WO2017177175A1 - Methods and compositions targeting retroviral latency - Google Patents

Abstract

Methods and compositions are provided for identifying cells latently infected with and harboring an occult reservoir of infectious virus. In certain embodiments, levels of IFITM1 expression by T cells are monitored to evaluate the efficacy of treatment for HIV. In other embodiments, treatment of latent HIV is provided by targeting cells expressing IFITM1 for antibody-mediated cytolysis. In other embodiments, CAR T-cell having monospecificity specificity for IFITM1 or bi-specificity for IFITM1 and CD4 are generated and used to treat HIV infected individuals.

Description

METHODS AND COMPOSITIONS TARGETING RETROVIRAL LATENCY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority based on US Provisional Application Serial No. 62/319,668 filed April 7, 2016, incorporated herein by reference in its entirety.

STATEMENT REGARDING GOVERNMENT INTERESTS

[0002] This invention was made with government support under Grant Nos. R21 AI093179, R01 AI076059, and UM1 AI26617 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] This invention relates generally to compositions and methods for reducing and eradicating reservoirs of latent viruses.

BACKGROUND OF THE INVENTION

[0004] Without limiting the scope of the invention, its background is described in connection with efforts to reduce the reservoir of latently infected cells in HIV infected individuals. Antiretroviral therapy (ART) potently suppresses HIV-1, but viral eradication remains unachievable due to the persistence of reservoirs. The introduction of highly-active antiretroviral therapy (HAART) has been a groundbreaking advance in the field as it effectively reduces HIV viral loads in plasma to undetectable levels. However, if patients interrupt therapy, the virus quickly rebounds, indicating the presence of silent or dormant forms of HIV in the body. To date there is no effective vaccine, regimen or strategy that can effectively target the latent reservoir.

[0005] Latent viral reservoirs are thought to be maintained by their intrinsic stability and homeostatic proliferation of latent cells, reseeding of newly infected cells by low levels of replicating HIV-1 in lymph nodes and gut-associated lymphoid tissue (GALT) and incomplete penetration of antiretroviral drugs into infected tissues. HIV-1 may continue to replicate and traffic in lymphoid tissue despite potent ART. Viral DNA persists in central memory and stem central memory CD4+ T-cells. Current eradication strategies rely on histone deacetylase inhibitors (HDACi) and related compounds, in conjugation with ART, to reactivate and kill infected cells within the reservoir. However, HDACi poorly reactivate the reservoir and may cause severe side effects due to the unspecific reactivation of host genes, such as oncogenes and other cancer related factors.

[0006] The use of antibody-mediated cell killing to effectively control HIV infection has gained significant interested in recent years. Recently, two papers described the use of dual- affinity re-targeting (DART) proteins aimed at improving T-cell-mediated clearance of HIV- 1-infected cells. See Sung JA, et al. Dual-Affinity Re-Targeting proteins direct T cell- mediated cytolysis of latently HIV-infected cells. J Clin Invest. 125(11) (2015) 4077-4090; Sloan DD, et al. Targeting HIV Reservoir in Infected CD4 T Cells by Dual- Affinity Retargeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells. PLoS Pathog. 11(11) (2015) el005233. In these studies, DARTs were specifically designed to recognize Env-expressing cells and to engage to CD3. Problematically for these approaches, they require T-cell activation and because viral antigens are involved the possibility remains that virus will escape killing by mutating.

[0007] From the foregoing, it appeared to the present inventors that identification and targeting of latently infected cells would be of key importance in the treatment of persistent viral infection including with HIV.

BRIEF SUMMARY OF THE INVENTION

[0008] The invention provides novel therapeutic methods and compositions directed to reactivating latent viral reservoirs and eliminating them. In one embodiment, one or more biomarkers are provided, including IFITM1, that identify the quantity of HIV latently infected cells during treatment and thus quantify the latent reservoir as a monitoring method that assesses the efficacy of treatment and directs modifications that are able to attain reduced expression of IFITM1.

[0009] In one embodiment, a method of treatment is provided that targets IFITM1+ HIV infected cells by the use of an antibody that binds to IFITM1. In particular embodiments the antibody that binds to IFITMl is not cross-reactive with IFITM2 and IFITM3. In certain embodiments the antibody is bi-specific. For one example, in certain embodiments the bi- specific antibody includes an anti-IFITMl moiety and an anti-CD4 moiety.

[0010] In certain embodiments the antibody is co-administered with an effective amount of an NK cell stimulator. The NK cell stimulator is selected from one or more of type I IFNs (mainly IFN-a and IFN-β), IL-2/anti-IL-2 monoclonal antibody complexes, IL-12, IL-15 and IL-18. The antibody to IFITM1 and the NK cell stimulator synergistically kill cells latently infected with the latent immunodeficiency virus. In certain embodiments the NK cell stimulator is an IL-15 superagonist. An example of an IL-15 superagonist is an IL-15 mutant bound to an IL-15 receptor a/IgGl Fc fusion protein.

[0011] In certain embodiments the antibody including specificity for IFITM1 is a chimeric molecule and is conjugated to a therapeutic agent. The therapeutic agent may be a cytotoxic agent selected from the group consisting of paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide.

[0012] Also provided herein is a treatment regimen for reducing a pool of latently infected cells in a patient infected with a latent immunodeficiency virus comprising co-administering an anti-IFITMl specific monoclonal antibody and one or more anti-viral agents selected from viral entry inhibitors, nucleotide/side reverse transcription inhibitors (NRTI), non-nucleoside reverses transcriptase inhibitors (NNRTI), integrase inhibitors and protease inhibitors. The treatment regimen may further comprise co-administering a drug that targets an immune checkpoint (IC) molecule, wherein the IC molecule is selected from Programed Cell Death- 1 (PD-1), Programed Cell Death Ligand-1 (PD-L1), T-cell Immunoregulator with Ig and ITEVI domains (TIGIT), Lymphocyte Activation Gene 3 (LAG-3), and combinations thereof. In addition, the treatment regimen may include co-administering and effective amount of an NK cell stimulator selected from one or more of type I IFNs (mainly IFN-a and IFN-β), IL- 2/anti-IL-2 monoclonal antibody complexes, IL-12, IL-15 and IL-18, and wherein the antibody to IFITM1 and the NK cell stimulator synergistically kill cells latently infected with the latent immunodeficiency virus.

[0013] In certain embodiments a cocktail of drugs is provided that is directed to killing cells latently infected with HIV wherein the cocktail comprises anti-IFITMl specific monoclonal antibody and a monoclonal antibody directed to an IC molecule selected from one or more of Programed Cell Death- 1 (PD-1), Programed Cell Death Ligand-1 (PD-L1), T-cell Immunoregulator with Ig and ITIM domains (TIGIT), and Lymphocyte Activation Gene 3 (LAG-3). [0014] In other embodiments, a CAR T cell is provided that has specificity for IFITM1. In still other embodiments a CAR T cell is bi-specific and includes an IFITM1 specific scFv joined in tandem to a CD4 specific scFv in the CAR extracellular domain. Whether mono or bi-specific, the exodomain is linked through a transmembrane domain to a signaling endodomain including one or more of signaling and co-stimulatory domains derived from CD2, CD 3, CD 27, CD28, CD30 (aka TNFRSF8), CD40 (aka TNFRSF5), CD! 34 (aka TNFRSF4 and OX40), CD137 (4-iBB), CD278 (ICOS, Inducible T-Cell CoStimulator), and glucocorticoid-induced T FR-related protein (GITR; also known as T FRSF18). In certain embodiments the IFITM1 specific CAR T cells are expanded ex vivo using IL-15 prior to administering the CAR modified T cells to the immunodeficiency virus-infected host.

[0015] The mono or bi-specific CAR T cell that includes IFITM1 specificity is administered to HIV infected patients to reduce the load of latently infected cells. In certain embodiments, the patient is co-administered a drug that targets an immune checkpoint (IC) molecule selected from Programed Cell Death- 1 (PD-1), Programed Cell Death Ligand-1 (PD-L1), T- cell Immunoregulator with Ig and ITIM domains (TIGIT), Lymphocyte Activation Gene 3 (LAG-3), and combinations thereof. The patient may be co-administered one or more antiviral agents selected from viral entry inhibitors, nucleotide/side reverse transcription inhibitors ( RTI), non-nucleoside reverses transcriptase inhibitors (N RTI), integrase inhibitors and protease inhibitors.

[0016] In one embodiment a method for treatment of a human individual infected with Human Immunodeficiency Virus (HIV) is provided wherein the method comprising the steps of: (a) obtaining a sample of peripheral blood from the individual; (b) determining an expression level of IFITMl on T cells in the sample; (c) administering a treatment regimen to the individual; (d) periodically repeating steps (a) and (b) to determine efficacy of the administered treatment; and (e) adjusting the treatment regimen if needed to attain a reducing in IFITMl expression of T cells from the individual. The treatment regimen may include administering one or more of: a cocktail of antiviral drugs, anti-IFITMl antibodies, IC antagonists, anti-IFITMl CAR T cells, and cytokines directed to maximizing NK function or CAR T cell activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For a more complete understanding of the present invention, including features and advantages, reference is now made to the detailed description of the invention along with the accompanying figures:

[0018] Fig. 1A illustrates a scheme for generating latently infected CD4+ T-cells according to one model. As shown in Fig. 1 A, CD4+ T-cells were negatively selected from PBMC and conditioned for 3 days with CCL19. CCL19-treated cells were infected with HIV-1NL4-3 for 6 days and resting CD4+ T-cells (CD25-/CD69-/HLA-DR-) were negatively selected by magnetic cell sorting. Resting CD4+ T-cells were reactivated with anti-CD3/CD28 for three days. Fig. IB shows representative intracellular staining of HIV-1 Gag (KC57) (n=3) in resting latent cells. Fig. 1C shows representative intracellular staining of HIV-1 Gag (KC57) (n=3) in reactivated cells.

[0019] Fig. 2A shows quantification of HIV-1 gag by real-time qPCR. Fig. 2B shows quantification of HIV-1 mRNA transcripts by real-time qPCR. Fig. 2C shows the fold- expression of selected antiviral genes in resting latent over reactivated cells. Data are plotted as mean ±SEM where n=3.

[0020] Figs. 3A - 3D show representative extracellular staining of IFITM1 (n=3) in HIV negative and resting/latent populations. In Fig. 3A, HIV negative cells are also stained for SSC-A. In Fig. 3B, the HIV negative cells are co-stained with anti-CD3/CD28. In Fig. 3C, resting latent cells are also stained for SSC-A. In Fig. 3D, the resting latent cells are co- stained with anti-CD3/CD28.

[0021] Figs. 4A - 4F represent ADCC of latently infected ACH-2. In Fig. 4A, ACH-2 (target) cells were incubated with anti-IFITMl or an isotype control prior to addition of effector cells. Target cell apoptosis was detected using the FLICA assay and a live/dead marker. Fig. 4A is a representative apoptosis plot gated on ACH-2 (n=4). Fig. 4B shows results at the end of the assay when cells were washed and stained with antibodies to detect NK cell degranulation and production of intracellular IFN-g with a representative functional assay gated on CD56+NK cells at different E:T ratios (n=4). Fig. 4C depicts the overall percentage of killed (dead/FLICA+) ACH-2 target cells. Fig. 4D depicts IFN- g+CD107a+NK cells. Fig. 4E depicts IFN-g+NK cells and Fig. 4F depicts CD107a+NK cells (n=4). Data are plotted as mean ± SD and significance was determined using a non- parametric two-tailed t-test. P<0.05 was considered significant.

[0022] Figs. 5 A - 5C show ADCC of IFITM1+CD4+ T-cells from ART-suppressed patients. Fig. 5 A depicts expression of IFITM1 in CD4+ T-cell subsets from healthy (n=6) and ART- suppressed patients (n=6). Naive, Stem central memory (TSCM), Central memory (TCM), Transitional memory (TTM) and Effector memory (TEM) cells are shown. Fig. 5B depicts purified CD4+ T-cells from ART-suppressed patients incubated with anti-IFITMl or isotype control prior to the addition of autologous effector cells at an E:T ratio of 10: 1. Target cell apoptosis was detected using the FLICA assay and a live/dead marker (n=6). Fig. 5C depicts the results when cells were washed and stained with anti-CD3, CD56, CD107a and IFN-g to detect NK cell degranulation and production of intracellular IFN-g. The overall percentage of IFN-g+CD107a+NK cells in autologous or heterologous conditions (n=6) is shown. Data are plotted as mean ± SD and significance was determined using a non-parametric two-tailed t-test.

[0023] Figs. 6A - 6G depict the results of reactivation of HIV-1 in ACH-2 cells. In Fig. 6A ACH-2 cells were treated for 5h with increasing concentrations of SAHA, panobinostat and romidepsin. After the initial pulse, cells were washed twice in complete media and cultured for an additional 18h. The frequency of reactivated cells was determined by intracellular HIV-1 Gag staining. Representative flow plots of three independent experiments are shown. Fig. 6B - 6E shows the pooled data from three different experiments (n=3) for cells treated with increasing concentrations of SAHA (Fig. 6B), panobinostat (Fig. 6C), and romidepsin (Fig. 6D), with Fig. 6E summarizing the results of Figs. 6B - 6D. Reactivated ACH-2 cells were calculated by subtracting the percentage of HIV-1 Gag+ cells by the percentage of HIV- 1 Gag+ cells in untreated control. Data are plotted as mean ±SEM. Fig. 6F shows the fold mRNA gene expression of IFITM1 in latent over reactivated cells (n=3). Data are plotted as mean + SD. Fig. 6G shows the effect of different HDACi on ACH-2 cell viability. Representative flow plots of three independent experiments are shown.

[0024] Figs. 7A - 7H show gating strategy for the detection of IFITM1 in different CD4+ T cell subsets. Fig. 7A shows the side scatter area (SSC-A) plotted against the Forward Scatter area (FSC-A) by flow cytometry with the cells to be analyzed within the drawn area. Fig. 7B shows the Forward Scatter area (FSC-A) plotted against the Forward Scatter height (FSC-H). PBMC were stained with antibodies against CD3 (Fig. 7C), CD4 (Fig. 7D and F), CD 8 (Fig. 7D), CD45RA (Fig. 7F), CCR7 (Figs. 7E and G), CD27 (Figs. 7E and G), CD95 (Fig. 7H) and IFITMl . Dead cells were excluded using a live/dead marker.

DETAILED DESCRIPTION OF THE INVENTION

[0025] HIV-1 persistence in latent reservoirs during antiretroviral therapy (ART) is the main obstacle to virus eradication. To date, there is no marker that adequately identifies latently infected CD4⁺ T-cells in vivo. Latency models have predominantly been used as means to study HIV-1 reactivation in hopes of providing data towards "shock-and-kill" strategies. We utilized a latently infected CD4⁺ T-cell model to obtain unique mechanistic insights into the role of intrinsic cellular immunity during ART and developed a treatment modality based on these findings. Using a quantitative PCR-based array capable of examining the expression of a predefined set of antiviral genes in primary cells, we explored transcriptional and epigenetic patterns found in latent and reactivated CD4⁺ T-cells to identify differential expression of restriction factors and other antiviral genes. Through this model utilizing latently infected CD4⁺ T-cells, we found Interferon Induced Transmembrane Protein 1 (IFITMl), a transmembrane antiviral factor, to be overexpressed in resting latent cells when compared to their reactivated counterparts.

[0026] By targeting IFITMl, we showed the efficient and specific killing of a latently infected cell line and CD4⁺ T-cells from ART-suppressed patients through IFITMl -antibody- dependent cell-mediated cytolysis (ADCC). Thus, in certain embodiment provided herein, IFITMl is used as a marker for natural HIV-1 reservoirs and provides an immune target for killing of latently infected cells through IFITMl -antibody-dependent cell-mediated cytolysis (ADCC) alone and combination with other agents.

[0027] In other embodiments, IFITMl is used as a bio-marker for detecting and quantitating latently infected T cells. Through measurement of IFITMl, antiviral regimens are tailored to maximize the efficacy of treatment in individual patients. Thus in one embodiment, monitoring of the efficacy of an anti-HIV regimen in a patient comprises: determining levels of expression of IFITMl on T cells from the patient, treating the patient with a treatment regimen, periodically re-determining levels of expression of IFITMl on T cells from the patient, and adjusting the therapeutic regimen to maximally affect reduced levels of IFITMl expression. The therapeutic regimen may include administering one or more of: a cocktail of antiviral drugs, anti-IFITMl antibodies, IC antagonists, anti-IFITMl CAR T cells, and cytokines directed to maximizing NK function or CAR T cell activity. [0028] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts which can be employed in a wide variety of specific contexts. The specific embodiment discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0029] ABBREVIATIONS: The following abbreviations are used throughout this application:

ACH-2 cells HIV-1 latent T cell clone with one integrated proviral copy.

ADCC Antibody -dependent-cell mediated cytolysis

ART Anti-retroviral therapy

BST2/tetherin Bone Marrow Stromal Antigen 2, a.k.a. tetherin (CD317)

CCL19 Chemokine (C-C motif) ligand 19, a.k.a. EBI1 ligand chemokine

(ELC) and macrophage inflammatory protein-3-beta (MTP-3-beta)

DART Dual-affinity re-targeting proteins

FLICA FAM Poly-caspase assay kit

GALT Gut-associated lymphoid tissue

HDACi Histone deacetylase inhibitors

IFITM1 Interferon Induced Transmembrane Protein 1

SAHA suberanilohydroxamic acid, a.k.a vorinostat is a histone deacetylase inhibitor marketed by Merck as ZOLINZA^®

SAMHD1 SAM domain and HD domain-containing protein 1, cellular enzyme that blocks HIV replication by converting dNTPs to inorganic phosphate and 2'-deoxynucleoside thus depelted available pool for RT

TRIM5 Triple motif-containing protein5

[0030] To facilitate the understanding of this invention, and for the avoidance of doubt in construing the claims herein, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. The terminology used to describe specific embodiments of the invention does not delimit the invention, except as outlined in the claims.

[0031] The terms such as "a," "an," and "the" are not intended to refer to a singular entity unless explicitly so defined, but include the general class of which a specific example may be used for illustration. The use of the terms "a" or "an" when used in conjunction with "comprising" in the claims and/or the specification may mean "one" but may also be consistent with "one or more," "at least one," and/or "one or more than one."

[0032] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives as mutually exclusive. Thus, unless otherwise stated, the term "or" in a group of alternatives means "any one or combination of the members of the group. Further, unless explicitly indicated to refer to alternatives as mutually exclusive, the phrase "A, B, and/or C" means embodiments having element A alone, element B alone, element C alone, or any combination of A, B, and C taken together.

[0033] Similarly, for the avoidance of doubt and unless otherwise explicitly indicated to refer to alternatives as mutually exclusive, the phrase "at least one of when combined with a list of items, means a single item from the list or any combination of items in the list. For example, and unless otherwise defined, the phrase "at least one of A, B and C," means "at least one from the group A, B, C, or any combination of A, B and C." Thus, unless otherwise defined, the phrase requires one or more, and not necessarily not all, of the listed items.

[0034] The terms "comprising" (and any form thereof such as "comprise" and "comprises"), "having" (and any form thereof such as "have" and "has"), "including" (and any form thereof such as "includes" and "include") or "containing" (and any form thereof such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0035] The term "effective" as used in the specification and claims, means adequate to provide or accomplish a desired, expected, or intended result.

[0036] The terms "about" or "approximately" are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, within 5%, within 1%, and in certain aspects within 0.5%.

[0037] The term "immunodeficiency virus" includes human immunodeficiency virus (HIV) as well as feline immunodeficiency virus and simian immunodeficiency virus. The term HIV as used herein includes human immunodeficiency virus- 1 (HIV-1), human immunodeficiency virus-2 (HIV-2), and other HIV subtypes.

[0038] As used herein the term "antibody" includes both intact immunoglobulin molecules as well as portions, fragments, and derivatives thereof, such as, for example, Fab, Fab', F(ab')2, Fv, Fsc, CDR regions, or any portion of an antibody that is capable of binding an antigen or epitope including chimeric antibodies that are bi-specific or that combine an antigen binding domain originating with an antibody with another type of polypeptide. For example, in one embodiment a bi-specific antibody is utilized that includes antigen specific binding for IFITMl on one Fab domain and antigen specific binding for CD4 on the other Fab domain. The term antibody includes monoclonal antibodies (mAb), chimeric antibodies, humanized antibodies, as well as fragments, portions, regions, or derivatives thereof, provided by any known technique including but not limited to, enzymatic cleavage and recombinant techniques. The term "antibody" as used herein also includes single-domain antibodies (sdAb) and fragments thereof that have a single monomelic variable antibody domain (VH) of a heavy-chain antibody. sdAb, which lack variable light (VL) and constant light (CL) chain domains are natively found in camelids (VHH) and cartilaginous fish (VNAR) and are sometimes referred to as "Nanobodies" by the pharmaceutical company Ablynx who originally developed specific antigen binding sdAb in llamas. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0039] The terms "co-administered" and "in combination with" include administration with two or more therapeutic agents either simultaneously, concurrently or sequentially in any order without specific time limits so long as the two or more "co-administered" drugs are present in measureable amounts in a single patient at a given time. In certain embodiments, the therapeutic agents are in the same composition or unit dosage form while in other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. The terms "active agent," "drug," "therapeutic agent," and synonymous terms according to those of skill in the art are used interchangeably herein.

[0040] An effective amount is an amount that reduces the reservoir of latent virus in an individual by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. Reductions in the reservoir of latently infected cells is can be determined using any known method, including by polymerase chain reaction (PCR), detecting and/or measuring viral polypeptides and antigens and measuring the CD4⁺ T cell count in the individual. [0041] In one embodiment, an eradication strategy is provided selectively targeting IFITM1 in conjugation with targeting immune checkpoint molecules shown to contribute to HIV persistence during ART to the end of effectively targeting and eliminate latently infected cells in vivo. Immune checkpoint molecules (ICs) act to up or down-modulate immune responses. Down-regulating (inhibitory) immune checkpoint molecules act normally to prevent hyper-immune activation, minimize collateral damage, and maintain peripheral self- tolerance. ICs are normally up-regulated with T-cell activation and act to limit the effector response by feedback inhibition. IC overexpression is associated with T-cell exhaustion and dysfunction in cancer and chronic viral infections, including HIV. Examples of immune check point molecules that may be targeted include those identified as expressed on cells harboring latent virus. To date, Programed Cell Death- 1 (PD-1), Programed Cell Death Ligand-1 (PD-L1), T-cell Immunoregulator with Ig and ITEVI domains (TIGIT), and Lymphocyte Activation Gene 3 (LAG-3), have been shown to be positively associated with CD4+ cells harboring latent HIV DNA. See Fromentin, R, et al. "CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART" PLoS Pathog 12(7) (2016) 1-19. Certain of these IC have been the target of drugs under development.

[0042] PD-1 is a cell surface receptor that is a member of the CD28 family of T-cell regulators. The human PD-1 gene is on chromosome 2q37 and the full-length PD-1 cDNA encodes a 288 amino acid protein. Several monoclonal antibodies targeting PD-1 have been developed to reverse PD-1 mediated T-cell down-regulation of antitumor responses. Anti PD-1 monoclonal developed to date include Nivolumab (previously termed BMS-936558, now FDA approved for certain cancers and marketed by Bristol-Myers Squibb as OPDIVO^¾), Pembrolizumab (previously termed MK-3475 now FDA approved for certain cancers and marketed by Merck as EYTRUDA*), and Pidilizumab (previously termed CT- 01 1 by CureTech and MDV9300 by Mediation). PD-1 has two ligands, PD-L1 and PD-L2, which are also transmembrane cell surface molecules that are typically expressed on antigen presenting cells and serve as immune system inhibitors. Expression of these ligands on tumor cells help the tumor ceils avoid immune mediated control.

[0043] Programed Death Ligand-1 (PD-L1) is also known as cluster of differentiation (CD) 274 or B7 homolog 1 is encoded in humans by the CD274 gene. The IC molecule PD-L! may be targeted by Atezolizumab (tradename TECENTRTG® by Genentech) and Avelumab (currently co-developed by Pfizer and Merck and also known as MSB0G1Q718C). [0044] LAG-3, CD designated as C ^'0223. is an immunoglobulin superfamiiy 503 -ami no acid transmembrane protein encoded on human chromosome 12 i 3 adjacent to the gene encoding CD4. Bristol-Myers Squibb has developed an anti-LAG3 monoclonal antibody called BMS- 986016 for tumor treatment. GlaxoSmith iine has also developed an anti -LAG-3 monoclonal antibody (GSK283178I) but for the indication of treating psoriasis.

[0045] TIGIT is a member of CD28 family and includes an extracellular IgV domain, a transmembrane domain, and a cytoplasmic tail containing two-immimoreceptor tyrosine- based inhibitor}' motifs (ITIM). TIGIT and PD-1 have been shown to be overexpressed on viral iy exhausted T cells. See Chew, GM, et al. "TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV infection" PLoS Pathog 12(1) (2016) 1-28. Antibodies to TIGIT for immunomodulation are under development by Genentech for reversal of T-cell exhaustion in cancer (MTIG7192A, RG6058)

[0046] The IFN-inducible transmembrane protein (IFITM) family includes several members including IFITM 1 (murine = fragilis 2), IFITM2 (murine = fragilis 3) and IFITM3 (murine = fragilis). These proteins are a family of interferon-induced antiviral cell-intrinsic restriction factors that are highly constitutively expressed in certain cells, including barrier epithelial cells. The IFITM 1 gene encodes the Leu-13 antigen, designated as CD225. As their name suggests, IFITM are strongly upregulated by both type I and II interferons. Certain of the IFITM family of proteins act to uniquely inhibit replication of members of the following diverse group of membrane-enveloped viruses: retroviruses (HIV), filoviruses (Marburg and Ebola), coronavirus (Severe Acute Respiratory Syndrome - SARS-CoV), phlebovirus (Rift Valley Fever - RVF), paramyxovirus (respiratory syncytial virus - RSV), flaviviruses (including Dengue (DenV), West Nile Virus (WNV), Zika, Yellow Fever Virus, and several hemorrhagic viruses)), influenza A virus and Hepatitis type C (HCV). In certain embodiments, targeting of IFITM is employed in conjunction with antiviral drugs shown to be effective against enveloped viruses.

[0047] Components of intrinsic immunity, such as restriction factors possess potent anti- HIV-1 activities by interfering with the virus life cycle. Restriction factor expression varies between cells and activation states, and in addition to acting directly against HIV-1, they elicit immune responses to accelerate the clearance of infected cells. Such roles have been described for BST2/tetherin and TRIM5. Type-I interferons, such as IFNa, are able to strongly induce the expression of most, but not all, restriction factors to further curb recurrent cycles of infection. Cell-specific intrinsic immune mechanisms that orchestrate the interferon response are likely to play a key role in the prevention of HIV- 1 infection in long- lived memory CD4+ T-cell subsets.

[0048] We determined that IFITM1 expressing CD4+ T cells are enriched in the central memory compartment (TCM) of ART-suppressed patients. TCM are the main source of latent virus in natural reservoirs. We were able to obtain efficient killing of latently infected CD4+ T cells by IFITM1 antibody-dependent cytolysis. In one embodiment provided herein selective targeting of IFITM1 in the TCM reservoir is employed to identify and destroy latently infected cells.

[0049] In certain embodiments, selectively targeting IFITM1 is conducted in conjunction with treatment with HIV/AIDS antivirals. HIV/AIDS antivirals include drugs in several categories that affect different aspects of the viral life cycle: viral entry inhibitors, nucleotide/side reverse transcription inhibitors (NRTI), non-nucleoside reverses transcriptase inhibitors (NNRTI), integrase inhibitors and protease inhibitors. Typically patients are treated with combinations that block more than one aspect of the viral life cycle. Combination therapies are variously called anti-retroviral therapy (ART), combination anti- retroviral therapy (cART) or highly active anti-retroviral therapy (HAART).

[0050] Drugs affecting viral entry include maraviroc (SELZENTRY®), which blocks the CCR5 receptor on helper T-cells, and enfuvirtide (FUZEON®), which is an injectable peptide drug that prevents infection of host cells by interacting with the N-terminal heptad repeat of HIV gp41 to form an inactive hetero six-helix bundle.

[0051] Nucleoside (tide) reverse transcriptase inhibitors (NRTI) act as substrates and competitively inhibit the action of the viral reverse transcriptase and include zidovudine, a.k.a. azidothymidine (RETROVIR®), abacavir (ZIAGEN®), lamivudine (EPIVIR®), emtricitabine (EMTRIVA®), and tenofovir (VIREAD®).

[0052] Non-nucleoside reverse transcriptase inhibitors (NNRTI) bind to the RT and thus inhibit its action. NNRTI include nevirapine (VIRAMUNE®), efavirenz (SUSTIVA®), etravirine (INTELENCE®) and rilpivirine (EDURANT®). [0053] Drugs that block HIV integration into the host chromosome act by inhibiting the viral integrase enzyme and include raltegravir (ISENTRESS^®), elvitegravir (VITEKTA^®) and dolutegravir (TIVICAY^®).

[0054] HIV protease inhibitors include ritonavir (NORVIR^®), tipranavir (APTIVUS^®), atazanavir (REYATAZ^®), darunavir (PREZISTA^®), indinavir (CRIXIVAN^®), nelfmavir (VIRACEPT^®), saquinavir (INVIRASE^®), lopinavir, amprenavir (AGENERASE^®) and its prodrug fosamprenavir (LEXIVA^®).

[0055] In one embodiment, methods and compositions are provided that enhance the activity of Natural Killer (NK) cells in the presence of an anti-IFITMl antibody. NK cells are large CD3^" CD56⁺ lymphocytes that are further segregated into CD56^{bri ht} and CD56^dim subsets. Approximately 90% of peripheral blood and spleen NK cells are CD56^dim and CD16⁺ with marked cytotoxic activity against target cells. The majority of NK cells found in lymph nodes and tonsils are CD56^bnghtCD16^" and perform immune regulation through production of cytokines such as interferon (IFN)-y in response to IL-12, IL-15 and IL-18 stimulation.

[0056] Early after infection and before an efficient HIV-specific adaptive immune response is raised, NK cells are activated and contribute to the initial control of HIV- 1 replication. We found however, that despite being able to kill target cells, autologous NK cells from ART- suppressed HIV-1 patients showed limited capacity to produce IFN-γ and to upregulate CD 107a in the presence of anti-IFITMl antibody. Thus, in one particular embodiment, the function of NK cells in the presence of an anti-IFITMl antibody is enhanced using stimulation with one or more of type I IFNs (mainly IFN-a and IFN-β), IL-2/anti-IL-2 monoclonal antibody complexes, IL-12, IL-15 and IL-18.

[0057] In certain embodiments, the function of NK cells in the presence of an anti-IFITMl antibody is enhanced by administration of IL-15 superagonists. One such IL-15 superagonist is ALT-803, which is an IL-15 mutant (IL-1 5N72D) bound to an IL-15 receptor α/IgGI Fc fusion protein developed by Altor Bioscience, and has been shown to potently activate NK cells, induce XFN-γ secretion by NK cells and enhance antibody-dependent cell-mediated cytotoxicity (A.DCC) of antibodies in various experimental models. Another II.- 15 super agonist is the fusion protein RLI, composed of the Li-terminal (amino acids 1-77, sushi+) domain of IL-15 receptor a coupled via a linker to IL-15. See Mortier, E., et al. Soluble Interleukin-15 Receptor a (IL-15Ra)-sushi as a Selective and Potent Agonist of IL-15 Action through IL-15Rp/a. Journal of Biological Chemistry 281(3 )(2006) 1612-1619. [0058] As provided herein, the identification of a latent viral reservoir in IFITM1 expressing cells provides a several treatment modalities. In certain embodiments, an antibody directed to IFITM1 and including an IgG Fc domain that binds to FcyRl receptors on K cells is administered to an HIV-1 positive patient. The IFITM1 specific antibody supports ADCC mediated killing of cells bearing latent HIV. In certain embodiments, the antibody is humanized. In certain embodiments the antibody is specific to IFITM1 and is not cross reactive with IFITM2 or IFITM3.

[0059] In certain embodiments, a monoclonal antibody to IFITM1 is utilized that includes an Fc domain that is selected for its effectiveness in fixing complement and promoting complement-dependent cytotoxicity (CDC) of antibody opsinized cells expressing IFITM1. In such embodiments, antibodies of the IgG3 and IgGl classes are preferred for complement activation through the classic cascade.

[0060] In other embodiments, a monoclonal antibody to IFITM1 is utilized that is optimized for ADCC by directed evolution with selection for increased killing of latently infected CD4+ cells.

[0061] In certain embodiments, the antibody is a bi-specific antibody that includes an anti- IFITMl moiety in addition to another moiety having a different target. In one embodiment the different target is CD4. In other embodiments the antibody is a chimeric molecule and is conjugated to a heterologous polypeptide. In such embodiments the IFITM1 antigen binding aspect of the antibody may be a Fab, Fv or antibody half molecule. The heterologous protein is a therapeutic agent. In particular embodiments the therapeutic agent is a cytotoxic agent selected from the group consisting of paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, or cyclophosphamide. By virtue of the cytotoxic agent the IFITM1 expressing cells are targeted for cytotoxic killing.

[0062] In certain embodiments, an anti-IFITMl specific monoclonal antibody is administered in addition to one or more anti-HIV agents selected from viral entry inhibitors, nucleotide/side reverse transcription inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), integrase inhibitors and protease inhibitors. [0063] In other embodiments, an anti-IFITMl specific monoclonal antibody is administered in addition to one or more of type I IFNs, IL-2/anti-IL-2 monoclonal antibody complexes, IL- 12, IL-15 and IL-18. In other embodiments, an anti-IFITMl specific monoclonal antibody is administered in addition to an IL-15 superagonist. In one embodiment, the IL-15 superagonist is selected from the group cons sting of RLI and ALT-803. In one embodiment, an infusion comprising an anti-IFITMl specific monoclonal antibody and an IL-15 superagonist is administered to an HIV-1 patient on ART or HAART therapy.

[0064] Also provided are IFITM1 Specific Chimeric Antigen Receptor (CAR) T cells in which immune effector T cells are modified to express an engineered T cell receptor having IFITMl specificity. In certain embodiments the CAR includes an IFITM1 specific exodomain formed by antibody VH and VL antigen binding domains separated by a linker to form a fusion protein termed a single-chain variable fragment (scFv) that is specific for binding to IFITMl .

[0065] In other embodiments, a bi-specific CAR T cell is employed that includes an IFITMl specific scFv joined in tandem to a CD4 specific scFv in the CAR exo (extracellular) domain.

[0066] Whether mono or bi-specific, scFv exodomain(s) are linked by a spacer to a transmembrane domain and an endo (intracellular) signaling domain. See e.g. G Dotti, et al. "Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells" Immunol Rev 257(1) (2014) 1-35. The signaling endodomain may include one or more of signaling and co-stimulatory domains derived from CD2, CD3, CD 27, CD28, CD30 (aka TNFRSF8), CD40 (aka TNFRSF5), CD] 34 (aka TNFRSF4 and OX40), CD137 (4- 1BB), CD278 (ICOS, Inducible T-Cell CoStimulator), glucocorticoid-induced TNFR-related protein (GITR; also known as T FRSF18) and combinations thereof.

[0067] For example the exodomain(s) may be linked to a CD3ζ (CD3-zeta) signaling domain of the T Cell Receptor complex, while in other embodiments the exodomain(s) may be linked to a CD28 ζ (zeta) signaling endodomain, in addition to co-stimulatory domains.

[0068] In one non-limiting example, the CAR T-cell signaling endodomain includes an intracellular signaling domain (CD3^) of the native T cell receptor complex including either intracellular signaling domains of CD28 or 4- IBB as costimulatory domains. In another non- limiting example, the CAR T-Cell endodomain combines CD3^ to provide cytotoxicity, a Pi3K binding domain derived from a CD28 ζ (zeta) signaling endodomain to confer proliferation and cytokine production, and a TRAF2 binding site from a 4-1BB or OX40 intracellular domain to augment survival of the CAR T cell.

[0069] In certain embodiments, IL-15 is used to expand the engineered CAR T-cells ex vivo and may be further administered to the recipient patient to support growth and expansion of the engineered CAR T-cells once administered to the patient. Further support for the CAR T-cells may be provided by co-administering a drug that targets one or more immune checkpoint (IC) molecules selected from Programed Cell Death- 1 (PD-1), Programed Cell Death Ligand-1 (PD-L1), T-cell Immunoregulator with Ig and ITIM domains (TIGIT), and Lymphocyte Activation Gene 3 (LAG-3). Certain drugs are available for such purpose although it is anticipated that other anti-IC drugs will be developed. Currently, the IC molecule PD-1 may be inhibited by ivolumab, Pembrolizumab, and Pidilizumab. The IC molecule PD-L1 may be targeted by Atezolizumab and Aveluniab. The IC molecule LAG-3 may be targeted by BMS-986016 and GSK2831781 , while the IC molecule TIGIT may be targeted by Genentech's MTIG7192A.

[0070] The following examples are included for the sake of completeness of disclosure and to illustrate the methods of making the compositions and composites of the present invention as well as to present certain characteristics of the compositions. In no way are these examples intended to limit the scope or teaching of this disclosure.

EXAMPLE 1 :

Materials and Methods

[0071] Generation of HIV-1 latently infected CD4⁺ T-cells: Latently infected CD4⁺ T-cells were generated beginning with PBMCs from healthy individuals isolated from buffy-coats as shown figuratively in Fig. 1A. CD4⁺ T-cell were isolated by negative selection (StemCell). Purified CD4⁺ T-cells were incubated with CCL19 (29 nM, R&D) for three days before HIV- 1 infection. Cells were infected with HIV-1NL4-3 for 4h at 37°C. The cells were then cultured in the presence of IL-2 (10 IU/ml, Roche) for six days. At day six post-infection, resting CD4⁺ T-cells were isolated by anti-PE magnetic bead depletion (Miltenyi Biotec) using a cocktail of antibodies to CD25-PE (BD Pharmingen, clone M-A251), CD69-PE (BD Pharmingen, clone FN50) and ULA-DR-PE (BD Pharmingen, clone G46-6). Resting latent CD4+ T-cells were then reactivated with anti-CD3/CD28 (StemCell) for three days in the presence of IL-2 (50 IU/ml), according to the manufacturer's specifications. [0072] ACH-2 cell line and reactivation with histone deacetylase inhibitors: ACH-2 cells were obtained from the NIH AIDS Reagents (catalogue #349) and were tested negative for mycoplasma contamination. Cells were typically propagated in RPMI1640, 2 mM L- Glutamine, 10% FBS in the presence of penicillin/streptomycin. The non-selective histone deacetylase inhibitors (HDACi) suberanilohydroxamic acid (SAHA, a.k.a. vorinostat, marketed by Merck as ZOLINZA®), panobinostat (marketed under the tradename FARYDAK®), and romidepsin (marketed under the tradename ISTODAX®) were purchased from Selleckchem. Cells were typically plated in 96-well plates (U-shape) and treated with different concentrations of FIDACi for 5h, washed twice and re-cultured for 18h prior to RNA extraction or flow cytometry.

[0073] Flow cytometry: To detect intracellular HIV-1 Gag, CD4⁺ T-cells were washed in FACS buffer (2 mM EDTA, 0.5% BSA in ice-cold PBS) and surface stained with antibodies against CD3 (1 : 100, ECD, clone UCHT1, Beckman Coulter), CD4 (1 :500, Alexa700, clone RPA-T4, Becton Dickinson), CD 8 (1 :400, BV605, clone RPA-T8, Biolegend), and a live/dead marker (1 :200, Invitrogen) emitting in the aqua wavelength, for 30 min at 4°C. After staining, the cells were washed three times, fixed in 2% paraformaldehyde for 20 min and stained with KC57-RD1 antibody (1 : 100, Beckman Coulter) in 0.1% saponin for 30 min. For the detection of IFITM1 in different CD4⁺ T-cell subsets, PBMCs were washed in FACS buffer and surface stained with monoclonal anti-IFITMl antibody (1 :200, 60074-1-Ig, monoclonal mouse anti-human, Proteintech) for 30 min at 4°C. After staining, cells were washed three times and stained with anti-mouse IgG2a-PE (1 :200, Biolegend). Following IFITM1 staining, PBMCs were washed and stained for CD3 (1 : 100, ECD, clone UCHT1, Beckman Coulter), CD4 (1 :500, Alexa700, clone RPA-T4, Becton Dickinson), CD8 (1 :400, BV605, clone RPA-T8, Biolegend), CD45RA (1 : 100, BV650, clone HI100, BD Pharmingen), CCR7 (1 : 100, PE-Cy7, cloneG043H7, Biolegend), CD27 (1 : 100, APC-Cy7, clone 0323, Biolegend) and CD95 (1 :50, PacificBlue, clone DX2, Biolegend) and a live/dead marker (1 :200, Invitrogen) for 30 min at 4°C. FMOs and single-stained polystyrene beads (BD Biosiences) were conducted for compensation purposes and the gating strategy can be observed in Figs. 7A - H. Cells were washed and events were collected on an LSRFortessa X-20 flow cytometer (BD Biosciences). Data were analyzed using Flow Jo software (version X.0.4, TreeStar). [0074] Depletion of IFITM1+ cells from bulk CD4+ T cells of ART-suppressed patients: Bulk CD4+ T cells were isolated from PBMCs of ART-suppressed patients by negative selection (StemCell) and an aliquot was used for isolation of gDNA. CD4+ T cells were incubated for 30min at 4°C with anti-IFITMl antibody (1 :200, 60074-1 -Ig, Proteintech) and after staining, cells were washed three times and stained with anti-mouse IgG2a-PE (1 :200, Biolegend) for an additional 30min at 4°C in the dark. Anti-PE microbeads (Miltenyi Biotec) were used to deplete IFITM1+ cells from bulk CD4+ T cells. IFITM1-CD4+ T cells were collected and genomic DNA was isolated using DNeasy Blood & Tissue kit (Qiagen), according to the manufacture's recommendations.

[0075] ADCC and functional assays: ACH-2 cells (NIH AIDS Reagents) or CD4⁺ T-cells, isolated from PBMC of ART-suppressed patients by negative selection (StemCell), were incubated with monoclonal anti-IFITMl antibody (1 :200, 60074-1-Ig, Proteintech) or IgG2a isotype control (Biolegend) prior to the addition of PBMC effector cells for 5h at the indicated effector to target ratio (20: 1 and 10: 1, in a 96-well U-shaped plate in a final volume of 200 μΐ). Autologous PBMCs or heterologous PBMCs from healthy donors were used as effector cells. Monensin (Golgi Stop, BD Biosciences) was added at the beginning of the assay. Target cell apoptosis was detected using the fluorescent inhibitor of caspases (FLIC A) flow cytometry -based assay as described by Leeanshya, E. et al. Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection. PLoS Pathog. 11(8) (2015) el005072. Briefly, the FLICA reagent (Vybrant FAM Poly-Caspases Assay Kit, Thermofisher) was added in the last hour to the cell culture media. When the assay was terminated, cells were washed and stained with a secondary antibody against the anti-IFITMl [anti-mouse IgG2a-PE (1 :200, Biolegend)], washed and surface stained for CD3 (1 : 100, ECD, clone UCHT1, Beckman Coulter), CD56 (1 :20, PE-Cy7, clone MEM-188, Biolegend), CD 107a (1 : 10, APC-H7, clone H4A3, BD Biosciences) and a live/dead marker (1 :200, Invitrogen) for 30 min at 4°C. After staining, cells were washed and fixed in Cytofix/Cytoperm for 20 min at 4°C. Intracellular staining was performed using anti-IFN-γ antibody (1 : 10, APC, clone B27, BD Biosciences) in Perm/Was (BD Biosciences). Samples were acquired on an LSRFortessa X-20 flow cytometer (BD Biosciences). Single-stained polystyrene beads (BD Biosiences) were used for compensation purposes. Data were analyzed using FlowJo software (version X.0.4, TreeStar). [0076] ADCC and viral reactivation: CD4+ T cells were isolated from PBMC of a ART- suppressed patient by negative selection (StemCell) and incubated with monoclonal anti- IFITM1 antibody (1 :200, 60074-1-Ig, Proteintech) or IgG2a isotype control (Biolegend) prior to the addition of autologous PBMC as effector cells for three days at an effector to target ratio of 10. At day 3, CD4+ T cells were re-purified by negative selection and stimulated with PHA (lOug/ml) and IL-2 (50U/ml) for an addition three days. Cells were harvested and cell-associated RNA was extracted using TRIzol reagent.

[0077] Quantification of antiviral gene expression: Total RNA from CD4⁺ T-cells was extracted using TRIzol reagent, followed by RNACleanup with in solution DNAasel treatment option using Qiagen RNase-Free DNase Set. DNase-treated clean RNA was transcribed into cDNA using random primers and the Superscript VILO cDNA Synthesis Kit (Invitrogen), according to manufacturer's instructions. Quantitative real-time PCR on blinded samples utilized custom-made TaqMan Low Density Array (TLDA) from Applied Biosystems and gene cards used for quantification as first developed in our laboratory. See Abdel-Mohsen M, et al. Expression profile of host restriction factors in HIV-1 elite controllers. Retrovir ology. 10 (2013) 106. Thermal cycling was performed using an ABI ViiA7 Real-Time PCR System. Complementary DNA (cDNA) in ΙΟΟμΙ of Applied Biosystems TaqMan Universal PCR Master Mix, with UNG was loaded onto the designated ports of the TLDA plates. Data was analyzed using ABI ViiA7 software. A panel of 6 housekeeping genes was included in the TLDA plates (GAPDH, 18S, ACTB, PPIA, RPLP0, and UBC). UBC (ubiquitin) was identified as the most stably expressed gene from those 6 housekeeping genes among the whole samples using the GeNorm algorithm. Raw cycle threshold numbers of amplified gene products were normalized to the housekeeping gene, UBC, to control for cDNA input amounts.

[0078] Quantification of HIV-1 RNA transcripts by real-time qPCR: HIV-1 Gag and mRNA transcripts were detected using the primers and probe described in Shan L, et al. A novel PCR assay for quantification of HIV-1 RNA. J Virol. 87(11) (2013) 6521-6525. CCR5 (Life Technologies, Hs99999149_sl) was used as endogenous control. Real-time PCR was performed in duplicate using TaqMan Universal PCR Master Mix (Applied Biosystems) on an ABI ViiA7 Real-Time PCR machine. Fold induction was determined using the delta delta Ct method. [0079] Statistical analysis: Statistical comparisons were performed using non-parametric two-tailed t-tests in GraphPad Prism. Data are plotted as means ± SEM or SD and P<0.05 were considered significant.

[0080] Study approval: Leukapheresis samples were obtained from HIV-1 -infected individuals with undetectable plasma viremia (<50 copies/ml) on stable ART through protocols approved by the Institutional Review Boards of participating Universities. Anonymous PBMCs from HIV-1 -seronegative individuals were obtained from Blood Banks.

EXAMPLE 2:

Immune Factor Activity During Latent Infection

[0081] To study the dynamics of intrinsic cellular immune factors during latent infection, primary HIV-1 latent cells were generated using an ex vivo model as depicted generally in Fig. 1A using the HIV-1+ molecular clone HIV-1. CD4+ T-cells were isolated from healthy donors, conditioned for three days in the presence of CCL19 and then infected with HIV-1. The initial absence of HIV-1 Gag protein was confirmed by flow cytometry (KC57 antibody). The KC57 monoclonal antibody available from Becton Coulter identifies the 55, 39, 33 and 24 kD proteins of the core antigens of Human Immunodeficiency Virus Type 1 (HIV-1). The 55 kD protein is the precursor protein for the core antigen. The 39 and 33 kD proteins are intermediate products and the 24 kD protein is the mature core protein that makes up the viral capsids. The 24 kD core protein is also known as the HIV gag (group- specific antigen) p24 protein. Fig. IB shows HIV-1 gag staining at day 6 post-infection in resting (CD25-, CD69- and HLA-DR-) latent CD4⁺ T-cells. Latent HIV-1 was then reactivated in resting cells with anti-CD3/CD28 for three days with a resulting dramatic increase in HIV-1 gag staining by flow cytometry as shown in Fig. 1C.

[0082] As sown in Figs. 2A-C, reactivation led to a 10-fold induction in HIV-1 gag (Fig. 2A) and HIV-1 mRNA transcripts (Fig. 2B), including unspliced (US), single spliced (SS) and multiple spliced (MS) mRNA, as measured by real-time qPCR. The expression of selected antiviral genes and host restriction factors was comprehensively analyzed and found that the expression of IFITM1 and SAMHDl were 4-fold and 2-fold increased, respectively, in resting latent cells when compared to reactivated cells (Fig. 2C). IFITM1 expression was over 2-fold increased in the latently infected ACH-2 T-cell line prior to reactivation with the HDACi romidepsin (Fig. 6A). Interestingly, APOBEC3F and APOBEC3G expression was found to be down-regulated in latent cells. [0083] Figs. 6A - G depict the results of reactivation of HIV-1 in ACH-2 cells. In Fig. 6A ACH-2 cells were treated for 5h with increasing concentrations of several histone deacetylase inhibitors (HDACi). The HDACi tested included SAHA (500, 1000 and 5000nM), panobinostat (10, 50, ΙΟΟηΜ) and romidepsin (10, 20, 50nM). SAHA is the acronym for suberanilohydroxamic acid, which is referred to by the generic term Vorinostat and is marketed by Merck under the tradename ZOLINDA®. Vorinostat is FDA approved for treatment of cutaneous T cell lymphoma (CTCL) but was recently shown to have both in vitro and in vivo effects against latently HIV infected T cells by inducing reactivation from latency in chronically infected cell lines and primary cells. See Contreras, X, et al. "Suberoylanilide Hydroxamic Acid Reactivates HIV from Latently Infected Cells" JBC 284 (11) (2009) 6782-6789.

[0084] In our studies, after the initial pulse, cells were washed twice in complete media and cultured for an additional 18h. The frequency of reactivated cells was determined by intracellular HIV-1 Gag staining. Figs. 6B - 6E show the pooled data from three different experiments (n=3) for cells treated with increasing concentrations of SAHA (Fig. 6B), panobinostat (Fig. 6C), and romidepsin (Fig. 6D), with Fig. 6E summarizing the results of Figs. 6B - 6D. Representative flow plots of three independent experiments are shown. Reactivated ACH-2 cells were calculated by subtracting the percentage of HIV-1 Gag+ cells by the percentage of HIV-1 Gag+ cells in untreated control. Data are plotted as mean ±SEM.

[0085] Fig. 6F shows the fold mRNA gene expression of IFITM1 in latent over reactivated cells (n=3). Data are plotted as mean + SD. Quantitative realtime PCR was performed using TaqMan probes and UBC was used identified as the most stably expressed gene across all conditions and was used for normalization of results by the comparative Ct method. Fig. 6G shows the effect of different HDACi on ACH-2 cell viability. The frequency of IFITM1+ CD4⁺ T-cells was further confirmed to be reduced upon HIV-1 reactivation (Figs. 3A - D).

[0086] Because IFITM1 is a transmembrane protein, the capacity of NK cells to recognize and kill IFITM1 -expressing cells by antibody-dependent cell-mediated cytotoxicity (ADCC) was tested. In one model the ACH-2 cell line was used. ACH-2 cells are a T-cell clonal line that is latently infected with HIV-1 and available through the NIH AIDS Reagent Program (Division of AIDS, NIAID, NIH: ACH-2 from Dr. Thomas Folks). ACH-2 cells are CD4-, CD5+, transferrin receptor+, Leu-1+, HIV-1+. ACH-2 cells constantly produce low levels of supernatant reverse transcriptase (RT) and p24. ACH-2 cells can be induced with phorbol myristate acetate or T F-α to secrete high levels of infectious HIV-1.

[0087] Latently infected ACH-2 cells were labeled with an anti-IFITMl antibody or control antibody and cultured with PBMCs from healthy donors at different effector to target (E:T) ratios. Dead cells were monitored by flow cytometry using a live/dead cell marker while the early stages of apoptosis were detected using the FAM Poly-Caspases assay kit (FLICA). A FLICA kit is available from Immunochemistry Technologies LLC and the assay employs the fluorescent inhibitor probe FAM-YVAD-FMK to label active caspase-1 enzyme in living cells or tissue samples and thereby quantitate apoptosis.

[0088] Representative flow plots of LIVE/DEAD Aqua vs. FLICA+ gated on the target cells (ACH-2) are depicted in Fig. 4A. In parallel, we investigated whether the cellular immune- complexes marked with anti-IFITMl antibody and latently infected ACH-2 led to NK cell (defined as CD3- and CD56+ lymphocytes) activation, by detecting the production of intracellular IFN-γ and degranulation (CD107a+). Representative flow plots are depicted in Fig. 4B. Significant killing of ACH-2 cells labeled with anti-IFITMl antibody (79±4.5%) was detected when compared to control (25±1.3%) (Fig. 4C), and this was associated with a significant increase (p=0.0286) in the frequency of IFN-y+ CD107a+ NK cells (1.345±1.3%) (Fig. 4D). When analyzed individually, IFN-γ production (Fig. 4E) and degranulation (Fig. 4F) were also significantly increased against ACH-2 cells labeled with anti-IFITMl antibody (p<0.05). These results indicate that targeting of latently infected ACH-2 cells with anti- IFITMl leads to efficient ADCC activity and killing.

[0089] Because HIV-1 predominantly persists in a reservoir of latently infected central memory CD4⁺ T-cells in patients on suppressive ART, we investigated the frequency of IFITM1+ CD4⁺ T-cells in different cell subsets. The gating strategy is depicted in Fig. 7A- 7H). Fig. 7A shows the side scatter area (SSC-A) plotted against the Forward Scatter area (FSC-A) by flow cytometry with the cells to be analyzed within the drawn area. Fig. 7B shows the Forward Scatter area (FSC-A) plotted against the Forward Scatter height (FSC-H) to identify single cells and exclude doublets with cells outside of the box excluded from analysis.

[0090] PBMC were stained with antibodies against CD3 (Fig. 7C), CD4 (Fig. 7D and F), CD 8 (Fig. 7D), CD45RA (Fig. 7F), CCR7 (Figs. 7E and G), CD27 (Figs. 7E and G), CD95 (Fig. 7H) and IFITM1. Dead cells were excluded using a live/dead marker. Of the cell populations present, naive cells are CD3⁺ CD4⁺ CD45RA⁺ CD27⁺ CCR7⁺ CD95^". Stem central memory (TSCM) cells are also CD3⁺ CD4⁺ CD45RA⁺ CD27⁺ CCR7⁺ CD95^". Central memory (TCM) cells are CD3⁺ CD4⁺ CD45RA^" CD27⁺ CCR7⁺. Transitional memory (TTM) cells are CD3⁺ CD4⁺ CD45RA^" CD27⁺ CCR7^". Effector memory (TEM) cells are CD3⁺ CD4⁺ CD45RA⁺ CD27^" CCR7^". TABLE 1 presents the population identifiers in tabular form.

TABLE 1

[0091] Interestingly, an overall increase of IFITM1⁺ CD4⁺ T-cells was found in patients on ART (2% vs. 0.9%), with a statistical significant increase of IFITM1⁺ CD4⁺ central memory T-cells (p=0.0260) (Fig. 5A).

[0092] To assess whether IFITM1⁺ CD4⁺ T-cells could be killed by ADCC, CD4⁺ T-cells from ART-suppressed patients were sorted, labeled with anti-IFITMl antibody, and cultured with effector cells from the same patient for 5h. IFITM1⁺ T-cells were found almost exclusively in the fraction positive for amine-reactive live/dead marker and FLICA (Fig. 5B), suggesting efficient and specific killing. To determine whether effector cells were able to produce intracellular IFN-γ and degranulate in the presence of anti-IFITMl antibody, we gated on K cells and found that autologous effector cells failed to do so despite efficient killing of target cells when compared to controls (Fig. 5C). The same experiments were then performed with heterologous effector cells from healthy donors, and overall a significant production of IFN-γ and upregulation of CD107a (p=0.0313) was found (Fig. 5C).

[0093] The results presented herein results show that IFITM1 is overexpressed in latently infected CD4+ T-cells and is employable as a marker for viral loads and a target for eliminating latently infected cells using anti-IFITMl antibodies and anti-IFITMl CAR T cells. [0094] All publications, patents and patent applications cited herein are hereby incorporated by reference as if set forth in their entirety herein. While this invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description. It is therefore intended that the appended claims encompass such modifications and enhancements.

Claims

We claim:

1. A method of killing cells harboring latent immunodeficiency virus in an immunodeficiency virus-infected host, the method comprising administering to the host an effective amount of an antibody to IFITMl .

2. The method of claim 1, wherein the antibody to IFITMl is bi-specific and further includes specificity for CD4.

3. The method of claim 1, wherein the antibody to IFITMl is a humanized monoclonal antibody that is optimized for ADCC by directed evolution with selection for increased killing of latently infected CD4+ cells.

4. The method of claim 1, wherein the antibody to IFITMl is a humanized monoclonal antibody that is optimized for promoting complement-dependent cytotoxicity (CDC) of antibody opsinized cells expressing IFITMl .

5. The method of any one of claims 1 - 4, wherein the antibody specific to IFITMl is not cross reactive with IFITM2 or IFITM3.

6. The method of any one of claims 1 - 4, further comprising administering to the host an effective amount of an NK cell stimulator.

7. The method of claim 5, wherein the NK cell stimulator is selected from one or more of type I IFNs (mainly IFN-a and IFN-β), IL-2/anti-IL-2 monoclonal antibody complexes, IL-12, IL-15 and IL-18, and wherein the antibody to IFITMl and the NK cell stimulator synergistically kill cells latently infected with the immunodeficiency virus.

8. The method of claim 6 wherein the NK cell stimulator is an IL-15 superagonist.

9. The method of claim 8, wherein the IL-15 superagonist is an 5 mutant bound to an IL-15 receptor ct/IgGl Fc fusion protein.

10. The method of claim 8, wherein the IL-15 superagonist is selected from ALT-803 and RLI

11. The method of claim 1, wherein the antibody to IFITMl is a chimeric molecule and is conjugated to a therapeutic agent.

12. The method of claim 1 1, wherein the therapeutic agent is a cytotoxic agent selected from the group consisting of paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide.

13. The method of any one of claims 1-4, further comprising co-administering a drug that targets an immune checkpoint (IC) molecule.

14. The method of claim 13, wherein the IC molecule is selected from Programed Cell Death- 1 (PD-1), T-cell Immunoregulator with Ig and ITIM domains (TIGIT), Lymphocyte Activation Gene 3 (LAG-3), and combinations thereof.

15. The method of claim 14, wherein the drug that targets IC molecule PD-1 is selected from one or more of Nivolumab, Pembrolizumab, and Pidilizumab.

16. The method of claim 14, wherein the drug that targets IC molecule LAG-3 is selected from one or more of BMS-986016 and GSK283 1781.

17. The method of claim 14, wherein the drug that targets IC molecule TIGIT is Genentech MTIG7192A.

18. A treatment regimen for reducing a pool of latently infected cells in a patient infected with a latent immunodeficiency virus comprising co-administering an antibody having specificity for IFITM1 and one or more anti -viral agents selected from viral entry inhibitors, nucleotide/side reverse transcription inhibitors (NRTI), non-nucleoside reverses transcriptase inhibitors (NNRTI), integrase inhibitors and protease inhibitors.

19. The treatment regimen of claim 18, further comprising co-administering a drug that targets an immune checkpoint (IC) molecule, wherein the IC molecule is selected from Programed Cell Death- 1 (PD-1), T-cell Immunoregulator with Ig and ITIM domains (TIGIT), Lymphocyte Activation Gene 3 (LAG-3), and combinations thereof.

20. The treatment regimen of any one of claims 18 - 19, further comprising coadministering and effective amount of an NK cell stimulator selected from one or more of type I IFNs (mainly IFN-a and IFN-β), IL-2/anti-IL-2 monoclonal antibody complexes, IL- 12, IL-15 and IL-18, and wherein the antibody to IFITM1 and the NK cell stimulator synergistically kill cells latently infected with the latent immunodeficiency virus.

21. A cocktail of drugs directed to killing cells latently infected with HIV wherein the cocktail comprises an antibody having specificity for IFITMl and an antibody directed to an IC molecule selected from one or more of Programed Cell Death- 1 (PD-1), T-cell Immunoregulator with Ig and ITEVI domains (TIGIT), and Lymphocyte Activation Gene 3 (LAG-3).

22. A chimeric antigen receptor (CAR) modified T cell, wherein the CAR comprises an exodomain including an IFITMl specific single-chain variable region fragment (scFv).

23. The CAR modified T cell of claim 22, wherein the CAR exodomain is bi-specific and further comprises a CD4 specific scFv.

24. The CAR modified T cell of claim 22 or 23, wherein the CAR comprises a signaling endodomain including one or more of signaling and co-stimulatory domains derived from CD2, CD 3, CD 27, CD28, CD30 (aka TNFRSF8), CD40 (aka TNFRSF5), CD! 34 (aka TNFRSF4 and OX40), CD137 (4-iBB), CD278 (ICOS, Inducible T-Cell CoStimulator), and glucocorticoid-induced T FR-r elated protein (GITR; also known as TNFRSF18).

25. A method of killing cells harboring latent immunodeficiency virus in an immunodeficiency virus-infected host, the method comprising administering to the host an effective amount of the CAR modified T cell of either of claim 22 or 23.

26. A method of killing cells harboring latent immunodeficiency virus in an immunodeficiency virus-infected host, the method comprising administering to the host an effective amount of the CAR modified T cell of claim 24.

27. The method of claim 25, further comprising co-administering a drug that targets an immune checkpoint (IC) molecule selected from Programed Cell Death- 1 (PD-1), Programed Cell Death- Ligandl (PD-L1), T-cell Immunoregulator with Ig and ITIM domains (TIGIT), Lymphocyte Activation Gene 3 (LAG-3), and combinations thereof.

28. The method of claim 26, further comprising co-administering a drug that targets an immune checkpoint (IC) molecule selected from Programed Cell Death- 1 (PD-1), Programed Cell Death- Ligandl (PD-L1), T-cell Immunoregulator with Ig and ITIM domains (TIGIT), Lymphocyte Activation Gene 3 (LAG-3), and combinations thereof.

29. The method of claim 25, further comprising expanding the CAR modified T cell ex vivo using IL-15 prior to administering the CAR modified T cell to the immunodeficiency virus-infected host.

28. The method of claim 26, further comprising expanding the CAR modified T cell ex vivo using IL-15 prior to administering the CAR modified T cell to the immunodeficiency virus-infected host.

29. A method for treatment of a human individual infected with Human Immunodeficiency Virus (HIV), the method comprising the steps of:

(a) obtaining a sample of peripheral blood from the individual;

(b) determining an expression level of IFITMl on T cells in the sample;

(c) administering a treatment regimen to the individual;

(d) periodically repeating steps (a) and (b) to determine efficacy of the administered treatment; and

(e) adjusting the treatment regimen if needed to attain a reducing in IFITMl expression of T cells from the individual.

30. The method of claim 29, wherein the treatment regimen comprises administering one or more of: a cocktail of antiviral drugs, anti-IFITMl antibodies, IC antagonists, anti- IFITM1 CAR T cells, and cytokines directed to maximizing NK function or CAR T cell activity.