TRAIL short antibodies and methods of use

Abstract

Antibodies against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) short are provided. The present invention provides humanized TRAIL short antibodies capable of neutralizing TRAIL shorts. For example, materials and methods are provided for inducing apoptosis (e.g., via TRAIL-mediated cell death, natural killer (NK) cytotoxicity, and / or CD8+ T cell killing) using one or more humanized TRAIL short antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 62 / 457,614, filed February 10, 2017, and U.S. Application No. 62 / 512,627, filed May 30, 2017. The disclosures of the prior applications are considered part of (and are incorporated by reference into) the disclosure of this application.

[0002] Federal Funding Statement This invention was made with Government support under AI120698 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 1. Technical Field This document relates to antibodies against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) shorts, more specifically humanized TRAIL short antibodies that can neutralize TRAIL shorts. For example, this document provides materials and methods for using one or more humanized TRAIL short antibodies to induce apoptosis. [Background technology]

[0004] 2. Background information TRAIL is an immunomodulatory protein expressed by immune and other cells that can kill virus-infected or malignant cells through binding to TRAIL receptor 1 (TRAIL-R1) or TRAIL-R2 on target cells. TRAIL-short is a splice variant of TRAIL that can block TRAIL-mediated cell death. TRAIL can bind to one of five cognate receptors, TRAIL-R1, R2, R3, R4, or osteoprotegerin, but only binding to TRAIL-R1 or R2 induces apoptotic death of receptor-bearing cells (Wang and El-Deiry, 2003 Oncogene 22:8628-8633). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Wang and El-Deiry, 2003, Oncogene 22:8628-8633 Summary of the Invention

[0006] TRAIL-short is a novel splice variant of TRAIL that can bind to TRAIL-R1 ("R1") and / or TRAIL-R2 ("R2"). When bound to R1 and / or R2, TRAIL-short prevents full-length TRAIL from inducing cell death (Schnepple et al., 2011, J Biol Chem 286:35742-35754).

[0007] This document provides antibodies against TRAIL short (e.g., anti-TRAIL short antibodies), more specifically, humanized TRAIL short antibodies that can neutralize TRAIL short but do not bind to full-length TRAIL. This document also provides materials and methods for making and using antibodies against TRAIL short. For example, one or more humanized TRAIL short antibodies can be used to modulate (e.g., increase or decrease) apoptosis (e.g., via TRAIL-mediated cell death, natural killer (NK) cytotoxicity, CD8+ T cell killing, chimeric antigen receptor (CAR) T cell killing, and / or oncolytic virotherapy). In some cases, one or more humanized TRAIL short antibodies can be used to treat cancer (bladder cancer, breast cancer, cervical cancer, esophageal cancer, head and neck cancer, renal cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, ovarian cancer, melanoma, pancreatic cancer, uterine cancer, and blood cancer, e.g., B-cell malignancies), and / or chronic viral infections (e.g., human immunodeficiency virus (HIV) infection).

[0008] As demonstrated herein, the anti-TRAIL effect of TRAIL-short can be effectively neutralized using TRAIL-short specific antibodies. Neutralization of TRAIL-short is effective in inducing TRAIL-mediated cell death, NK cytotoxicity, CD8+ T cell killing, CAR T cell killing, and / or apoptosis by oncolytic virotherapy.

[0009] In general, one embodiment of this document features an antibody that binds to TRAIL short. The TRAIL short antibody may comprise a heavy chain variable region (VH) domain comprising the complementarity determining regions (CDRs) set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and a light chain variable region (VL) domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. The VH domain may comprise a sequence having at least 75 percent sequence identity to SEQ ID NO:6. The antibody may be a humanized antibody comprising the VH domain sequence set forth in SEQ ID NO:6. The heavy chain may comprise a sequence comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. The VL domain may comprise a sequence having at least 75 percent sequence identity to SEQ ID NO:20. The antibody may be a humanized antibody comprising the VL domain sequence set forth in SEQ ID NO:20. The VL domain may comprise a sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. The antibody may be a chimeric antibody (e.g., a humanized antibody). The antibody may be an antigen-binding fragment of an antibody. The antibody may be a monoclonal antibody. The antibody may bind to a TRAIL short. The antibody may neutralize a TRAIL short. In some cases, the antibody may be bispecific (e.g., may contain an antigen-binding fragment that binds to a TRAIL short and an antigen-binding fragment that binds to another antigen, such as a cell surface antigen (also called a cell surface marker)).

[0010] In another aspect, this document features a method of neutralizing a TRAIL short in a mammal. The method comprises or consists essentially of administering to the mammal an antibody that binds to a TRAIL short described herein (e.g., an antibody comprising a VH domain comprising the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and a VL domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16). The mammal can be a human. The antibody can be a humanized antibody.

[0011] In another aspect, this writing features a method of inducing apoptosis in a mammalian cell. The method comprises or consists essentially of administering to a mammal an antibody that binds a TRAIL short described herein (e.g., an antibody comprising a VH domain comprising the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and a VL domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16), under conditions effective for the antibody to induce apoptosis in the mammalian cell. The mammal can be a human. The antibody can be a humanized antibody. The apoptosis includes TRAIL-induced apoptosis.

[0012] In another aspect, this document features a method of increasing NK cell cytotoxicity, CD8+ T cell killing, CAR T cell killing, and / or oncolytic virotherapy in mammalian cells. The method comprises or consists essentially of administering to a mammal an antibody that binds to a TRAIL short described herein (e.g., an antibody comprising a VH domain comprising the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and a VL domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16), under conditions effective for the antibody to increase NK cell cytotoxicity, CD8+ T cell killing, CAR T cell killing, and / or oncolytic virotherapy in mammalian cells. The mammal can be a human. The antibody can be a humanized antibody.

[0013] In another aspect, this document features a method of treating an infectious disease. The method includes or consists essentially of administering to a mammal having an infectious disease an antibody that binds to a TRAIL short (e.g., an antibody comprising a VH domain comprising the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and a VL domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16), under conditions in which the antibody increases NK cytotoxicity, CD8+ T cell killing, CAR T cell killing, and / or oncolytic virotherapy. The infectious disease can be a chronic infectious disease (e.g., an HIV infection). The mammal can be a human. The antibody can be a humanized antibody. The method can also include administering to the mammal a TRAIL agonist (e.g., recombinant TRAIL, an anti-TRAIL-R1 antibody, an anti-TRAIL-R2 antibody, or a TRAIL oligomer). The method can also include administering to the mammal an antiretroviral therapy (e.g., abacavir, didanosine, emtricitabine, entecavir, lamivudine, stavudine, tenofovir disoproxil fumarate, zalcitabine, zidovudine, delavirdine, efavirenz, etravirine, nevirapine, rilpivirine, adefovir, tenofovir, enfuvirtide, maraviroc, dolutegravir, elvitegravir, raltegravir, bevirimat, amprenavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, atazanavir, darunavir, tipranavir, TRIM5 alpha, tat antagonists, or trichosanthin).

[0014] In another aspect, this document features a method of treating cancer. The method comprises or consists essentially of administering to a mammal having cancer an antibody that binds to a TRAIL short (e.g., an antibody comprising a VH domain comprising the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and a VL domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16) under conditions in which the antibody induces apoptosis in cancer cells from the cancer. The cancer can be, for example, cervical cancer, head and neck cancer, lymphoma, leukemia, ovarian cancer, melanoma, pancreatic cancer, or B-cell malignancies. The mammal can be a human. The antibody can be a humanized antibody. The method can also include administering to the mammal a TRAIL agonist (e.g., recombinant TRAIL, an anti-TRAIL-R1 antibody, an anti-TRAIL-R2 antibody, or a TRAIL oligomer). The method can also include administering to the mammal a cancer treatment (e.g., a chemotherapeutic agent, radiation therapy, brachytherapy, or surgery).When the cancer treatment is a chemotherapy agent, the chemotherapy agent can be altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, thiotepa, 5-fluorouracil (5-FU), 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, and pemetrexed, daunorubicin, doxorubicin, epirubicin, idarubicin. , actinomycin-D, bleomycin, mitomycin-C, mitoxantrone, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, vinorelbine, prednisone, ethylprednisolone, dexamethasone, L-asparaginase, bortezomib, retinoids, tretinoin, bexarotene, arsenic trioxide, CHOP, R-CHOP, trastuzumab, pertuzumab, ipilimumab, dinutuximab Mab, siltuximab, cetuximab, panitumumab, necitumumab, ramucirumab, bevacizumab, pembrolizumab, nivolumab, atezolizumab, olaratumab, denosumab, blinatumomab, rituximab, ofatumumab, obinutuzumab, daratumumab, alemtuzumab, elotuzumab, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab, emtansine, apatinib, cabozantinib, alectinib, crizotinib, dasatinib, imatinib, nilotinib, erlotinib, gefitinib, la It may be selected from the group consisting of patinib, sorafenib, sunitinib, tofacitinib, cobimetinib, trametinib, bortezomib, disulfiram, lactacystin, tamoxifen, obatoclax, navitoclax, gossypol, iniparib, olaparib, perifosine, dabrafenib, vemurafenib, trametinib, abemaciclib, palbociclib, ribociclib, trilaciclib, fulvestrant, temsirolimus, everolimus, vemurafenib, trametinib, dabrafenib, and vintafolide.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although the present invention can be practiced using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. The present invention also relates to the following: [Item 1] An antibody that binds to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) short, a heavy chain variable region (VH) domain comprising the complementarity determining regions (CDRs) set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and A light chain variable region (VL) domain comprising the CDRs set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. The antibody comprising: [Item 2] The antibody of item 1, wherein the VH domain comprises a sequence having at least 75 percent sequence identity to SEQ ID NO:6. [Item 3] The antibody of item 2, wherein the antibody is a humanized antibody comprising a VH domain sequence set forth in SEQ ID NO:6. [Item 4] 2. The antibody of item 1, wherein the heavy chain comprises a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. [Item 5] The antibody of item 1, wherein the VL domain comprises a sequence having at least 75 percent sequence identity to SEQ ID NO:20. [Item 6] The antibody of item 5, wherein the antibody is a humanized antibody comprising the VL domain sequence set forth in SEQ ID NO: 20. [Item 7] 2. The antibody of item 1, wherein the light chain comprises a sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. [Item 8] The antibody of item 1, wherein the antibody is a chimeric antibody. [Item 9] The antibody of item 8, wherein the chimeric antibody is a humanized antibody. [Item 10] The antibody of item 1, wherein the antibody is an antigen-binding fragment of the antibody. [Item 11] The antibody of item 1, wherein the antibody is a monoclonal antibody. [Item 12] The antibody of item 1, wherein the antibody neutralizes TRAIL short. [Item 13] 1. A method for neutralizing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) short in a mammal, comprising: Administering the antibody according to item 1 to the mammal. The method comprising: [Item 14] 14. The method of claim 13, wherein the mammal is a human. [Item 15] The method of claim 14, wherein the antibody is a humanized antibody. [Item 16] 1. A method for inducing apoptosis in a mammalian cell, comprising: The method comprises administering to a mammal the antibody of item 1, wherein the antibody is effective to induce apoptosis in the mammalian cells. [Item 17] 17. The method of claim 16, wherein the mammal is a human. [Item 18] 18. The method of claim 17, wherein the antibody is a humanized antibody. [Item 19] 17. The method of claim 16, wherein the apoptosis comprises apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). [Item 20] 1. A method for increasing natural killer (NK) cell cytotoxicity in a mammalian cell, comprising: The method comprises administering to a mammal the antibody of item 1, wherein the antibody is effective to increase NK cytotoxicity in the mammalian cells. [Item 21] 21. The method of claim 20, wherein the mammal is a human. [Item 22] 22. The method of claim 21, wherein the antibody is a humanized antibody. [Item 23] 1. A method of treating an infectious disease, comprising: administering to the mammal having the infection an antibody that binds to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) short, the antibody comprising a heavy chain variable region (VH) domain comprising complementarity determining regions (CDRs) set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a light chain variable region (VL) domain comprising CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; The method, wherein the antibody increases natural killer (NK) cytotoxicity. [Item 24] 24. The method of claim 23, wherein the infection is a chronic infection. [Item 25] 25. The method of claim 24, wherein the infectious disease is selected from the group consisting of a human immunodeficiency virus infection, a hepatitis B virus infection, and a hepatitis C virus infection. [Item 26] 24. The method of claim 23, wherein the mammal is a human. [Item 27] 27. The method of claim 26, wherein the antibody is a humanized antibody. [Item 28] 24. The method of claim 23, further comprising administering to the mammal a TRAIL agonist. [Item 29] 29. The method of claim 28, wherein the TRAIL agonist is selected from the group consisting of recombinant TRAIL, anti-TRAIL-R1 antibody, anti-TRAIL-R2 antibody, and TRAIL oligomers. [Item 30] 24. The method of claim 23, further comprising administering to the mammal an antiretroviral therapy. [Item 31] The antiretroviral therapy may be abacavir, didanosine, emtricitabine, entecavir, lamivudine, stavudine, tenofovir disoproxil fumarate, zalcitabine, zidovudine, delavirdine, efavirenz, etravirine, nevirapine, rilpivirine, adefovir, tenofovir, enfuvirtide, maraviroc, dolutegravir, elvitegravir, raltegravir, bevirimat, amprenavir, fosamprenavir, indinavir, lopinavir, nelfinavir, rifavir ... 31. The method of claim 30, wherein the agonist is selected from the group consisting of adalimumab, saquinavir, atazanavir, darunavir, tipranavir, TRIM5 alpha, tat antagonist, trichosanthin, pegylated interferon alpha (PEG-IFN-a), daclatasvir, elbasvir, grazoprevir, glecaprevir, pibrentasvir, ledipasvir, sofosbuvir, ombitasvir, paritaprevir, ritonavir, dasabuvir, simeprevir, velpatasvir, and voxilaprevir. [Item 32] 1. A method of treating cancer, comprising: administering to the mammal having cancer an antibody that binds to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) short, the antibody comprising a heavy chain variable region (VH) domain comprising complementarity determining regions (CDRs) set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a light chain variable region (VL) domain comprising CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; The method, wherein the antibody induces apoptosis in cancer cells derived from the cancer. [Item 33] 33. The method of claim 32, wherein the cancer is selected from the group consisting of ovarian cancer, melanoma, pancreatic cancer, T-cell malignancies, B-cell malignancies, bladder cancer, non-small cell lung cancer, squamous non-small cell lung cancer, non-squamous non-small cell lung cancer, colorectal cancer, melanoma, hormone receptor positive breast cancer, HER2 positive breast cancer, triple negative breast cancer, cervical cancer, esophageal cancer, head and neck cancer, papillary renal cell carcinoma, clear cell renal carcinoma, transitional renal cell carcinoma, chromophobe renal cell carcinoma, hepatocellular carcinoma, myeloma, and uterine cancer. [Item 34] 33. The method of claim 32, wherein the mammal is a human. [Item 35] 35. The method of claim 34, wherein the antibody is a humanized antibody. [Item 36] 33. The method of claim 32, further comprising administering to the mammal a TRAIL agonist. [Item 37] 37. The method of claim 36, wherein the TRAIL agonist is selected from the group consisting of recombinant TRAIL, anti-TRAIL-R1 antibody, anti-TRAIL-R2 antibody, and TRAIL oligomers. [Item 38] 33. The method of claim 32, further comprising administering to the mammal a cancer treatment. [Item 39] 39. The method of claim 38, wherein the cancer treatment is selected from the group consisting of chemotherapy, radiation therapy, brachytherapy, and surgery. [Item 40] The chemotherapeutic agent may be selected from the group consisting of altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, thiotepa, 5-fluorouracil (5-FU), 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, and pemetrexed, daunorubicin, doxorubicin, epirubicin, idarubicin, actinomycin-D, bleomycin, mitogen-activated receptor antagonist ... Mycin-C, mitoxantrone, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, vinorelbine, prednisone, ethylprednisolone, dexamethasone, L-asparaginase, bortezomib, retinoids, tretinoin, bexarotene, arsenic trioxide, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone), R-CHOP (rituximab, cyclophosphamide, doxorubicin, Vincristine, and prednisolone), trastuzumab, pertuzumab, ipilimumab, dinutuximab, siltuximab, cetuximab, panitumumab, necitumumab, ramucirumab, bevacizumab, pembrolizumab, nivolumab, atezolizumab, olaratumab, denosumab, blinatumomab, rituximab, ofatumumab, obinutuzumab, daratumumab, alemtuzumab, elotuzumab, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab, emtansine, apatinib, cabozantinib, alectinib, crizotinib , dasatinib, imatinib, nilotinib, erlotinib, gefitinib, lapatinib, sorafenib, sunitinib, tofacitinib, cobimetinib, trametinib, bortezomib, disulfiram, lactacystin, tamoxifen, obatoclax, navitoclax, gossypol, iniparib, olaparib, perifosine, dabrafenib, vemurafenib, trametinib, abemaciclib, palbociclib, ribociclib, trilaciclib, fulvestrant, temsirolimus, everolimus, vemurafenib, trametinib, dabrafenib,40. The method of claim 39, wherein the agonist is selected from the group consisting of vintafolide and vintafolide. [Item 41] 1. An antibody that blocks sk-TRAIL-mediated cell death, TRAIL short extracellular domain; and Immunoglobulin Fc domain The antibody comprising: [Item 42] 42. The antibody of claim 41, wherein the immunoglobulin Fc domain is an IgG Fc domain. [Item 43] 1. A method of treating apoptosis-related diseases and / or conditions, comprising: administering to a mammal having said disease and / or condition associated with undesired apoptosis an antibody that prevents sk-TRAIL-mediated cell death, said antibody comprising an extracellular domain of TRAIL-short and an immunoglobulin Fc domain; The method, wherein the antibody reduces apoptosis in cells from the disease and / or condition associated with unwanted apoptosis. [Item 44] 44. The method of claim 43, wherein the immunoglobulin Fc domain is an IgG Fc domain. [Item 45] 44. The method of claim 43, wherein the disease and / or condition associated with undesired apoptosis is selected from the group consisting of myocardial infarction, ischemia-reperfusion injury, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, liver damage following alcohol consumption, liver damage following acetaminophen ingestion, and non-alcoholic steatohepatitis. [Item 46] 44. The method of claim 43, wherein the mammal is a human. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 shows that TRAIL-short is produced in both uninfected and HIV-infected cells. Single cell gene expression in primary CD4 T cells from HIV-positive patients (HIV+; N=6) and HIV-negative controls (HIV-; N=3) was assessed by Fluidigm technology. A) Representative heatmap of TRAIL, TRAIL-short, mRNA levels, and expression of mRNA transcripts of cell surface markers in single cells from a single HIV-positive patient. B) HIV mRNA gene expression in cells from HIV+ and HIV- patients (Pts). C) HLA-DR gene expression in cells from HIV+ and HIV-Pts. D) Expression of TRAIL-short message in cells from HIV+ and HIV-Pts. E) TRAIL-short expression in HIV+ and HIV- cells as determined by cellular expression of HIV genes. F) TRAIL-short expression in single cells expressing or not expressing TRAIL. G) TRAIL short expression in resting (CD25-, CD38- and HLA-DR-) or activated (expressing either CD25, CD38, and / or HLA-DR). H) TRAIL short expression in T cell subsets - RTE: recent thymic emigrant; TCM: central memory T cells; TEM: effector memory T cells; TTM: transitional memory T cells. [Diagram 2]Figure 2 shows that type I interferons drive the production of TRAIL-short. For A, B, and C, primary uninfected resting (CD25-, CD69-, HLA-DR-) CD4 T cells were either unstimulated or stimulated with interferon (A), interleukin (B), or other bioactive proteins (C), and TRAIL-short mRNA was measured by qRT-PCR. D) Concomitant TRAIL and TRAIL-short mRNA expression was compared between samples. E) Resting CD4 T cells were stimulated as indicated, and TRAIL-short protein expression was assessed by Western blot. F) PBMCs were treated with vehicle control, TLR7, 8, or 9 agonists, or TLR9 inactive control for 24 hours, and TRAIL-short mRNA expression was measured. G) Resting isolated CD4 T cells were treated as in (F), measuring PBMCs and TRAIL-short mRNA. H) PBMCs were treated as in (F) and 24 hours later, CD4 T cells were isolated and TRAIL short mRNA was measured in the CD4 subset. Data represent the mean (SEM) of 5 independent experiments per treatment. P<0.05 was considered statistically significant. [Diagram 3]FIG. 3 shows that the C-terminus of TRAIL short is extracellular and interacts with TRAIL "death" receptors but not with TRAIL "decoy" receptors. A) Schematic alignment of the exons, cytosolic, transmembrane, extracellular, and 3' untranslated regions (UTRs) of TRAILFL and TRAIL short. B) 293T cells transiently expressing TRAIL short with c-myc cloned into the EC domain were stained with anti-c-myc antibody or isotype control. Key: gray fill (eGFP+TRAIL short myc with isotype control); dashed line (eGFP, anti-myc); solid line (eGFP+TRAIL short, anti-myc). C) Lysates of 293T cells transiently expressing C-terminal FLAG-tagged TRAIL R1 or R2 were combined with lysates from cells expressing N-terminal HA-tagged TRAILFL or N-terminal HA-tagged TRAIL short (indicated at the top of the panel) and then immunoprecipitated with anti-HA antibody. Complexed proteins were separated and immunoblotted for FLAG or HA. Total input lysates are shown in the bottom two panels. D) Lysates of 293T cells expressing decoy TRAIL receptors R3 and R4 were combined with lysates from cells expressing N-terminally HA-tagged TRAILFL or N-terminally HA-tagged TRAIL short (shown in the top panel) and then immunoprecipitated with anti-HA antibody. Complexed proteins were separated and immunoblotted for FLAG or HA. Total input lysates are shown in the bottom two panels. E) Binding of TRAIL short to TRAIL receptors R1, R2, R3 and R4 was determined by ELISA. F) Binding of TRAIL short to TRAIL receptors R1, R2, R3 and R4 visualized by confocal microscopy. Data shown are representative of four independent experiments. [Figure 4]FIG. 4 shows that TRAIL-short is contained in extracellular vesicles. A) Jurkat T cells transiently expressing GFP-TRAIL-short were analyzed by confocal microscopy and cells were counterstained with DAPI. B) Membrane or cytoplasmic fractions of HIV-infected and non-infected (NI) Jurkat T cells were analyzed for HSP 70 (a cytosol-selective protein) or TRAIL-short. C) Cytoplasmic, membrane, and supernatant fractions were prepared from HIV-infected and non-infected (NI) Jurkat T cells and analyzed by Western blotting with anti-TRAIL-short antibody. D) Extracellular vesicles of PHA-stimulated CD4+ cells were fixed, stained with phosphotungstic acid, and then analyzed by electron microscopy. Scale bar, 2 μm. E) Extracellular vesicles from PHA-stimulated and non-stimulated CD4+ cells were analyzed by Western blotting for TRAIL-short. F) Western blot analysis of extracellular vesicle preparations purified from 293T cells transiently expressing HA-TRAILFL and HA-TRAIL short. G) Flow cytometry analysis of extracellular vesicle preparations from the supernatant of 293T cells transfected with eGFP-TRAIL short alone, Ruby-TRAILFL alone. H) Extracellular vesicles from the supernatant of 293T cells transfected with HA-TRAILFL alone, HA-TRAIL short alone, or co-transfected with HA-TRAILFL / HA-TRAIL short at a ratio of 1:1 or 1:2 were used to treat target Jurkat T-cells. Apoptotic activity was determined by staining Jurkat cells for cleaved caspase 3. I) Western blot confirmation of HA-TRAILFL and HA-TRAIL short expression in extracellular vesicles of 293T cells from (H). J) Supernatants from control, HIV-infected, and mock-infected primary CD4 T cells in culture were separated into microvesicle or exosome fractions as indicated and analyzed by Western blot using a TRAIL-short specific monoclonal antibody. [Diagram 5]Figure 5 shows that TRAIL-short protection from TRAIL can be transferred to neighboring cells. A) Effector 293T cells expressing either TRAILFL, TRAIL-short, or both were mixed with CTO-labeled target Jurkat T cells. After incubation, cells were analyzed by flow cytometry, gated on the Cell Tracker Orange (CTO) positive (Jurkat) population, and stained for live / dead cells. B) 293T cells were transfected with 1, 2, 5, 10, or 20 μg of N-terminally HA-tagged TRAILFL alone and analyzed by Western with an anti-HA antibody. C) 293T cells were co-transfected with 1, 2, 5, or 10 μg of TRAIL-short and a constant 10 μg of TRAILFL and analyzed by Western blot. D) 293T effector cells were transfected with increasing amounts of TRAILFL from (B) and co-cultured with CTO+ target Jurkat cells, and the percentage of killing of CTO+ Jurkat cells was determined. E) 293T effector cells from (D) co-transfected with increasing amounts of TRAIL short with constant TRAILFL were co-cultured with target cells and analyzed as in (C). F) Schematic of plasmids encoding N-terminally Ruby-tagged-TRAIL short (Ruby-TRAIL short), a construct lacking the transmembrane domain (Ruby-TRAIL short ΔTM), and the Ruby tag alone (lower panel). Western analysis using anti-TRAIL short antibody and anti-actin control of lysates from 293T cells transfected with these constructs (upper panel). G) 293T cells were transfected with constructs from (F) and representative micrographs are shown (upper panel). Supernatants from 293T transfected cells were collected and used to treat target HeLa cells, which were then analyzed by confocal microscopy. Representative micrographs of HeLa nuclei stained with DAPI and overlaid with ruby-TRAIL short or Ruby-TRAIL short ΔTM filter channels are shown (bottom panel). Only HeLa cells treated with supernatant from 293T cells expressing Ruby TRAIL short have punctate, surface-localized Ruby TRAIL short on the plasma membrane.H) HeLa cells as in (G) pretreated with supernatants from 293T cells transfected with Ruby, Ruby-TRAIL short, or Ruby-TRAIL short ΔTM were treated with superkiller (sk)-TRAIL. Cell death was quantified by active caspase 3 staining over time. At the rightmost time point, the four traces are, from top to bottom, sk-TRAIL+Ruby, sk-TRAIL+Ruby-TRAIL short ΔTM, sk-TRAIL+medium, and sk-TRAIL+Ruby-TRAIL short ΔTM. Representative data from four independent experiments. P<0.05 was considered statistically significant by linear regression. [Figure 6]Figure 6 shows that TRAIL-short contains a PEST domain and is ubiquitinated and degraded by the proteasome. A) 293T cells were transfected with N-terminally HA-tagged TRAIL-short, treated with cycloheximide (100 μg / mL) and analyzed by Western analysis with an anti-HA antibody at the indicated times. B) The putative PEST domain of TRAIL-short is indicated by a "+" below the peptide sequence of TRAIL-short. C) A proline 76 to alanine (P76A) mutation was engineered into the PEST domain of TRAIL-short and cells were transfected and analyzed in the presence of cycloheximide as in (A). D) HA-tagged TRAIL-short was transfected into 293T cells and immunoprecipitated with anti-HA antibody, and then immunoprecipitates were treated with various deubiquitinases (DUBs) that distinguish between ubiquitin linkages between Lys48 and Lys63 (OTUB1, AMSH, OOTUD3, TRABID, OTUD7B, YOD1, Otulin, or UP2). Treated samples were resolved by SDS-PAGE and immunoblotted for ubiquitin. E) 293T cells transfected with TRAIL-short were incubated in the presence of MG132 for the indicated times, then harvested and analyzed by Western with anti-TRAIL-short. F) TRAIL-short expression is increased in the P76A mutant. TRAIL-short and P76A TRAIL-short were transfected into 293T cells and analyzed for TRAIL-short by flow cytometry (lower panel) or with an isotype control antibody (upper panel). All data are representative of at least three independent experiments. [Figure 7]FIG. 7 shows that TRAIL short is necessary and sufficient to cause TRAIL resistance. A) Jurkat T cells constitutively expressing TRAIL short were induced to die by addition of sk-TRAIL in the presence or absence of increasing amounts of anti-TRAIL short antibody. Cell death was measured by active caspase 3 staining. Control cells were treated with increasing amounts of anti-TRAIL short antibody alone. At the far right time point, the top four traces are, from top to bottom, 5 μg / mL anti-TRAIL short, 2.5 μg / mL anti-TRAIL short, 1 μg / mL anti-TRAIL short, and 0 μg / mL anti-TRAIL short. B) HeLa cells not expressing TRAIL short were stimulated to die by addition of sk-TRAIL in the absence or presence of increasing amounts of a fusion protein consisting of the extracellular domain of TRAIL short fused to Fc (Ts-ECD:Fc) or stimulated with bovine serum albumin (BSA) control and analyzed as in (A). Additional control cells were treated with increasing amounts of TRAIL short-ECD:Fc alone. At the far right time points, the top five traces are, from top to bottom, 0 μg / mL anti-TRAIL short-ECD:Fc, 10 μg / mL BSA, 5 μg / mL anti-TRAIL short-ECD:Fc, 2.5 μg / mL anti-TRAIL short-ECD:Fc, and 10 μg / mL anti-TRAIL short-ECD:Fc. Data are representative of six independent experiments. [Figure 8] Figure 8 shows that overexpression of TRAIL-short reduces NK cell cytotoxicity. A) Western blot shows overexpression of TRAIL-short-EGFP-C1 in Jurkat cells. B) NK cells were co-cultured with CTO-labeled Jurkat cells and stained for activated caspase-3. C) Cytotoxicity assays were performed with Jurkat cells in the presence of TRAIL-short or control plasmids at increasing E:Tt ratios. EGFP-C1 is on the left and TRAIL-short-EGFP-C1 is on the right in all cases. [Figure 9]Figure 9 includes flow cytometry showing that HIV IIIB induces surface expression of TRAIL-short on NK cells. NK cells were incubated with culture supernatants containing HIV IIB virus or Gp120 recombinant protein and stained for surface expression of TRAIL-short and TRAIL-FL. [Figure 10] Figure 10 contains graphs showing that TRAIL-short does not affect NK activation. NK cells were co-cultured with Jurkat cells overexpressing TRAIL-short (gray bars), isolated, and stained for CD16, perforin, CD107, and CD69. [Figure 11] FIG. 11 includes schematic diagrams showing: A) how TRAIL-short occupies TRAIL receptors 1 and / or 2 on target cells, thus preventing TRAIL-mediated cell death; B) how TRAIL-short antibodies can bind to and block TRAIL-short, allowing TRAIL to bind to R1 or R2 and induce apoptosis. [Figure 12] Figure 12 includes amino acid sequences of heavy chain variable region (VH) and light chain variable region (VL) of representative anti-TRAIL short antibodies. A. Amino acid sequence alignment including mouse VH (VH0) sequence (SEQ ID NO: 4) aligned with four humanized VH variants: VH1 (SEQ ID NO: 5), VH2 (SEQ ID NO: 6), VH3 (SEQ ID NO: 7), and VH4 (SEQ ID NO: 8). CDRs are underlined. B. Amino acid sequence alignment including mouse VL (VL0) sequence (SEQ ID NO: 17) aligned with four humanized VL variants: VL1 (SEQ ID NO: 18), VL2 (SEQ ID NO: 19), VL3 (SEQ ID NO: 20), and VL4 (SEQ ID NO: 21). CDRs are underlined. [Figure 13-1]Figure 13 includes amino acid sequences of humanized anti-TRAIL short antibodies: A. Amino acid sequences of humanized heavy chains HC0 (SEQ ID NO:9), HC1 (SEQ ID NO:10), HC2 (SEQ ID NO:11), HC3 (SEQ ID NO:12), and HC4 (SEQ ID NO:13). B. Amino acid sequences of humanized heavy chains LC0 (SEQ ID NO:22), LC1 (SEQ ID NO:23), LC2 (SEQ ID NO:24), LC3 (SEQ ID NO:25), and LC4 (SEQ ID NO:26). [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14-1] FIG. 14 shows that in vivo administration of anti-TRAIL short antibody (2.2) can reduce the number of leukemia cells and extend the survival time of mice. A) Representative photographs showing luciferase expression at day 52 in mice treated with anti-TRAIL short antibody (left panel) or isotype control (IC; right panel) on day 27 and anti-TRAIL receptor 2 (anti-DR5) antibody the next day (e.g., day 28). B) Photographs optimized for grayscale display showing luciferase expression at day 45 in mice treated with anti-TRAIL short antibody (left panel) or isotype control (IC; right panel) on day 27 and anti-TRAIL receptor 2 (anti-DR5) antibody the next day (e.g., day 28). C) Graph showing luciferase expression in mice treated with IC or anti-TRAIL short antibody on day 27, indicated by dotted line, and anti-TRAIL receptor 2 (anti-DR5) antibody on day 28, indicated by dashed line. D) Graph showing percent survival in mice treated with IC or anti-TRAIL short antibody on day 27, indicated by dotted line, and anti-TRAIL receptor 2 (anti-DR5) antibody on day 28, indicated by dashed line. E) Graph showing luciferase expression in mice treated with IC or anti-TRAIL short antibody on days 20, 27, and 34, indicated by dotted line. F) Graph showing percent survival in mice treated with IC or anti-TRAIL short antibody on days 20, 27, and 34, indicated by dotted line. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 15-1]FIG. 15 includes graphs showing the effect of anti-TRAIL short antibodies on sk-TRAIL cytotoxicity in different cancer cell lines. Cancer cells were preincubated with anti-TRAIL short antibody 2.2 (Ts 2.2) or IgG3 control, and then sk-TRAIL was added to the cells. Cell death (caspase 3 / 7+) was measured over time in A) Jeko and HBL1 lymphoma leukemia cells, B) L3.6, BxPc3, and Miapaca pancreatic cancer cells, C) MeLa 15, sk-mel-28, C32TG, and A375 melanoma cells, and D) TYKNU, Kuramochi, hOvtax2, CaOv3, COV362, PEO1, OVCAR5, and Ovcar8 ovarian cancer cells. Where indicated, p-values ​​are based on sk-TRAIL with anti-TRAIL short antibody 2.2 vs. sk-TRAIL alone. [Figure 15-2] This is a continuation of Figure 15-1. [Figure 15-3] This is a continuation of Figure 15-1. [Figure 16] 16 includes graphs showing quantification of TRAIL-short (TRAIL-s) mRNA in various cancer cells. Using qRTPCR, the copy number of TRAIL-s was calculated using TRAIL-s standards. [Figure 17] Figure 17 includes immunohistochemistry images showing tissue stained using TRAIL short 2.2 antibody. Shown are examples of normal and corresponding cancer tissue stained positive for TRAIL short (upper panel). Also shown is a 40x magnification of a malignant sample (lower panel). Pancreatic, gastric and prostate cancer tissues show significantly elevated expression of TRAIL short compared to non-malignant tissue. [Figure 18]Figure 18 shows that TRAIL-short expression is observed by immunohistochemistry of tissue microarrays. The figure is an overview of eight tissue microarrays (TMA) performed using anti-TRAIL-short antibody 2.2 at a 1:400 dilution. The inner circle represents the percent of cases in that array that are positive for TRAIL-short staining. The outer circle represents the immunoreactivity score calculated as follows: percent tissue positivity (0-25%=1, 26-50%=2.51-75%=3, and 76-100%=4) x intensity of staining (negative=0, weak=1, moderate=2, and strong=3). [Figure 19] Figure 19 shows that TRAIL shorts are found in many human tumors. TRAIL short levels quantified as reads per million kilobases (RPKM) are shown in various human tumors. Data was determined from the Cancer Genome Atlas (TCGA). [Figure 20-1] Figure 20 shows that increased levels of TRAIL short are associated with decreased survival in some human tumors. Survival was assessed by Kaplan-Meier analysis in patients with diffuse large B-cell lymphoma (A), colon and rectal adenocarcinoma (B), chromophobe renal cell carcinoma (C), pancreatic adenocarcinoma (D), and thymoma (E). P values ​​are as indicated. [Figure 20-2] This is a continuation of Figure 20-1. [Figure 21] FIG. 21 validates that anti-TRAIL short antibodies interact with the TRAIL short antigen. HEK293T cells were transfected with plasmids encoding green fluorescent protein (GFP) (top row), GFP-TRAIL short (middle row), or GFP-TRAILFL (bottom row). Cells were stained with anti-TRAIL short antibodies, secondary goat anti-mouse phycoerythrin (PE) (middle row, left column), and 4',6-diamidino-2-phenylindole (DAPI) (middle row, right column). Cells were analyzed by confocal microscopy for PE labeling (red; far left column), GFP (green; middle row, left column), and DAPI stained nuclei (blue; middle row, right column). Overlaid composite images of all three colors are also shown (overlay, far right column). [Figure 22] FIG. 22 shows that the anti-TRAIL short antibody clone HC2LC3 exhibited an affinity of 3.8 pM and demonstrated significant synergistic killing in the presence of sk-TRAIL (1 ng / ml). [Figure 23-1] Figure 23 shows that some patient-derived cells were responsive to anti-TRAIL short antibody + sk-TRAIL. Freshly isolated cells from the spleen of a patient who underwent splenectomy for suspected hematological malignancies were treated with nothing (control), superkiller TRAIL (sk-TRAIL, an oligomerized TRAIL agonist), humanized anti-TRAIL short antibody (clone HC2LC3), or isotype control antibody (IgG4) at the indicated concentrations (and alone or in the indicated combinations). Cell death was monitored over time by analyzing active caspase 3 / 7 activity over time using the Incucyte live cell imaging platform. A) Results from a patient with angioimmunoblastic T-cell lymphoma (lower panel shows IHC of TRAIL short in different cases with the same diagnosis, showing significant expression of TRAIL short). B) Results from a patient with Hodgkin's lymphoma (lower panel shows IHC of TRAIL short in different cases with the same diagnosis, showing minimal expression of TRAIL short). C) Results from a patient with relapsed B cell lymphoma (lower panel shows flow cytometry of TRAIL short, TRAIL, DR4, DR5 of the same cells). D) Results from a patient with mantle cell lymphoma (lower panel shows flow cytometry of TRAIL short, TRAIL, DR4, DR5 of the same cells). E) Results from a patient with non-Hodgkin's lymphoma B cell lineage / cyclin D positive. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 23-3] This is a continuation of Figure 23-1. [Figure 23-4] This is a continuation of Figure 23-1. [Figure 24-1]FIG. 24 includes graphs showing the effect of anti-TRAIL short antibodies on sk-TRAIL cytotoxicity in different human patient-derived cancer cell lines. Cells from patient spleens were received fresh in RPMI and tested for sensitivity to cytotoxicity in the presence of a 1 ng / mL dose of sk-TRAIL in the presence or absence of increasing doses (1.25, 2.5, or 5 μg) of the anti-TRAIL-short clone HC2LC3. Controls were treated with the same dose of human IgG4. Cell death was monitored every 2 hours for 72 hours by the number of cleaved caspase 3 / 7 positive cells. Cancer types shown in panels A-I are as labeled. [Figure 24-2] This is a continuation of Figure 24-1. [Figure 24-3] This is a continuation of Figure 24-1. [Diagram 25] Figure 25 shows that responsiveness to anti-TRAIL short plus sk-TRAIL is higher than that of sk-TRAIL alone in multiple human patient derived cancer cell lines. Shown are the number of dead cells 48 hours after treatment with 1 ng / mL dose of sk-TRAIL alone or sk-TRAIL (same dose) + 5 μg / mL dose of anti-TRAIL short clone HC2LC3. Responsiveness was defined as a statistically significant increase in the number of dead cells after addition of anti-TRAIL short antibody. Paired t-test statistics are shown. [Figure 26] Figure 26 shows summary results of the responsiveness of human patient-derived cell lines to anti-TRAIL-short, both alone and in combination with sk-TRAIL. Responsiveness was defined as a statistically significant increase in the number of dead cells compared to treatment with sk-TRAIL alone. [Figure 27] Figure 27 shows increased TRAIL short expression in cell lines responding to anti-TRAIL short. A) Quantification of TRAIL short mRNA copy number using real-time qPCR in various responding and non-responsive cell lines. B) Percentage of TRAIL short positives measured by staining three cell lines using flow cytometry with anti-TRAIL-short 2.2 or isotype conjugated to CF555 or isotype. [Figure 28-1] Figure 28 shows TRAIL short expression in 293T cells and tumor tissues from patients. Immunohistochemistry slides stained for TRAIL short using anti-TRAIL short antibody at 1:400 dilution. A) The left column shows 293T cells transfected with EGFP (20x magnification), the right column shows 293T cells transfected with TRAIL short EGFP (20x magnification) stained with an antibody specific for TRAIL short (seen in brown). The top row is a composite image, the middle row is a component of the image stained in blue, and the bottom row is a component of the image stained in brown (stained for TRAIL short). B) Tissues from a patient with pancreatic adenocarcinoma at 20x (left) and 40x (right) magnification. The top row is a composite image, the middle row is a component of the image stained in blue, and the bottom row is a component of the image stained in brown (stained for TRAIL short). C) Tissues from a patient with squamous cell carcinoma tonsil at 20x (left) and 40x (right) magnification. The top row is the composite image, the middle row is the blue stained component of the image, and the bottom row is the brown stained component of the image (stained for TRAIL short). [Figure 28-2] This is a continuation of Figure 28-1. [Figure 28-3] This is a continuation of Figure 28-1. [Figure 29] Figure 29 shows that NSG mice implanted with human Jurkat T-cell lymphoma cells are effectively treated with a combination of anti-DR5 and anti-TRAIL short antibodies. Established tumors were treated every 2 weeks as indicated with 10 mg / kg every 14 days of either (i) TRAIL short isotype control and DR5 antibody isotype (referred to as isotype), (ii) anti-TRAIL short antibody clone 2.2 + anti-DR5 (referred to as anti-TRAIL short), (iii) anti-TRAIL short antibody + anti-DR5 antibody isotype control (referred to as isotype control + anti-DR5), or (iv) anti-TRAIL short (2.2) + anti-DR5. A) Over time, mice were analyzed for luciferase expression by whole body imaging. B) Survival assessed by Kaplan-Meier analysis. [Diagram 30] Figure 30 shows that anti-TRAIL short antibody + isotype control or anti-TRAIL short antibody + anti-DR5 antibody results in the suppression of tumor growth in mouse xenograft models of human cancer. NSG mice were implanted subcutaneously with human diffuse large B-cell lymphoma (HBL-1) cell line and tumor size was measured daily. When tumors reached a size greater than or equal to 100 cubic mm, mice received the indicated treatments by weekly IP injection as indicated by either (i) TRAIL short and DR5 antibody isotype control (IC+IC), (ii) anti-TRAIL short antibody clone 2.2 + anti-DR5 isotype control (2.2+IC), (iii) anti-TRAIL short antibody + anti-DR5 antibody isotype control (IC+DR5), or (iv) anti-TRAIL short (2.2) + anti-DR5 antibody (2.2+DR5) at 10 mg / kg every 14 days. A) Over time, mice were analyzed for tumor size and fold change calculated from baseline. B) Survival assessed by Kaplan-Meier analysis. [Figure 31-1] FIG. 31 shows that TRAIL short can be detected in tissues by in situ hybridization (ISH). Macaque tissues infected with simian immunodeficiency virus (SIV) were stained with either a TRAIL full-length-specific probe or a TRAIL short-specific probe and visualized at the following magnifications: in A-D, the left column is at 10× magnification, the middle column is at 20× magnification, and the right column is at 40× magnification. In A-D, the top row is the composite image, the middle row is the components of the composite image represented in red (TRAIL in (A) and (C), and TRAIL short in (B) and (D)), and the bottom row is the components of those composite images represented in blue. A) SIV-infected macaque axillary lymph node tissue stained with a TRAIL-specific probe. B) SIV-infected macaque axillary lymph node tissue stained with a TRAIL short-specific probe. C) SIV-infected macaque spleen tissue stained with a TRAIL-specific probe. D) SIV-infected macaque spleen tissue stained with a TRAIL short-specific probe. [Figure 31-2] This is a continuation of Figure 31-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] This document provides TRAIL short antibodies (e.g., humanized TRAIL short antibodies) capable of neutralizing TRAIL shorts, as well as materials and methods for making and using TRAIL short antibodies. In some cases, one or more TRAIL short antibodies can be used to treat a disease (e.g., cancer and / or liver conditions) and / or an infection (e.g., chronic infection). For example, one or more TRAIL short antibodies can be administered to a mammal having a disease and / or infection under conditions that reduce the number of diseased cells (e.g., cancer cells) and / or infected cells. In some cases, one or more TRAIL short antibodies can be used to modulate (e.g., increase or decrease) apoptosis (e.g., TRAIL-mediated cell death, NK cytotoxicity, CD8+ T cell killing, CAR T cell killing, and / or oncolytic virotherapy). For example, one or more TRAIL short antibodies can be administered to a mammal having diseased cells (e.g., cancer cells) and / or infected cells (e.g., virus-infected cells) under conditions that induce apoptosis in one or more of the diseased cells (e.g., cancer cells) and / or in one or more of the infected cells. This document also provides nucleic acids encoding the TRAIL short antibodies described herein, as well as constructs expressing nucleic acids encoding the TRAIL short antibodies described herein.

[0019] When treating a disease as described herein, the disease may be, for example, cancer (e.g., carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma, and metastasis) or a liver condition. Examples of cancers that may be treated as described herein include, but are not limited to, ovarian cancer, melanoma, pancreatic cancer, hematological malignancies (e.g., T-cell malignancies and B-cell malignancies), bladder cancer, lung cancer (e.g., non-small cell lung cancer, squamous non-small cell lung cancer, and non-squamous non-small cell lung cancer), colon cancer, melanoma, breast cancer (e.g., hormone receptor positive breast cancer, HER2 positive breast cancer, and triple negative breast cancer), cervical cancer, esophageal cancer, head and neck cancer, kidney cancer (e.g., papillary cell renal carcinoma, clear cell renal carcinoma, transitional cell renal carcinoma, and chromophobe renal cell carcinoma), hepatocellular carcinoma, myeloma, and uterine cancer.

[0020] When treating infectious diseases as described herein, the infectious disease may be, for example, chronic infectious disease and / or viral infectious disease. Examples of infectious diseases that can be treated as described herein include, but are not limited to, HIV, SIV, endogenous retrovirus, anellovirus, circovirus, human herpes virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, polyoma virus, adeno-associated virus, herpes simplex virus, adenovirus, hepatitis B virus, hepatitis C virus, hepatitis D virus, GB virus C, papilloma virus, human T-cell leukemia virus, xenotropic murine leukemia virus-related virus, polyoma virus, rubella virus, parvovirus, measles virus, and coxsackie virus infection. In some cases, the infectious disease to be treated as described herein may be HIV infection.

[0021] When treating undesired apoptosis as described herein, the undesired apoptosis may be associated with, for example, any suitable disease and / or condition. Examples of diseases and / or conditions associated with undesired apoptosis that can be treated as described herein include, but are not limited to, myocardial infarction, ischemia-reperfusion injury, neurodegenerative diseases (e.g., multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease), liver damage (e.g., after alcohol consumption, after drug (e.g., acetaminophen) intake, and non-alcoholic steatohepatitis).

[0022] Any type of mammal having a disease and / or infection or at risk of developing a disease and / or infection can be treated as described herein. For example, humans and other primates, such as monkeys, having a disease and / or infection can be treated with one or more TRAIL short antibodies. In some cases, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats can be treated with one or more TRAIL short antibodies described herein.

[0023] Any suitable method can be used to identify a mammal having a disease and / or infection or at risk of developing a disease (e.g., cancer) and / or infection. For example, imaging techniques (with or without contrast; e.g., computed tomography (CT) scan or magnetic resonance imaging (MRI)), biopsy techniques, bone marrow aspiration, colonoscopy, sigmoidoscopy, digital rectal examination, blood tests, platelet tests, fecal tests, urine tests, endoscopic techniques, ELISA techniques, PCR-based techniques, blotting techniques (e.g., Western blot), and histological techniques can be used to identify a mammal (e.g., human) having a cancer. For example, imaging techniques (with or without contrast; e.g., CT scan or MRI), biopsy techniques (e.g., liver tissue examination), and blood tests can be used to identify a mammal (e.g., human) having a liver condition. For example, antibody techniques, viral antigen detection tests, culture techniques, ELISA techniques, PCR-based techniques (e.g., viral load tests), blotting techniques (e.g., Western blots), hybridization techniques (e.g., ISH), and histological techniques (e.g., immunohistochemistry (IHC)) can be used to identify mammals (e.g., humans) that have an infectious disease.

[0024] In some cases, one or more TRAIL short antibodies can be used to identify mammals that are likely to respond well to TRAIL-based therapy. For example, one or more TRAIL short antibodies can be administered to a mammal with a disease and / or infection to identify mammals that are likely to respond well to TRAIL-short based therapy. In some cases, a mammal with a disease and / or infection or at risk of developing a disease and / or infection can be evaluated for the presence or absence of TRAIL short and / or TRAIL. For example, the presence, absence, or level of TRAIL short and / or TRAIL in a sample obtained from a human with a disease and / or infection can be used to determine whether the human is likely to respond to a TRAIL-based therapy. Any suitable sample can be evaluated for the presence of TRAIL short and / or TRAIL. For example, biological samples, such as tissue samples and body fluid samples (e.g., blood, serum, plasma, or urine), can be obtained from a mammal and the presence, absence, or level of TRAIL short and / or TRAIL can be evaluated. Any suitable method can be used to detect the presence, absence, or level of TRAIL short and / or TRAIL. For example, antibody techniques, viral antigen detection tests, culture techniques, ELISA techniques, PCR-based techniques (e.g., viral load tests), blotting techniques (e.g., Western blots), hybridization techniques (e.g., ISH), and histological techniques (e.g., IHC) can be used to determine the presence, absence, or level of TRAIL short and / or TRAIL in a sample obtained from a mammal. In some cases, the TRAIL short antibodies described herein (e.g., comprising a VH domain comprising the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a VL domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16) can be labeled (e.g., with a radionuclide to enable PET or SPECT imaging) to determine the presence, absence, or level of TRAIL short and / or TRAIL in a sample obtained from a mammal.A mammal can be identified as likely to respond to TRAIL-based therapy if a sample obtained from the mammal has detectable levels of TRAIL-short and / or TRAIL. In some cases, a mammal identified as likely to respond to TRAIL-based therapy can be treated as described herein. For example, one or more anti-cancer agents that inhibit IL-6, IL-8, and EGF can be administered to a human identified as having triple-negative breast cancer (e.g., TNBC) to treat the human.

[0025] Once identified as having a disease and / or infection, at risk of developing a disease and / or infection, or likely to respond well to a TRAIL-based therapy, the mammal may be administered or instructed to self-administer one or more TRAIL short antibodies (e.g., a composition comprising one or more TRAIL short antibodies that neutralize the TRAIL short). In some cases, a mammal (e.g., a human) may be identified as having a TRAIL short. For example, the mammal may be evaluated for the presence or absence of a TRAIL short using any suitable method. For example, techniques for detecting the level of a TRAIL short include ELISA techniques, PCR-based techniques, blotting techniques (e.g., Western blot), hybridization techniques (e.g., ISH), and / or histological techniques (e.g., IHC). The presence or absence of a TRAIL short can be used, for example, to determine that the mammal is likely to respond to a TRAIL short antibody therapy.

[0026] Any suitable method can be used to administer one or more TRAIL short antibodies to a mammal. For example, one or more TRAIL short antibodies can be administered to a mammal and / or a nucleic acid encoding one or more TRAIL short antibodies can be administered to a mammal. When a nucleic acid encoding one or more TRAIL short antibodies is administered to a mammal, the nucleic acid can be included in a vector (e.g., for vector-mediated antibody gene transfer).

[0027] The TRAIL short antibodies provided herein specifically bind to an epitope on a TRAIL short. The term "antibody" as used herein refers to intact antibodies as well as antibody fragments (e.g., antigen-binding fragments) that retain some ability to bind to a TRAIL short. In some cases, the TRAIL short antibodies provided herein do not bind to full-length TRAIL. The TRAIL short antibodies can be monoclonal or polyclonal antibodies. The TRAIL short antibodies can be chimeric (e.g., partially humanized or fully humanized) antibodies. The TRAIL short antibodies can be non-immunogenic antibodies. In some cases, the TRAIL short antibodies can be neutralizing antibodies. As used herein, a TRAIL short "neutralizing" antibody is an antibody that binds to a TRAIL short and neutralizes the biological effect of the TRAIL short. In some cases, the TRAIL short antibody can induce (e.g., increase) apoptosis. For example, a TRAIL short neutralizing antibody binds to a TRAIL short and prevents the TRAIL short from binding to R1 and / or R2, thus making R1 and R2 available for TRAIL binding and induction of apoptosis. For example, a TRAIL short neutralizing antibody binds to a TRAIL short and releases the bound TRAIL short from R1 and / or R2, thus making R1 and R2 available for TRAIL binding and induction of apoptosis. In some cases, a TRAIL short antibody can recapitulate a TRAIL short function. As used herein, "TRAIL short function" includes the ability to reduce and / or eliminate TRAIL-mediated cell death. In some cases, a TRAIL short antibody can inhibit (e.g., reduce) apoptosis. For example, a TRAIL short antibody provided herein can include an extracellular domain of a TRAIL short fused to an Fc domain (e.g., an IgG Fc domain). For example, an extracellular domain of a TRAIL short fused to an IgG Fc domain can be used to reduce and / or eliminate apoptosis (e.g., undesired apoptosis).

[0028] The TRAIL short antibodies provided herein can be prepared using any suitable method (see, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), pp. 1-5 (Humana Press 1992); Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols In Immunology, section 2.4.1 (1992); Coligan et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994; Kohler & Milstein, Nature vol. 256: p. 495 (1975); Coligan et al., section 2.5.1 2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, p. 726 (Cold Spring Harbor Pub. 1988)). For example, a sample containing a TRAIL short can be used as an immunogen to induce an immune response in an animal so that a specific antibody is produced.

[0029] The TRAIL short antibodies provided herein can be humanized and / or deimmunized (e.g., made non-immunogenic) using any suitable method. For example, a humanized monoclonal antibody can be generated by transferring mouse CDRs from the heavy and light variable chains of a mouse immunoglobulin into a human variable domain, and then substituting human residues into the framework regions of the mouse counterpart. The use of antibody components derived from a humanized monoclonal antibody can obviate potential problems associated with the immunogenicity of mouse constant regions when treating humans. General techniques for cloning mouse immunoglobulin variable domains are described, for example, in Orlandi et al., Proc. Nat'l. Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Carter et al., Proc. Nat'l. Acad. Sci. USA 89:4285 (1992); and Sandhu, Crit. Rev. Biotech. 12:437 (1992); Singer et al., J. Immunol. 150:2844 (1993). In some cases, humanization, e.g., superhumanization, can be used as described elsewhere (Hwang et al., Methods, 36:35-42 (2005)).In some cases, SDS grafting (Kashmiri et al., Methods, 36:25-34 (2005)), human string content optimization (Lazar et al., Mol. Immunol., 44:1986-1998 (2007)), framework shuffling (Dall'Acqua et al., Methods, 36:43-60 (2005); and Damschroder et al., Mol. Immunol., 44:3049-3060 (2007)), and phage display approaches (Rosok et al., J. Biol. Chem., 271:22611-22618 (1996); Radar et al., Proc. Natl. Acad. Sci. USA 95:8910-8915 (1998); and Huse et al., Science 246:1275-1281 (1989)) can be used to obtain TRAIL short antibody preparations. In some cases, the TRAIL short antibody can be humanized as described in Examples 3 and 4. The TRAIL short antibody can be the antibody Ab866.

[0030] The TRAIL short antibody heavy chain can include an immunoglobulin Fc domain (e.g., a humanized immunoglobulin Fc domain). The Fc domain can be from any isotype (e.g., IgG, IgA, IgD, IgE, or IgM). The Fc domain can be from any isotype subclass. For example, if the Fc domain is an IgG isotype, the Fc domain can be an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0031] A TRAIL short antibody (e.g., antibody Ab866) VH domain can include the complementarity determining regions (CDRs) set forth below: TIFF0007604428000001.tif24113

[0032] Examples of TRAIL short antibody VH domains comprising VH CDR1, VH CDR2R, and VH CDR3 include, but are not limited to, the VH domains set forth below: TIFF0007604428000002.tif177159

[0033] In some cases, a TRAIL short antibody VH domain may have an amino acid sequence that is at least 75 percent (e.g., at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to a sequence set forth in any one of SEQ ID NOs: 4-8. For example, a TRAIL short antibody heavy chain VH domain may have an amino acid sequence that is at least 75% identical to SEQ ID NO: 4. For example, a TRAIL short antibody heavy chain VH domain may have an amino acid sequence that is at least 75% identical to SEQ ID NO: 6.

[0034] An example of a TRAIL short antibody heavy chain is set forth below: TIFF0007604428000003.tif235156TIFF0007604428000004.tif154156

[0035] A TRAIL short antibody (e.g., antibody Ab866) VL domain can include the CDRs set forth below: TIFF0007604428000005.tif23103

[0036] Examples of TRAIL short antibody VL domains comprising VL CDR1, VL CDR2R, and VL CDR3 include, but are not limited to, the VL domains set forth below: TIFF0007604428000006.tif176157

[0037] In some cases, a TRAIL short antibody VL domain may have an amino acid sequence that is at least 75 percent (e.g., at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to a sequence set forth in any one of SEQ ID NOs: 17-21. For example, a TRAIL short antibody heavy chain VL domain may have an amino acid sequence that is at least 75% identical to SEQ ID NO: 17. For example, a TRAIL short antibody heavy chain VL domain may have an amino acid sequence that is at least 75% identical to SEQ ID NO: 20.

[0038] An example of a TRAIL short antibody light chain is set forth below: TIFF0007604428000007.tif241155TIFF0007604428000008.tif16156

[0039] The TRAIL short antibodies described herein can include any combination of the heavy chains described herein and the light chains described herein. In some cases, the TRAIL short antibodies can include HC2 and LC3.

[0040] In some cases, the TRAIL short antibodies described herein can include (e.g., fused or conjugated with) one or more labels (e.g., detectable labels). The labels can be, but are not limited to, fluorescent labels (e.g., fluorophores), radioactive labels, or enzymes. Examples of detectable labels include, but are not limited to, R-phycoerythrin (PE), CTO, GFP, fluorogenic activating protein (FAP), Gaussia luciferase (GLuc), Cypridina luciferase (Cluc), radionuclides, and biotin.

[0041] In some cases, one or more TRAIL short antibodies (e.g., one, two, three, four, five, or more TRAIL short antibodies) can be administered to a mammal to treat a disease and / or infection. For example, two or more TRAIL short antibodies can be administered to a mammal (e.g., a human) to treat a disease and / or infection.

[0042] In some cases, a composition comprising one or more TRAIL short antibodies can be administered as the sole active ingredient to a mammal having a disease and / or infection.

[0043] In some cases, a composition comprising one or more TRAIL short antibodies can be administered to a mammal having a disease and / or infection as a combination therapy with one or more additional drugs / therapies used to treat the disease and / or infection. For example, a combination therapy used to treat a mammal having a disease and / or infection can include administering to a mammal (e.g., a human) one or more TRAIL short antibodies combined with one or more cell-based therapies and / or one or more TRAIL-based therapies. TRAIL-based therapies can include, for example, TRAIL modulators, such as, but not limited to, recombinant TRAIL (e.g., dulanermin), anti-TRAIL-R1 antibodies (e.g., mapatumumab), anti-TRAIL-R2 antibodies (e.g., conatumumab, lexatumumab, tigatuzumab, droditumab, LBY-135), TRAIL oligomers (e.g., ABBV-621), and / or TRAIL short extracellular domain:Fc fusions. In some cases, the TRAIL modulator can be a TRAIL agonist. In some cases, the TRAIL modulator may include (e.g., be fused or conjugated to) one or more additional domains (e.g., domains that enhance stability and / or function, such as polyhistidine, a FLAG epitope, an isoleucine zipper motif (LZ), the Fc portion of a human immunoglobulin, albumin, and / or a nanoparticle). When two or more (e.g., two, three, four, or more) TRAIL modulators are administered to a mammal, the TRAIL modulators can be administered alone or in any combination. Cell-based therapies can include, for example, adoptive cell transfer therapy (e.g., adoptive T cell therapy), such as, but not limited to, adoptive transfer of tumor-infiltrating lymphocytes (TILs), including autologous TILs, TILs engineered with alpha-beta T cell receptors, TILs engineered with chimeric antigen receptors (CAR T cell therapy), and / or engineered NK or NK / T cells.

[0044] Combination therapies used to treat a mammal with cancer include administering to the mammal (e.g., a human) a composition comprising one or more TRAIL short antibodies and one or more cancer therapies, such as chemotherapeutic agents, including, but not limited to, alkylating agents (e.g., altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, and thiotepa), Antimetabolites (e.g., 5-FU), 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, and pemetrexed), anthracycline antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, and idarubicin), non-anthracycline antibiotics (e.g., actinomycin-D, bleomycin, mitomycin-C, and mitoxantrone), topoisomerase inhibitors (e.g., cyclosporine ... These include but are not limited to: topoisomerase I inhibitors (e.g., topotecan and irinotecan (e.g., CPT-11)), topoisomerase II inhibitors (e.g., etoposide (e.g., VP-16), teniposide, and mitoxantrone), mitotic inhibitors (e.g., docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, and vinorelbine), corticosteroids (e.g., prednisone, ethylprednisolone, and dexamethasone), enzyme inhibitors (e.g., ribavirin ... L-asparaginase), and / or proteasome inhibitors (e.g., bortezomib); differentiation inducers, such as, but not limited to, retinoids, tretinoin, bexarotene, and arsenic trioxide; cocktail therapy (e.g., a combination of cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP), or a combination of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP));Monoclonal antibodies, such as, but not limited to, anti-HER2 antibodies (e.g., trastuzumab and pertuzumab), anti-CTLA-4 antibodies (e.g., ipilimumab), anti-GD2 antibodies (e.g., dinutuximab), anti-IL-6 antibodies (e.g., siltuximab), anti-EGFR antibodies (e.g., cetuximab, panitumumab, and necitumumab), anti-VEGF antibodies (e.g., ramucirumab and bevacizumab), anti-PD-1 antibodies (e.g., pembrolizumab and nivolumab), anti-PD-L1 antibodies (e.g., atezolizumab), anti-PDGF antibodies (e.g., for example, olaratumab), anti-RANK antibodies (e.g., denosumab), anti-CD3 antibodies (e.g., blinatumomab), anti-CD19 antibodies (e.g., blinatumomab), anti-CD20 antibodies (rituximab, ofatumumab, and obinutuzumab), anti-CD38 antibodies (e.g., daratumumab), anti-CD52 antibodies (e.g., alemtuzumab), and anti-SLAMF7 antibodies (e.g., elotuzumab); antibody-drug conjugates, for example, but not limited to, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab, and emtansine;Targeted small molecules, such as, but not limited to, tyrosine kinase inhibitors (e.g., VEGF inhibitors (e.g., apatinib), c-Met inhibitors (e.g., cabozantinib), ALK inhibitors (e.g., alectinib and crizotinib), Brc-Abl inhibitors (e.g., dasatinib, imatinib, and nilotinib), EGFR inhibitors (e.g., erlotinib and gefitinib), HER2 inhibitors (e.g., lapatinib), multi-targeting Tyrosine kinase inhibitors (e.g., sorafenib and sunitinib), JAK1 inhibitors (e.g., tofacitinib), MEK inhibitors (e.g., cobimetinib and trametinib), proteasome inhibitors (e.g., ixazomib, carfilzomib, bortezomib, disulfiram, and lactacystin), estrogen receptor modulators (e.g., tamoxifen), Bcl-2 inhibitors (e.g., obatoclax, navitoclax, and gossypol), PARP inhibitors (e.g., iniparib and olaparib), PI3K inhibitors (e.g., perifosine), BRAF inhibitors (e.g., dabrafenib and vemurafenib), MEK inhibitors (e.g., trametinib), CDK inhibitors (e.g., abemaciclib, palbociclib, ribociclib, and trilaciclib), selective estrogen receptor degraders (e.g., fulvestrant), BET inhibitors, serine / threonine kinase inhibitors (e.g., temsirolimus, everolimus, vemurafenib, trametinib, and dabrafenib); small molecule drug conjugates, such as, but not limited to, vintafolide; radiation therapy (e.g., external beam therapy, intensity modulated radiation therapy, image-guided radiation therapy, proton beam therapy); brachytherapy; and / or surgery (e.g., surgical resection of the tumor, a portion of a tumor, or a metastasis);

[0045] Combination therapies used to treat a mammal having a hepatic condition can include administering to a mammal (e.g., a human) a composition comprising one or more TRAIL short antibodies and one or more liver disease treatments, such as, but not limited to, weight loss, vaccination against liver disease (e.g., Hepatitis A, Hepatitis B, and Hepatitis C), vitamin E, and / or coffee.

[0046] Combination therapies used to treat a mammal having an infection (e.g., a viral infection) include compositions comprising one or more TRAIL short antibodies, and one or more agents to treat the infection, such as antiretroviral therapies, including, but not limited to, nucleoside reverse transcriptase inhibitors (e.g., abacavir, didanosine, emtricitabine, entecavir, lamivudine, stavudine, tenofovir disoproxil fumarate, zalcitabine, and zidovudine); non-nucleoside reverse transcriptase inhibitors (e.g., delavirdine, efavirenz, etravirine, nevirapine, and rilpivirine); nucleoside reverse transcriptase inhibitors (e.g., adefovir and tenofovir); fusion inhibitors (e.g., enfuvirtide); entry inhibitors (e.g., maraviroc); integrase inhibitors (e.g., dolutegravir, elvitegravir (ritovir and / or ritovir); and / or translation inhibitors (e.g., trimethoprim-peptide with or without covisistat, and raltegravir); maturation inhibitors (e.g., bivirimat); protease inhibitors (e.g., amprenavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, atazanavir, darunavir, and tipranavir); uncoating inhibitors (e.g., TRIM5 alpha); transcription inhibitors (e.g., tat antagonists); and / or translation inhibitors (e.g., trichosanthin); pegylated interferon alpha (PEG-IFN-a); daclatasvir; elbasvir; grazoprevir; glecaprevir; pibrentasvir; ledipasvir; sofosbuvir; ombitasvir; paritaprevir; ritonavir; dasabuvir; simeprevir; velpatasvir; and voxilaprevir may be administered to a mammal (e.g., a human).

[0047] When one or more TRAIL short antibodies are used in combination with one or more additional agents used to treat a disease and / or an infection, the one or more additional agents can be administered simultaneously or independently. For example, a composition comprising one or more TRAIL short antibodies can be administered first and the one or more additional agents can be administered second, or vice versa. In embodiments where one or more TRAIL short antibodies are used in combination with one or more additional therapies used to treat a disease and / or an infection, the one or more additional therapies can be administered simultaneously or independently with the one or more TRAIL short antibodies. For example, a composition comprising one or more TRAIL short antibodies can be administered before, during, or after the one or more additional therapies are administered.

[0048] In some cases, one or more TRAIL short antibodies can be formulated into a pharma- ceutically acceptable composition for administration to a mammal with a disease and / or infection. For example, a therapeutically effective amount of a TRAIL short antibody can be formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. The pharmaceutical composition can be formulated for administration in solid or liquid form, including, but not limited to, sterile solutions, suspensions, sustained release formulations, tablets, capsules, pills, powders, and granules. Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in the pharmaceutical compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0049] Pharmaceutical compositions comprising one or more TRAIL short antibodies may be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, and intradermal) or inhalation administration. When administered orally, pharmaceutical compositions comprising one or more TRAIL short antibodies may be in the form of pills, tablets, or capsules. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. Compositions for inhalation may be delivered, for example, using an inhaler, nebulizer, and / or dry powder inhaler. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0050] In some cases, a pharma- ceutically acceptable composition comprising one or more TRAIL short antibodies can be administered locally or systemically, for example, a composition comprising a TRAIL short antibody can be administered systemically by oral administration or inhalation to a mammal (e.g., a human).

[0051] The effective dose may vary depending on the severity of the disease and / or infection, the route of administration, the age and general health of the subject, the use of excipients, combination with other therapeutic agents, e.g., the possible use of other drugs, and the judgment of the treating physician.

[0052] In some cases, an effective amount of a composition comprising one or more TRAIL short antibodies may be any amount that reduces the severity or occurrence of symptoms of the disease and / or infection being treated without causing significant toxicity to the mammal. In some cases, an effective amount of a composition comprising one or more TRAIL short antibodies may be any amount that reduces the number of diseased cells (e.g., cancer cells) and / or infected cells without causing significant toxicity to the mammal. The effective amount may be constant or may be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors may affect the actual effective amount used in a particular application. For example, frequency of administration, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease and / or infection may require an increase or decrease in the actual effective amount administered.

[0053] In some cases, the administration frequency can be any frequency that reduces the severity or occurrence of symptoms of the disease and / or infection being treated without causing significant toxicity to the mammal. In some cases, the administration frequency can be any frequency that reduces the number of diseased cells (e.g., cancer cells) and / or infected cells without causing significant toxicity to the mammal. For example, the administration frequency can be about once a month to once every two weeks, about once a week to about three times a day, about twice a month to about six times a day, or about twice a week to once a day. In some cases, the administration frequency can be once a week. In some cases, the administration frequency can be every two weeks. The administration frequency can be constant or can be changed during the treatment period. A course of treatment with a composition comprising one or more TRAIL short antibodies can include a rest period. For example, a composition comprising one or more TRAIL short antibodies can be administered daily for two weeks, followed by a two-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can affect the actual administration frequency used for a particular application. For example, the effective amount, the duration of treatment, the use of multiple therapeutic agents, the route of administration, and the severity of the disease and / or infection may require increased or decreased frequency of administration.

[0054] In some cases, the effective period for administering a composition comprising one or more TRAIL short antibodies can be any period that reduces the severity or occurrence of symptoms of the disease and / or infection being treated without causing significant toxicity to the mammal. In some cases, the effective period for administering a composition comprising one or more TRAIL short antibodies can be any period that reduces the number of diseased cells (e.g., cancer cells) and / or infected cells without causing significant toxicity to the mammal. For example, the effective period can vary from days to weeks, months, or years. In some cases, the effective period for treating a disease and / or infection can range from about one month to about ten years. Several factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the condition being treated.

[0055] In certain instances, the course of treatment and the severity of the disease and / or infection being treated can be monitored. Any suitable method can be used to determine whether the severity of the disease and / or infection is being reduced. For example, the severity of the disease (e.g., cancer) can be assessed using imaging techniques (with or without contrast), biopsy techniques, bone marrow aspiration, colonoscopy, sigmoidoscopy, digital rectal examination, blood tests, platelet tests, fecal tests, urine tests, endoscopic techniques, ELISA techniques, PCR-based techniques, blotting techniques (e.g., Western blot), flow cytometry, genetic analysis (e.g., for gene rearrangement), and / or histological techniques at different time points. For example, the severity of the infection can be assessed using antibody techniques, viral antigen detection tests, culture techniques, ELISA techniques, PCR-based techniques (e.g., viral load tests), blotting techniques (e.g., Western blot), and / or histological techniques at different time points. Any suitable method can be used to monitor the response to TRAIL short antibody therapy or combination therapy including TRAIL short antibody. For example, techniques for detecting levels of TRAIL and / or TRAIL short include ELISA techniques, PCR-based techniques, blotting techniques (e.g., Western blot), hybridization techniques (e.g., ISH) and / or histological techniques (e.g., IHC).

[0056] In some cases, theranostics can be included in monitoring the response to TRAIL short antibody therapy or combination therapy including TRAIL short antibody. For example, one or more TRAIL short antibodies can be administered to a mammal having a disease and / or infection, and the disease and / or infection can be monitored simultaneously. For example, one or more TRAIL short antibodies described herein (e.g., antibodies comprising a VH domain comprising the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a VL domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16) can be labeled with a radionuclide to allow monitoring by PET or SPECT imaging.

[0057] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0058] Example 1: Resistance to TRAIL is conferred by microvesicle-associated TRAIL shorts and reversed by antibody neutralization overview TRAIL can bind to TRAIL-R1 or TRAIL-R2 and induce apoptosis in tumor cells and / or virus-infected cells without harming normal cells. Unfortunately, the clinical success of therapies targeting TRAIL receptors has been limited. The following data show that type I interferon signaling induces the expression of TRAIL-short, which can be released into the cellular microenvironment in microvesicles and taken up by bystander cells. TRAIL-short binds to TRAIL-R1 and R2, but not to the TRAIL decoy receptors R3 and R4, preventing full-length TRAIL from inducing cell death. TRAIL-short-mediated protection against TRAIL killing occurs in trans, as cells that do not produce TRAIL-short are protected against TRAIL after binding to TRAIL-short. Recombinant TRAIL-short is sufficient to protect cells against TRAIL-induced killing, whereas depletion of TRAIL-short by specific antibodies restores TRAIL sensitivity. These results establish a paradigm for understanding and overcoming TRAIL resistance.

[0059] result TRAIL-short is produced by both uninfected and HIV-infected cells TRAIL-short production in HIV-infected versus uninfected cells was assessed using single-cell mRNA analysis. In these experiments, single CD4 T cells from HIV-infected or uninfected donors were isolated with an integrated fluidic circuit, and individual cell analysis was performed using PCR for HIV-specific targets, TRAIL-short and TRAIL receptors / ligands, as well as markers of cell lineage and activation state (Figure 1A).

[0060] The specificity of the assay was confirmed by observing that there were no cells containing HIV transcripts in HIV-uninfected donors and that most of the CD4 T cells from HIV-infected donors did not contain HIV transcripts (Figure 1B). The inappropriate and excessive immune activation observed in HIV-infected patients was also observed; i.e., more cells from HIV-infected donors expressed the activation marker HLA-DR than cells from HIV-uninfected donors (Figure 1C). Cells from both HIV-infected and uninfected donors contained transcripts for TRAIL-short (Figure 1D), and both HIV-infected (HIV mRNA+) and HIV-uninfected (HIV mRNA-) cells from HIV-infected donors expressed TRAIL-short (Figure 1E). This indicates that HIV infection is not required for the production of TRAIL-short. The majority of individual cells expressed TRAIL-short. FL or TRAIL short, or FL Neither TRAIL short nor TRAIL short was expressed. FL To gain insight into what stimuli induce TRAIL-short expression, we assessed whether such expression correlated with cell activation and found that TRAIL-short message was not associated with immune activation as determined by the presence of CD25, CD38, or HLA-DR transcripts (Figure 1G). Within the CD4 T cell subsets represented in this analysis, we found that TRAIL-short message was not associated with immune activation as determined by the presence of CD25, CD38, or HLA-DR transcripts (Figure 1G). EM ), or transitional memory CD4 T cells (T TM ) than central memory CD4 T cells (T CM ) contained the TRAIL-short transcript (Fig. 1H). Thus, the data indicate that TRAIL-short can be expressed in individual non-HIV-infected cells.

[0061] Type I interferons drive the production of TRAIL-short Since HIV-uninfected cells are able to make TRAIL-short, it became reasonable to understand which stimuli drive the production of TRAIL-short. HIV proteins, HIV-induced cytokines, and / or IFNs may be involved in the induction of TRAIL-short expression. HIV infection produces a number of bioactive HIV-encoded proteins that have pleiotropic effects on host cells. HIV infection of individual cells can be recognized by a series of pattern recognition receptors (PRRs), including TLRs and RIGs, which lead to the production of type I IFNs and the subsequent induction of IFN-stimulated genes (ISGs) (Zitvogel et al., 2015 Nat Rev Immunol 15:405-414). To determine which of these stimuli drive TRAIL-short production, we screened a panel of cytokines, IFNs, and HIV proteins present in the plasma of infected patients to assess whether any of these stimuli can induce resting (CD25-, CD69-, HLA-DR-) CD4 T cells to produce TRAIL-short. IFNα14 and IFNβ significantly induced TRAIL short message by approximately 50-fold (Fig. 2A and E), whereas a panel of cytokines including inflammatory cytokines modestly, but not significantly, increased the expression of TRAIL short message by several fold (Fig. 2B). Similarly, TNFα, LPS, and gp120 minimally affected TRAIL short expression (Fig. 2C). A similar effect of type I interferons compared to other stimuli was found when TRAIL short protein levels were analyzed in treated samples (Fig. 2E). In the same samples, TRAIL FL The message is measured and TRAIL FL A close linear correlation between the expression of TRAIL-short and that of TRAIL-short was found (Figure 2D), which was consistent with Figure 1F. Taken together, these data support the conclusion that TRAIL-short is a de facto interferon-stimulated gene and is therefore likely expressed in diverse conditions characterized by type I interferon signaling.

[0062] The carboxyl terminus of TRAIL short is externalized on the plasma membrane and interacts with TRAIL receptors R1 and R2, but not with TRAIL receptors R3 and R4 TRAIL FL is expressed as a single-pass type II transmembrane protein with an extracellular C-terminal domain. TRAIL contains approximately amino acids 95-281. FL The C-terminal region of TRAIL is involved in proapoptotic functions and forms homotrimers coordinated around a central zinc ion (Cha et al., 1999 Immunity 11:253-261). TRAIL short arises as a result of a splicing event that deletes exons 3 and 4 and introduces a frameshift in exon 5, resulting in a premature stop codon and a novel 11 amino acid C-terminus (Figure 3A).

[0063] We assessed whether TRAIL-short is transported to the plasma membrane and present on the outer surface of cells. To do so, a myc tag was inserted within the novel C-terminus of TRAIL-short and flow cytometry was used to analyze epitope accessibility in non-permeabilized or permeabilized transfected cells. While permeabilized control transfected cells contained detectable myc (likely reflecting endogenous myc), TRAIL-short-myc transfected cells showed additional myc staining consistent with transfected protein expression (Figure 3B, left panel). Although non-permeabilized samples of the same cells showed no surface myc staining of control transfected cells, a distinct population of surface myc-positive cells could be detected after TRAIL-short-myc expression (Figure 3B, right panel), supporting the interpretation that the carboxyl terminus of TRAIL-short is accessible in the extracellular environment.

[0064] TRAIL FLTRAIL exerts its proapoptotic effects by binding to members of the TRAIL receptor family, of which there are four distinct member proteins. TRAIL-R1 and R2 contain a death domain that, following ligand binding, recruits the death domain (FADD) and Fas-associated protein, leading to caspase-8 activation. TRAIL-R3 and R4 do not contain a functional death domain, do not lead to caspase-8 activation, and are often considered "decoy" receptors. Members of the TRAIL receptor family also bind TRAIL FL One isothermal titration calorimetry study demonstrated that TRAIL binds to TRAIL-R2. FL One showed that TRAIL-R2 has a binding affinity at least 35-fold higher than TRAIL-R1 and 100-fold higher than TRAIL-R3 (TRAIL-R4 was not assessed) (Truneh et al., 2000, J Biol Chem 275:23319-23325), while another showed TRAIL R4>R2>R3>R1, with dissociation constants ranging from 0.869 to 4.08 nM (Lang et al., 2016, J Biol Chem 291:5022-5037). These studies suggest that TRAIL-R2 is more efficient at binding to TRAIL FL This highlights that it is the apoptosis-inducing receptor with the highest affinity for .

[0065] To address whether TRAIL-R1 also interacts with any other TRAIL receptor family members, cell lysates from 293T cells expressing C-terminally FLAG-tagged TRAIL-R1, R2, R3, or R4 were incubated with N-terminally HA-tagged TRAIL FL FLAG-TRAIL-R1 and R2 were combined with cell lysates from cells expressing HA-TRAIL or N-terminally HA-tagged TRAIL short. The mixtures were immunoprecipitated with anti-HA and analyzed for FLAG-containing proteins. Both FLAG-TRAIL-R1 and R2 suppressed the expression of HA-TRAIL. FL FLAG-TRAIL-R1 and R2 also coimmunoprecipitated HA-TRAIL short to a similar extent (Fig. 3C). However, TRAIL FLAlthough TRAIL-R1 and TRAIL-R2 were associated with both TRAIL-R3 and R4, TRAIL-short specifically did not (Figure 3D). Thus, TRAIL-short preferentially binds death-inducing TRAIL-R1 and R2 while sparing the decoy receptors R3 and R4. This suggests that selective receptor binding may be central to the biological effects of TRAIL-short.

[0066] TRAIL-short is contained within extracellular vesicles How TRAIL-short is released into its compartment remains unclear. To investigate this process, Jurkat T cells transfected with GFP or GFP-TRAIL-short were analyzed by confocal microscopy. TRAIL-short was observed to localize to the plasma membrane (Fig. 4A), confirmed by immunoblotting heavy membrane fractions of HIV-infected or control cells (Fig. 4B). Purity of the fractions was confirmed by cytosol-specific HSP70. TRAIL-short was found in the supernatant fraction of infected cultures. Moreover, supernatant-associated TRAIL-short migrated similarly to cell-associated TRAIL-short. This suggests that supernatant-associated TRAIL-short is unlikely to have been generated by a cleavage event (Fig. 4C).

[0067] To examine whether TRAIL-short could be incorporated within extracellular vesicles, extracellular vesicles from PHA-stimulated and unstimulated CD4+ cells were collected by ultracentrifugation, fixed, and analyzed for size by electron microscopy, revealing a range of 50 nm to approximately 400 nm with a median diameter of 136 nm (Figure 4D). These results suggest the presence of both microvesicles (>100 nm) and exosomes (<100 nm). Western blot analysis of the same extracellular vesicles was performed using an anti-TRAIL-short antibody. This revealed a band of approximately 15 kDa (Figure 4E), consistent with extracellular vesicles containing TRAIL-short. HA-TRAIL FLWhole cell lysates and purified extracellular vesicle preparations from 293T cells transfected with eGFP-TRAIL and HA-TRAIL-short produced similar patterns by Western blot (Figure 4F). The presence of TRAIL-short-associated extracellular vesicles suggests that 293T cells were transfected with eGFP-TRAIL. FL This was further verified by transfecting cells with eGFP-TRAIL and / or Ruby-TRAIL short and analyzing the supernatants by flow cytometry. Untransfected cell supernatants were virtually free of GFP or Ruby signals, whereas eGFP-TRAIL FL Cells transfected with eGFP-TRAIL or Ruby-TRAIL short alone showed detectable expression of each protein individually. FL and when co-transfected with Ruby-TRAIL short, eGFP-TRAIL FL only, Ruby-TRAIL short only, eGFP-TRAIL FL and Ruby-TRAIL short, vesicles were detected ( Fig. 4G ).

[0068] Microvesicle localization TRAIL FL has been shown to be biologically active (Huber et al., 2005, Gastroenterology 128:1796-1804), therefore, it was investigated whether TRAIL-short in extracellular vesicles would be biologically active as well. FL alone, HA-TRAIL short alone, both in a 1:1 ratio, or 1:2 HA-TRAIL FLJurkat cells were transfected with both TRAIL-short and HA-TRAIL-short ratios and concentrated supernatants containing TRAIL-short microvesicles were collected. Jurkat cells were incubated with either fresh medium, medium conditioned from untransfected 293T cells, or medium conditioned from one of four varieties of transfected 293T cells and analyzed by flow cytometry for cleaved caspase-3 as a marker of apoptotic cell death. Jurkat cells incubated with fresh medium or medium from untransfected 293T cells had low levels of basal apoptosis (approximately 5%). In contrast, TRAIL-short microvesicles were not associated with apoptosis. FL Jurkat cells incubated with medium from 293T cells transfected with TRAIL showed greater than 20% apoptosis and FL Importantly, TRAIL-1 was activated in 293T cells. FL When cotransfected with and TRAIL-short, the extent of killing of Jurkat T cells by the microvesicle preparation was reduced and was lowest when TRAIL-short was present in greater amounts (approximately 3% to 13%; Fig. 4H and I ).

[0069] Because TRAIL-short is a membrane-associated protein with a transmembrane domain, we tested whether TRAIL-short is similarly associated with microvesicles rather than exosomes. Supernatants from HIV-infected primary CD4 T cell cultures were fractionated into microvesicles by ultracentrifugation or into exosomes by resin-based extraction as described elsewhere (see, e.g., Taylor et al., 2011 Methods Mol Biol 728:235-246). Microvesicle preparations and exosomes were analyzed by immunoblot using our TRAIL-short specific antibody, which identified TRAIL-short only in microvesicle preparations (Figure 4J).

[0070] TRAIL short protection from TRAIL can be transferred to adjacent cells To evaluate the biological effects of TRAIL short expression,FL TRAIL-R2, TRAIL-short, or both were expressed in 293T cells used as effector cells. Target Jurkat T cells expressing TRAIL-R2 were labeled with Cell Tracker Orange (CTO), and effector 293T cells were mixed with target CTO+ Jurkat cells and analyzed by flow cytometry gating on the CTO-positive population (Figure 5A). Target Jurkat cells were incubated with 1 μg to 20 μg of HA-TRAIL FL Transfection of HA-TRAIL increased the amount of transfected HA-TRAIL in the range of 7.1% to 32.8%. FL The effector 293T cells were treated with 10 μg of HA-TRAIL. FL and increasing amounts of HA-TRAIL short, co-cultured with CTO-labeled Jurkat T cells and analyzed in a similar manner. Increasing amounts of the TRAIL short plasmid led to increased TRAIL short expression (Figure 5D). This in turn led to a decrease in the killing of Jurkat target cells (Figure 5E). Thus, TRAIL FL Co-expression of TRAIL-short with TRAIL-1 inhibited TRAIL-2 in a dose-dependent manner. FL It antagonizes the apoptosis-inducing activity of

[0071] To examine whether TRAIL-short-mediated resistance to TRAIL killing could be transferred from TRAIL-short-producing cells to bystander cells present in the microenvironment that do not produce TRAIL-short, ruby-tagged TRAIL-short (ruby-TRAIL-short) and ruby-tagged TRAIL-short lacking the transmembrane domain (ruby-TRAIL-shortΔTM) constructs were generated, and robust expression of each construct was confirmed (Figure 5F). 293T cells transfected with ruby-TRAIL-short showed peri-membrane expression of ruby, whereas ruby-TRAIL-shortΔTM expressing cells showed diffuse cytoplasmic expression (Figure 5G, top panel). Supernatants from transfected cells were collected and used to treat HeLa cells. Treated HeLa cells showed uptake of ruby-TRAIL-short but not ruby-TRAIL-shortΔTM (Figure 5G, bottom panel). This is consistent with the understanding that the transmembrane domain is required for membrane localization of TRAIL-short and further indicates that microvesicles containing TRAIL-short are taken up by neighboring cells that do not produce TRAIL-short. We tested whether these microvesicle-treated HeLa cells acquired resistance to TRAIL-mediated killing. Untreated HeLa cells died in a time-dependent manner after treatment with sk-TRAIL. Addition of supernatants from 293T cells transfected with ruby ​​or with ruby-TRAIL-shortΔTM did not appreciably alter sk-TRAIL killing, whereas addition of supernatants from cells transfected with ruby-TRAIL-short reduced sk-TRAIL-induced killing by about 20% (Figure 5H). Thus, expression of TRAIL-short by one cell can confer TRAIL resistance to another cell mediated by microvesicle translocation of TRAIL-short.

[0072] TRAIL-short contains a PEST domain and is ubiquitinated and degraded by the proteasome During studies investigating the subcellular localization of TRAIL-short (Figure 1A), we noticed a seemingly short half-life of TRAIL-short. To investigate this observation, 293T cells were transfected with HA-TRAIL-short and the half-life of the protein was assessed in the absence and presence of the protein synthesis inhibitor cycloheximide (Figure 6A). Approximately 50% of the protein was lost within the first approximately 60 min, indicating rapid protein turnover. A putative PEST domain in TRAIL-short was identified between amino acids 59 and 81 (Figure 6B). Substitution of proline 76 to alanine significantly extended the half-life of TRAIL-short (Figure 6C), confirming a functional PEST domain and suggesting subsequent ubiquitination and proteasome-mediated degradation.

[0073] To confirm that TRAIL-short is ubiquitinated, HA-tagged TRAIL-short was expressed in 293T cells and anti-HA pull-down and ubiquitin immunoblotting were performed. These analyses revealed abundant ubiquitinated proteins migrating at sizes corresponding to polyubiquitinated HA-TRAIL-short (Figure 4D). Modification of proteins with ubiquitin at Lys48 linkages can drive proteasomal degradation, whereas ubiquitination at Lys63 linkages leads to modification of protein:protein interactions and alterations in signaling pathways (Deng et al., 2000 Cell 103:351-361). To assess whether ubiquitinated TRAIL-short is targeted for Lys48-mediated degradation, we used a specific deubiquitinating enzyme (DUB) that distinguishes between Lys48 and Lys63 ubiquitin linkages (see, e.g., Komander et al., 2009 Nat Rev Mol Cell Biol 10:550-563). Ubiquitination of HA-TRAIL-short was reversed by the Lys48-specific DUB USP2 (Figure 6D), indicating that TRAIL-short is ubiquitinated consistent with being targeted to the proteasome for degradation. This was confirmed by measuring TRAIL-short levels in cells treated with or without the proteasome inhibitor MG132, revealing that proteasome inhibition significantly increased TRAIL-short expression levels (Figure 6E). Consistent with these observations and the hypothesis that the ubiquitin-proteasome pathway governs the expression levels of TRAIL-short, expression of the PEST mutant P76A TRAIL-short led to increased levels of TRAIL-short in individual cells, without a significant increase in the total number of cells containing TRAIL-short (Figure 6F).

[0074] TRAIL short is both sufficient and necessary to cause TRAIL resistance These data indicate that TRAIL-short fundamentally affects cell death / survival of both cells producing TRAIL-short and bystander cells that take up TRAIL-short from microvesicle-mediated translocation. To assess whether TRAIL-short alone is responsible for these effects, recombinant constructs of TRAIL-short were generated by cloning the extracellular domain of TRAIL-short fused to the Fc domain of immunoglobulin G. After purification, recombinant TRAIL-short extracellular domain:Fc fusion (TRAIL-short ECD:Fc) was added to Jurkat T cells alone or to Jurkat T cells pretreated with sk-TRAIL. TRAIL-short ECD:Fc was not toxic but prevented sk-TRAIL-mediated killing of Jurkat T cells in a dose-dependent manner (Figure 7A).

[0075] We evaluated whether depletion of TRAIL-short is sufficient to alleviate TRAIL resistance. The generation of TRAIL-short antibodies specific for the 11 C-terminal amino acids unique to TRAIL-short has been described elsewhere (see, for example, Schnepple et al., 2011 J Biol Chem 286:35742-35754). Jurkat T cells constitutively expressing TRAIL-short were stimulated with IFNα14 to induce maximal expression of TRAIL-short and then induced death by sk-TRAIL. Consistent with our previous observations, sk-TRAIL treatment resulted in potent killing of Jurkat T cells (Figure 7B). Consistent with TRAIL-short being an antagonist of TRAIL, sk-TRAIL treatment in the presence of increasing doses of TRAIL-short antibodies induced a dose-dependent increase in Jurkat death. This effectively doubled the number of dead cells at 5 μg / ml, the highest dose of antibody tested, despite the lack of intrinsic cytotoxicity of the TRAIL-short antibody alone. Thus, in this model system, the presence of TRAIL-short is necessary and sufficient for TRAIL resistance, and our data indicate that the anti-TRAIL effect of TRAIL-short can be effectively inhibited by a TRAIL-short specific antibody.

[0076] Example 2: TRAIL-Short reduces the cytotoxicity of NK cells NK cells play a key role in the antiviral response to HIV infection. NK cells can produce TRAIL as an effector molecule to kill tumor cells, but its role in antiviral defense is just beginning to emerge. TRAIL short produced by HIV-infected cells may be a major factor in reducing the ability of these cells to mount important innate immune responses against the virus. To determine the effect of TRAIL short on NK function, we assessed the role of TRAIL short in reducing the cytotoxic function of NK cells during HIV infection.

[0077] method Jurkat cells were transfected with control or TRAIL-short expressing plasmids and incubated with primary NK cells from uninfected donors (N=10) at various effector:target ratios (1:1-20:1). A flow cytometry-based assay was used to determine the cytotoxic effect of NK cells against target Jurkat cells. The effect of TRAIL-short overexpression on NK cell activity and function was determined by staining for CD69, perforin, CD16 and CD107a. Furthermore, the effect of supernatants containing gp120 or HIV-1 IIIB strain on NK expression of TRAIL and TRAIL-short was measured by surface staining.

[0078] result Overexpression of TRAIL-short in target cells significantly reduced NK cytotoxic function against these cells across a range of E:T ratios compared to control cells (Figure 8; p=0.006 at an E:T ratio of 20:1). These results demonstrated that TRAIL-short expression significantly reduced NK-mediated cytotoxicity.

[0079] Pretreatment of NK cells with culture supernatant containing HIV IIIB virus significantly increased the surface expression of TRAIL-short, whereas gp120 alone had no effect on TRAIL expression (Fig. 9). These results demonstrated that HIV-containing supernatant alone increases the surface expression of TRAIL-short in NK cells.

[0080] TRAIL-short did not alter the expression of CD69, CD16, perforin, or CD107a in NK cells (Figure 10). These results demonstrated that TRAIL-short can affect NK function and play an important role in the innate immune response to HIV infection.

[0081] Example 3: Humanization of anti-TRAIL short antibodies The mouse monoclonal antibody TRAILs 2.2 was humanized to generate antibody Ab866.

[0082] Heavy chain The sequence of the VH domain of mouse TRAILs 2.2 is set forth below, with the CDRs in bold: TIFF0007604428000009.tif22159

[0083] The CDRs of mouse VH were transferred to the human acceptor framework to generate humanized variants VH1-VH4: TIFF0007604428000010.tif139156

[0084] An alignment of humanized variants VH1-4 with the VH0 mouse sequence is shown in Figure 12 A. The homology of humanized variants VH1-4 to the VH0 mouse sequence is shown in Table 1 below.

[0085] [Table 1]

[0086] Light chain The sequence of the VL domain of murine TRAILs 2.2 is set forth below, with the CDRs in bold: TIFF0007604428000012.tif30155

[0087] The CDRs of mouse VL were transferred to the human acceptor framework to generate humanized variants VL1-VL4: TIFF0007604428000013.tif66155TIFF0007604428000014.tif68155

[0088] The alignment of humanized variants VL1-4 with the VL0 mouse sequence is shown in Figure 12B. The homology of humanized variants VL1-4 to the VL0 mouse sequence is shown in Table 2 below.

[0089] [Table 2]

[0090] All humanized variants conformed to the World Health Organization (WHO) definition of a humanized antibody.

[0091] Example 4: Anti-TRAIL short antibody V H Each of the domains is synthesized in frame with the human IgG1 isotype constant domain sequence. The entire heavy chain sequence is codon optimized (DNA2.0, USA) and the DNA sequence can be verified. The amino acid sequence of the IgG1 constant domain (allotype G1m17,1) is: TIFF0007604428000016.tif53156

[0092] V L Each of the domains is synthesized in frame with the human IgK isotype constant domain sequence. The entire light chain sequence is codon optimized (DNA2.0, USA) and the DNA sequence can be verified. The amino acid sequence of the IgK constant domain (allotype Km3) is: TIFF0007604428000017.tif23155

[0093] Each of the mutant chains is verified by DNA sequencing analysis. The 16 humanized antibodies can include any combination of humanized variable domains shown in Table 3.

[0094] [Table 3]

[0095] The complete amino acid sequences of each of the heavy and light chains are shown in FIG.

[0096] Example 5: Anti-TRAIL short antibodies for treating cancer To examine the effect of anti-TRAIL short antibodies in vivo, NSG mice were injected with Jurkat T cell leukemia cells that constitutively express luciferase, and luciferase expression was measured twice weekly. For the experiment shown in Figure 14A-C, mice were injected with either IC antibody or anti-TRAIL short antibody clone 2.2 (e.g., murine TRAILs 2.2 described in Example 4) at 10 mg / kg on day 27. Mice were injected with anti-TRAIL receptor 2 (anti-DR5) antibody (10 mg / kg) 24 hours later (e.g., on day 28). Representative luciferase expression was decreased in mice treated with anti-TRAIL short antibodies compared to mice treated with isotype control (Figures 14A and 14B). Luciferase expression was significantly decreased in mice treated with anti-TRAIL short antibodies and anti-DR5 (Figure 14C). All of the mice treated with anti-TRAIL short antibody and anti-DR5 survived, whereas the control mice did not, but this difference was not statistically significant (Figure 14D). These results indicate that TRAIL short inhibition in the presence of a TRAIL agonist (anti-DR5) results in improved tumor control and improved survival compared to isotype control + anti-DR5. For the experiment shown in Figures 14D-E, mice were injected with either IC antibody or anti-TRAIL short antibody clone 2.2 at 10 mg / kg on days 20, 27, and 34. Luciferase expression was significantly reduced in mice treated with anti-TRAIL short antibody (Figure 14E), and survival was significantly increased in mice treated with anti-TRAIL short antibody compared to mice treated with isotype control antibody (Figure 14F). These results indicate that in vivo administration of anti-TRAIL short antibody alone or in the presence of anti-DR5 can reduce the number of leukemic cells in mice and extend the survival of mice.

[0097] The cytotoxic effect of the anti-TRAIL short antibody sk-TRAIL was examined in various cancer cell lines. Cells were plated in 96-well plates at 1 × 10 per well. 4Cells were seeded at a density of 100x100 / mL. Cells were then pre-incubated with neutralizing anti-TRAIL short antibody clone 2.2 or IgG3 control at variable concentrations (1-20 μg / mL) as indicated for 1 h at 37°C. sk-TRAIL was then added to the cells at doses of 1-10 ng / mL as indicated. To measure cell death, caspase 3 / 7 apoptosis assay reagent was added to the cells at a dilution of 1:1000. For live-time analysis of cell death, real-time images of cells were captured every 2 h using the IncuCyte® system. Lymphoma leukemia cells (Figure 15A), pancreatic cancer cells (Figure 15B), melanoma cells (Figure 15C), and ovarian cancer cells (Figure 15D) were examined. Each treatment was performed in triplicate. These results demonstrated that in some cell lines, anti-TRAIL short antibodies act in concert with recombinant TRAIL to induce apoptosis in cancer cells.

[0098] [Table 4]

[0099] To investigate TRAIL expression in different cancer cells, TRAIL mRNA was quantified in different cancer cells by qRTPCR. RNA was isolated using the RNeasy mini kit from Qiagen. 1 μg of RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit. The cDNA was diluted and purified using TRAIL short and TRAIL FL Using standards for , the copy number of each gene was calculated in lymphoma leukemia cells, pancreatic cancer cells, melanoma cells, and ovarian cancer cells (Figure 16).

[0100] To further investigate the expression of TRAIL in different cancer cells, tissues of tissue microarray were stained by immunohistochemistry with TRAIL short 2.2 antibody at a concentration of 1:400. As shown in Figure 17, examples of normal tissues and corresponding cancer tissues of pancreas, stomach, and prostate were positively stained for TRAIL short. These results showed that cancer tissues had significantly elevated expression of TRAIL short compared with non-malignant tissues.

[0101] Example 6: Anti-TRAIL short antibodies for treating cancer TRAIL-short expression in various cancer types was observed using tissue microarray immunohistochemistry. Eight tissue microarrays (TMAs) performed using anti-TRAIL-short antibody 2.2 at a dilution of 1:400 are summarized in Figure 18 and Table 5 below.

[0102] [Table 5] TIFF0007604428000021.tif228160

[0103] TRAIL short was found in many human tumors (Figure 19). TRAIL short levels were quantified as reads per million kilobases (RPKM). Data was determined from The Cancer Genome Atlas (TCGA).

[0104] Increased levels of TRAIL short were associated with poor survival in some human tumors (Figure 20). Survival was assessed by Kaplan-Meier analysis in patients with diffuse large B-cell lymphoma (Figure 20A), colon and rectal adenocarcinoma (Figure 20B), pigmented renal cell carcinoma (Figure 20C), pancreatic adenocarcinoma (Figure 20D), and thymoma (Figure 20E), stratified by TRAIL short levels expressed as transcripts per kilobases million (TPM). Survival curves were determined from the Cancer Genome Atlas (TCGA).

[0105] Anti-TRAIL short antibody interacted with the TRAIL short antigen. HEK293T cells were transfected with green fluorescent protein (GFP), GFP-TRAIL short, or GFP-TRAIL short. FL and analyzed by confocal microscopy (Figure 21).

[0106] Codon optimization yielded 16 anti-TRAIL short antibody variants. The anti-TRAIL short antibody variants, in combination with superkiller (sk)-TRAIL and with different affinities, had different effects on TRAIL-induced cytotoxicity against Jurkat cells (Table 6). Affinity was analyzed using surface plasmon resonance. Clone HC2LC3 showed an affinity of 3.8 pM and significant synergistic killing in the presence of sk-TRAIL (1 ng / ml; Figure 22).

[0107] [Table 6]

[0108] Some of the patient-derived cells were responsive to anti-TRAIL short antibody + sk-TRAIL, whereas others were not. Freshly isolated cells from the spleen of a patient who underwent splenectomy for suspected hematological malignancy were treated with nothing (control), superkiller TRAIL (sk-TRAIL, an oligomerized TRAIL agonist), humanized anti-TRAIL short antibody (clone HC2LC3), or isotype control antibody (IgG4). Cell death over time was monitored by analyzing active caspase 3 / 7 activity over time using the Incucyte live cell imaging platform (Figure 23).

[0109] The effect of anti-TRAIL short antibodies on sk-TRAIL cytotoxicity in different human patient-derived cancer cell lines was evaluated. Cells from the patient's spleen were freshly received in RPMI and tested for sensitivity to cytotoxicity in the presence of sk-TRAIL at a dose of 1 ng / mL in the presence or absence of increasing doses of the anti-TRAIL-short clone HC2LC3 (1.25, 2.5, or 5 μg). Controls were treated with the same dose of human IgG4. Cell death was monitored every 2 hours over 72 hours by the number of cells positive for cleaved caspase 3 / 7 using an Incucyte (Figure 24).

[0110] Responsiveness to anti-TRAIL short + sk-TRAIL was greater than sk-TRAIL alone in multiple human patient derived cancer cell lines. The number of dead cells at 48 hours after treatment with sk-TRAIL alone at a dose of 1 ng / mL or sk-TRAIL (same dose) + 5 μg / mL of the anti-TRAIL short clone HC2LC3 is shown in Figure 25. Responsiveness was defined as a statistically significant increase in the number of dead cells after addition of anti-TRAIL short antibody. Paired t-test statistics are shown.

[0111] The responsiveness of human patient-derived cell lines to anti-TRAIL-short alone and in combination with sk-TRAIL is shown in Figure 26 and Table 7. Responsiveness was defined as a statistically significant increase in the number of dead cells in response to treatment with sk-TRAIL.

[0112] [Table 7]

[0113] Increased TRAIL-short expression in cell lines responsive to anti-TRAIL-short was observed by quantification of TRAIL-short mRNA copy number using real-time qPCR in various responsive and non-responsive cell lines (Figure 27A) and the percentage of TRAIL-short positives measured by staining of the three cell lines with anti-TRAIL-short 2.2 or isotype conjugated to CF555 by flow cytometry (Figure 27B).

[0114] TRAIL short expression was examined in 293T cells and tumor tissue from patients. Immunohistochemistry slides stained for TRAIL short using anti-TRAIL short antibody at a dilution of 1:400 are shown in FIG.

[0115] NSG mice implanted with human Jurkat T-cell lymphoma cells were effectively treated with a combination of anti-DR5 + anti-TRAIL short antibodies. NSG mice were implanted with luciferase-expressing Jurkat T cells by IV injection. Tumors were allowed to establish and treated every 2 weeks with 10 mg / kg of either the following as indicated every 14 days: (i) TRAIL short isotype control and DR5 antibody isotype (referred to as isotype), (ii) anti-TRAIL short antibody clone 2.2 + anti-DR5 isotype control (referred to as anti-TRAIL short), (iii) anti-TRAIL short antibody isotype control + anti-DR5 antibody (referred to as isotype control + anti-DR5), or (iv) anti-TRAIL short (2.2) + anti-DR5. Over time, mice were analyzed for luciferase expression by whole body imaging (Figure 29A). Survival was assessed by Kaplan-Meier analysis (Figure 29B).

[0116] Anti-TRAIL short antibody + isotype control, or anti-TRAIL short antibody + anti-DR5 antibody caused tumor growth inhibition in mouse xenograft models of human cancer (Figure 30). NSG mice were subcutaneously implanted with human diffuse large B-cell lymphoma (HBL-1) cell line, and tumor size was measured daily. When tumors reached a size greater than or equal to 100 cubic mm, mice received the indicated treatment every 14 days by IP injection at 10 mg / kg weekly of either of the following, as indicated: (i) TRAIL short and DR5 antibody isotype control (IC+IC), (ii) anti-TRAIL short antibody clone 2.2 + anti-DR5 isotype control (2.2+IC), (iii) anti-TRAIL short antibody isotype control + anti-DR5 antibody (IC+DR5), or (iv) anti-TRAIL short (2.2) + anti-DR5 antibody (2.2+DR5). Over time, mice were analyzed for tumor size and fold change calculated from baseline (Figure 30A). Survival was assessed by Kaplan-Meier analysis (Figure 30B).

[0117] The toxicity of anti-TRAIL short antibody clone 2.2 and the reversibility of any toxic effects were investigated. Ten C57BL / 6 mice (5 males and 5 females) were used. The animals received anti-TRAIL short antibody clone 2.2 by intravenous bolus at a dose of 10 mg / kg and 5 ml / kg on day 1. Parameters evaluated during the study included effects on skin, fur, eyes and mucous membranes, respiratory system, circulatory system, autonomic central nervous system, somatomotor activity, locomotor activity and behavioral pattern changes, weight changes, and mortality. At the end of the survival period, all animals were weighed, humanely euthanized, blood was collected for clinical chemistry and hematology analysis, and gross necropsy was performed. There were no abnormalities or changes in the physical condition, activity, or behavior of the animals. Clinical observations of all study animals were performed daily. All study animals were observed twice daily for 7 days. All animals survived for the duration of the study. All animals lost weight during the study; however, final weights were collected after fasting and before sacrifice, so it is unclear whether the weight loss was due to the fasting treatment, the anti-TRAIL short antibody clone 2.2, or a combination. Clinical pathology investigations for hematology and clinical chemistry were performed on all surviving study animals on day 8. Terminal blood samples were collected from the vena cava or cardiac puncture. In a cohort of 10 healthy C57 BL / 6 mice, no adverse toxicity was observed over a 7-day observation period following IV injection of anti-TRAIL short antibody on day 1 in either male or female animals.

[0118] TRAIL short can be detected in tissues by in situ hybridization (ISH; FIG. 31). SIV-infected macaque tissues were stained with either a TRAIL full-length or a TRAIL short-specific probe from Advanced Cell Diagnostics (having the sequence 383 bp 5'-tcgttaga aagactccaa gaatgaaaag gctctgggcc gca -3' 423 bp (SEQ ID NO: 31)) and visualized at 10x, 20x, and 40x magnification (FIG. 31). SIV-infected macaque axillary lymph node tissues were stained with both the TRAIL and TRAIL short-specific probes. Similarly, SIV-infected macaque spleen tissues were stained with both the TRAIL and TRAIL short-specific probes.

[0119] Other embodiments Although the invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is illustrative and does not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. [Sequence table] TIFF0007604428000024.tif199141TIFF0007604428000025.tif199141TIFF0007604428000026.tif199141TIFF0007604428000027.tif199141TIFF0007604428000028.tif199141TIFF0007604428000029.tif199141TIFF0007604428000030.tif199141TIFF0007604428000031.tif199141TIFF0007604428000032.tif199141TIFF0007604428000033.tif199141TIFF0007604428000034.tif199141TIFF0007604428000035.tif199141TIFF0007604428000036.tif199141TIFF0007604428000037.tif199141TIFF0007604428000038.tif199141TIFF0007604428000039.tif199141TIFF0007604428000040.tif199141TIFF0007604428000041.tif199141TIFF0007604428000042.tif199141TIFF0007604428000043.tif199141TIFF0007604428000044.tif199141TIFF0007604428000045.tif199141TIFF0007604428000046.tif199141TIFF0007604428000047.tif199141TIFF0007604428000048.tif199141TIFF0007604428000049.tif199141TIFF0007604428000050.tif199141TIFF0007604428000051.tif199141TIFF0007604428000052.tif199141TIFF0007604428000053.tif199141TIFF0007604428000054.tif199141

Claims

1. A pharmaceutical composition for treating a viral infection, comprising: The antibody binds to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) short. The antibody comprises a heavy chain variable region (VH) domain comprising the complementarity determining regions (CDRs) set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and having at least 90% sequence identity to SEQ ID NO:6; and a light chain variable region (VL) domain comprising the CDRs set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, and having at least 90% sequence identity to SEQ ID NO:20, the pharmaceutical composition is administered to a mammal having the viral infection; The pharmaceutical composition, wherein the antibody increases natural killer (NK) cytotoxicity.

2. The pharmaceutical composition of claim 1, wherein the viral infection is a chronic infection.

3. 2. The pharmaceutical composition of claim 1, wherein the viral infection is selected from the group consisting of a human immunodeficiency virus infection, a hepatitis B virus infection, and a hepatitis C virus infection.

4. The pharmaceutical composition of claim 1, wherein the mammal is a human.

5. The pharmaceutical composition of claim 1 , wherein the antibody is a humanized antibody.

6. The pharmaceutical composition of claim 1, wherein a TRAIL agonist is further administered to the mammal.

7. The pharmaceutical composition of claim 6, wherein the TRAIL agonist is selected from the group consisting of recombinant TRAIL, an anti-TRAIL-R1 antibody, an anti-TRAIL-R2 antibody, and a TRAIL oligomer.

8. The pharmaceutical composition of claim 1, wherein an antiretroviral therapy is further administered to the mammal.

9. The antiretroviral therapy is selected from the group consisting of abacavir, didanosine, emtricitabine, entecavir, lamivudine, stavudine, tenofovir disoproxil fumarate, zalcitabine, zidovudine, delavirdine, efavirenz, etravirine, nevirapine, rilpivirine, adefovir, tenofovir, enfuvirtide, maraviroc, dolutegravir, elvitegravir, raltegravir, bevirimat, amprenavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir ...

9. The pharmaceutical composition of claim 8, wherein the antiviral agent is selected from the group consisting of cefovir, saquinavir, atazanavir, darunavir, tipranavir, TRIM5 alpha, tat antagonists, trichosanthin, pegylated interferon alpha (PEG-IFN-a), daclatasvir, elbasvir, grazoprevir, glecaprevir, pibrentasvir, ledipasvir, sofosbuvir, ombitasvir, paritaprevir, ritonavir, dasabuvir, simeprevir, velpatasvir, and voxilaprevir.

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