Patent Application
20080226548
The present invention relates to the treatment of HIV infection using radioimmunotherapy.
Throughout this application various publications are referred to in parenthesis. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
The human immunodeficiency virus (HIV) epidemic is a major threat to health in the developing and western world. HIV induces acquired immune deficiency syndrome (AIDS). An estimated 40 million people world-wide are infected with the virus. After more than 20 years into the epidemic, not a single person has been cured of the infection (Hamer, 2004). Highly active antiretroviral therapy (HAART), a combination of drugs that inhibits enzymes essential for HIV replication, can reduce the viremia and opportunistic infections in most patients, and prolong survival. However, HAART regimens are expensive, complicated and often accompanied by significant toxicity (Carr, 2003). Furthermore, the virus persists in infected cells (Chun et al., 1997), and HIV can rapidly evolve resistance to HAART drugs (Little et al., 2002). It has been argued that latent HIV infection in cellular reservoirs renders the infection intrinsically incurable by antiretroviral therapy alone (Persaud et al., 2003). Hence, HIV infection is often manageable but not curable. To combat this problem, several approaches have been tried (Hamer, 2004), among them the use of immunotoxins that specifically recognize HIV-encoded membrane proteins and thereby potentiate the destruction of infected cells (Bera et al., 1998; Goldstein et al., 2000; Pincus et al., 2003; Saavedra-Lozano et al., 2002, 2004; Till et al., 1987). Although promising, none of these strategies has yet been shown to be effective in humans, and there is theoretical concern for the suitability of those approaches for repeated dosing. Clinical trials of the toxin CD4-Pseudomonas exotoxin (CD4-PE), which targets the HIV envelope glycoprotein gp120, were not successful due to high nonspecific toxicity and lack of therapeutic effect at maximum tolerated doses (Davey et al., 1994; Ramachandran et al., 1994). Thus, there remains a long-felt need for treatment of individuals with HIV infection, especially for new treatment options.
Radioimmunotherapy (RIT) is a therapeutic modality which uses antibody-antigen interaction and antibodies radiolabeled with therapeutic radioisotopes. Radiolabeled antibodies have been used to treat experimental murine cryptococcosis and pneumococcal bacterial infection (Dadachova et al., 2003, 2004a-c; U.S. Patent Application Publication No. U.S. 2004/0115203). However, since HIV (Hernigou et al., 2000), and certain other types of microorganisms (e.g., many fungi, bacterium Deinococcus radiodurans, and yeasts Saccharomyces ellipsoideus and Saccharomyces cerevisiae (Casarett, 1968; Komarova et al., 2002; Mironenko et al., 2000; Sayeg et al., 1959; Schmidt et al., 2002; Shvedenko et al., 2001)), are extremely resistant to gamma radiation, it has not been apparent whether or not HIV would be susceptible to radioimmunotherapy. In addition, antibody-dependent enhancement of HIV infection has been reported (Robinson et al., 1990, 1991). Furthermore, antibodies to C. neoformans radiolabeled with 125-Iodine are known to quickly lose their radiolabel in vivo (Goldman et al., 1997). Accordingly, the likelihood of success of using radioimmunotherapy to treat individuals infected with HIV was not apparent prior to the present disclosure.
The present invention is directed to the combination of immune and radiation therapy for the treatment of human immunodeficiency virus (HIV) infection. Surprisingly, it was found that although radiolabeled antibodies to HIV envelope proteins are not effective at killing HIV particles, such therapy is effective at killing cells that harbor HIV. The present invention, which targets and kills HIV infected cells, is expected to have a major impact on the treatment of acute HIV exposure and elimination of persistent reservoirs of HIV-infected cells, which serve as sites of viral synthesis and latency.
The present invention provides a method for treating a subject infected with HIV which comprises administering to the subject an amount of a radiolabeled antibody effective to kill HIV infected cells, wherein the antibody is specific for a HIV envelope glycoprotein and wherein the radiolabeled antibody specifically binds to cells that are infected with HIV virus and that express the HIV envelope glycoprotein to which the antibody specifically binds.
The invention also provides a pharmaceutical composition formulated in dosage form, comprising a radiolabeled antibody and/or a radiolabeled agent, such as a peptide or an aptamer, and a pharmaceutically acceptable carrier, wherein the antibody and the agent are specific for a HIV envelope glycoprotein and the dosage is appropriate to kill cells infected with HIV in a subject.
The subject invention is directed to a method for treating a subject infected with human immunodeficiency virus (HIV) which comprises administering to the subject an amount of a radiolabeled antibody effective to kill HIV infected cells, wherein the antibody is specific for a HIV envelope antigen (protein or polysaccharide) and wherein the radiolabeled antibody specifically binds to cells that are infected with HIV virus and that express the HIV envelope antigen (protein or polysaccharide) to which the antibody specifically binds.
As used herein, the term “treat” a subject with an HIV infection means to kill cells within the subject that contain HIV, to reduce the number of HIV particles causing the infection in the subject, to prevent the HIV infection from spreading in the subject, to reduce the further spread of HIV infection in the subject, to prevent the establishment of HIV infection in the subject, to treat the HIV infection, to improve symptoms associated with HIV infection, to reduce or prevent opportunistic infection associated with HIV infection, and/or to eliminate the HIV infection. The treatments disclosed herein are also expected to reduce the likelihood of spread of HIV infection to new subjects.
The invention also provides a pharmaceutical composition formulated in dosage form, comprising a radiolabeled antibody and a pharmaceutically acceptable carrier, wherein the antibody is specific for a HIV envelope protein and wherein the dosage is appropriate to kill cells infected with HIV in a subject.
The subject can be any animal that is infected with HIV and is preferably a human.
The invention also provides a method for killing cells infected with human immunodeficiency virus (HIV) which comprises contacting the cells with an amount of a radiolabeled antibody effective to kill HIV infected cells, wherein the antibody is specific for a HIV envelope antigen (protein or polysaccharide) and wherein the radiolabeled antibody specifically binds to cells that are infected with HIV virus and that express the HIV envelope antigen (protein or polysaccharide) to which the antibody specifically binds.
As used herein, the term “antibody” encompasses whole antibodies and fragments of whole antibodies wherein the fragments specifically bind to a HIV envelope protein. Antibody fragments include, but are not limited to, F(ab′)2 and Fab′ fragments and single chain antibodies. F(ab′)2 is an antigen binding fragment of an antibody molecule with deleted crystallizable fragment (Fc) region and preserved binding region. Fab′ is ½ of the F(ab′)2 molecule possessing only ½ of the binding region. The term antibody is further meant to encompass polyclonal antibodies and monoclonal antibodies. The antibody can be, e.g., a neutralizing antibody or a non-neutralizing antibody. Preferably, the antibody is a non-neutralizing antibody, since neutralizing antibodies often bind to highly variable motifs in viral antigens that are vulnerable to antigenic variation.
The antibody can be, e.g., any of an IgA, IgD, IgE, IgG, or IgM antibody. The IgA antibody can be, e.g., an IgA1 or an IgA2 antibody. The IgG antibody can be, e.g., an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4 antibody. A combination of any of these antibodies subtypes can also be used. One consideration in selecting the type of antibody to be used is the desired serum half-life of the antibody. IgG has a serum half-life of 23 days, IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days (Abbas et al., 2000). Another consideration is the size of the antibody. For example, the size of IgG is smaller than that of IgM allowing for greater penetration of IgG into tissues. IgA, IgG, and IgM are preferred antibodies.
The antibody can be specific for any HIV envelope protein, e.g. glycoprotein gp120, gp41 or gp160. Glycoprotein gp160 is a precursor polypeptide, which when cleaved forms gp120 and gp41 (e.g., Kibler et al., 2004). The antibody can target protein or polysaccharide epitopes. Combinations of different antibodies can be used, where each different antibody binds to a different epitope. The HIV can be any subtype of HIV, e.g. HIV type 1 or HIV type 2. HIV type 1 induces AIDS. HIV type 2 also leads to immune suppression; however, HIV-2 is not as virulent as HIV-1. Numerous antibodies that bind to a HIV envelope protein have been described (e.g., Gorny and Zolla-Pazner, 2000; Nádas et al., 2004; Nyambi et al., 2000; Pincus et al., 2003; Till et al., 1989; Xu et al., 1991; Zolla-Pazner, 2004; U.S. Pat. Nos. 5,731,189, 6,241,986 and 6,395,275).
The antibody is preferably a human antibody. However, the antibody can be a non-human antibody such as a goat antibody or a mouse antibody. Non-human antibodies can be used in subjects infected with HIV due to the immune system suppression that occurs in HIV infected subjects. In fact, the molecule carrying the radioactive isotope need not be immunoglobulin since all that is required is a molecule with specificity for binding to a viral antigen expressed on a virally infected cell. Although such molecules are usually proteins, there is no exclusionary requirement for this type of compound and it is conceivable that polysaccharides, lipids, and even small synthetic molecules can be designed to deliver targeted cytotoxic radiation.
Antibodies can be “humanized” using standard recombinant DNA techniques. By transferring the mouse antibody binding site coding region into a human antibody gene, a “human antibody” can be engineered which retains the specificity and biological effects of the original mouse antibody but has the potential to be nonimmunogenic in humans. Additionally, antibody effector functions can be improved through manipulation of the antibody constant region genes (e.g., Clark, 2000; Jolliffe, 1993; LoBuglio et al., 1989). Humanized monoclonal antibodies to gp120 have been described (Dezube et al., 2004; Major et al., 1994). An anti-gp120 humanized monoclonal antibody has been shown to be well tolerated in human subjects in a phase I study (Dezube et al., 2004).
The invention can also be practiced using a radiolabeled agent effective to kill HIV infected cells, wherein the agent is specific for a HIV envelope antigen and wherein the radiolabeled agent specifically binds to cells that are infected with HIV virus and that express the HIV envelope antigen to which the agent specifically binds. The chemical composition of the antigen can be, e.g., protein or polysaccharide. Examples of agents that bind to HIV envelope antigens include peptides and aptamers. The agent can be, e.g., a neutralizing agent or a non-neutralizing agent. Preferably, the agent is a non-neutralizing agent.
Examples of HIV envelope glycoprotein-binding peptides include Fuzeon® and retrocyclin-1. Fuzeon® (also known as T-20 or enfuvirtide) is a C-peptide derived from the gp41 C-terminal heptad repeat (CHR) region and is the first member of a new class of anti-HIV drugs known as HIV fusion inhibitors. T-20 may inhibit HIV-1 entry by targeting multiple sites in gp41 and gp120 (Liu et al., 2005). Retrocyclin-1 is a theta-defensin peptide which binds to gp120 (Owen et al., 2004). Neutralizing (Khati et al., 2003) and non-neutralizing (Sayer et al., 2002) aptamers that bind to gp120 have been described. A neutralizing antibody or agent is one that reacts with a HIV envelope protein and destroys or inhibits the infectivity and/or virulence of the HIV virus. Methods for generating peptides (Valadon et al., 1996) and aptamers (U.S. Pat. No. 5,756,291) have been described.
The antibody or agent could also target an antigen that is expressed in HIV-infected cells, but not in non-HIV-infected cells, where the antigen may have viral, mammalian, or combined origin.
The invention also provides a pharmaceutical composition formulated in dosage form, comprising a radiolabeled agent and a pharmaceutically acceptable carrier, wherein the agent is specific for a HIV envelope antigen and wherein the dosage is appropriate to kill cells infected with HIV in a subject.
Apart from cost and availability, two characteristics are important in the choice of a radioisotope—emission range in the tissue and half-life. Preferably, the antibody or agent is radiolabeled with an alpha emitter or a beta emitter. Alpha emitters have a short emission range in comparison to beta emitters. Examples of alpha emitters include 213-Bismuth (half-life 46 minutes), 223-Radium (half-life 11.3 days), 224-Radium (half-life 3.7 days), 225-Radium (half-life 14.8 days), 225-Actinium (half-life 10 days), 212-Lead (half-life 10.6 hours), 212-Bismuth (half-life 60 minutes), 211-Astatine (half-life 7.2 hours), and 255-Fermium (half-life 20 hours). A preferred alpha emitter is 213Bi, which emits a high LET α-particle with E=5.9 MeV with a path length in tissue of 50-80 μm. Theoretically a cell can be killed with one or two α-particle hits. 213Bi is the only α-emitter that is currently available in generator form, which allows transportation of this isotope from the source to clinical centers within the United States and abroad.
Examples of beta emitters include 188-Rhenium (half-life 16.7 hours), 32-Phosphorous (half-life 14.3 days), 47-Scandium (half-life 3.4 days), 67-Copper (half-life 62 hours), 64-Copper (half-life 13 hours), 77-Arsenic (half-life 38.8 hours), 89-Strontium (half-life 51 days), 105-Rhodium (half-life 35 hours), 109-Palladium (half-life 13 hours), 111-Silver (half-life 7.5 days), 131-Iodine (half-life 8 days), 177-Lutetium (half-life 6.7 days), 153-Samarium (half-life 46.7 hours), 159-Gadolinium (half-life 18.6 hours), 186-Rhenium (half-life 3.7 days), 166-Holmium (half-life 26.8 hours), 166-Dysprosium (half-life 81.6 hours), 140-Lantanum (half-life 40.3 hours), 194-Irridium (half-life 19 hours), 198-Gold (half-life 2.7 days), 199-Gold (half-life 3.1 days), 90-Yttrium (half-life 2.7 days), 177-Lutetium (half-life 6.7 days) and 131-Iodine (half-life 8 days). Preferred beta emitters include 131-Iodine, 90-Yttrium, 188-Rhenium, 186-Rhenium, 177-Lutetium, 166-Holmium, 67-Copper, and 64-Copper, with the high-energy β-emitter 188-Rhenium (Emax=2.12 MeV) being most preferred. 188Re has the additional advantage that it emits γ-rays which can be used for imaging studies.
The radioisotope can be attached to the antibody or agent using any known means of attachment used in the art, including interactions such as avidin-biotin interactions, “direct” radiolabeling (Dadachova and Mirzadeh, 1997) and radiolabeling through a bifunctional chelating agent (Saha, 1997). Preferably, the radioisotope is attached to the antibody or agent before the radioisotope or the antibody or agent is administered to the subject.
The invention also includes the use of a combination of antibodies and/or agents radiolabeled with different radiolabels. Preferably, the radioisotopes are isotopes of a plurality of different elements. In a preferred embodiment, at least one radioisotope in the plurality of different radioisotopes is a long range (beta) emitter and at least one radioisotope is a short range (alpha) emitter. Preferably, the beta emitter is 188-Rhenium. Preferably, the alpha emitter is 213-Bismuth.
It is known from radioimmunotherapy studies of tumors that whole antibodies usually require from 1 to 3 days time in circulation to achieve maximum targeting. While slow targeting may not impose a problem for radioisotopes with relatively long half-lives such as 188Re (t1/2=16.7 hours), faster delivery vehicles may be preferred for short-lived radioisotopes such as 213Bi (t1/2=46 min). The smaller F(ab′)2 and Fab′ fragments or domain-deleted antibodies provide much faster targeting which matches the half-lives of short-lived radionuclides (Milenic, 2000; Buchsbaum, 2000). A ‘domain-deleted’ antibody is an anitbody from which a particular domain, e.g. CH2, has been deleted and replaced with a peptide linker for the purpose of optimizing its therapeutic potential (Milenic, 2000).
In order to calculate the dose of the radioisotope which can significantly decrease or eliminate infection burden without radiotoxicity to vital organs, a diagnostic scan of the patient with the antibody or agent radiolabeled with diagnostic radioisotope or with low activity therapeutic radioisotope can be performed prior to therapy, as is customary in nuclear medicine. The dosimetry calculations can be performed using the data from the diagnostic scan (Early and Sodee, 1995).
Clinical data (Sgouros et al., 1999; Paganelli et al., 1999) indicate that fractionated doses of radiolabeled antibodies are more effective than single doses against tumors and are less radiotoxic to normal organs. Depending on the status of a patient and the effectiveness of the first treatment with RIT, the treatment may consist of one dose or several subsequent fractionated doses.
The dose of the radioisotope for humans will typically be between about 1-500 mCi.
The radiolabeled antibody or agent can be delivered to the subject by a variety of means. Preferably, the radiolabeled antibody or agent is administered parenterally. The radiolabeled antibody or agent can be injected, for example, into the bloodstream, into a muscle or into an organ such as the spleen.
The HIV-infected cell that is targeted and killed by the radiolabeled antibody or agent can be any of, e.g., but not limited to, a lymphocyte, such as a T lymphocyte or a CD4+ T lymphocyte, a monocyte, a macrophage, an astrocyte and/or a microglial cell.
Despite the effectiveness of the radiolabeled antibodies in killing cells infected with HIV, the radiolabeled antibody does not kill more than 50% of free HIV virus particles in vitro in a solution containing free HIV particles. Typically, no killing of free viral particles can be detected under in vitro conditions.
The invention also provides a method of making a composition effective to treat a subject infected with HIV which comprises admixing a radiolabeled antibody or agent and a carrier, wherein the antibody or agent specifically binds to a HIV envelope protein and is effective to kill HIV-infected cells.
As used herein, the term “carrier” encompasses any of the standard pharmaceutical carriers, such as a sterile isotonic saline, phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsions.
The invention further provides for the use of a radiolabeled antibody or agent for the preparation of a composition for treating a subject infected with human immunodeficiency virus (HIV), wherein the antibody or agent is specific for a HIV envelope protein and wherein the radiolabeled antibody or agent specifically binds to cells that are infected with HIV virus and that express the HIV envelope protein to which the antibody or agent specifically binds.
The methods of treatment described herein can be used in combination with other therapies against HIV (e.g., Hamer, 2004). For example, agents that induce transcription of latent provirus can be used to express viral proteins in latently infected resting CD4 T cells. HAART therapy can be used to prevent the spread of infection by virus released from cells killed by radioimmunotherapy.
This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.
Antibodies. Goat polyclonal antibody (Ab) against gp-120 (IgG1) was purchased from Biodesign International (Saco, Me.). Murine 18B7 monoclonal antibody (mAb) (IgG1) specific for cryptococcal polysaccharide (Casadevall et al., 1998) was used as an isotype-matching control. Human anti-gp41 (cluster I) mAb 246D was obtained from Dr. Susan Zolla-Pazner, New York University School of Medicine. The 246 D antibody was produced as described in Nyambi et al. (2000a). The 246 D antibody was previously described in publications by Dr. Zolla-Pazner and her colleagues (Gorny and Zolla-Pazner, 2000; Nyambi et al., 2000a; Robinson et al., 1991; Xu et al., 1991; U.S. Pat. No. 5,731,189). As described in U.S. Pat. No. 5,731,189, lymphoblastoid cell line 126-6 producing human monoclonal antibodies directed against gp41 was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 21110-2209) on Feb. 24, 1989 and received ATCC Accession number CRL 10037. Human mAb 1418 (IgG1) to parvovirus B19 (Gigler et al., 1999) was used as an irrelevant control for mAb 246D, and human mAb 447 (IgG3) to the V3 loop of HIV-1 gp120 (Conley et al., 1994) was used as a positive control in the FACS studies. Prior to use the antibodies were purified by affinity chromatography.
Radioisotopes and quantification of radioactivity. 188Re in the form of Na perrhenate (Na188ReO4) was eluted from a 188W/188Re generator (Oak Ridge National Laboratory (ORNL), Oak Ridge, Tenn.). Actinium-225 (225Ac) for construction of a 225Ac/213Bi generator was acquired from the Institute for Transuranium Elements, Karlsruhe, Germany. The 225Ac/213Bi generator was constructed using MP-50 cation exchange resin, and 213Bi was eluted with 0.15 M HI (hydroiodic acid) in the form of 213BiI52− as described in Boll et al. (1997). A gamma counter (Wallac) with an open window was used to count the 188Re and 213Bi samples.
Radiolabeling of antibodies with 188Re and 213Bi. Antibodies were radiolabeled with beta-emitter 188Re (half-life 17.0 h) or alpha-emitter 213Bi (half-life 45.6 min). Abs were labeled “directly” with 188Re via reduction of antibody disulfide bonds by incubating the antibody with 75-fold molar excess of dithiothreitol (Dadachova and Mirzadeh, 1997) for 40 min at 37° C. followed by centrifugal purification on Centricon-30 or -50 microconcentrators with 0.15 M NH4OAc, pH 6.5. Simultaneously 3-10 mCi (110-370 MBq) 188ReO4− in saline was reduced with SnCl2 by incubation in the presence of Na gluconate, combined with purified reduced antibodies and kept at 37° C. for 60 min. Radioactivity not bound to the antibody was removed by centrifugal purification on Centricon microconcentrators.
For radiolabeling with 213Bi, Abs were conjugated to bifunctional chelator N-[2-amino-3-(p-isothiocyanatophenyl)propyl]-trans-cyclohexane-1,2-diamine-N, N′, N″, N′″, N″″-pentaacetic acid (CHXA”) as in Boll et al., 1997, Chappell et al., 2000, Dadachova et al., 1997, and Mirzadeh et al., 1990. The average final number of chelates per antibody molecule was determined by the Yttrium-Arsenazo III spectrophotometric method (Pippin et al., 1992). CHXA”-conjugated Abs were radiolabeled with 213Bi by incubating them for 5 min with 213BiI52− at room temperature. If required, the radiolabeled antibodies were purified by size exclusion HPLC (TSK-Gel® G3000SW, TosoHaas, Japan).
In vitro killing of ACH-2 cells. An ACH-2 cell line, a latent T-cell clone infected with HIV-IIIB that produces steady low levels of supernatant RT and p24, was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: ACH-2, catalogue #349 from Dr. Thomas Folks. HIV-1 chronically infected human T-cells ACH-2 (phytohaemagglutinin (PHA)-stimulated, phorbol myristate (PMA)-stimulated, and non-stimulated) were treated with 0-50 μCi of 188Re-labeled Abs, 0-20 μCi 213Bi-labeled Abs or with matching amounts (2.5-12.5 μg) of “cold” Abs. Approximately 2×105 cells per sample were used. The cells were incubated with radiolabeled or “cold” Abs at 37° C. for 3 h, transferred into fresh cell culture medium and then incubated in 5% CO2 at 37° C. for 72 h. The number of viable cells 72 h post-treatment was assessed using blue dye exclusion assay.
Treatment of HIV1-infected and non-infected peripheral blood mononuclear cells (PBMCs) with radiolabeled mAbs. Human Peripheral Blood Mononuclear Cells (PBMCs) obtained from the New York Blood Center (New York, N.Y.) were stimulated with PHA and interleukin-2 (IL-2) 48 h prior to infection with HIV-1 strain JR-CSF (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1JR-CSF, catalogue #394 from Dr. Irvin S. Y. Chen). While the number of ACH-2 cells infected with HIV-1 was almost 100%, only a fraction (˜10-30%) of the PBMCs were infected with HIV-1 as determined by limiting dilution co-culture technique (Ho et al., 1989). Cells exposed to HIV-1 are referred to as “infected” cells and those which were not exposed to the virus are referred to as “non-infected” cells. At 48 h after infection with HIV-1, infected PBMCs were treated with 0-20 μCi 213Bi-labeled Abs or with matching amounts (2.5-12.5 μg) of “cold” Abs. Approximately 2×105 cells per sample were used. As controls, non-infected PBMCs were treated with 213Bi-anti-gp41 mAb. The cells were incubated with radiolabeled or “cold” Abs at 37° C. for 3 h, transferred into fresh cell culture medium and then incubated in 5% CO2 at 37° C. for 72 h. The number of viable cells 72 h post-treatment was assessed using blue dye exclusion assay.
Flow cytometric analysis of mAbs binding to virus infected cells. Binding studies of human mAbs to the surface of hPBMCs infected with the JR-CSF strain of HIV-1 were performed as described previously (Zolla-Pazner et al., 1995). Briefly, PHA-stimulated hPBMCs were infected with 1 ml of stock HIV-1JR-CSF virus and cultured for 13 days in medium supplemented with human recombinant IL-2 (20 U/ml, Boehringer Mannheim Biochemicals, Indianopolis, Ind.). The cells were incubated with each human mAb at 10 μg/ml for 1 h on ice, washed and reincubated with PE-labeled goat F(ab′)2 anti-human IgG(γ) (Caltag Laboratories, Burlingame, Calif.). Using a FACScan flow cytometer (Becton Dickinson), live lymphocytes were selected for analysis by gating with forward and 90° scatter. The negative control consisted of cells from infected cultures stained with the conjugated anti-IgG in the absence of a human mAb.
Treatment of HIV infected PBMCs pre-incubated with HIV positive blood. Human PBMCs were grown and infected with JR-CSF strain of HIV1 as described above. 2×105 infected PBMCs were incubated for 1 h at 37° C. with 200 μL of undiluted serum from a HIV1-positive patient, or with the same volume of 1:10 or 1:100 diluted HIV1-positive serum using HIV-negative serum as a diluent, or with HIV-negative serum only. Following the incubation the cells were washed with PBS, 1 mL PBS per sample was added and the cells were treated with 20 μCi 213Bi-anti-gp41 mAb (˜12.5 μg) or left untreated. The cells were incubated with radiolabeled mAb at 37° C. for 3 h, transferred into fresh cell culture medium and then incubated in 5% CO2 at 37° C. for 72 h. The number of viable cells 72 h post-treatment was assessed using blue dye exclusion assay.
Treatment of naked HIV1 virus with radiolabeled anti-gp41 mAb. Viral particles were incubated with mAbs for 3 h, followed by infection of healthy PBMCs. On Day 6 post-infection the cultures were analyzed for the presence of HIV core protein p24 by core Profile ELISA (DuPont-NEN).
Determination of splenic uptake of radiolabeled mAbs. Two groups of SCID mice were used in this experiment. One group was injected intrasplenically with HIV-1 infected PBMCs and the second group was injected with non-infected PBMCs (25 million cells per mouse). One hour later 20 μCi (20 μg) 188Re-246-D mAb was given IP to each mouse. Three hours post-injection the animals were sacrificed, their spleens removed, blotted from blood, weighed, counted in a gamma counter, and the percentage of injected dose per gram (ID/g) was calculated.
Determination of platelet counts in mice treated with radiolabeled mAbs. Platelet counts were used as a marker of RIT toxicity in treated animals. For measurement of platelet counts, the blood of SCID mice injected intrasplenically with HIV-1-infected hPBMCs and either treated with 100 μCi (20 μg) 213Bi-246-D or 160 μCi (20 μg) 188Re-246-D IP 1 hour after infection with PBMCs or untreated was collected from the tail vein into 200 μL 1% ammonium oxalate on day 0, 4, 8 and 15 days post-therapy. Platelets were counted in a hemocytometer, using phase contrast, at 400 times magnification, as described in Miale (1982).
Treatment of HIV1-infected mice with radiolabeled mAbs. Human PBMCs were stimulated with PHA and IL-2 48 h prior to infection with HIV-1 strain JR-CSF. At 48 h after infection with HIV-1, infected PBMCs were injected intrasplenically (25 million cell per animal) into groups of SCID mice (10 mice per group). Mice received either 20 μg “cold” anti-gp41 mAb 246D, 100 μCi (20 μg) 213Bi-1418 or 80 μCi (20 μg) 188Re-1418 as isotype-matching controls, 80 μCi (20 μg) 188Re-246D, or 100 μCi (20 μg) 213Bi-246D IP 1 hour after infection with PBMCs. In some experiments mice were given 80 μCi (20 μg) 188Re-246D IP 1 h prior to infection with PBMCs. The SCID mice were sacrificed 72 hours after treatment and the spleens were harvested and processed. A limiting dilution co-culture of the splenocytes was performed using freshly activated PBMCs as described in Wang et al. (2002). Supernatants were harvested on day 8 after initiation of co-culture and analyzed for the presence of HIV-1 core protein p24 by core Profile ELISA (DuPont-NEN). Data are reported as infected splenocytes/106 splenocytes. The number of HIV-1-infected cells present in the spleen was measured using limiting dilution quantitative co-culture as described by Ho et al. (1989). This technique measures the number of cells capable of producing infectious HIV-1. Five-fold dilutions of cells isolated from each spleen (in the range 1×106−3.2×102 cells) were cultured in duplicate at 37° C. in 24-well culture plates with PHA-activated hPBMCs (1×106 cells) in 2.0 mL of RPMI 1640 medium containing fetal calf serum (10% vol/vol) and interleukin-2 (32 U/mL). The HIV-1 p24 antigen content of the supernatant was measured 1 week later, using the HIV-1 p24 core profile ELISA (DuPont-NEN). The lowest number of added cells that infected at least half the duplicate cultures with HIV-1 was determined and represented the frequency of cells productively infected with HIV-1 in each spleen, reported as TCID50/106 splenocytes. In dose response experiment the groups of infected mice were given 40, 80 or 160 μCi (20 μg) 188Re-246-D IP, 20 μg “cold” mAb 246-D or left untreated and the efficacy of the therapy was assessed.
Statistical analysis. Prism software (GraphPad, San Diego, Calif.) was used for statistical analysis of the data. Student's t-test for unpaired data was employed to analyze differences in the number of viable ACH-2 cells, PBMCs or infected splenocytes/106 splenocytes between differently treated groups during in vitro and in vivo therapy studies, respectively. Differences were considered statistically significant when P values were <0.05.
In vitro killing of HIV-infected ACH-2 cells with radiolabeled mAbs. To determine the capacity of RIT to kill HIV-1 infected cells, goat polyclonal anti-gp120 Ab was labeled with radioisotopes with distinctly different emission characteristics—213-Bismuth (213Bi, a radioisotope that emits alpha-particles which are He atoms with the charge of +2 and mass of 4) and 188-Rhenium (188Re, a radioisotope that emits high energy beta-particles (electrons)). 213Bi and 188Re have different emission ranges in tissue—50-80 μm for 213Bi versus 10 mm (average) for 188Re. Both radioisotopes have been used in pre-clinical and clinical settings.
HIV-1-infected ACH-2 cells were incubated with 188Re-anti-gp120 Ab, 188Re-control Ab (irrelevant murine mAb 18B7) or “cold” anti-gp120 Ab. Significant killing of HIV-infected ACH-2 cells was observed with 188Re-anti-gp120 Ab (
Since the relative biological effectiveness (RBE) of α-particles is significantly higher than that of β particles by virtue of their mass, charge and energy (Casarett, 1968, Wheldon 1994), the study was repeated using 2.5 times lower radioactivity in the incubation of ACH-2 cells with 213Bi-labeled antibodies than in 188Re experiments. Five μCi 213Bi-anti-gp120 per 2×105 ACH-2 cells eradicated virtually 100% of the cells (
In vitro killing of HIV-1 infected human PBMCs with radiolabeled mAb. Human anti-gp41 mAb 246-D was used for these studies. This mAb binds specifically to HIV-1-infected cells as demonstrated by flow cytometry of hPBMCs infected with HIV-1JR-CSF (
Sparing of PBMCs not infected with HIV-1 following treatment with radiolabeled mAbs. As shown in
Killing of HIV infected PBMCs pre-incubated with HIV positive blood. Human PBMCs infected with HIV were incubated with serum from a HIV1-positive patient before exposure of the PBMCs to 213Bi-anti-gp41 mAb. 100% killing of the PBMCs was observed following treatment with 213Bi-anti-gp41 mAb (results not shown). Thus, antibodies to gp41 glycoprotein in HIV1-positive serum, which can potentially compete with radiolabeled anti-gp41 mAb, were not able to block the binding of 213Bi-anti-gp41 mAb to the PBMCs which resulted in their killing.
Sparing of naked HIV-1 virus following treatment with radiolabeled anti-gp41 mAb. Table 1 compares the effects of treating HIV-1 viral particles with radiolabeled (“hot”) anti-gp41 Ab, cold anti-gp41, and hot irrelevant mAb. Radiolabeled Abs to the gp41 HIV envelope protein were not effective in killing HIV viral particles.
Elimination of HIV-1 infected PBMCs in mice by RIT. Human anti-gp41 mAb 246D was used for in vivo experiments. Targeting gp41 has the advantage that this protein is reliably expressed on the surface of chronically infected cells. In addition to the advantages of using human mAb relative to goat polyclonal sera with regards to specific activity and specificity, published data indicate that immunotoxins are more efficient against HIV-infected cells when delivered to the cells by anti-gp41 mAbs rather than anti-gp120 mAbs (Pincus et al., 2003). In the present mouse model, HIV-infected cells are residing in the spleen, which is one of the significant reservoirs of HIV-harboring cells in humans, and thus this model has advantages over more artificial lymphoma tumor-type models (Pincus et al., 2003).
Human PBMCs infected with HIV-1JR-CSF were injected into the spleens of SCID mice and the mice were treated as indicated. Doses of 80 μCi dose 188Re-labeled 246-D and 100 μCi 213Bi-246-D were chosen because these doses were therapeutic and safe in experimental RIT of fungal and bacterial infections (Dadachova et al., 2004a,b). The mice were evaluated 72 hours later for the presence of residual HIV-1-infected cells by quantitative co-culture (Conley et al., 1994). The 72 hour time period was chosen to give sufficient time for 188Re-labeled mAb to deliver a lethal dose of radioactivity to the cells as the 188Re half-life is 16.9 hr and several half-lives are required for a given radionuclide to deliver the dose to the target.
The results of RIT of SCID mice infected intrasplenically with JR-CSF-harboring human PBMCs are presented in
188Re-246-D was more effective in Vivo than 213Bi-246-D due to the longer physical half-life of 188Re (16.9 hours versus 46 minutes) allowing the labeled mAb to reach infected cells while still carrying high activity “payload”. To investigate the dose-response effect, the mice were treated with 40, 80 and 160 μCi 188Re-246-D, corresponding to 50, 100 and 200% of the therapeutic dose, respectively. While 40 μCi 188Re-246-D was not effective in killing infected PBMCs in vivo, 160 μCi dose essentially eliminated infected cells (
To further investigate the specificity of radiolabeled mAb binding to gp41 HIV-infected hPBMCs, the splenic uptake of 188Re-246-D mAb was compared in mice injected intrasplenically with hPBMCs and HIV-1 infected hPBMCs. The uptake expressed as percentage of injected dose (ID) per gram of spleen was 8±4 and 57±10% ID/g (P<0.001) for non-infected and infected PBMCs, respectively. This result establishes in vivo targeting of 188Re-246-D to HIV-1-infected cells.
Lack of hematological toxicity of RIT of HIV infection. The hematological toxicity of radiolabeled 246-D mAb during HIV-1 infection was evaluated in the SCID mice by platelet counts. The platelet count nadir usually occurs 1 week after radiolabeled antibody administration to tumor-bearing animals (Behr et al., 1999; Sharkey et al., 1997). No changes were observed in platelet counts in mice treated with 100 μCi 213Bi-246D mAb on days 4, 8 and 15 post-treatment in comparison to non-treated infected controls, with platelet counts being stable at (1.5±0.2)×109 platelet/mL blood (data not shown). For mice given 160 μCi 188Re-246-D (the highest dose used in this study), a slight drop in platelet count was noted on day 7 post-treatment with counts returning to normal by day 15 (
The present application discloses the efficacy of radioimmunotherapy (RIT) in treating HIV infection using radiolabeled antibodies directed to HIV envelope proteins. The β-emitter 188-Rhenium (188Re) and α-particle emitter 213-bismuth (213Bi) were used herein as examples of therapeutic radionuclides for RIT of HIV infection. 188Re (T1/2=16.7 h) is a high-energy β-emitter (Emax=2.12 MeV) and has the additional advantage that it emits γ-rays which can be used for imaging studies. 213Bi (T1/2=45.6 min) emits a high linear energy transfer (LET) α-particle with E=5.9 MeV with a path length in tissue of 50-80 μm. Theoretically a cell can be killed with one or two α-particle hits.
The results disclosed herein demonstrate that RIT is effective against cells harboring HIV both in vivo and in vitro, but not against naked HIV particles as tested in vitro. In contrast, RIT is efficient against fungal and bacterial pathogens (Dadachova et al., 2003, 2004a-c; U.S. Patent Application Publication No. U.S. Ser. No. 2004/0115203) despite the fact that the track range in tissue of radiation emitted by 213Bi and especially by 188Re is much longer than a fungal or bacterial cell diameter. The apparent inability of RIT to kill naked HIV virus is probably a combination of the extremely small size of viral particles (nanometer range) and their extreme radioresistance.
RIT has several advantages over an immunotoxin approach for treatment of HIV infection. First, the antibody used for delivery of radiation does not need to be internalized to deliver its toxic payload to the cell, since radiation emitted by radioisotopes is cytotoxic without the need for internalization. Second, not every infected cell in the body needs to be targeted by the antibody as particulate radiation kills neighboring cells via “cross-fire” effect (i.e., radiation emanating from a radiolabeled cell hits adjacent cells). Consistent with this mechanism 188Re-labeled mAbs were more effective in vivo (
One of the advantages of using RIT against infections as opposed to cancer is that, in contrast to tumor cells, cells expressing microbial antigens are antigenically very different from host tissues and thus provide the potential for exquisite specificity and low cross-reactivity. A large therapeutic window is available because the therapeutic effect disclosed herein was achieved with activities that were significantly lower than the reported maximum tolerated activity (MTA) for 188Re (800 μCi for IV injection; Sharkey et al., 1997) and 213Bi-labeled IgGs (in excess of 1,000 μCi when given IP; Milenic et al, 2004).
In the clinic, RIT may be most effective when used in combination with highly active antiretroviral therapy (HAART) (Berger et al., 1998), which blocks virus replication in newly infected cells. An exciting use of RIT combined with HAART would be to prevent HIV infection when administered to individuals within the first days of exposure to HIV. In addition, initial treatment of patients soon after infection may reduce the number of HIV-1-infected cells and thereby reduce viral set-point. Moreover, RIT may be a useful adjunct for protocols designed to “flush out” quiescent, latently infected lymphocytes by the administration of factors that promote HIV replication such as valproic acid (Lehrman et al., 2005). The availability of RIT is envisioned to provide a novel treatment for the eradication of HIV-1 infection.
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This application claims the benefit of U.S. Provisional Application No. 60/659,582, filed Mar. 7, 2005, the content of which is hereby incorporated by reference in its entirety into the subject application.
The invention disclosed herein was made with U.S. Government (National Institutes of Health) support under Albert Einstein College of Medicine (AECOM) Center for AIDS Research grant number 9526-9814 and by grant numbers AI60507, AI033142, A1033774 and HL059842. Accordingly, the U.S. Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/07961 | 3/6/2006 | WO | 00 | 4/29/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60659582 | Mar 2005 | US |
