytokines and their receptors, integrin and adhesion molecules, as well as checkpoint molecules.
[0092] In one aspect of any of the embodiments, described herein is a method of treating or preventing a hematopoietic disease, Graft-versus-host disease (GvHD), T cell dysfunction, and/or a condition caused by or associated with T cell dysfunction, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising: (a) a CD80/86 inhibitor. As described herein, administration of such reagents inhibits the expression or activity of one or more immune checkpoint polypeptides in T cells and promotes reconstituting a functional T cell population.
[0093] In one aspect of any of the embodiments, described herein is a method of treating hematopoietic disease, the method comprising administering to a subject i) a bone marrow or HCT transplant and ii) a therapeutically effective amount of a pharmaceutical composition comprising: (a) a CD80/86 inhibitor. The steps i) and ii) can be conducted sequentially or concurrently. If conducted sequentially, step i) can be performed first, followed by step ii). Or step ii) can be performed first, followed by step i). Or step ii) can be performed first, followed by step i), followed by one or more reptitions of step ii).
[0094] As used herein, the term“inhibitor” refers to an agent which can decrease the expression and/or activity of the target molecule or activity or process, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more. The efficacy of an inhibitor of, for example, CD80/86, e.g. its ability to decrease the level and/or activity of CD80/86 can be determined, e.g. by measuring the level of interaction of CD80/86 with one of its interaction partners e.g. CD28 and/or by measuring the level of CD80/86 protein (or its mRNA).
Methods for measuring the level of interaction of polypeptides are known to one of skill in the art, e.g. Immunoprecipitation with antibodies directed against CD80/86, followed by Western Blotting against the interacting polypeptide e.g. CD28 can be used to determine the level of interaction between two polypeptides. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RT-PCR with primers can be used to determine the level of RNA, and Western blotting with an antibody can be used to determine the level of a polypeptide. In some embodiments of any of the aspects, an inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule. Exemplary inhibitors, e.g., of CD80/86 can include a small molecule, nucleic acid (e.g., inhibitory nucleic acid), polypeptide, antibody reagent, or genome editing system. As used herein, the terms“compound” or“agent” are used interchangeably and refer to molecules and/or compositions. The compounds/agents include, but are not limited to, drugs, chemical compounds, and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; peptides; aptamers; and antibodies and intrabodies, or fragments thereof. In some embodiments,“drug” as used herein refers to an agent approved for medical use, e.g., by the FDA.
[0095] In some embodiments of any of the aspects, an inhibitor of can be an antibody or antigen binding fragment thereof, an aptamer, or an inhibitory fragment of a natural receptor or ligand of the target. In some embodiments of any of the aspects, the inhibitor binds specifically to the target. In some embodiments of any of the aspects, the inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. In some embodiments of any of the aspects, the inhibitor binds specifically to the target. In some embodiments of any of the aspects, the inhibitor does not bind to CTLA-4 and/or CD28 and/or the subject is not administered a therapy that binds to CTLA-4 and/or CD28.
[0096] As used herein,“CD80” or“cluster of differentiation 80” refers to a Ig superfamily membrane protein found on certain immune cells and which binds to CD28 or CTLA-4 on T cells. The sequences of CD80 expression products are known for a number of species, e.g., human CD80 (NCBI Gene ID No: 941) mRNA (NCBI Ref Seq: NM_005191.4) and polypeptide (NCBI Ref Seq:
NP_005182.1). As used herein,“CD86” or“cluster of differentiation 86” refers to a closely realted Ig superfamily membrane protein found on certain immune cells and which binds to CD28 or CTLA-4 on T cells. The sequences of CD86 expression products are known for a number of species, e.g., human CD86 (NCBI Gene ID No: 942) mRNA (NCBI Ref Seqs: NM)001206924.1, NM OO 1206925.1, NM_175862.5, NM_176892. lor NM_006889.4) and polypeptide (NCBI Ref Seqs: NP 001193853.1, NP 001193854.1, NP_787058.5, NP_795711.1, or NP_008820.3).
[0097] In some embodiments of any of the aspects, a CD80/86 inhibitor inhibits both CD80 and CD86. In some embodiments of any of the aspects, a CD80/86 inhibitor inhibits only CD80. In some embodiments of any of the aspects, a CD80/86 inhibitor inhibits only CD86. In some embodiments of any of the aspects, a CD80/86 inhibitor binds specifically to both CD80 and CD86. In some
embodiments of any of the aspects, a CD80/86 inhibitor binds specifically to only CD80. In some embodiments of any of the aspects, a CD80/86 inhibitor binds specifically to only CD86.
[0098] An exemplary but non-limiting CD80/86 inhibitor is abatacept or a reagent comprising the six CDRs of abatacept. In some embodiments of any of the aspects, the CD80/86 inhibitor is belatacept. [0099] In some embodiments of any of the aspects, the inhibitor is targeted to T cells, e.g., it comprises a T cell targeting moiety. In some embodiments of any of the aspects, the inhibitor is administered or provided in a composition which further comprises a T cell targeting molecule or moiety. Such a moiety can be, e.g., an antibody or antigen-binding fragment thereof, an aptamer, or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
[00100] Targeting can be achieved, e.g. by conjugating the inhibitor to a targeting group or including the inhibitor in a composition comprising a targeting group (e.g. a nanoparticule). Targeting groups can include, e.g., a T cell targeting agent, e.g., an antibody, that binds to T cells, or a cell permeation agent. Non-limiting examples of T cell targeting groups can include antibodies to T cell surface markers (e.g., CD3, CD4, CD8, CD25, CD127, and/or CD196).
[00101] In some embodiments of any of the aspects, the CD80/86 inhibitor inhibits the expression or activity in T cells of one or more genes or polypeptides selected from Table 1.
[00102] In some embodiments of any of the aspects, the subject can be further administered an immune checkpoint inhibitor (e.g., an inhibitor of the expression or activity of one or more immune checkpoint polypeptides), a calcineurin inhibitor, or a dihydrofolate reductase (DHFR) inhibitor.
[00103] In some embodiments of any of the aspects, the calcineurin inhibitor is an antibody or antigen-binding fragment thereof, an aptamer, or a calcineurin inhibitor fragment of a natural receptor or ligand of calcineurin.
[00104] As used herein,“calcineurin” or“CaN” refers to a serine/threonine protein phosphtase which is calcium and calmodulin dependent. Calcineurin positively resgulates T cells. Calcineurin is a homodimer of Calcinuerin A (NCBI Gene ID: 5530, 5532, or 5533) and Calcineurin B (NCBI Gene ID: 5534 or 5535). Exemplary but non-limiting calcineurin inhibitors include cyclosporine and tacrolimus.
[00105] As used herein,“dihydrofolate reductase” or“DHFR” refers to an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid as part of nucleic acid synthesis. The sequences of DHFR expression products are known for a number of species, e.g., human DHFR (NCBI Gene ID No: 1719) mRNA (NCBI Ref Seqs: NM 00791.4, NM_001290354.2, and NM_001290357.2) and polypeptide (NCBI Ref Seqs: NP 001277286.1, NP 001277283.1, and NP_000782.1). Exemplary but non-limiting DHFR inhibitors include methotrexate, trimethoprim, pyrimethamine, diaminopteridines,
diaminotriazines, diaminopyrroloquinazolines, stilbenes, chalcones, deoxybenzoins, diaminoquinazolines, diaminopyrimidines, and diaminotriazines.
[00106] The immune system has multiple inhibitory pathways that are critical for maintaining self tolerance and modulating immune responses. For example, in T-cells, the amplitude and quality of response isinitiated through antigen recognition by the T-cell receptor and is regulated by immune checkpoint proteins that balance co-stimulatory and inhibitory signals. In some embodiments of any of the aspects, a subject or patient is treated with at least one inhibitor of an immune checkpoint protein. As used herein, “immune checkpoint protein” refers to a protein which, when active, exhibits an inhibitory effect on immune activity, e.g., T cell activity. Exemplary immune checkpoint proteins can include PD-1 (e.g., NCBI Gene ID: 5133); PD-L1 (e.g, NCBI Gene ID: 29126); PD-L2 (e.g., NCBI Gene ID: 80380); TIM-3 (e.g, NCBI Gene ID: 84868); CTLA4 (e.g., NCBI Gene ID: 1493); TIGIT (e.g, NCBI Gene ID: 201633); KIR (e.g., NCBI Gene ID: 3811); LAG3 (e.g., NCBI Gene ID: 3902); DDl-a (e.g., NCBI Gene ID: 64115); A2AR (e.g, NCBI Gene ID: 135); B7-H3 (e.g, NCBI Gene ID: 80381); B7-H4 (e.g, NCBI Gene ID: 79679); BTLA (e.g, NCBI Gene ID: 151888); IDO (e.g, NCBI Gene ID: 3620); TDO (e.g, NCBI Gene ID: 6999); HVEM (e.g., NCBI Gene ID: 8764); GAL9 (e.g., NCBI Gene ID: 3965); 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, gd, and memory CD8+ (ab) T cells) (e.g., NCBI Gene ID: 51744); CD160 (also referred to as BY55) (e.g., NCBI Gene ID: 11126); and various B-7 family ligands. B7 family ligands include, but are not limited to, B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7.
[00107] Non-limiting examples of immune checkpoint inhibitors (with checkpoint targets and manufacturers noted in parantheses) can include :MGA271 (B7-H3: MacroGenics); ipilimumab (CTLA-4; Bristol Meyers Squibb); pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb) ; atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3: Immuntep); BMS-986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137; Bristol-Meyers Squibb); PF-05082566 (CD137; Pfizer); IPH2101 (KIR; Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX-1127 (CD27; CellDex); MEDI-6769 (0x40; Medlmmune); CP-870,893 (CD40; Genentech); tremelimumab (CTLA-4; Medimmune); pidilizumab (PD-1; Medivation); MPDL3280A (PD-L1; Roche); MEDI4736 (PD-L1; AstraZeneca); MSB0010718C (PD-L1; EMD Serono); AUNP12 (PD-1; Aurigene); avelumab (PD-L1; Merck); durvalumab (PD-L1; Medimmune); IMP321, a soluble Ig fusion protein (Brignone et ak, 2007, J. Immunol. 179:4202-4211); the anti-B7-H3 antibody MGA271 (Loo et ak, 2012, Clin. Cancer Res. July 15 (18) 3834); TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et ak, 2010, J. Exp. Med. 207:2175-86 and Sakuishi et ak, 2010, J. Exp. Med. 207:2187-94); anti-CTLA-4 antibodies described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238; tremelimumab, (ticilimumab, CP-675,206); ipilimumab (also known as 10D1, MDX- D010); PD-1 and PD-L1 blockers described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: W003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699; nivolumab (MDX 1106, BMS 936558, ONO 4538); lambrolizumab (MK-3475 or SCH 900475); CT-011; AMP-224; and BMS-936559 (MDX- 1105-01). The foregoing references are incorporated by reference herein in their entireties.
[00108] As described elsewhere herein, the methods described herein (e.g., the administration of an CD80/86 inhibitor), can result in inhibition of one or more immune checkpoint polypeptides. In some embodiments of the aspects, the immune checkpoint polypeptides are selected from CTLA4, Lag-3, HAVCR2 (TIM-3), PDCD1, Ki-67, CD-28. In some embodiments of the aspects, the immune checkpoint polypeptides are selected from CTLA4, Lag-3, HAVCR2 (TIM-3), PDCD1, Ki-67, CD-28.
[00109] As further described elsewhere herein, the methods described herein (e.g., the administration of an CD80/86 inhibitor), can result in reconstitution of a T cell population. In some embodiments of any of the aspects, the T cell population comprises CD3+, CD4+ and/or CD8+ T cells. In some embodiments of any of the aspects, the T cell population comprises Regulatory T cells (TregS). As used herein, "regulatory T cell" or "Treg" refers to those T cells (lymphocytes) that have immunoregulatory properties and the ability to suppress the proliferation and/or effector function of other T cell populations. A number of cell surface molecules are used to characterize and define Treg cells as described below herein.
Regulatory T cells or Treg cells play an important role for the maintenance of immunological tolerance by suppressing the action of autoreactive effector cells and have been shown to be critically involved in preventing the development of autoimmune reactions.
[00110] In some embodiments of any of the aspects, a Treg cell can be a T cell expressing one or markers selected from the group consisting of CTLA4; PDL1; LAP; GARP; CD25; and CD27. In some embodiments of any of the aspects, a Treg cell can be a CD8- (e.g., NCBI Gene ID: 925) CD4+ (e.g., NCBI Gene ID: 920) CD3+ cell.
[00111] "X+", wherein "X" is a cell surface marker, indicates the marker is present in the indicated cell, while "X-" indicates the marker is not present. One skilled in the art will be capable of assessing the molecules present on a cell using standard techniques, for example using immunofluorescence to detect commercially available antibodies bound to the marker molecules. Such designators are often used when sorting or identifying cells by FACS, in which gates can be established to divide cells based on the level of expression of the marker.
[00112] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a hematopoietic disease with one or more of the compositions or agents described herein. Subjects having a hematopoietic disease, e.g., cancer can be identified by a physician using current methods of diagnosing cancer.
[00113] As used herein, the term“hematopoietic disease” refers to any blood disorder including but not limited to hematopoietic malignancy, hematopoietic cancer, hemoglobinopathy, and immunodeficiency. In some embodiments of any of the aspects, the hematopoietic disease can be hematopoietic cancer or leukemia.
[00114] As used herein,“T cell lymphopenia” refers to a temporary or continuous decrease in lymphocytes (including but not limited to T cells, B cells and natural killer cells) in a subject. In some embodiments of any of the aspects, the T cells are Tregs. Exemplary conditions caused by or associated with T cell lymphopenia include but are not limited to Graft-versus-host disease (GvHD), non-relapse mortality (NRM), infection, and leukemia. In some embodiments, the subject is a subject who will or has received a bone marrow transplant. In some embodiments, the subject is a subject who will or has received a hematopoietic cell transplant (HCT), e.g, from an unrelated donor (URD).
[00115] As used herein,“T cell dysfuction” refers to a temporary or continuous decrease in T cell function in a subject. A temporary or continues descrease in T cell function includes but is not limited reduced proliferative capacity, decreased effector function, and overexpression of multiple inhibitory receptors on T cells. In some embodiments of any of the aspects, the T cells are Tregs. Exemplary conditions caused by or associated with T cell dysfunction include but are not limited to Graft-versus-host disease (GvHD), non-relapse mortality (NRM), infection, and leukemia. In some embodiments, the subject is a subject who will or has received a bone marrow transplant. In some embodiments, the subject is a subject who will or has received a hematopoietic cell transplant (HCT), e.g, from an unrelated donor (URD).
[00116] GvHD can be acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), or chronic Graft-versus-host disease (cGvHD).
[00117] In some embodiments of any of the aspects, the administration described herein can be prophylactic. In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein“prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to organ rejection or immune activity of a graft against the host. For example, the subject can be a subject who will or has received a bone marrow transplant but who has not exhibited a sign or symptom of GvHD at the time administration is commenced. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
[00118] The compositions and methods described herein can be administered to a subject having or diagnosed as having, e.g., a hematopoetic disease. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of e.g., a hematopoetic disease. As used herein, "alleviating a symptom" is ameliorating any condition or symptom associated with a disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
[00119] The term“effective amount" as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the composition that is sufficient to provide a particular effect when administered to atypical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact“effective amount". However, for any given case, an appropriate“effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
[00120] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e.. the concentration of the active ingredient, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for Treg activity or levels, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. [00121] In one aspect of any of the embodiments, described herein is a composition comprising a CD80/86 inhibitor; and at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor. Compositions comprising all possible pairwise, 3-way, and 4- way combinations are contemplated herein. In some embodiments of any of the aspects, the composition comprises a CD80/86 inhibitor and an immune checkpoint inhibitor. In some embodiments of any of the aspects, the composition comprises a CD80/86 inhibitor and a Calcineurin inhibitor. In some
embodiments of any of the aspects, the composition comprises a CD80/86 inhibitor and a dihydrofolate reductase (DHFR) inhibitor.
[00122] In one aspect of any of the embodiments, described herein is a combination comprising a CD80/86 inhibitor; and at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor, e.g., for use in a method described herein. Combinations comprising all possible pairwise, 3-way, and 4-way combinations are contemplated herein. In some embodiments of any of the aspects, the combination comprises a CD80/86 inhibitor and an immune checkpoint inhibitor. In some embodiments of any of the aspects, the combination comprises a CD80/86 inhibitor and a Calcineurin inhibitor. In some embodiments of any of the aspects, the combination comprises a CD80/86 inhibitor and a dihydrofolate reductase (DHFR) inhibitor. The two or more reagents of the combination can be present in the same solution, formulation, mixture, vial, or container. Alternatively, the two or more reagents of the combination can be present in separate solutions, formulations, mixtures, vials, or containers. For example, they can be provided in separate solutions in a single kit with instructions to administer them to the same patient, e.g., concurrently or sequentially.
[00123] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a CD80/86 inhibitor and one or more additional agents as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise a CD80/86 inhibitor as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of a CD80/86 inhibitor as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of a CD80/86 inhibitor as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent as described herein.
[00124] In some embodiments, the pharmaceutical composition comprising a CD80/86 inhibitor as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.
[00125] Suitable vehicles that can be used to provide parenteral dosage forms of a CD80/86 inhibitor as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a CD 80/86 inhibitor as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.
[00126] Pharmaceutical compositions comprising a CD80/86 inhibitor can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water- in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
[00127] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the a CD80/86 inhibitor can be administered in a sustained release formulation.
[00128] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled- release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
[00129] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
[00130] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza
Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
[00131] Im some embodiments of any of the apsects, the composition or combiantion described herein is administered as a monotherapy, e.g., another treatment for the disease or condition is not administered to the subject.
[00132] In some emboidments of any of the aspects, the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A;
bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophore s), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone -Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;
NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC -alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[00133] In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
[00134] In certain embodiments, an effective dose of a composition comprising a CD80/86 inhibitor as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a CD80/86 inhibitor can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising a CD80/86 inhibitor, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
[00135] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
[00136] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active ingredient. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. [00137] The dosage ranges for the administration of a CD80/86 inhibitor, according to the methods described herein depend upon, for example, the form of the active ingredient, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for immune system activity, inflammation, or the extent to which, for example, Tregs are desired to be induced. The dosage should not be so large as to cause adverse side effects, such as pathological immune system suppression. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
[00138] The efficacy of a CD80/86 inhibitor in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered“effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. Treg activity or checkpoint protein activity. Efficacy can also be measured by a failure of an individual to worsen as assessed by
hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. Treg activity or levels). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of acute and chronic GvHD. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a GVHD marker is observed, e.g. alteration in one ore more immune cell populations (including but not limited to increased CD3+ T cells, Thl7, CD4+ and CD8+ effector memory cells, monocytes, CD86 expression, BAFF/B cell ratio, and deficiency of Treg, NK cells, and naive CD8+ T cells) and/or alterations in one or more inflammatory and immunomodulatory polypeptides (including but not limited to TNFa, IL-6, IL-Ib, IL-8, sIL-2R, and IL-IRa, BAFF, anti-dsDNA, sIL-2Ra, and sCD13).
[00139] In vitro and animal model assays are provided herein which allow the assessment of a given dose of a CD80/86 inhibitor. By way of non-limiting example, the effects of a dose of a CD80/86 inhibitor can be assessed by measuringthe expression or activity of one or more inflammatory and immunomodulatory polypeptides (including but not limited to TNFa, IL-6, IL-Ib, IL-8, sIL-2R, and IL- lRa, BAFF, anti-dsDNA, sIL-2Ra, and sCD13). A non-limiting example of a protocol for such an assay is as follows assessing mRNA or protein levels of TNFa via, e.g., PCR-based assays, an enzyme-linked immunosorbent assay and/or western blotting, respectively. The efficacy of a given dosage combination can also be assessed in an animal model, e.g. mouse models of acute and chronic GvHD. For example, using sclerodermatous (pro-fibrotic) cGvHD models that are characterized by fibrotic changes in the dermis, which can involve the lung, liver and salivary glands. Fibrosis in the mouse models begins within 30 days of transplantation. The pathology is dependent on CD4+ T cells that release Th2 cytokines, which can stimulate other cells to release fibrosing cytokines (including but not limited to IL-13 and TϋRb) resulting in the sclerodermatous changes. Thefeore, the effects of a dose of a CD80/86 inhibitor can be assessed by measuring the fibrotic changes using immunohistochemical methods and by measuring the expression or activity of fribrosing cytokines including but not limited to IL-13 and TGFD e.g. using PCR-based assays, an enzyme-linked immunosorbent assay and/or western blotting, respectively.
[00140] By way of non-limiting example, the effects of a dose of a CD80/86 inhibitor can be assessed by measuring the expression or activity of one or more marker including CD4+cells and/or CD8+T cells. For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
[00141] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
[00142] The terms“decrease”,“reduced”,“reduction”, or“inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments,“reduce,”“reduction" or“decrease" or“inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein,“reduction” or“inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.“Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[00143] The terms“increased”,“increase”,“enhance”, or“activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms“increased”,“increase”, “enhance”, or“activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a“increase” is a statistically significant increase in such level.
[00144] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,”“patient” and“subject” are used interchangeably herein.
[00145] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease. A subject can be male or female.
[00146] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
[00147] A“subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
[00148] As used herein, the term“cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
[00149] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e.. division beyond normal limits), invasion (i.e.. intrusion on and destruction of adjacent tissues), and metastasis (i.e. , spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a“metastatic tumor” or a“metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term“benign” or“non- malignanf’ refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
[00150] A“cancer cell” or“tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g. , leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
[00151] As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm. [00152] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
[00153] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma;
rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome
[00154] A“cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.
Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
[00155] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
[00156] As used herein, the terms "treat,” "treatment," "treating,” or“amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term“treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a condition. Treatment is generally“effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective" if the progression of a disease is reduced or halted. That is,“treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
[00157] As used herein, the term“pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the
pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature. [00158] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
[00159] As used herein,“contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
[00160] The term“statistically significant" or“significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
[00161] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term“about.” The term“about” when used in connection with percentages can mean ±1%.
[00162] As used herein, the term“comprising” means that other elements can also be present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.
[00163] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[00164] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[00165] As used herein, the term“specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. [00166] In some embodiments of any of the aspects, the inhibitor is an inhibitory nucleic acid. In some embodiments of any of the aspects, inhibitors of the expression of a given gene can be an inhibitory nucleic acid. As used herein,“inhibitory nucleic acid” refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like. In some embodiments of any of the aspects, the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA).
[00167] Double -stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
[00168] As used herein, the term“iRNA” refers to an agent that contains RNA (or modified nucleic acids as described below herein) and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In some embodiments of any of the aspects, an iRNA as described herein effects inhibition of the expression and/or activity of a target, e.g. CD80/86. In some embodiments of any of the aspects, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA. In some embodiments of any of the aspects, administering an inhibitor (e.g. an iRNA) to a subject results in a decrease in the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the subject without the presence of the iRNA.
[00169] In some embodiments of any of the aspects, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target, e.g., it can span one or more intron boundaries. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive. In some embodiments of any of the aspects, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a“part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
[00170] Exemplary embodiments of types of inhibitory nucleic acids can include, e.g,. siRNA, shRNA,miRNA, and/or amiRNA, which are well known in the art. One skilled in the art would be able to design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of, e.g., CD80 or CD86, e.g., using publically available design tools. siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).
[00171] In some embodiments of any of the aspects, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and/or modified by methods well established in the art, such as those described in“Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In some embodiments of any of the aspects, the modified RNA will have a phosphorus atom in its intemucleoside backbone.
[00172] Modified RNA backbones can include, for example, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones;
sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular— CH2— NH— CH2— , —CH2—N(CH3)—0—CH2— [known as a methylene (methylimino) or MMI backbone], --CH2--0-- N(CH3)— CH2— ,— CH2— N(CH3)— N(CH3)— CH2— and -N(CH3)-CH2-CH2-[wherein the native phosphodiester backbone is represented as— O— P— O— CH2— ] .
[00173] In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
[00174] The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This stmcture effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
[00175] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein can include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, orN-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. Exemplary suitable modifications include 0[(CH2)nO] mCH3, 0(CH2).n0CH3, 0(CH2)nNH2, 0(CH2) nCH3,
0(CH2)n0NH2, and 0(CH2)n0N[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br,
CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments of any of the aspects, the modification includes a 2' methoxyethoxy (2'-0— CH2CH20CH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0— CH2— O— CH2— N(CH2)2, also described in examples herein below.
[00176] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
[00177] An inhibitory nucleic acid can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein,“unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6- azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3- deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the invention. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
[00178] The preparation of the modified nucleic acids, backbones, and nucleobases described above are well known in the art.
[00179] Another modification of an inhibitory nucleic acid featured in the invention involves chemically linking to the inhibitory nucleic acid to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et ah, Biorg. Med. Chem. Let., 1994, 4: 1053-1060), athioether, e.g., beryl-S-tritylthiol (Manoharan et ah, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et ah, Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol
(Oberhauser et ah, Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et ah, EMBO J, 1991, 10: 1111-1118; Kabanov et ah, LEBS Lett., 1990, 259:327-330; Svinarchuk et ah, Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethyl -ammonium l,2-di-0-hexadecyl-rac-glycero-3-phosphonate (Manoharan et ah, Tetrahedron Lett., 1995, 36:3651-3654; Shea et ah, Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino- carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
[00180] As used herein,“ligand” refers to a molecule which can bind, e.g., specifically bind, to a second molecule or receptor. In some embodiments, a ligand can be, e.g., an polypeptide, an antibody, antibody fragment, antibody portion, and/or affibody. [00181] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "enzyme,"
"amino acid chain," and "contiguous amino acid sequence" are all encompassed within the definition of a "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures. For example, one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; lie), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys). Amino acid residues provided herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue provided the desired properties of the polypeptide are retained.
[00182] The term "homology" as used herein refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue ( e.g similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is "unrelated" or "non- homologous" shares less than 40% identity. Determination of homologs of the genes or peptides described herein may be easily ascertained by the skilled artisan.
[00183] The sequences provided here can be modified, comprise conservative amino acid
substitutions, or have additional amino acids that can improve targeting or efficacy of the composition described herein. In some embodiments of any of the aspects, the first polypeptide has an amino acid sequence with at least 99% homology to the second polypeptide. In some embodiments of any of the aspects, the third polypeptide has an amino acid sequence with at least 99% homology to the fourth polypeptide. In some embodiments of any of the aspects, the first polypeptide has an amino acid sequence that is non-homologous to the second polypeptide. In some embodiments of any of the aspects, the third polypeptide has an amino acid sequence that is non-homologous to the fourth polypeptide. In some embodiments of any of the aspects, the first or second polypeptide has an amino acid sequence that is non-homologous to the third and/or fourth polypeptides.
[00184] The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity, fore examples, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. "Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Thus, a "conservative substitution" of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not substantially alter activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984).) In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservative substitutions." Insertions or deletions are typically in the range of about 1 to 5 amino acids.
[00185] Conservative substitutions that permit the formation of the tetrameric coiled coil structure described herein can be used. For example, directed evolution can be used to subject the polypeptides described herein to random mutagenesis and the resulting polypeptides are screened for desired qualities (e.g, using circular dichroism or binding assays). These methods are known in the art. See Wang et al. Cell, Volume 160, Issue 4, 2015, Pages 785-797; or Daugherty et al. Protein Engineering, Design and Selection, Volume 11, Issue 9, 1998, Pages 825-832.
[00186] As used herein the term,“aptamer” refers to single-stranded nucleic acids that are capable of binding to cells and target molecules. Nucleic acid aptamers include RNA, DNA, and/or synthetic nucleic acid analogs (e.g., PNA) capable of specifically binding target molecules. Aptamers are an attractive alternative to antibodies for cell selection because of their high level of specificity and affinity for cell surface markers. As used herein, the term "nucleic acid" includes one or more types of:
polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites).
[00187] The term "nucleic acid," as used herein, also includes polymers of ribomicleosides or deoxyribomicleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like. "Nucleic acids" include single- and double -stranded DNA, as well as single- and double -stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.
[00188] The term "antibody" or“antibody reagent” broadly refers to any immunoglobulin (Ig) molecule or compositions of Igs and/or immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen) comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Nonlimiting embodiments of which are discussed below, and include but are not limited to a variety of forms, including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a human antibody, a humanized antibody, a single chain antibody, a Fab, a F(ab’), a F(ab’)2, a Fv antibody, fragments produced by a Fab expression library, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope -binding fragment thereof, bifunctional hybrid antibodies ( e.g ., Fanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains ( e.g ., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference) and/or antigen-binding fragments of any of the above (See, generally, Hood et al., Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Fane, Antibodies. A Laboratory Manual, Cold Spring Harbor Faboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference). Antibodies also refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen or target binding sites or“antigen-binding fragments.” The antibody or immunoglobulin molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2,
IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule, as is understood by one of skill in the art. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.
[00189] In a full-length antibody, each heavy chain is comprised of a heavy chain variable domain (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains: CHI, CH2, and CH3. Each light chain is comprised of a light chain variable domain (abbreviated herein LCVR as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well-known to those skilled in the art. The chains are usually linked to one another via disulfide bonds.
[00190] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain, and a CH3 domain, and optionally comprises a CH4 domain.
Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, for example, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgGl and IgG3, mediate ADCC and CDC via binding to Fc.gamma.Rs and complement Clq, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half- life of antibodies. In still another embodiment at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered.
[00191] The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD80 or CD86). Antigen-binding functions of an antibody can be performed by fragments of a full-length antibody. Such antibody fragment embodiments may also be incorporated in bispecific, dual specific, or multi-specific formats such as a dual variable domain (DVD-Ig) format; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature, 341: 544-546; PCT Publication No. WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
[00192] Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123); Kontermann and Dubel eds., Antibody Engineering, Springer- Verlag, N.Y. (2001), p. 790 (ISBN 3-540-41354-5). As used herein, a“bispecific antibody” refers to an antibody that can simultaneously bind to two different types of antigen.
[00193] In addition, single chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. (1995) Protein Eng. 8(10): 1057-1062; and U.S. Pat. No. 5,641,870). An immunoglobulin constant (C) domain refers to a heavy (CH) or light (CL) chain constant domain. Murine and human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.
[00194] In some embodiments of any of the aspects, the antibody reagent is a humanized antibody.
[00195] The term "humanized antibody" refers to antibodies that comprise heavy and light chain variable domain sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. Accordingly, "humanized" antibodies are a form of a chimeric antibody, that are engineered or designed to comprise minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). As used herein, a“composite human antibody” or“deimmunized antibody” are specific types of engineered or humanized antibodies designed to reduce or eliminate T cell epitopes from the variable domains.
[00196] One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. Also "humanized antibody" is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab'). sub.2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In an embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments of any of the aspects, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments of any of the aspects, a humanized antibody only contains a humanized light chain. In some embodiments of any of the aspects, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain. A humanized antibody may be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype including without limitation IgGl, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.
[00197] The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In an exemplary embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.
[00198] A "human antibody,”“non-engineered human antibody,” or“fully human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art.
In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al. Proc. Natl. Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous mouse immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications:
Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).
Alternatively, the human antibody can be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (such B lymphocytes can be recovered from an individual or can have been immunized in vitro). See, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol., 147 (l):86-95 (1991); and U.S. Pat. No. 5,750,373.
[00199] As described herein, an "antigen" is a molecule that is bound by a binding site on a polypeptide agent, such as a binding protein, an antibody or antibody fragment, or antigen-binding fragment thereof. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule. In the case of conventional antibodies and fragments thereof, the antibody binding site as defined by the variable loops (LI, L2, L3 and HI, H2, H3) is capable of binding to the antigen. The term "antigenic determinant" refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.
[00200] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by a binding protein. An epitope may be determined by obtaining an X-ray crystal structure of an antibody: antigen complex and determining which residues on the antigen are within a specified distance of residues on the antibody of interest, wherein the specified distance is, 5Ά or less, e.g., 5 A, 4A, 3 A, 2A, lA or any distance in between. In some embodiments of any of the aspects, an“epitope” can be formed on a polypeptide both from contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. An "epitope" includes the unit of structure conventionally bound by an immunoglobulin VH VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation. The terms "antigenic
determinant" and "epitope" can also be used interchangeably herein. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. In some embodiments of any of the aspects, an epitope comprises of 8 or more contiguous or non-contiguous amino acid residues in the target sequence in which at least 50%, 70% or 85% of the residues are within the specified distance of the antibody or binding protein in the X-ray crystal structure.
[00201] The term "antibody fragment,” or“antigen-binding fragment” as used herein, refer to a protein fragment that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain;
(v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al, Nature 341, 544-546 (1989)) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870).
[00202] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00203] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00204] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978- 0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek,
David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.
[00205] One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see
Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).
[00206] Other terms are defined herein within the description of the various aspects of the invention.
[00207] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[00208] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[00209] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
[00210] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A method for treating or preventing T cell lymphopenia or a condition caused by or
associated with T cell lymphopenia, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor abatacept, thereby inhibiting the expression or activity of one or more immune checkpoint polypeptides in T cells and reconstituting the T cell population.
2. The method of paragraph 1, wherein the condition caused by or associated with T cell lymphopenia is selected from Graft-versus-host disease (GvHD) or leukemia.
3. A method for treating or preventing GvHD, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor.
4. The method of paragraph 3, wherein the administration is prophylactic.
5. The method of any of paragraphs 1-4, wherein the subject is a subject who is or has
received a bone marrow transplant.
6. A method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a bone marrow transplant to the subject; and
b. administering a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor.
7. The method of paragraph 6, wherein the hematopoietic disease is a hematopoietic cancer.
8. The method of paragraph 6, wherein the hematopoietic disease is leukemia. The method of any of paragraphs 1-8, wherein the CD80/86 inhibitor is an antibody or antigen-binding fragment thereof, an aptamer, or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86. The method of any of paragraphs 1-9, wherein the CD80/86 inhibitor binds specifically to CD80 and/or 86. The method of any of paragraphs 1-10, wherein the CD80/86 inhibitor is abatacept. The method of any of paragraphs 1-11, wherein the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. The method of paragraph 12, wherein the receptor is CTLA-4 and/or CD28 The method of any of paragraphs 1-13, wherein the immune checkpoint polypeptides are selected from CTLA4, Ki-67, CD-28. The method of any of paragraphs 1-14, wherein the immune checkpoint polypeptides are selected from CTLA4, Ki-67, CD-28, PD-1, TIM-3, and LAG-3. The method of any of paragraphs 1-15, wherein the T cell population comprises CD3+, CD4+ and/or CD8+ T cells. The method of any of paragraphs 1-16, wherein the T cell population comprises
Regulatory T cells (TregS). The method of any of paragraphs 1-17, wherein the inhibitor is targeted to T-cells. The method of paragraph 18, wherein the inhibitor comprises a T cell targeting moiety. 20. The method of paragraph 19, wherein the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide.
21. The method of paragraph 20, wherein the T cell-specific cell surface polypeptide is
selected from the group consisting of CD3, CD4, CD8, CD25, CD127, and/or CD196.
22. The method of any of paragraphs 19-21, wherein the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof, an aptamer, or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
23. The method of any of paragraphs 1-22, further comprising administering a therapeutically effective amount of an inhibitor of the expression or activity of one or more immune checkpoint polypeptides.
24. The method of any of paragraphs 1-23, wherein the condition caused by or associated with T cell lymphopenia is selected from Graft-versus-host disease (GvHD) or leukemia.
[00211] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A method for treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction, the method comprising:
administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor;
thereby inhibiting the expression or activity of one or more immune checkpoint polypeptides in T cells and reconstituting the T cell population.
2. The method of paragraph 1, wherein the condition caused by or associated with T cell
dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia and lymphoma relapse. A method for treating or preventing GvHD, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CD 80/86 inhibitor. The method of paragraph 3, wherein the administration is prophylactic. The method of any of paragraphs 1-4, wherein the subject is a subject who is in need of or has received a bone marrow transplant. A method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a bone marrow transplant to the subject; and
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor. The method of paragraph 6, wherein the hematopoietic disease is a hematopoietic cancer. The method of paragraph 6, wherein the hematopoietic disease is leukemia. The method of any of paragraphs 1-8, wherein the CD80/86 inhibitor is an antibody or antigen binding fragment thereof; an aptamer; or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86. The method of any of paragraphs 1-9, wherein the CD80/86 inhibitor binds specifically to CD80 and/or 86. The method of any of paragraphs 1-10, wherein the CD80/86 inhibitor is abatacept. The method of any of paragraphs 1-11, wherein the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. The method of paragraph 12, wherein the receptor is CTLA-4 and/or CD28. The method of any of paragraphs 1-13, wherein the immune checkpoint polypeptides are selected from the group consisting of CTLA4, Lag-3, HAVCR2 (TIM-3), and/or PDCD1. The method of any of paragraphs 1-14, wherein the immune checkpoint polypeptides are selected from the group selected from Lag-3, HAVCR2 (TIM-3), and/or PDCD1. The method of any of paragraphs 1-15, wherein the T cell population comprises CD3+, CD4+, and/or CD8+ T cells. The method of any of paragraphs 1-16, wherein the T cell population comprises Regulatory T cells (TregS). The method of any of paragraphs 1-17, wherein the inhibitor is targeted to T-cells. The method of paragraph 18, wherein the inhibitor comprises a T cell targeting moiety. The method of paragraph 19, wherein the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide. The method of paragraph 20, wherein the T cell-specific cell surface polypeptide is selected from the group consisting of CD3, CD4, and/or CD8. The method of any of paragraphs 19-21, wherein the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof; an aptamer; or a natural ligand that specifically binds the T cell-specific cell surface polypeptide. The method of any of paragraphs 1-22, further comprising administering a therapeutically effective amount of an inhibitor of the expression or activity of one or more immune checkpoint polypeptides. The method of any of paragraphs 1-23, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia, and lymphoma relapse. The method of any of the preceding paragraphs, further comprising administering a
therapeutically effective amount of a Calcineurin inhibitor. The method of paragraph 25, wherein the Calcineurin inhibitor binds specifically to Calcineurin. The method of any of paragraphs 25-26, wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus. The method of any of paragraphs 25-27, wherein the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. The method of any of the preceding paragraphs, further comprising administering a
therapeutically effective amount of a dihydrofolate reductase (DHFR) inhibitor. The method of paragraph 29, wherein the dihydrofolate reductase (DHFR) inhibitor is an antibody or antigen-binding fragment thereof; an aptamer; or a dihydrofolate reductase (DHFR) inhibitor fragment of a natural receptor or ligand of dihydrofolate reductase (DHFR). The method of any of paragraphs 29-30, wherein the dihydrofolate reductase (DHFR) inhibitor binds specifically to dihydrofolate reductase (DHFR). The method of any of paragraphs 29-31, wherein the dihydrofolate reductase (DHFR) inhibitor is methotrexate. The method of any of paragraphs 29-32, wherein the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. The method of any of the preceding paragraphs, wherein the CD80/86 inhibitor inhibits the expression or activity of one or more T cell polypeptides selected from the polypeptides listed in Table 1. The method of any of the preceding paragraphs, wherein the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus- host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD). The method of any of the preceding paragraphs, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of non-relapse mortality (NRM) and infection. The method of any of the preceding paragraphs, wherein the subject is a subject who is in need of or who has received a Hematopoietic Cell Transplant (HCT). The method of paragraph 37, wherein the Hematopoietic Cell Transplant (HCT) originates from an Unrelated Donor (URD). A composition comprising:
a. a CD80/86 inhibitor; and
b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a
dihydrofolate reductase (DHFR) inhibitor. The composition of paragraph 39, wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus. The composition of any of paragraphs 39-40, wherein the dihydrofolate reductase (DHFR) inhibitor is methotrexate. The composition of any of paragraphs 39-41, wherein the CD80/86 inhibitor is abatacept. A method of treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction in a subject in need thereof, the method comprising administering to the subject a composition of any of the preceding paragraphs. The method of paragraph 43, wherein the condition caused by or associated with T cell dysfunction is selected from the group of Graft-versus-host disease (GvHD) and leukemia. 45. The method of paragraph 44, wherein the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
46. A method of treating or preventing a hematopoietic disease in a subject in need thereof, the
method comprising administering to the subject a composition of any of the preceding paragraphs.
47. A method of treating or preventing a non-relapse mortality (NRM) in a subject who is need of or who has received a Hematopoietic Cell Transplant (HCT), the method comprising administering to the subject a composition of any of the preceding paragraphs.
48. The method of paragraph 47, wherein the Hematopoietic Cell Transplant (HCT) originates from an Unrelated Donor (URD).
49. A method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a hematopoietic stem transplant to the subject;
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor; and
c. further administering a therapeutically effective amount of a Calcineurin inhibitor and/or a therapeutically effective amount of a dihydrofolate reductase inhibitor (DHFR).
50. The method of paragraph 49, wherein the hematopoietic disease is a hematopoietic cancer.
51. The method of paragraph 49, wherein the hematopoietic disease is leukemia.
[00212] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A method for treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction, the method comprising:
administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor; thereby inhibiting the expression or activity of one or more immune checkpoint polypeptides in T cells and reconstituting the T cell population. The method of paragraph 1, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia and lymphoma relapse. A method for treating or preventing GvHD, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CD 80/86 inhibitor. The method of paragraph 3, wherein the administration is prophylactic. The method of any of paragraphs 1-4, wherein the subject is a subject who is in need of or has received a bone marrow transplant. A method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a bone marrow transplant to the subject; and
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor. The method of paragraph 6, wherein the hematopoietic disease is a hematopoietic cancer. The method of paragraph 6, wherein the hematopoietic disease is leukemia. The method of any of paragraphs 1-8, wherein the CD80/86 inhibitor is an antibody or antigen binding fragment thereof; an aptamer; or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86. The method of any of paragraphs 1-9, wherein the CD80/86 inhibitor binds specifically to CD80 and/or 86. The method of any of paragraphs 1-10, wherein the CD80/86 inhibitor is abatacept. The method of any of paragraphs 1-11, wherein the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. The method of paragraph 12, wherein the receptor is CTLA-4 and/or CD28. The method of any of paragraphs 1-13, wherein the immune checkpoint polypeptides are selected from the group consisting of CTLA4, Lag-3, HAVCR2 (TIM-3), and/or PDCD1. The method of any of paragraphs 1-14, wherein the immune checkpoint polypeptides are selected from the group selected from Lag-3, HAVCR2 (TIM-3), and/or PDCD1. The method of any of paragraphs 1-15, wherein the T cell population comprises CD3+, CD4+, and/or CD8+ T cells. The method of any of paragraphs 1-16, wherein the T cell population comprises Regulatory T cells (TregS). The method of any of paragraphs 1-17, wherein the inhibitor is targeted to T-cells. The method of paragraph 18, wherein the inhibitor comprises a T cell targeting moiety. The method of paragraph 19, wherein the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide. The method of paragraph 20, wherein the T cell-specific cell surface polypeptide is selected from the group consisting of CD3, CD4, and/or CD8. The method of any of paragraphs 19-21, wherein the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof; an aptamer; or a natural ligand that specifically binds the T cell-specific cell surface polypeptide. The method of any of paragraphs 1-22, further comprising administering a therapeutically effective amount of an inhibitor of the expression or activity of one or more immune checkpoint polypeptides. The method of any of paragraphs 1-23, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia, and lymphoma relapse. The method of any of the preceding paragraphs, further comprising administering a
therapeutically effective amount of a Calcineurin inhibitor. The method of paragraph 25, wherein the Calcineurin inhibitor binds specifically to Calcineurin. The method of any of paragraphs 25-26, wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus. The method of any of paragraphs 25-27, wherein the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. The method of any of the preceding paragraphs, further comprising administering a
therapeutically effective amount of a dihydrofolate reductase (DHFR) inhibitor. The method of paragraph 29, wherein the dihydrofolate reductase (DHFR) inhibitor is an antibody or antigen-binding fragment thereof; an aptamer; or a dihydrofolate reductase (DHFR) inhibitor fragment of a natural receptor or ligand of dihydrofolate reductase (DHFR). The method of any of paragraphs 29-30, wherein the dihydrofolate reductase (DHFR) inhibitor binds specifically to dihydrofolate reductase (DHFR). The method of any of paragraphs 29-31, wherein the dihydrofolate reductase (DHFR) inhibitor is methotrexate. The method of any of paragraphs 29-32, wherein the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor. The method of any of the preceding paragraphs, wherein the CD80/86 inhibitor inhibits the expression or activity of one or more T cell polypeptides selected from the polypeptides listed in Table 1. The method of any of the preceding paragraphs, wherein the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus- host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD). The method of any of the preceding paragraphs, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of non-relapse mortality (NRM) and infection. The method of any of the preceding paragraphs, wherein the subject is a subject who is in need of or who has received a Hematopoietic Cell Transplant (HCT). The method of paragraph 37, wherein the Hematopoietic Cell Transplant (HCT) originates from an Unrelated Donor (URD). A composition comprising:
a. a CD80/86 inhibitor; and
b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a
dihydrofolate reductase (DHFR) inhibitor. The composition of paragraph 39, wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus. The composition of any of paragraphs 39-40, wherein the dihydrofolate reductase (DHFR) inhibitor is methotrexate. The composition of any of paragraphs 39-41, wherein the CD80/86 inhibitor is abatacept. A method of treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction in a subject in need thereof, the method comprising administering to the subject a composition of any of the preceding paragraphs. The method of paragraph 43, wherein the condition caused by or associated with T cell dysfunction is selected from the group of Graft-versus-host disease (GvHD) and leukemia. The method of paragraph 44, wherein the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD). A method of treating or preventing a hematopoietic disease in a subject in need thereof, the method comprising administering to the subject a composition of any of the preceding paragraphs. A method of treating or preventing a non-relapse mortality (NRM) in a subject who is need of or who has received a Hematopoietic Cell Transplant (HCT), the method comprising administering to the subject a composition of any of the preceding paragraphs. The method of paragraph 47, wherein the Hematopoietic Cell Transplant (HCT) originates from an Unrelated Donor (URD). A method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a hematopoietic stem transplant to the subject;
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor; and
c. further administering a therapeutically effective amount of a Calcineurin inhibitor and/or a therapeutically effective amount of a dihydrofolate reductase inhibitor (DHFR). The method of paragraph 49, wherein the hematopoietic disease is a hematopoietic cancer. The method of paragraph 49, wherein the hematopoietic disease is leukemia. A pharmaceutical composition formulated for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction, comprising:
a. a CD 80/86 inhibitor; and
b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor; and
c. a pharmaceutically acceptable carrier. A pharmaceutical composition of paragraph 52 for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft- versus-host disease (GvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD), leukemia and lymphoma relapse. The pharmaceutical composition of paragraph 52 for the treatment or prevention of a
hematopoietic disease in a subject in need thereof. The pharmaceutical composition of paragraph 52 for the treatment of a hematopoietic disease, wherein the hematopoietic disease is a hematopeitic cancer. The pharmaceutical composition of paragraph 52 for the treatment or prevention of a non-relapse mortality (NRM) in a subject who is need of or who has received a Hematopoietic Cell
Transplant (HCT) comprising administering to the subject a composition of any of the preceding paragraphs.
EXAMPLES
Example 1: Control of relapse when abatacept is combined with calcineurin inhibition and methotrexate: an unexpected discovery made with the ABA2 trial.
[00213] The ABA2 trial was designed by the inventors to improve survival after unrelated-donor hematopoietic stem cell transplantation (HCT) by adding the T cell costimulation blockade agent, abatacept, to background immunosuppression with a standard regimen containing a calcineurin inhibitor and methotrexate. Abatacept is an immunosuppressive drug, marketed by Bristol Myers Squibb, which is approved for Rheumatoid Arthritis. The hoped-for effect in the ABA2 trial was that the addition of abatacept would decrease the most deadly complication of HCT, which is a syndrome called“Acute Graft-versus-Host Disease” (AGVHD). Severe AGVHD is caused by over-activation of the immune system after transplant, which leads to organ damage in patients that is caused by the transplanted T cells.
[00214] Results: The ABA2 had the hoped for outcome with respect to AGVHD: Patients prophylaxed with abatacept demonstrated significantly less AGVHD than patients who received standard calcineurin inhibition + methotrexate alone. The magnitude of this effect has been significant, and has led to the FDA granting abatacept a Breakthrough Therapy Designation for prevention of GVHD after unrelated donor HCT. While improvement in AGVHD was the anticipated outcome of ABA2, the inventors have made an additional discovery in the course of analyzing the data for ABA2 that was totally unexpected given the previously -understood mechanism of action of abatacept. The inventors found that the addition of abatacept for AGVHD prophylaxis did not increase relapse after transplant. In one cohort of ABA2 patients (called the“7/8 cohort”), the one-year relapse rates were 9.3% in the
CNI/MTX+abatacept patients compared to 15.4% in CNI/MTX patients. Notably, there were no relapses in this abatacept cohort after 6 months post-transplant, with the 2-year relapse rates for these patients remaining at 9.3% versus 21.4% in the CNI/MTX patients. In the second cohort of ABA2 patients (called the“8/8 cohort”) the one-year relapse rates were 13.8% for patients receiving abatacept and 20.5% for patients receiving placebo. To begin to determine the underlying mechanisms that could explain the control of relapse in the ABA2 trial, the inventors analyzed the transcriptomes of CD4+ T cells reconstituting after transplant in patients on this study. This analysis revealed a previously undocumented, and unexpected finding: CD4+ T cells reconstituting in abatacept-prophylaxed patients demonstrated downregulation of multiple checkpoint molecules (including CTLA4, PD1, LAG3 and TIM-3) compared to patients receiving placebo. This observation indicates that T cells that cause AGVHD are unable to provide anti-leukemic effects due to their upregulation of these checkpoint molecules. The fact that abatacept not only decreased the expression of these checkpoint molecules, but also uncoupled their linkage with AGVHD indicates that this downregulation of checkpoint pathways account for the control of relapse in patients treated with CNI/MTX + abatacept, despite the fact that these patients received more immunosuppression than the patients treated with placebo.
Example 2: Transcriptomic Analysis of CD4+ T Cell Dysfunction during GVHD: Evidence for Profound Reprograming of T Cell Signaling during Acute Gvhd That Is Controlled during CD28:CD80/86 Costimulation Blockade with Abatacept
[00215] Although acute graft-versus-host-disease (AGVHD) is one of the major causes of non-relapse mortality after hematopoietic stem cell transplant (HCT), no one can predict which patients will develop the most severe form of this disease, or which molecular pathways are dysregulated in the T cells that cause disease. Thus, understanding the molecular features of AGVHD is a critical unmet need. To address this, the inventors have performed a companion mechanistic study as a part of our completed Phase 2 trial of abatacept, a CD28:CD80/86 costimulation blockade agent, for severe AGVHD prevention
(Clinicaltrials.gov # NCT01743131,‘ABA2’). ABA2 has demonstrated significant improvement in AGVHD in patients prophylaxed with abatacept in addition to calcineurin inhibition (CNI) +
Methotrexate (MTX) compared to controls receiving CNI/MTX alone. To begin to uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug, the inventors interrogated the transcriptome of CD4+ T cells reconstituting in patients prophylaxed with abatacept compared to CNI/MTX.
[00216] To perform this analysis, the inventors flow cytometrically sorted CD4+ T cells on Days 21- 28 post-transplant from all patients on ABA2, as well as a cohort of 12 untransplanted healthy controls, and subsequently performed mRNA-sequencing on these cells. Weighted Gene Correlation Network Analysis (W GCNA) was performed on the top 6000 most variant transcripts from the resulting sequencing data. Hierarchical clustering of the WGCNA co-expression matrix enabled the identification of self-assembling modules (SAMs) that met a threshold of coexpression (FIGs. 1A-1D). For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), +/- prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0-4), relapse, non-relapse mortality, and all-cause mortality.
[00217] The WGCNA clustering analysis resulted in the identification of 4 discrete SAMs, which were highly correlated with clinical variable metamodules. This analysis revealed a strong positive correlation of a 476-gene SAM (the Turquoise module) in patients prophylaxed with CNI/MTX + placebo and anti-correlation of this module in patients prophylaxed with CNI/MTX + abatacept, as demonstrated in both the WGCNA heatmap and through Gene Set Enrichment Analysis (FIGs. 1A-1D). These opposing correlations suggested that interrogation of this module would reveal mechanistic correlates with standard prophylaxis that were decoupled by abatacept. Pathway analysis using the Reactome database (FIGs. 1A-1D) revealed the turquoise SAM to be dominated by four types of pathways: (1) Those that define canonical cell-cycle pathways (2) Those involved in T cell metabolism (3) Those involved in apoptosis and (4) Those involved in T cell activation, consistent with upregulation of these transcripts in placebo versus abatacept patients.
[00218] In addition to being highly correlated with patients receiving placebo, the expression of a subset of the transcripts in the Turquoise module were also directly correlated with the severity of AGVHD in these patients. Thus, linear regression analysis of the 476 transcripts in this module identified a subset of 93 genes for which transcript expression level was increased both in placebo compared to abatacept, and for which expression level also positively correlated with Grade of AGVHD. As with the Turquoise module as a whole, this subset of genes also formed a highly correlated network, linking transcripts involved in T cell proliferation, apoptosis, activation, metabolism as well as the T cell checkpoint (FIGs. 1A-1D).
[00219] This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from
AGVHD severity. These results indicate a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD.
Example 3: Costimulation Blockade with Abatacept for severe acute GVHD
[00220] Allogeneic HCT is an effective treatment for aggressive hematologic malignancies, often representing the only option for cure. For patients lacking HFA-matched related donors, unrelated donors (URD) are often used. The major disadvantage of URD HCT is an increased risk for non-relapse mortality (NRM) mediated by severe acute GVHD (AGVHD), chronic GVHD (CGVHD) and infection1 7. The use of an HFA mismatched URD accentuates these risks, and carries a higher incidence of both severe AGVHD (up to 37%3) and NRM (up to 45%3). Notably, the majority of patients in ethnic minorities will not have an HFA-matched URD, but will have a 7/8 URD donor available8, underscoring the importance of improving the safety of these high-risk transplants.
[00221] The T cell-mediated immune activation that causes AGVHD mirrors that occurring during organ rejection and autoimmunity. Studies in these diseases have led to the development of T cell costimulation blockade agents that inhibit T cell activation.9 One of the most potent approaches occurs through blockade of CD28:CD80/CD86 signaling10, with the first costimulation blockade agent,
CTLA4Ig, or‘abatacept,’ approved for use in rheumatoid arthritis11 14 and the related compound, belatacept, approved for renal transplantation15 16.
[00222] Preclinical data demonstrate that CTLA4-Ig can prevent GVHD 17-24. These results provided the rationale for the first-in-disease trial of abatacept for GVHD prevention (ClinicalTrials.org
#NCT01012492)25 which established the feasibility and general safety of the approach. This Phase 2 trial of abatacept for the prevention of severe AGVHD (‘ABA2’, #NCT01743131) was designed to test the hypothesis that abatacept could lower the risk of severe AGVHD for patients receiving either 7/8 URD HCT or 8/8 URD HCT, thereby improving transplant outcomes.
[00223] Methods
[00224] Patients and Treatment: ABA2 is a Phase 2 study of abatacept in addition to standard calcineurin inhibition (‘CNT, using either cyclosporine or tacrolimus) and methotrexate (‘MTX’, 4 doses, 15 mg/m2 on Day +1 and 10 mg/m2 on Days +3, +6 and +11 relative to transplant). CNI was continued through day 100, and weaned between days 100-180 post-transplant as tolerated. For patients receiving abatacept, four doses were delivered, 10 mg/kg/dose, on Days -1, +5, +14, +28. Pre-transplant conditioning used one of four regimens: busulfan/fludarabine, busulfan/cyclophosphamide, total body irradiation/cyclophosphamide, fludarabine/melphalan (details in the ABA2 protocol (see, e.g. Example 4).
[00225] This trial had two strata: For 8/8 patients, a randomized double-blind placebo-controlled design was utilized, with patients randomized 1: 1 to abatacept or placebo. For 7/8 patients, a single arm open label design was used, with a pre-specified matched analysis comparing outcomes with patients from the CIBMTR registry, transplanted between 2008-2015. Details of control selection are provided in the ABA2 protocol (see also Example 4). It is important to note that the 7/8 stratum was initially designed as a randomized, double-blind placebo-controlled trial. However, after more than a year of open enrollment, the 7/8 stratum was experiencing very slow enrollment (FIGs. 13A-13B) due to reluctance on the treating physicians part to randomize such high risk patients to placebo. The trial was therefore amended and all patients receiving 7/8 URD HCT were subsequently assigned to CNI/MTX+abatacept as an open-label single arm stratum. The matched-control parameters for the CIBMTR registry controls were stipulated at the time of this amendment.
[00226] Study participants included pediatric (>6 years old) and adult patients with hematologic malignancies who met disease eligibility criteria (see Supplemental Materials). The trial was open from 3/1/2013 to 11/15/2017, with the 7/8 stratum completing accrual on 11/30/2016 and the 8/8 stratum completing accrual on 11/15/2017. ABA2 was open to accrual at 21 sites with 14 sites enrolling patients. [00227] Endpoints: The primary endpoint for this trial was the cumulative incidence (Cl) of severe (Gr 3-4) AGVHD at Day +100. Secondary endpoints included: (1) Gr 3-4 AGVHD at Day +180; (2) Severe (Gr 3-4) AGVHD-Free Survival (‘SGFS’) at Day 180; (3) Gr 2-4 AGVHD at Days +100 and +180; (4) CGVHD at 1 year; (5) NRM at Day +180 and 1 Year, (6) Relapse at One Year; (7) Relapse- Free Survival (RFS) at Day +180 and 1 Year; (6) OS at Day +180 and One Year; (8) CMV Reactivation and Disease at Day 180; (9) EBV Reactivation and post-transplant lymphoproliferative disease (PTLD) at Day 180; (10) Hematologic (neutrophil and platelet) recovery and (11) Donor engraftment. During post- hoc analysis, the Cl of steroid-refractory AGVHD (defined by the need for second-line therapy, or lack of response to steroids at day 28 after start of AGVHD treatment) was also compared in 8/8s (the necessary treatment data was not available for the CIBMTR controls for 7/8 analysis). Details of GVHD grading, chimerism analysis, adverse event grading, and study oversight, including immune reconstitution, biomarker and transcriptomic analysis: details of these analyses can be found e.g. in the ABA2 protocol (see, e.g. Example 4).
[00228] Statistical Analysis
[00229] Statistical analysis was performed in the modified intent-to treat (ITT) population, which included all patients who received at least on dose of study medication. A screening Phase-2 design (Bayesian Type I error probability <0.2 and Bayesian power > 0.8) was used for sample size
calculations26. The decision rule was to reject the null hypothesis if p8/8 > 0.8 for the 8/8 HLA-matched URD and p7/8 > 0.8 for the 7/8 HLA-mismatched URD. For the 8/8 stratum, a sample size of 70 per arm was calculated to achieve 80% Bayesian power to detect a reduction in severe AGVHD from 20% to 10% (Bayesian Type I probability < 0.2). Patients in the 8/8 cohort were randomized at a 1: 1 ratio using a non- adaptive randomization with a block size of 8 stratified by patient age (patients <21 years of age versus patients > 21 years). For the 7/8 stratum, a sample size of 40 patients was calculated to achieve 80% Bayesian power to detect a reduction in severe AGVHD from 30% to 10% (Bayesian Type I probability < 0.2). The control arm was constructed using a cohort from the CIBMTR. Up to 4 controls per patient were selected, with matching on disease type, disease status, age within 10 years, or 50% of age (whichever was smaller), and preference for a match on performance score if more than 4 controls were available.
Five subjects from the abatacept cohort did not have an available matched control from CIBMTR patients receiving CNI/MTX, and four did not have an available match receiving CNI/MTX+ATG. Thus, the comparative data presented shows outcomes for 38 or 39 abatacept patients (compared to CNI/MTX or CNI/MTX+ATG, respectively) as well as the ITT data. As shown throughout the results, the outcomes for the comparator groups of patients were indistinguishable from the outcomes for the ITT population. For all analyses of the 7/8 stratum described below, we report the outcomes for the ITT cohort; the outcomes for the 38- or 39-patient abatacept comparative cohorts for CNI/MTX or CNI/MTX+ATG, respectively, are reported in the relevant Tables and Supplementary Tables. Risk-set sampling with replacement was used to more efficiently utilize controls with rare disease and demographic characteristics27 29. Statistical tests and associated p-values for the primary and secondary outcomes analyses were stratified by risk-set to account for the matched pair design and imbalances in matching. Further details of these analyses can be found e.g. in the ABA2 protocol (see, e.g. Example 4).
[00230] RESULTS:
[00231] Patients: As shown in Supplementary Figure 1, within the 7/8 non-randomized cohort, 40 out of 41 patients enrolled (97.6%) were included in the modified ITT analysis. One 7/8 patient was found to meet one of the exclusion criteria and was removed from study prior to treatment. In addition, 3 patients who were randomized to abatacept prior to protocol amendment were included, resulting in 43 evaluable patients in the 7/8 cohort Of those 43 patients, 97.7% received all scheduled abatacept doses. As also shown in Supplementary Figure 1, within the 8/8 cohort, 95.9% (142) of 148 patients who were enrolled and randomized were included in the modified ITT analysis. Of the 142 patients treated, 91.5% received all scheduled study drug doses and 97.8% received at least 3 of the 4 scheduled doses. Patient characteristics are shown in Table 1. The 7/8 cohort enrolled more minorities (30%) than the 8/8 stratum (13%), reflective of the previously documented increased representation of minorities in 7/8 URD HCT8. Within the 8/8 cohort, the abatacept and placebo groups were balanced with respect to all parameters tested (Table 1). The 7/8 cohort was well-matched for all variables with the exception of overall disease- type, resulting from non-uniform case: control matching. This imbalance is accounted for in the matched pair analysis.
[00232] Efficacy Outcomes:
[00233] Acute GVHD: ABA2 demonstrated improvement in AGVHD in both 7/8 and 8/8 URD HCT patients receiving abatacept. The primary endpoint was the Cl of severe AGVHD at Day +100. As shown in FIG. 2 and FIG.10A, in the 7/8 stratum, patients receiving abatacept demonstrated a substantial decrease in the Day +100 Cl of severe AGVHD, with 2.2% severe AGVHD in patients receiving CNI/MTX+abatacept versus 30.2% in CNI/MTX CIBMTR controls, (p<0.001), greatly exceeding the expectations of the trial’s statistical design. The 8/8 stratum also demonstrated a decrease in severe AGVHD, from 14.8% in placebo to 6.8% in patients receiving abatacept, p = 0.13 (FIG. 3, FIG. 10B). Because the sample size for ABA2 was designed with an□ error of 0.2, the 8/8 stratum did reach the protocol-specified statistical endpoint, thus allowing secondary end-points to be evaluated, which demonstrated improvement in multiple AGVHD end-points. Thus, Gr 2-4 AGVHD was significantly improved with abatacept: In the 7/8 and 8/8 cohorts, respectively, patients receiving abatacept demonstrated Day +180 Gr 2-4 AGVHD of 41.9% and 44.8%, compared with 57.4% and 63.7% for CIBMTR and placebo controls (Figure 1 C-D, p = 0.03 and 0.006 respectively). Moreover, in the 8/8s abatacept reduced steroid-refractory AGVHD at Day 180 (4.2% with abatacept versus 16.2% with placebo, FIG. 10F p =0.02).
[00234] The inventors also performed the same analyses to compare recipients of 7/8 HCT who received CNI/MTX+abatacept to CIBMTR controls receiving CNI/MTX+ ATG (FIG. 7). While ATG is not considered a consensus prophylaxis agent for 7/8 URD transplants (with multiple center-specific variations in both dose and timing of this drug30), it is frequently added to CNI/MTX prior to transplantation. Thus, this analysis provided an important real-world comparison with an agent often used in these high-risk transplants. As shown in FIGs. 15A-15F and FIG. 8, 7/8 URD patients receiving abatacept demonstrated improved Gr 3-4 AGVHD compared to controls receiving ATG (2.2 vs 22.4%, p=0.003). Day 180 Severe AGVHD-Free Survival (‘SGFS’): Because abatacept is a novel agent for AGVHD prophylaxis, in addition to measuring efficacy, we also measured early treatment failure using the composite SGFS end=point, which includes both severe AGVHD and death within 180 days as events. A similar endpoint (SGFS through 100 days) has been described in a study of ATG for GVHD prevention31. As shown in FIGs. 10G-H, FIG. 3, and FIG. 8, in both the 7/8 and 8/8 cohorts, SGFS outcomes for patients receiving abatacept were superior to those receiving standard GVHD prophylaxis. In the 7/8 cohort, SGFS was 97.7% (abatacept ITT) versus 58.7% (CNI/MTX) (p <0.001), and 62.2% (CNI/MTX/ATG) (p <0.001). In the 8/8 cohort SGFS was 93.2% (abatacept) versus 82.0% (placebo) (p = 0.05).
[00235] GVHD biomarkers using the Mount Sinai Acute GVHD International Consortium (MAGIC) algorithm to predict 6-month NRM: Consistent with the decrease in AGVHD and the improvement in SGFS, patients receiving abatacept also demonstrated improvement in serum biomarkers at GVHD onset 32, with a MAGIC algorithm probability (MAP) of 6-month NRM32 =0.06 in 7/8 ABA2 patients versus 0.10 in MAGIC controls, p= 0.01) and 0.05 in 8/8 abatacept patients versus 0.09 in placebo, p=0.001,
FIG. 10E).
[00236] Chronic GVHD:
[00237] The four-dose abatacept prophylaxis regimen did not improve CGVHD. Thus, for the 7/8 and 8/8 strata, patients receiving abatacept demonstrated a 1-year mild-severe CGVHD Cl of 62.0% and 51.9%, respectively, compared with 45.9% and 45.3% for CIBMTR and placebo controls (p=0.74 and p=0.55 respectively). While the rate of moderate-severe CGVHD was not available for CIBMTR controls, this comparison was made for the 8/8 stratum, demonstrating 44.6% and 34.6% for patients receiving abatacept and placebo controls (p=0.25, FIG. 10J). The 1 -year moderate-severe CGVHD Cl for the 7/8 abatacept patients was 57.9% (FIG. 10J).
[00238] Hematologic Reconstitution and Donor Engraftment:
[00239] As shown in FIG. 5 and FIGs. 14A-14H, 14K-14L, both the 7/8 and 8/8 strata demonstrated successful neutrophil and platelet reconstitution, donor engraftment, and leukocyte reconstitution post transplant (as measured by multi -parameter flow cytometry).
[00240] Viral Reactivation and Disease:
[00241] There was no difference in CMV or EBV viral reactivation, end-organ disease or PTLD in patients receiving abatacept versus controls (FIGs. 14A I-14J, FIG. 5). Relapse: The additional immune suppression with abatacept did not increase relapse (FIG. 3). In 7/8s, one-year relapse was 9.3% (ITT), compared to 15.4% (CNI/MTX) and 10.5% (CNI/MTX+ATG) CIBMTR controls (p=0.37 and p = 0.83 respectively). Of note, there were no relapses in the 7/8 abatacept cohort between 6 months and 2 years, with 2-year relapse rates remaining 9.3% versus 21.4% (CNI/MTX, p=0.21) and 17.4%
(CNI/MTX+ATG, p=0.36) for CIBMTR controls. In the 8/8 stratum, one-year relapse was 13.8% for abatacept versus 20.5% for placebo (p =0.33).
[00242] Survival Endpoints:
[00243] In addition to Day 180 SGFS, other survival indicators were also tracked in ABA2, including NRM, RFS and OS (FIG. 3, FIG. 8, FIGs. l lA-B FIGs. 15 D-15F). In 7/8s, abatacept significantly improved each of these outcomes (Figure 2 A, C, E). Thus, 1-year NRM was 9.3% in ABA (ITT) versus 34.1% (CNI/MTX, p =0.009) and 27.4% (CNI/MTX+ATG, p= 0.03). One-year RFS was 81.4% in ABA (ITT) versus 50.4% (CNI/MTX, p =0.002) and 62.1% (CNI/MTX+ATG, p 0.02) and one-year OS was 88.4% in ABA (ITT) versus 57.5% (CNI/MTX, p = 0.002) and 68.0% (CNI/MTX+ATG, p = 0.03). Importantly, all of the survival advantages in the 7/8 cohort were stable through 2 years for the ABA2 versus CNI/MTX CIBMTR control comparisons (FIG. 3), underscoring the major impact that controlling AGVHD makes to long-term transplant success in this patient population. In the 8/8 stratum, Day 180 NRM and OS were significantly improved in the ABA patients compared to placebo (FIG. 3) with one- year survival statistics not significantly different between the two cohorts (FIG. 3, FIGs. 1 IB, 1 ID, 1 IF).
[00244] Transcriptomic analysis reveals control of CD4+ T cell proliferation and activation with abatacept.
[00245] To uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug25, the inventors interrogated the transcriptome of CD4+ T cells reconstituting in patients who received abatacept compared to placebo. To perform this analysis, the inventors flow cytometrically sorted CD4+ T cells on Days 21-28 post-transplant from ABA2 patients (both 7/8 and 8/8 cohorts), as well as a cohort of 12 healthy controls, and subsequently performed RNASeq on these cells (analysis strategy shown in FIG. 12A). Weighted Gene Correlation Network Analysis (WGCNA)33 followed by unsupervised hierarchical clustering identified self-assembling gene modules (SAMs, FIG. 12B) that were highly correlated with clinical variables, including prophylaxis regimen and AGVHD (FIG. 9). The most pertinent SAM, color- coded Turquoise, encapsulates a 476-gene module (FIG. 12B) that strongly correlated with patients receiving CNI/MTX prophylaxis and away from patients receiving CNI/MTX+abatacept prophylaxis (FIG. 12C), providing the first list of genes controlled by costimulation blockade during HCT. Even more noteworthy, embedded within the Turquoise module was a subset of 93 genes that not only correlated strongly with CNI/MTX prophylaxis, but which also demonstrated increasing expression with increasing grade of AGVHD, thus identifying the first human T cell transcriptional network associated with AGVHD severity (Table 1). These 93 genes included a highly correlated network, consisting of pathways controlling T cell proliferation, apoptosis, activation, checkpoint, and metabolism (FIG. 12D), all of which were down-regulated with the addition of abatacept. These data identify a robust mechanistic link between the decrease in clinical AGVHD with abatacept and its impact on T cell transcriptional programming.
[00246] DISCUSSION
[00247] There is a critical unmet need in HCT for safe and effective transplant strategies for patients lacking an HLA -matched sibling donor. This is especially true for patients who also lack an 8/8 HLA matched URD, a population that is highly skewed towards those in specific ethnic groups (Black, Hispanic, Asian Pacific Islander, etc.) While both cord blood and haploidentical transplants are reasonable alternatives for these patients, challenges with these graft sources remain, including slow immune recovery, infections with both graft sources, and potentially higher relapse risk with
haploidentical transplant34 39. The ABA2 study was designed to test whether targeted in vivo CD80/86 costimulation blockade with abatacept could improve outcomes for both 7/8 and 8/8 URD HCT.
[00248] We found that the addition of 4 peri-transplant doses of abatacept improved AGVHD outcomes, with strikingly positive results in 7/8 URD HCT. Thus, for 7/8 patients, the abatacept cohort demonstrated substantial improvements in Gr 3-4 AGVHD, Gr 2-4 AGVHD, and Day 180 SGFS compared with CNI/MTX controls, and improved Gr 3-4 AGVHD and Day 180 SGFS compared to CNI/MTX+ATG controls. As noted earlier, sample size calculations for ABA2 used a screening Phase-2 design, with a = 0.2. The 7/8 cohort therefore far exceeded statistical expectations for the primary endpoint, with Gr 3-4 AGVHD markedly reduced in comparisons to both CNI/MTX and
CNI/MTX+ATG CIBMTR controls, generating a p value <0.001 and 0.003, respectively. Moreover, in extended follow-up, the 7/8 ABA2 cohort continued to demonstrate superiority: Patients receiving abatacept demonstrated improved 2 -year NRM, OS and RFS when compared to the pre-specified
CNI/MTX matched control cohort. The significant improvement in outcomes for 7/8 patients receiving prophylaxis with abatacept generated an additional, unexpected observation: the NRM, RFS and OS statistics from the 7/8 abatacept cohort compared favorably to the 8/8 cohort receiving
CNI/MTX+placebo (FIG. 3). This indicates that a regimen containing CNI/MTX+ abatacept can‘level the playing field’ for those patients (often ethnic minorities) for whom the only URD option is HLA- mismatched. Moreover, while not a controlled analysis, the survival results for the 7/8 ABA2 patients also compare favorably to a retrospective study of post-transplant cyclophosphamide and ATG in HLA- mismatched URD transplants recently published by the European Bone Marrow Transplant Consortium40.
[00249] For 8/8 patients, abatacept significantly improved Grade 2-4 AGVHD, steroid-refractory AGVHD and Day 180 SGFS compared to placebo controls, with improvements in Gr 3-4 AGVHD meeting the statistical expectations of the trial design. NRM, OS and RFS were all statistically- significantly improved in the abatacept versus placebo cohorts at Day 180, with these outcomes becoming more similar at 1 year. Together, these results show that the addition of abatacept significantly improves both short- and long-term outcomes for patients undergoing high-risk 7/8 URD HCT, and improves AGVHD-related and other short-term outcomes for 8/8 URD HCT.
[00250] The improvement in survival outcomes in ABA2 was driven by the combined decrease in AGVHD and the favorable safety profile, with the placebo-controlled analysis of safety endpoints in the 8/8 stratum adding rigor to this analysis. Most notably, abatacept did not appear to increase the risk of disease relapse in either 7/8 or 8/8 HCT, a key safety outcome when adding an adjunctive
immunomodulating agent to transplants for hematologic malignancies. Indeed, one of the most noteworthy observations in the 7/8 cohort was the fact that with a median follow-up of 684 days and with the last enrolled patient followed for 355 days, no patient had relapsed later than 6 months post-transplant. While the mechanisms driving this encouraging relapse profile have not yet been determined, three prevailing hypotheses exist: First, the prevention of severe AGVHD decreases exposure to steroids and other non-specific immunosuppressive agents, which protected against relapse41. Second, the
development of CGVHD in patients receiving abatacept may have had a protective impact on relapse42 44. Third, the downregulation of checkpoint molecule transcription in abatacept-treated patients compared to placebo controls have a salutary effect on the anti -leukemic surveillance activity of reconstituting T cells.
[00251] One notable exception to the improvement in outcomes with abatacept was in the area of CGVHD, with abatacept not improving CGVHD outcomes in either the 7/8 and 8/8 cohorts compared to controls. This outcome may be expected given the short-course of abatacept that was administered in this trial, (four doses, with the final dose on Day +28, with lack of drug exposure expected by Day +10025). The improvement in one-and two-year survival statistics despite equivalent rates of CGVHD in the 7/8 stratum underscores the impact that severe AGVHD makes on survival after URD transplant, especially after HLA mismatched HCT. Recent smaller studies have suggested that abatacept may have activity in treating CGVHD and that extending the dosing of abatacept could provide protection against chronic as well as AGVHD45,46. Given the encouraging safety profile of the four-dose abatacept regimen used in this trial, it is contemplated that extending the dosing of abatacept can improve chronic as well as acute GVHD outcomes, while preserving the beneficial graft-versus-leukemia effect observed in the ABA2 trial.
[00252] Since abatacept’ s primary biologic effect is on CD4+ T cells, we performed a transcriptomic analysis comparing CD4+ T cells isolated from patients receiving CNI/MTX+abatacept to those purified from untransplanted healthy controls, and from patients receiving CNI/MTX+placebo. The results of this analysis provide the first comprehensive transcriptomic analysis of reconstituting CD4+ T cells during HCT as well as a compelling snapshot of the early impact of abatacept on T cell activation. Multivariate analysis identified a group of 93 genes (encompassing a network of proliferation, activation, apoptosis, metabolic and checkpoint pathways) from CNI/MTX+placebo patients for which expression increased with increasing severity of AGVHD. Abatacept not only downregulated the expression of these genes compared to CNI/MTX, but also de-coupled their expression from AGVHD severity. These results show a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD.
[00253] In summary, the ABA2 trial demonstrated the safety and efficacy of in vivo costimulation blockade with abatacept in preventing severe AGVHD after 7/8 URD HCT, with a significant impact on moderate-severe AGVHD and steroid-refractory AGVHD in 8/8 patients. Based on the results of this trial, abatacept has been granted a Breakthrough Therapy Designation by the FDA for the prevention of AGVHD after URD HCT. The substantial improvement in one- and two-year survival indicators in the 7/8 cohort demonstrate that the addition of abatacept is practice-changing for these otherwise high-risk transplants.
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[00255] Table 1: Gene symbols and NCBI Gene ID number of human Transcripts Correlated with AGVHD Grade. The genes listed in this table correlate with the severity of Acute GVHD in patients undergoing a hematopoietic stem cell transplant.
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
[00256] The nucleic acid sequences of the genes described herein have been assigned NCBI and ENSBL accession numbers for different species such as human, mouse and rat. The sequences for any of the genes described herein can be readily retrieved from either database by one of ordinary skill in the art. In some embodiments of any of the aspects, the sequence of a gene, transcript, or polypeptide described herein is the sequence available in the NCBI or ENSMBL database as of the filing date of this application. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
[00257] SUPPLEMENTARY MATERIAL
[00258] Methods:
[00259] Control Selection: For the 7/8 stratum, pre-specified controls from the CIBMTR were selected from a cohort of patients who received 7/8 URD HCT for hematologic malignancy, and who received CNI/MTX for GVHD prophylaxis, without the use of either anti-thymocyte globulin (ATG) or alemtuzumab. A secondary analysis using CIBMTR controls who had received CNI/MTX+ATG was also performed. Matching was based on age (within 50% or 10 years, whichever was smaller), diagnosis, CIBMTR disease status, and performance score when >4 matches were available, with all controls having a performance score >80%.
[00260] Human Studies Ethics Statement: The patients described in this manuscript were enrolled on a clinical trial that was conducted according to the principles set forth in the Declaration of Helsinki, and which was approved by both the institutional review board (IRB) at the data-coordinating center, and at participating site IRBs. For comparative immune reconstitution and transcriptomic analysis, blood was also collected from a cohort of healthy untransplanted participants.
[00261] GVHD grading, chimerism analysis and adverse event grading: The NIH consensus criteria were used for the grading of acute 1 and chronic GVHD2. Lineage specific (CD33+ and CD3+) donor chimerism analysis was performed to monitor donor engraftment. The National Cancer Institute Common Terminology Criteria for Adverse Events version 4 was used to classify adverse events. Review and adjudication of the grading of AGVHD and CGVHD was performed centrally by blinded members of the protocol team, using the Blood and Marrow Transplant Clinical Trial Network (BMTCTN) Manual of Procedures, as described in Section 3.10 of the protocol.
[00262] Immune reconstitution Studies: Longitudinal flow cytometry analysis was performed on all patients, and included the enumeration of CD3+ T cells (CD3+/CD20- lymphocytes), CD20+ B cells (CD3-/CD20+ lymphocytes), natural killer (NK) cells (CD3-/CD20-/CD16+ [CD56+ and -], CD4+ (CD4+/CD8-) T cells, and CD8+ (CD8+/CD4-) T cells. A complete description of the antibodies used for flow cytometry analysis is provided in FIG. 4.
[00263] Transcriptomic analysis: Flow cytometric CD4+ T cell sorting was performed on patients enrolled at two laboratory sites (Emory University and Seattle Children’s Research Institute) at Days 21 and 28 post-transplant, using either a FACSJazz or FACSAria flow cytometric cell sorter. For transcriptomic analysis, only samples that were sorted immediately after the blood draw (without shipping) from patients at the two laboratory sites were included, since shipping of samples from other sites resulted in a significant impact on the transcriptomic signature. This resulted in 65 analyzed samples from patients receiving CNI/MTX+abatacept and 41 analyzed samples from patients receiving
CNI/MTX+placebo at Day 21, and 67 analyzed samples from patients receiving CNI/MTX+abatacept and 45 analyzed samples from patients receiving CNI/MTX+placebo at Day 28. For transcriptomic analysis, samples from patients in the 7/8 and 8/8 cohorts who received abatacept were analyzed together. CD4+ T cells were also sorted from the peripheral blood from a cohort of 12 healthy individuals to use as controls for transcriptomic analysis.
[00264] RNA was purified from the sorted CD4 T cells using Qiagen RNeasy Micro columns with on-column DNase treatment, and RNA quality was assessed using an Agilent Bioanalyzer. Two nanograms of total RNA were used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit according to the manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed bar codes using the Illumina NexteraXT DNA Library Preparation kit. Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled, and sequenced on an Illumina HiSeq3000 at 100SR at an average read depth of 25 million reads/sample. Sequence data were extracted and demultiplexed using Illumina bcl2fastq v2. Resulting FASTQ files were aligned to the human genome (hg38) using STAR 3. Read counts per gene were calculated using the GenomicAlignments 4 package and statistical modeling and a variance stabilizing transformation was performed using the DESeq2 5 package. Weighted Gene Correlation Network Analysis (WGCNA)6 as well as Gene Set Enrichment Analysis7 were performed on the top 6000 most variant normalized counts. For WGCNA, parameters for signed-hybrid network construction included a soft-thresholding power of 6, mean connectivity of 10, and minimum module size of 50. Enrichment analysis was performed using the piano4 package using Reactome8 pathway terms from the MSigDB v6.17,9. For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0- 4), relapse, non-relapse mortality, and all-cause mortality. Self-Assembling Modules (SAMs) were determined as follows: First we performed an unsupervised hierarchical clustering of the gene dissimilarity scores and then applied a cut height of 0.93 on the clustering output to identify gene modules. Those modules with a minimum module size of 100 were identified and, using Principal Component Analysis (PCA), similar modules were identified and merged based on a module dissimilarity threshold of 0.310.
[00265] Linear Regression Analysis of the Turquoise SAM: To identify those genes in the Turquoise SAM for which expression was positively correlated with grade of AGVHD, a linear regression analysis was performed. To accomplish this, all gene expression values were first log-transformed to better approximate normality. Then, patients’ AGVHD severity scores were binned as follows: no AGVHD (‘O'), Grade 1-2 AGVHD ('2') or Grade 3-4 AGVD ('4'). The significance of the correlation between expression level and AGVHD score was determined using a linear regression fit (lm function in R) of log(gene expression) versus the standardized AGVHD score. Relationships were considered significant if P < 0.05.
[00266] Transcriptional Network Visualization: Genes in the Turquoise SAM for which expression level was positively correlated with the grade of AGVHD through linear regression were then analyzed using ingenuity pathway analysis (IPA, http://www.ingenuity.com) to identify direct and indirect interactions between genes, supported by a meta-analysis of scientific literature contained in the Ingenuity Knowledge Base. Default search parameters were used, including all direct and indirect gene-gene interactions supported by human studies with highest confidence.
[00267] GVHD biomarker analysis using the Mount Sinai Acute GVHD International Consortium (MAGIC) biomarker algorithm: In patients who developed AGVHD, the prognostic biomarkers ST2 and REG3a were analyzed by enzyme-linked immunoabsorbent assay, as previously described11 13. For the 7/8 cohort, for which serum samples were not available from the CIBMTR controls, a control sample group of 58 patients was constructed from the MAGIC database 14, with controls chosen based on both age (controls were older than 6 years of age), and GVHD prophylaxis regimen used (controls received GVHD prophylaxis with CNI +MTX, with or without ATG). For MAGIC analyses, if AGVHD was treated with systemic steroids, a sample collected at the time of treatment (within a window of 7 days prior to treatment to 3 days after start of treatment) was used for analysis. If lower-grade AGVHD was diagnosed but never systemically treated, a sample drawn at the time of GVHD diagnosis (+/- 7 days) was used for analysis. The MAGIC algorithm probability (MAP) of NRM was calculated using the previously published model 14 and the median MAP was compared between the groups.
[00268] Statistical Analysis:
[00269] Demographic, disease, and transplant-related factors were compared between treatment groups using c2 for categorical variables and Wilcoxon rank-sum tests for continuous variables. Time- dependent outcomes without the presence of competing events, including OS, Severe AGVHD-free survival (SGFS)15, and RFS, were examined using Kaplan-Meier survival curves, and resulting survival distributions were compared using a log-rank test. For time-dependent outcomes in the presence of competing events, cumulative incidence functions (CIF) were generated to account for the competing events, and Gray’s test was used to compare CIF between treatment groups. Estimates of NRM were calculated while accounting for relapse as a competing event, and estimates of relapse and GVHD were calculated while accounting for NRM as a competing event. In addition, for GVHD outcomes and SGFS, patients were censored at relapse; otherwise patients were censored at last follow-up. Survival and cumulative incidence estimates are presented for time points of interest and accompanied by 95% confidence intervals. For the 7/8 cohort, the stratified Gray’s test of equality of cumulative incidence functions and the log-rank stratified test of equality were used to account for matched pairs when comparing CIF and survival distributions, respectively. Cox proportional hazards regression models were used to estimate the effects of abatacept on GVHD, relapse, NRM, RFS, and OS. For the 7/8 cohort, results were adjusted for matched pairs using stratification. Analysis was conducted using SAS v. 9.4 (SAS Institute, Cary, NC, USA) or R (r-project.org, Vienna, Austria).
[00270] Matching between enrolled patients and CIBMTR controls for the 7/8 stratum: The 7/8 patient cohort and the CIBMTR control cohorts were well-matched for all variables with the exception of overall disease-type. Because disease-type was a matching variable, imbalances in the distributions resulted from a non-uniform case:control matching ratio for the abatacept cohort (i.e., some abatacept patients were matched 1:2 vs. 1:3 or 1:4). While this covariate imbalance is present overall, analysis of time-dependent outcomes accounting for the matched pair design eliminates the effect of this imbalance on parameter estimation.
[00271] Supplementary Results:
[00272] Hematologic Reconstitution and Donor Engraftment: As shown in FIG. 5 and FIG. 8, there was no difference in neutrophil engraftment between patients receiving abatacept and controls in either the 7/8 (100% vs 99.2%, p=0.22) or 8/8 strata (98.6% vs 100%, p=0.44). Platelet engraftment was similar in the two arms of the 8/8 stratum (97.3% in the abatacept arm versus 97% in the placebo arm, p=0.50).
In the 7/8 stratum, abatacept patients had significantly more complete platelet engraftment by day 100 (97.7% versus 88.2%, p=0.04). None of the subjects in either the 7/8 or 8/8 strata demonstrated primary graft failure or graft rejection. One subject with myelodysplastic syndrome in the 8/8 abatacept cohort demonstrated secondary graft failure. The subject underwent a second transplant with successful engraftment. This rate of secondary graft failure is consistent with published rates with standard GVHD prophylaxis 16. While chimerism could not be compared between the abatacept and control cohorts for the 7/8 stratum (given a lack of chimerism data for the CIBMTR controls), this analysis could be performed in the 8/8 cohort, with similar proportions of complete (>95%) donor myeloid and lymphoid engraftment at 1 year between the two cohorts (Supplemental Figure 2 K-L). Flow cytometric analysis of granulocyte-, lymphocyte-, B cell-, NK cell-, total T cell-, CD4+ and CD8+ T cell- reconstitution also did not detect significant differences in the abatacept or placebo cohorts (FIGs. 14A-14H).
[00273] Viral Reactivation and Disease: [00274] The inventors prospectively measured CMV reactivation and end-organ disease, as well as EBV reactivation and PTLD (FIGs. 14I-14J, FIG. 5). CMV and EBV reactivation rates are described for >300 IU/ml for CMV and >1000 IU/ml for EBV because all centers were able to quantify values that exceeded these cut-offs. While a comparison of viral reactivation could not be made in the 7/8 cohort (given a lack of viral load data for the CIBMTR controls), this comparison could be performed in the 8/8 randomized stratum, which demonstrated no difference in CMV reactivation to >/= 300 IU/ml between abatacept and placebo cohorts (FIG. 5 and FIG. 14 I, ABA = 47.3%, placebo = 33.3% p = 0.16, 32.6% 7/8 ABA patients reactivated CMV to >/= 300 IU/ml). There were 6 cases of CMV end-organ disease (4 colitis, 1 gastritis, 1 retinitis) in the 8/8 abatacept cohort and 2 cases ( 1 colitis, 1 ileitis) in the 8/8 placebo cohort (FIG. 5, p=0.19). In the 7/8 abatacept cohort there were 2 cases of CMV colitis (these occurred on Days 211 and 238 so are not included in Supplemental Table 2 which describes events through Day 180). All cases of CMV end organ disease were successfully treated with standard therapies. There was also no difference in EBV reactivation to >/= 1000 IU/ml for the abatacept versus placebo arms in the 8/8 stratum (FIG. 5, FIG. 14 J, ABA = 6.5%, placebo = 6.5%, p = 0.87). For the 7/8 abatacept cohort, 25.6% ABA patients reactivated EBV >1000 IU/ml. Rates of PTFD were also similar between the two cohorts: ABA 2.8%, Placebo 0%, p = 0.18. The rate of PTFD was 4.7% in the 7/8 stratum. All PTFD cases resolved completely (2 treated with rituximab alone and 2 treated with PTFD-directed combination therapy). Of note, of the 185 enrolled on ABA2, 4 patients developed PTFD, 2 in the 7/8 and 2 in the 8/8 stratum. Three of these patients were from a single center, for which acyclovir prophylaxis was standardly discontinued at day 30 post-transplant. Given the cluster of PTFD at this site, and after a survey of other ABA2 sties revealed that the majority of the sites continued acyclovir through day 180 post-transplant (in addition to published reports suggesting that acyclovir may have a role in preventing EBV disease 17, 18) a protocol amendment was enacted which mandated the administration of acyclovir prophylaxis through day 180 for all enrolled patients. Subsequent to this change in supportive care, no further cases of PTFD were reported in ABA2 patients at that center, or for the remaining 71 patients enrolled in the study.
[00275] Severe Adverse Events (SAEs):
[00276] SAEs were reported less frequently among abatacept-treated patients (47.9%) as compared to placebo-treated subjects in the 8/8 cohort (55.1%). The percentage of SAEs in the 7/8 abatacept cohort was similar to that of the 8/8 placebo group (53.5%) (FIG. 6).
[00277] SEQUENCES:
[00278] Sequences for CD80 are known for a number of species, e.g., human CD80 (NCBI Gene ID: 941), mRNA (NCBI Ref Seq: NM 005191.4), and polypeptide (NCBI Ref Seq: NP_005182.1). CD80 refers to all naturally occuring variants or isoforms of CD80. In one embodiment, the CD80 polypeptide sequence is presented in SEQ ID NO: 1. In some embodiments of any of the aspects, the CD80 polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 1.
1 mghtrrqgts pskcpylnff qllvlaglsh fcsgvihvtk evkevatlsc ghnvsveela
61 qtriywqkek kmvltmmsgd mniwpeyknr tifditnnls ivilalrpsd egtyecvvlk
121 yekdafkreh laevtlsvka dfptpsisdf eiptsnirri icstsggfpe phlswlenge
181 elnainttvs qdpetelyav sskldfhmtt nhsffncliky ghlrvnqtfn wnttkqehfp
241 dnllpswait lisvngifvi ccltycfapr crerrmerl rresvrpv (SEQ ID: 1)
[00279] Sequences for CD86 are known for a number of species, e.g., human CD86 (NCBI Gene ID: 942), mRNA (NCBI Ref Seq: NM_001206924.1), and polypeptide (NCBI Ref Seq: NP_001193853.1). CD86 refers to all naturally occuring variants or isoforms of CD86. In one embodiment, the CD86 polypeptide sequence is presented in SEQ ID NO: 2. In some embodiments of any of the aspects, the CD86 polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 2.
1 mdpqctmgls nilfVmafll sanfsqpeiv pisnitenvy inltcssihg ypepkkmsvl
61 lrtknstiey dgimqksqdn vtelydvsis lsvsfpdvts nmtifcilet dktrllsspf
121 sieledpqpp pdhipwitav lptviicvmv fclilwkwkk kkrpmsykc gtntmerees
181 eqtkkrekih ipersdeaqr vfkssktssc dksdtcf (SEQ ID: 2)
[00280] Sequences for Calcineurin are known for a number of species, e.g., human Calcineurin (NCBI Gene ID: 5533), mRNA (NCBI Ref Seq: NM_001243974.1), and polypeptide (NCBI Ref Seq: NP_001230903.1). Calcineurin refers to all naturally occuring variants or isoforms of Calcineurin. In one embodiment, the Calcineurin polypeptide sequence is presented in SEQ ID NO: 3. In some embodiments of any of the aspects, the Calcineurin polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 3.
1 msgrrfhlst tdrvikavpf pptqrltfke vfengkpkvd vlknhlvkeg rleeevalki
61 indgaailrq ektmievdap itvcgdihgq ffdlmklfev ggspsntryl flgdyvdrgy
121 fsiecvlylw slkinhpktl fllrgnhecr hltdyftfkq ecrikyseqv ydacmetfdc
181 lplaallnqq flcvhggmsp eitslddirk ldrfteppaf gpvcdllwsd psedygnekt
241 lehythntvr gcsyfysypa vceflqnnnl lsiiraheaq dagyrmyrks qatgfpslit 301 ifsapnyldv ynnkaavlky ennvmnirqf ncsphpywlp nfmdvftwsl pfVgekvtem
361 lvnvlnicsd delisddeae dhyipsyqkg sttvrkeiir nkiraigkma rvfsilrqes
421 esvltlkglt ptgtlplgvl sggkqtieta tveavearea irgfslqhki rsfeeargld
481 rinermpprk dsihaggpmk svtsahshaa hrsdqgkkah s (SEQ ID NO: 3)
[00281] Sequences for dihydrofolate reductase are known for a number of species, e.g., human dihydrofolate reductase (NCBI Gene ID: 1719), mRNA (NCBI Ref Seq: NM_000791.4), and polypeptide (NCBI Ref Seq: NP_000782.1). Dihydrofolate reductase refers to all naturally occuring variants or isoforms of dihydrofolate reductase. In one embodiment, the dihydrofolate reductase polypeptide sequence is presented in SEQ ID NO: 4. In some embodiments of any of the aspects, the dihydrofolate reductase polypeptide can be an ortholog, variant, and/or allele of SEQ ID NO: 4.
1 mvgslnciva vsqnmgigkn gdlpwpplm efryfqrmtt tssvegkqnl vimgkktwfs
61 ipeknrplkg rinlvlsrel keppqgahfl srslddalkl teqpelankv dmvwivggss
121 vykeamnhpg hlklfVtrim qdfesdtffp eidlekykll peypgvlsdv qeekgikykf
181 evyeknd (SEQ ID NO: 4)

Claims

What is claimed herein is:
1. A method for treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction, the method comprising:
administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CD80/86 inhibitor;
thereby inhibiting the expression or activity of one or more immune checkpoint polypeptides in T cells and reconstituting the T cell population.
2. The method of claim 1, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia and lymphoma relapse.
3. A method for treating or preventing GvHD, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CD 80/86 inhibitor.
4. The method of claim 3, wherein the administration is prophylactic.
5. The method of any of claims 1-4, wherein the subject is a subject who is in need of or has
received a bone marrow transplant.
6. A method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a bone marrow transplant to the subject; and
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor.
7. The method of claim 6, wherein the hematopoietic disease is a hematopoietic cancer.
8. The method of claim 6, wherein the hematopoietic disease is leukemia.
9. The method of any of claims 1-8, wherein the CD80/86 inhibitor is an antibody or antigen binding fragment thereof; an aptamer; or a CD80/86-binding fragment of a natural receptor or ligand of CD80/86.
10. The method of any of claims 1-9, wherein the CD80/86 inhibitor binds specifically to CD80 and/or 86.
11. The method of any of claims 1-10, wherein the CD80/86 inhibitor is abatacept.
12. The method of any of claims 1-11, wherein the CD80/86 inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
13. The method of claim 12, wherein the receptor is CTLA-4 and/or CD28.
14. The method of any of claims 1-13, wherein the immune checkpoint polypeptides are selected from the group consisting of CTLA4, Lag-3, HAVCR2 (TIM-3), and/or PDCD1.
15. The method of any of claims 1-14, wherein the immune checkpoint polypeptides are selected from the group selected from Lag-3, HAVCR2 (TIM-3), and/or PDCD1.
16. The method of any of claims 1-15, wherein the T cell population comprises CD3+, CD4+, and/or CD8+ T cells.
17. The method of any of claims 1-16, wherein the T cell population comprises Regulatory T cells (TregS).
18. The method of any of claims 1-17, wherein the inhibitor is targeted to T-cells.
19. The method of claim 18, wherein the inhibitor comprises a T cell targeting moiety.
20. The method of claim 19, wherein the T cell targeting moiety comprises a moiety that specifically binds to a T cell-specific cell-surface polypeptide.
21. The method of claim 20, wherein the T cell-specific cell surface polypeptide is selected from the group consisting of CD3, CD4, and/or CD 8.
22. The method of any of claims 19-21, wherein the T cell targeting moiety comprises an antibody or antigen-binding fragment thereof; an aptamer; or a natural ligand that specifically binds the T cell-specific cell surface polypeptide.
23. The method of any of claims 1-22, further comprising administering a therapeutically effective amount of an inhibitor of the expression or activity of one or more immune checkpoint polypeptides.
24. The method of any of claims 1-23, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), leukemia, and lymphoma relapse.
25. The method of any of the preceding claims, further comprising administering a therapeutically effective amount of a Calcineurin inhibitor.
26. The method of claim 25, wherein the Calcineurin inhibitor binds specifically to Calcineurin.
27. The method of any of claims 25-26, wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus.
28. The method of any of claims 25-27, wherein the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
29. The method of any of the preceding claims, further comprising administering a therapeutically effective amount of a dihydrofolate reductase (DHFR) inhibitor.
30. The method of claim 29, wherein the dihydrofolate reductase (DHFR) inhibitor is an antibody or antigen-binding fragment thereof; an aptamer; or a dihydrofolate reductase (DHFR) inhibitor fragment of a natural receptor or ligand of dihydrofolate reductase (DHFR).
31. The method of any of claims 29-30, wherein the dihydrofolate reductase (DHFR) inhibitor binds specifically to dihydrofolate reductase (DHFR).
32. The method of any of claims 29-31, wherein the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
33. The method of any of claims 29-32, wherein the Calcineurin inhibitor does not bind to a receptor and/or the subject is not administered a therapy that binds to a receptor.
34. The method of any of the preceding claims, wherein the CD80/86 inhibitor inhibits the expression or activity of one or more T cell polypeptides selected from the polypeptides listed in Table 1.
35. The method of any of the preceding claims, wherein the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus- host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
36. The method of any of the preceding claims, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of non-relapse mortality (NRM) and infection.
37. The method of any of the preceding claims, wherein the subject is a subject who is in need of or who has received a Hematopoietic Cell Transplant (HCT).
38. The method of claim 37, wherein the Hematopoietic Cell Transplant (HCT) originates from an Unrelated Donor (URD).
39. A composition comprising:
a. a CD80/86 inhibitor; and
b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a
dihydrofolate reductase (DHFR) inhibitor.
40. The composition of claim 39, wherein the Calcineurin inhibitor is selected from the group of cyclosporine and tacrolimus.
41. The composition of any of claims 39-40, wherein the dihydrofolate reductase (DHFR) inhibitor is methotrexate.
42. The composition of any of claims 39-41, wherein the CD80/86 inhibitor is abatacept.
43. A method of treating or preventing T cell dysfunction or a condition caused by or associated with T cell dysfunction in a subject in need thereof, the method comprising administering to the subject a composition of any of the preceding claims.
44. The method of claim 43, wherein the condition caused by or associated with T cell dysfunction is selected from the group of Graft-versus-host disease (GvHD) and leukemia.
45. The method of claim 44, wherein the Graft-versus-host disease (GvHD) is selected from the group of acute Graft-versus-host disease (AGvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus-host disease (cGvHD).
46. A method of treating or preventing a hematopoietic disease in a subject in need thereof, the
method comprising administering to the subject a composition of any of the preceding claims.
47. A method of treating or preventing a non-relapse mortality (NRM) in a subject who is need of or who has received a Hematopoietic Cell Transplant (HCT), the method comprising administering to the subject a composition of any of the preceding claims.
48. The method of claim 47, wherein the Hematopoietic Cell Transplant (HCT) originates from an Unrelated Donor (URD).
49. A method of treating a hematopoietic disease in a subject in need thereof, the method comprising: a. administering a hematopoietic stem transplant to the subject;
b. administering a therapeutically effective amount of a pharmaceutical composition
comprising a CD80/86 inhibitor; and
c. further administering a therapeutically effective amount of a Calcineurin inhibitor and/or a therapeutically effective amount of a dihydrofolate reductase inhibitor (DHFR).
50. The method of claim 49, wherein the hematopoietic disease is a hematopoietic cancer.
51. The method of claim 49, wherein the hematopoietic disease is leukemia.
52. A pharmaceutical composition formulated for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction, comprising:
a. a CD 80/86 inhibitor; and
b. at least one of: an immune checkpoint inhibitor, a Calcineurin inhibitor, and/or a dihydrofolate reductase (DHFR) inhibitor; and
c. a pharmaceutically acceptable carrier.
53. A pharmaceutical composition of claim 52 for the treatment or prevention of T cell dysfunction or a condition caused by or associated with T cell dysfunction, wherein the condition caused by or associated with T cell dysfunction is selected from the group consisting of Graft-versus-host disease (GvHD), severe acute Graft-versus-host disease (aAGVHD), and chronic Graft-versus- host disease (cGvHD), leukemia and lymphoma relapse.
54. The pharmaceutical composition of claim 52 for the treatment or prevention of a hematopoietic disease in a subject in need thereof.
55. The pharmaceutical composition of claim 52 for the treatment of a hematopoietic disease,
wherein the hematopoietic disease is a hematopeitic cancer.
56. The pharmaceutical composition of claim 52 for the treatment or prevention of a non-relapse mortality (NRM) in a subject who is need of or who has received a Hematopoietic Cell
Transplant (HCT) comprising administering to the subject a composition of any of the preceding claims.
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