CN101065144B - Methods and compositions for treatment of hematologic cancers - Google Patents

Abstract

The present invention relates to oncolytic picornaviruses, and methods and compositions for treating subjects with hematological cancers. It includes the use of the disclosed picornaviruses, such as Coxsackieviruses, methods and compositions for treating myeloma, administered directly or indirectly to subjects, and ex vivo in autologous grafts prior to transplantation Purify malignant cells.

Description

Methods and compositions for treating hematological cancers

technical field

The present invention relates to oncolytic picornaviruses and methods and compositions for treating subjects with hematological cancers.

Background technique

Blood cancers are cancers of the "blood system". These cancers usually affect white blood cells (disease- and infection-fighting cells) rather than red blood cells (oxygen-carrying cells). Some of these cancers develop in the bone marrow, where all blood cells are made. Some occur in lymph nodes and other lymphoid tissues through which white blood cells flow. Common cancers of white blood cells are leukemia, Hodgkin lymphoma, other lymphomas, and multiple myeloma.

Multiple myeloma (MM) is a B-cell malignancy that occurs in approximately 1% of all types of human cancer and is more common than myeloid leukemia or Hodgkin's disease.

Multiple myeloma can cause anemia, severe bone pain, and in some cases pathological fractures, increased chances of infection, hypercalcemia, and renal failure. Although chemotherapy is the preferred initial treatment for symptomatic MM, the disease is highly resistant to chemotherapeutic agents and most patients who initially respond to this treatment eventually relapse.

A new approach to controlling cancer progression is through the use of oncolytic viruses. Oncolytic viruses are viruses that selectively destroy or "lyse" malignant cells while leaving normal host cells intact. Many human solid tumors are susceptible to the oncolytic activity of a large number of viruses, each with unique biological properties to mediate selective oncolysis. Reovirus (Alain T et al., Blood.2002; 100:4146-4153; Thirukkumaran CM et al., Blood.2003; 102:377-387), measles virus (Grote D et al., Blood.2001; 97: 3746-3754), and Newcastle disease virus (Schirrmacher V et al., Int J Oncol. 2001; 18: 945-952) are three of the oncolytic viruses known to be effective against some malignancies.

A common cold virus, Coxsackievirus A21 (CVA21), was recently shown to be effective against human melanoma xenografts in an immunodeficient mouse model (Shafren DR et al., Clin Cancer Res. 2004; 10:53-60 ). CVA21 is an enterovirus known to selectively utilize the cell surface molecules intercellular adhesion molecule 1 (ICAM-1) and decay accelerating factor (DAF) to mediate cell infection. Although CVA21 can bind DAF, ICAM-1 is a key receptor for CVA21 cell infection—without ICAM-1 expression on the cell surface, CVA21 cannot gain cellular entry and infect host cells under normal conditions.

The applicant has previously developed a novel method of treating solid malignancies using an oncolytic virus that recognizes ICAM-1 (PCT/AU00/01461 (WO01/37866), entitled: "Methods of treating malignancies in subjects and methods for their use" Pharmaceutical composition (A Method of Treating a Malignancy in a Subject and a Pharmaceutical Composition For Use in Same)"). Excellent therapeutic results have been obtained using various strain A coxsackievirus strains on a large number of solid tumor cell types. The applicants have also developed new methods of treating abnormal mammalian cells (such as cancer cells) using viruses that recognize integrin _α2β1 for cell infection, such as echovirus (PCT/ _AU2003 /001688 (WO2004/ 054613), entitled "A method of treating a malignancy in a subject via direct Picornaviral-mediated oncolysis"). In order to expand the possible cancer treatments and even provide more effective treatments, the present inventors have surprisingly found that picornavirus isolates may also be suitable as oncolytic agents for hematological cancers.

Contents of the invention

In a first aspect, the present invention provides a method of treating and/or preventing hematological cancer in a subject, the method comprising administering a therapeutically effective amount of a picornavirus or a modified form thereof such that at least some cancer cells undergo viral oncolysis.

In a second aspect, the present invention provides a method of treating and/or preventing a blood cancer in a subject selected from the group consisting of multiple myeloma, B cell lymphoma, B prolymphocytic leukemia and monocytic leukemia, the The method comprises administering a therapeutically effective amount of a picornavirus or a modified form thereof such that at least some cancer cells undergo viral oncolysis.

In a preferred embodiment of the invention the subject is a human.

The picornavirus may be any picornavirus, including known and classified picornaviruses, and picornaviruses yet to be classified. Preferably, the Picornavirus is selected from prototype and clinical isolates. In a preferred method, the picornavirus is an enterovirus, including coxsackieviruses, echoviruses, polioviruses, and enteroviruses unclassified, or other picornavirus genera, including rhinoviruses, paraviruses, Paraechoviruses, Hepatoviruses, Cardioviruses, Foot-and-Mouthviruses, Erboviruses, Koboviruses, and Teschoviruses.

In a preferred embodiment, the Picornavirus is a Coxsackievirus. Preferably, the Group A Coxsackievirus is selected from the group consisting of CVA13, CVA15, CVA18, CVA20, CVA21, modified forms thereof and combinations thereof. More preferably, the group A Coxsackievirus is selected from CVA13, CVA15, CVA18, CVA20 or CVA21.

In a preferred embodiment, the Group A Coxsackievirus is CVA15 or CVA21. Preferably, the Group A Coxsackievirus is CVA15. More preferably, CVA15 is G-9.

In another preferred embodiment, the Group A Coxsackievirus is CVA21. More preferably, CVA21 is a Kuykendall strain.

The term "blood cancer" as used herein includes non-solid tumors such as leukemia, multiple myeloma, Hodgkin's disease, non-Hodgkin's disease, myelodysplasia, and lymphomas such as B-cell lymphoma. Examples of leukemias include, but are not limited to, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), chronic and lymphocytic leukemia (CLL), B prolymphocytic leukemia, and monocytic leukemia. In a preferred embodiment, the hematological cancer is multiple myeloma.

In preferred embodiments, the hematological cancer is, or comprises cells resistant to one or more chemotherapeutic drugs.

In preferred embodiments, the Picornavirus is administered to a human subject in any suitable manner. For example, virus can be administered intravenously, intratumorally, intraperitoneally, intramuscularly, intraocularly, subcutaneously, orally, topically, or ex vivo to decontaminate malignant cells in autologous grafts prior to autologous stem cell transplantation. In one embodiment, the method includes prophylactic treatment, eg, for MGUS. In one embodiment, the method includes systemic antineoplastic agents for hematological cancers, such as multiple myeloma. In one embodiment, the method comprises ex vivo decontamination of malignant cells within the autologous graft prior to transplantation. Preferably, the autologous graft includes hematopoietic stem cells.

In a preferred embodiment, the amount of virus used may range from about 0.01 to about 1000 infectious virus units per cell.

In a preferred embodiment, the virus is administered to the subject in combination with an effective amount of a chemotherapeutic drug.

In another preferred embodiment, the virus is administered to the subject in combination with an effective amount of a probiotic.

Cells of hematological cancers can overexpress the virus-cell entry receptor molecules intercellular adhesion molecule-1 (ICAM-1) and/or decay accelerating factor (DAF).

Hematological cancer cells can constitutively express NF- _κB .

In a third aspect, the present invention provides a method for treating and/or preventing hematological cancer in a subject, the method comprising administering a therapeutically effective amount of a picornavirus-derived nucleic acid molecule or a modified form thereof, such that at least some cancer cells Killed by the virus.

The nucleic acid molecule can be single-stranded RNA or complementary DNA from a virus.

In a preferred embodiment, the Picornavirus is a Coxsackievirus. Preferably, the Coxsackievirus is type A, more preferably Coxsackievirus A21 (CVA21). Preferably, CVA21 is a Kuykendall strain.

In a fourth aspect, the present invention provides a pharmaceutical composition for treating and/or preventing blood cancer in a subject, the composition comprising an effective amount of a picornavirus capable of lytically infecting blood cancer or a modified form thereof, And a pharmaceutically acceptable excipient, diluent or carrier.

Pharmaceutically acceptable excipients, diluents or carriers are well known to those skilled in the art and include, but are not limited to, any inactive substances that can be used as vehicles for the administration of therapeutic drugs. In a preferred embodiment, the pharmaceutically acceptable carrier may be a liposome.

In a fifth aspect, the present invention provides a pharmaceutical composition for treating and/or preventing hematological cancer in a subject, the composition comprising an effective amount of a picornavirus derived from a hematological cancer capable of lytically infecting a hematological cancer or a modified one thereof. Form nucleic acid molecules, and pharmaceutically acceptable excipients, diluents or carriers.

In a preferred embodiment, the pharmaceutical composition further comprises liposomes (which may also contain monoclonal antibodies that bind specific tumor markers), allowing targeting of nucleic acid-liposome complexes.

In a sixth aspect, the present invention provides a method of treating and/or preventing hematological cancer in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition according to the third or fourth aspect of the present invention, such that at least some Cancer cells undergo viral oncolysis.

In the seventh aspect, the present invention provides a Picornavirus or its modified form capable of lytically infecting blood cancer, and a pharmaceutically acceptable excipient or diluent, in the treatment and/or prevention of blood cancer in a subject application in the method.

In an eighth aspect, the present invention provides nucleic acid molecules derived from picornaviruses capable of lytically infecting hematological cancers or improved forms thereof, and pharmaceutically acceptable excipients or diluents, for use in the treatment and/or prevention of Application of the method in subjects with blood cancers.

In the ninth aspect, the present invention provides the use of a picornavirus capable of lytically infecting hematological cancer or an improved form thereof in the preparation of a medicament for treating and/or preventing hematological cancer in a subject.

In a tenth aspect, the present invention provides the use of a nucleic acid molecule derived from a picornavirus capable of lytically infecting blood cancer or a modified form thereof in the preparation of a medicament for treating and/or preventing blood cancer in a subject.

In an eleventh aspect, the present invention provides a method for inducing an immune response in a mammal to hematological tumors or cancer cells, the method comprising infecting the cells of the mammal with a therapeutically effective amount of a picornavirus or a modified form thereof, such that Viral oncolysis occurs in at least some cancer cells.

In this specification, unless the context requires otherwise, the word "comprise" or variations thereof such as "comprises" or "comprising" are to be understood as implying the inclusion of stated elements, integers or steps, or group of elements, integers or steps, without excluding any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, statutes, substances, devices, articles or the like is included in this specification solely to provide the context of the invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in Australia or elsewhere in the field relevant to the present invention before the priority date of this application.

For a clearer understanding of the present invention, preferred embodiments are described with reference to the following figures and examples.

abbreviation

The abbreviation "MM" is used herein to denote multiple myeloma.

The abbreviation "CVA" is used herein to denote group A Coxsackieviruses, eg CVA21 is an abbreviation for Coxsackievirus A21. Documents in the art use the different abbreviations "CAV" and "CVA" to refer to Group A Coxsackieviruses, it being understood for the purposes of this application that these abbreviations refer to the same organism and are therefore interchangeable.

The abbreviation "MOI" is used herein to denote multiplicity of infections.

The abbreviation "MGUS" is used herein for monoclonal gammopathy of undetermined significance.

The abbreviation "BM" is used herein to denote bone marrow.

The abbreviation "CFU-GM" is used herein to denote granulocyte/macrophage colony forming unit.

The abbreviation "MTT assay" is used herein to denote the microculture tetrazolium salt assay using the compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide .

The abbreviation "PI" is used herein to denote propidium iodide.

The abbreviation "PBMC" is used herein to denote peripheral blood mononuclear cells.

The abbreviation "BMSC" is used herein to denote bone marrow stromal cells.

The abbreviation "mAb" is used herein to denote monoclonal antibody.

The abbreviation " _TCID50 " is used herein to denote the tissue culture 50% infectious dose.

The abbreviation "PFU" is used herein to denote plaque forming unit.

The abbreviation "CPE" is used herein to denote cytopathic effect.

The abbreviation "ICAM-1" is used herein to denote intercellular adhesion molecule 1.

The abbreviation "DAF" is used herein to denote decay accelerating factor.

The abbreviation "kb" is used herein to denote kilobase pairs.

The abbreviation "DNA" is used herein to denote deoxyribonucleic acid.

The abbreviation "RNA" is used herein to denote ribonucleic acid.

The abbreviation "ELISA" is used herein to denote enzyme-linked immunosorbent assay.

Description of drawings

Figure 1. Flow cytometric analysis of ICAM-1 and DAF on multiple myeloma cell lines.

Multiple myeloma cell lines RPMI-8226, U266 and NCI-H929 cells and normal PBMC were double stained with anti-ICAM-1 and anti-DAF mAb. Anti-ICAM-1 mAb was directly cross-linked with FITC, and anti-DAF mAb was indirectly stained via a secondary conjugate linked to PE.

Figure 2. Cytopathic effect of CVA21 on multiple myeloma cell lines. To determine the cytopathic effect of CVA21 on multiple myeloma cell lines and normal PBMCs, cultures of RPMI-8226, U266, NCI-H929 and PBMCs were cultured in 6-well plates with or without CVA21 at 37°C for 48 h (MOI ~1). Carefully aspirate the cell supernatant from each well, and the cells are stained with 0.01% trypan blue solution. Nonviable cells destroyed by CVA infection stained positively with trypan blue, while viable cells were able to exclude the dye. Photomicrographs were taken at ×100 magnification.

Figure 3. Detection of oncolytic effect of CVA21 on MM cell lines and normal PBMCs using the MMT assay.

The MM cell lines RPMI-8226, U266 and NCI-H929 as well as normal PBMCs demonstrated differential sensitivity to continuous exposure to CVA2148h. Error bars represent the mean of the standard error of four wells/group.

Figure 4. Production of CVA21 after infection of multiple myeloma cell lines.

Ability of MM cell lines NCI-H929, U266 and RPMI-8226 to produce infectious virus particles 24h and 48h after infection with CVA21. After infection with ~1 MOI of CVA21, an increase in virus was detectable in each of the multiple myeloma cell lines, as opposed to normal PBMC.

Figure 5. Viral growth curve of CVA21 in multiple myeloma cells. Viral growth of CVA21 in U266(·), NCI-H929(o) and RPMI-8226(▼) cell lines measured after simultaneous infection (infection multiplicity approximately 1) and collection of viral progeny at indicated time intervals curve. Titration of samples was performed in triplicate and the average virus yield at each time point was plotted.

Figure 6. Analysis of DNA fragmentation in RPMI-8226, NCI-H929 and U266 cells infected with CVA21.

RPMI-8226, NCI-H929 and U266 cells were infected with CVA21 (MOI～10TCID ₅₀ /cell) for 24h. Total DNA was extracted from infected and uninfected cells, and fragments were detected by agarose gel electrophoresis. DNA samples from the myeloma cell lines RPMI-8226 (lanes 1 and 2), NCI-H929 (lanes 3 and 4) and U266 (lanes 5 and 6) are shown. Lane "M" contains a 1-kb DNA ladder. DNA extracted from MM cell lines cultured alone in medium is shown in lanes 1, 3 and 5, while lanes 2, 4 and 6 contain cellular DNA extracted from cell lines treated with CVA21.

Figure 7. Ex vivo purification of multiple myeloma cells from admixture with PBMCs.

Normal PBMC and the mixture of multiple myeloma cells RPMI-8226 and U266 were cultured together, infected with CVA21 for 3 days, and the effect of multiple myeloma purification was determined. Viable myeloma cells (CD138 ⁺ /PI ⁻ ) and PBMCs (CD138 ⁻ /PI ⁻ ) were analyzed by flow cytometry. Flow cytometry plots of purified ("CVA21") and non-purified ("virus-free") samples of RPMI-8226 and U266 cell lines are shown.

Figure 8. Analysis of ICAM-1 expression and CVA21 growth inhibitory effect on clinical multiple myeloma samples.

(A) A patient's bone marrow aspirate (clinical sample #001) was obtained from a multiple myeloma patient and processed to obtain a single cell suspension. Cells were double stained with anti-CD138 and anti-ICAM-1 antibodies. ICAM-1 positive myeloma cells (CD138 ⁺ /ICAM-1 ⁺ ) appeared in the upper right quadrant of the dot matrix. Approximately 37% of cells in primary tumor samples consisted of CD138 ⁺ plasma cells.

(B) Clinical bone marrow cells from patients were then infected with CVA21 at various concentrations and growth inhibition of cancer cells was detected by MTT assay. The graph shows the percentage of cell survival for sample #001 at different virus inputs.

Figure 9. Ability of CVA21 to purify multiple myeloma plasma cells from clinical bone marrow samples.

(A) Bone marrow samples from patient #001 were cultured with virus-free, ~2.75 TCID ₅₀ /cell, ~5.5 TCID ₅₀ /cell, or ~11 TCID ₅₀ /cell virus for 72 h before analysis by flow cytometry to test for viability percentage of myeloma cells. Cells were double stained with propidium iodide, and anti-CD138-FITC antibody. Surviving myeloma cells after purging with different concentrations of virus can be seen in the lower right quadrant of each dot plot (CD138 ⁺ /PI ⁻ ).

(B) Photomicrographs of CVA21-infected primary tumor samples infected with 0, -2.75 and _-5.5 TCID50/cell for 48 h. Cell aggregation was seen in all virus-treated samples compared to no-virus controls (×40 magnification).

(C) The percentage of viable myeloma cells was calculated from the flow cytometry data above after challenge with different concentrations of virus.

Figure 10. CVA21 selectively purifies plasma cells from MM and MGUS BM.

(A) ICAM-1 expression on CD138 ⁺ cells. Representative dot plot of patient #005 after staining with anti-CD138 and anti-ICAM-1.

(B)(i) from patients with relapsed MM (#001, #002 and #008), partial remission MM (#003), monoclonal gammopathy of undetermined significance (MGUS) (#004, #006) and in vitro infection and killing of BM samples from a patient diagnosed with MM (#007). BM samples from seven patients were infected in vitro with CVA21 at a multiplicity of infection (MOI) of 0 (sham), 3 or 10 _TCID50 /cell. Samples were collected 48 hours after infection and analyzed by flow cytometry after CD138 staining. 10,000 events are recorded per sample; percentage of viable cells positive for CD138 is shown.

(B)(ii). Combined results of a total of 19 clinical samples within the range of conditions shown in Table 1 herein.

(C) BM progenitors are resistant to CVA. Residual cells (approximately 10,000 cells) in three BM samples infected for 48 hours with CVA21 at an MOI of 0 (sham), 3 or 10 _TCID50 /cell, in 3 ml MethoCult GF4434 complete methylcellulose medium (Stem Cell Technologies, Cultured in Vancouver, Canada). CFU-GM cultures were performed in wells of a 6-well plate at 37 °C, 5% _CO2 for 14 days. Colonies consisting of 20 or more cells of granulocytes and/or macrophages were recorded using an inverted microscope for CFU-GM. The mean number of colonies after infection with each concentration of CVA21 is shown. Error bars represent standard error of the mean of three samples. Colony numbers shown are per 1 x ¹⁰⁴ seeded cells. CFU-GM: Granulocyte/macrophage colony forming unit.

(D) The PBMC (CD138 ⁻ ) population was still alive after CVA21 purification as detected by flow cytometry. ^CD138- cells were also quantified after infection of 7 patient BM samples as described in B(i) above. This population remained relatively unchanged following CVA21 infection.

(E) In vitro infection and killing of BM samples from patients described in Figure 10B(ii), expressed as percent reduction of CD138 ⁺ cells.

Figure 11 (A and B). Trafficking of ICAM-1 and DAF on B-cell lymphoma, B prolymphocytic leukemia, acute promyelocytic leukemia (APML), monocytic leukemia and multiple myeloma cell lines cell analysis.

(A) B-cell lymphoma cell line SCOTT, B prolymphocytic leukemia cell line JVM13, acute promyelocytic leukemia (APML) cell line NB4 and (B) HL-60, monocytic leukemia cell line U937 and multiple Myeloma cell line H929, double stained with anti-ICAM-1 and anti-DAF mAb. Anti-ICAM-1 mAb was directly cross-linked with FITC, and anti-DAF mAb was indirectly stained through a secondary cross-linker attached to PE.

Figure 12. Detection of oncolytic effects of CVA21, CVA18, CVA15 and CVA13 on selected hematological cancer cell lines using the MTT assay.

(A) MM cell line RPMI-8226, (B) monocytic leukemia cell line U937 and (C) acute promyelocytic leukemia (APML) cell line HL-60 demonstrated differential exposure to the virus for 48 h of continuous exposure sensitivity.

Figure 13. Production of CVA21 following infection of selected hematological cancer cell lines.

After 24 and 48 h infection with CVA21, (◇) MM cell line RPMI-8226, (□) monocytic leukemia cell line U937 and (Δ) acute promyelocytic leukemia (APML) cell line HL-60 were infectious capacity of virus particles. An increase in virus was only detected in the MM cell line RPMI-8226 after infection with ~ ₁₀ TCID50/cell of CVA21.

Detailed ways

Although some naturally occurring picornaviruses and other viruses, such as reoviruses, are known to be suitable for the treatment of limited types of cancer, there is still a need to develop improved treatments.

As described herein, the present inventors have discovered that picornaviruses can be used to lytically infect hematological tumors or cancers. "Susceptible" cells are those cells that have been shown to induce cytopathic effects, viral protein synthesis and/or virus production.

Based on these findings, the present inventors have developed methods and compositions for treating and/or preventing hematological cancers in mammals. The mammal can be any mammal in need of treatment according to the present invention. A mammal can be a human or an individual of any species of social, economic or research importance, including but not limited to mice, dogs, cats, sheep, goats, cows, horses, pigs, non-human primates, and humans. In a preferred embodiment, the mammal is a human.

Cell death typically results from viral infection of the cell and may be caused by cytolysis due to intracellular replication of the virus, or infection that triggers apoptosis, most likely as a result of activation of cellular caspases. Once lysed, the cytosolic contents of infected cells can escape from the ruptured membrane and can release antigens, including cell surface antigens, that can elicit an immune response to the abnormal cell. Thus, treating hematological tumors or cancer cells in a mammal according to the methods of the present invention can boost the immune response of the mammal against these cells.

The picornavirus may be any picornavirus, including known and classified picornaviruses, and picornaviruses yet to be classified. Picornaviruses can be selected from prototype and clinical isolates. Representative types of human picornaviruses include enteroviruses, coxsackieviruses, echoviruses, polioviruses, and unclassified enteroviruses, rhinoviruses, paretroviruses, hepatoviruses, and cardioviruses belongs to. In a preferred method, the picornavirus is an enterovirus, including coxsackieviruses, echoviruses, polioviruses, and enteroviruses unclassified, or picornaviruses from other genera, which may include rhinoviruses Viruses, Paretroviruses, Hepaviruses, Cardioviruses, Foot-and-Mouthviruses, Equine Rhinoviruses, Rigiviruses, and Teshinviruses. In a preferred embodiment, the Picornavirus is a Coxsackievirus. Preferably, the Coxsackievirus is type A, more preferably Coxsackievirus A21.

Ideally, the virus may be selected from Group A Coxsackieviruses. CVA21 is preferred, especially CVA21 (Kuykendall) (Sickles G.M., Proc. Soc. Exp. Biol. Med. 102: 742; Shafren D. et al., J. Virol 1997, 71: 4736; Hughes et al., J. Gen Virol. 1989 , 70:2943; Schmidt, NJ. et al., Proc.Soc.Exp.Biol.Med., 1961, 107:63. CVA21 (Kuykendall) is available from American Type Culture Collection (ATCC) 10801 University Boulevard, Manassas, Virginia 20110-2209 , United States of America, Accession No. VR-850. Other preferred Group A Coxsackieviruses are also available from ATCC, including CVA13 (Accession No. VR-171), CVA15 and especially G-9 (Registration No. No. VR-1021), and CVA18 (Registration Nos. VR-1024 and VR-176).

Picornaviruses can be naturally occurring or modified. A picornavirus is "of natural origin" when it has been isolated from a natural source and has not been artificially modified by humans in the laboratory. For example, picornaviruses can be obtained from a "field source": ie, from a human patient.

Picornaviruses can be modified and still be able to lytically infect hematological tumors or cancers.

Preferably, the picornaviruses used in the methods or compositions described herein will cause little or only mild clinical symptoms of viral infection in the recipient.

A picornavirus may be a recombinant picornavirus derived from two or more types of picornaviruses with different pathogenic phenotypes such that it contains different antigenic determinants, thereby reducing or avoiding previous exposure to picornaviruses Mammalian immune responses to viral subtypes.

In the methods of the invention, the picornavirus can be administered to a hematological tumor or cancer in a single subject. Different serotypes and/or different strains and/or different species and/or different genera of picornaviruses may be used, such as coxsackieviruses originating from different animal species. If desired, the picornavirus can be chemically or biochemically pretreated (eg, treated with a protease, such as chymotrypsin or trypsin) prior to administration to the tumor. These pretreatments remove the viral envelope, which can lead to better viral infectivity.

Picornaviruses can be administered or used in combination with other therapeutic agents. For example, a picornavirus can be administered with one or more different strains or serotypes or species or genus of picornaviruses. Picornaviruses of other strains or serotypes or species or genera may have the same or different receptor requirements for cell infection as the picornaviruses of the present invention.

As a further example, picornaviruses, or combinations thereof, may be administered in combination with one or more drugs capable of modulating or suppressing the immune response of the individual being treated. In this way, an individual's natural immune response to viral infection can be altered, preferably resulting in a more effective viral infection and/or oncolytic and/or therapeutic outcome. Drugs that alter the immune response are typically drugs that suppress the immune response. Drugs capable of modulating or suppressing the immune response of an individual, such as a human individual, as described, for example, in The Merck Index, Thirteenth Edition, Merck & Co. Inc, Whitehouse Station, NJ, USA., the contents of which are incorporated herein by reference .

Picornaviruses, or combinations thereof, may be used in combination with one or more chemotherapy drugs (also known as antineoplastic drugs). For example, antineoplastic agents are described in The Merck Index, Thirteenth Edition, Merck & Co. Inc, Whitehouse Station, NJ, USA. For example, picornaviruses can be administered with chemotherapeutic drugs such as: doxorubicin, paclitaxel, fluorouracil, melphalan, cisplatin, alpha interferon, COMP (cyclophosphamide, vincristine, methylamine pterin and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone), PROMACE/MOPP (strong pine, methotrexate (w/ leucovin rescue), doxorubicin, cyclophosphamide, paclitaxel, etoposide/nitrogen mustard, vincristine, prednisone, and procarbazine), Vincristine, vinblastine, angioinhibin, TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, Thalidomide, SP-PG and the like. Other chemotherapeutic agents include alkylating agents, such as nitrogen mustards, including mechlorethamine, melphalan, chlorambucil, cyclophosphamide, and ifosfamide; nitrosoureas , including carmustine, cyclohexylnitrosourea, methylcyclohexylnitrosourea, and streptozotocin; alkyl sulfonates, including busulfan; triazines, including dacarbazine; Amines (ethyenimines), including thiotepa and hexamethylmelamine; folic acid analogs, including methotrexate; pyrimidine analogs, including 5-fluorouracil, cytarabine; purine analogs, including 6-mercaptopurine and 6 - Thioguanine; antineoplastic antibiotics, including actinomycin D; anthracyclines, including doxorubicin, bleomycin, mitomycin C, and methramycin; hormones and hormone antagonists , including tamoxifen and corticosteroids, and miscellaneous drugs, including cisplatin and brequinar. Picornaviruses may be used in combination with one or more of bleomycin, deacetylvinblastine, vincristine, dactamycin, procarbazine, nitrosourea, or dacarbazine, for example, in the treatment of melanoma . Picornaviruses can be used in combination with one or more of cisplatin and carboplatin, eg, to treat ovarian cancer. Further examples of chemotherapeutic drugs used in combination with picornaviruses, such as for the treatment of breast cancer, include cyclophosphamide (Cytoxan), methotrexate (Amethopterin, Mexate, Folex) and fluorouracil (Fluorouracil, 5-FU, Adrucil) [ CMF]; Cyclophosphamide, Adriamycin, and Fluorouracil [CAF]; Adriamycin and Cyclophosphamide [AC]; Adriamycin and Cyclophosphamide with paclitaxel (Taxol); doxorubicin (Adriamycin), then CMF; cyclophosphamide, epirubicin (Ellence), and fluorouracil. Other chemotherapy drugs used to treat women with breast cancer include, for example, docetaxel (Taxotere), vinorelbine (Navelbine), gemcitabine (Gemzar), and capecitabine (Xeloda).

It is understood that picornaviruses or combinations thereof may be used in combination with known treatments for hematological cancers, such as peptidylpiperidone, proteasome inhibitors, and arsenic trioxide for myeloma.

It is to be understood that picornaviruses and one or more additional drugs, such as one or more other picornaviruses, one or more drugs that modulate or stimulate an immune response, or one or more antineoplastic "Combined" use or administration of drugs refers to any manner of use or administration in which the picornavirus and the additional drug have a therapeutic effect, such as a temporary overlapping effect. The members of the combination may be administered simultaneously or separately in any order to achieve the desired therapeutic effect. When contemplated for combination therapy, the Picornavirus and the additional drug may be provided as a physical mixture thereof or separately, eg, in kit form, with or without instructions for administration. The kits according to the invention may also comprise other components necessary or required for carrying out the methods of the invention, such as buffers and/or diluents. In the methods of the invention, kits typically include containers for the various components and instructions for using the kit components.

It is to be understood that the pharmaceutical compositions of the present invention include compositions in the form of a physical mixture of a picornavirus and one or more other therapeutic agents, as well as compositions comprising a picornavirus as the only therapeutically active agent.

Picornaviruses can be administered in combination with an effective amount of a probiotic. Probiotics may include, but are not limited to, Lactobacillus acidophilus, L. gasseri, L. confusus, Streptococcus thermophilus, Bifidobacterium breve, and B. longum.

Cancers of the blood—such as leukemia, lymphoma, and myeloma—are cancers that arise from cells in the bone marrow or lymphoid tissue. Blood cancers can grow as solid tumors or as single cells, in which case they appear in the blood as leukemia.

Typically, at least some hematological cancer cells express ICAM-1 and/or DAF. Typically at least some hematological cancer cells overexpress ICAM-1 and/or DAF compared to non-malignant cells. Hematologic neoplasms or cancers that are particularly susceptible to treatment by the methods of the invention include leukemia, multiple myeloma, Hodgkin's disease, non-Hodgkin's disease, myelodysplasia, and lymphoma. Examples of leukemia include, but are not limited to, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL).

Picornaviruses are typically administered to hematological cancers in a physiologically acceptable carrier or vehicle, such as phosphate buffered saline. "Administered to a hematological cancer" means that the picornavirus is administered in such a way that it comes into contact with hematological cancer cells. The route of administration of the picornavirus, as well as the dosage form, carrier or vehicle, depend on the site and type of the tumor. A variety of routes of administration can be employed. For example, for accessible solid tumors, picornaviruses can be administered by direct injection into the tumor. For example, for hematopoietic tumors, picornaviruses are typically administered intravenously or intravascularly. For example, intravenous delivery may be by single or multiple dose boluses or by slow infusion into the intravenous system, eg, using a drip device. For tumors that are not easily accessible in vivo, such as metastases or brain tumors, the picornavirus can be delivered systemically through the mammalian body to reach the tumor (eg, intrathecally, intravenously or intramuscularly). Alternatively, picornaviruses can be administered directly to a single solid tumor, from where they are carried systemically through the body to metastases. Picornaviruses can also be administered subcutaneously, intraperitoneally, topically, orally, rectally, vaginally, nasally, or by inhalation of a spray.

In general, suitable compositions will be prepared according to methods known to those of ordinary skill in the art and thus may include pharmaceutically acceptable carriers, diluents and/or adjuvants.

The compositions can be administered by standard routes. In general, compositions can be administered parenterally (eg, intravenously, intrathecally, subcutaneously, or intramuscularly), orally, or topically. More preferably administration is by parenteral route.

Carriers, diluents and adjuvants must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the recipient thereof.

Examples of pharmaceutically acceptable carriers or diluents are demineralized or distilled water; saline solution; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oils such as peanut oil, safflower oil, Oil, olive oil, cottonseed oil, corn oil, sesame oil, arachis oil, or coconut oil; silicone oils, including silicones such as methylpolysiloxane, phenylpolysiloxane, and methylbenzene volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, carboxy sodium methylcellulose or hydroxypropylmethylcellulose; lower alkanols, such as ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, For example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butanediol or glycerol; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyethylene Pyrrolidone; Agar Agar; Gum Tragacanth or Arabic and Petrolatum. Typically the carrier or carriers form 10% to 99.9% by weight of the composition.

The composition may be in a dosage form suitable for injectable administration, a dosage form suitable for oral ingestion (such as capsules, tablets, caplets, elixirs), an ointment, cream or lotion suitable for topical administration, suitable for use as eye drops formulations suitable for administration by inhalation, aerosol formulations suitable for administration by inhalation (eg, by intranasal inhalation or oral inhalation), formulations suitable for parenteral administration, ie subcutaneous, intramuscular or intravenous injection.

For administration of injectable solutions or suspensions, nontoxic parenterally acceptable diluents or carriers can include Ringer's solution, isotonic saline, phosphate-buffered saline, ethanol and 1,2 propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvants for oral administration include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, acacia, tragacanth, dextrose , Sucrose, Sorbitol, Mannitol, Gelatin and Lecithin. In addition, these oral dosage forms may contain suitable flavoring and coloring agents. When used in capsule form, the capsules can be coated with a compound which delays disintegration, such as glyceryl monostearate or glyceryl distearate.

Adjuvants typically include lubricants, emulsifiers, thickeners, preservatives, bactericides and buffers.

Solid dosage forms for oral administration may contain binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants and/or time-delaying agents acceptable in human and veterinary pharmaceutical practice. agent (time delay agent). Suitable binders include acacia, gelatin, corn starch, tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweetening agents include sucrose, lactose, dextrose, aspartame or saccharin. Suitable disintegrants include cornstarch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavoring agents include oil of peppermint, oil of wintergreen, cherry, citrus or raspberry flavorings. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, a-tocopherol, ascorbic acid, methylparaben, propylparaben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Liquid dosage forms for oral administration may contain, in addition to the above drugs, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol , ethanol, propanol, isopropanol, glycerin, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further include dispersing and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, sodium alginate or acetyl ethanol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate, polyoxyethylene Ethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.

Emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifiers include the dispersants exemplified above or natural gums such as guar, acacia or tragacanth.

Methods for preparing parenteral compositions will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pa., here Incorporated by reference.

The topical dosage forms of this invention comprise the active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Dosage forms suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the area in need of treatment, such as liniments, lotions, creams, ointments or pastes, and drops suitable for ophthalmic, ear or nasal administration agent.

Drops according to the invention may comprise sterile aqueous or oily solutions or suspensions. These dosage forms can be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal and/or any other suitable preservative, optionally including a surfactant. The resulting solution can then be clarified by filtration, transferred to a suitable container and sterilized. Sterilization can be accomplished by autoclaving or holding at 90°C-100°C for half an hour, or by filtration followed by transfer to containers by aseptic technique. Examples of bactericides and fungicides suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for preparing oily solutions include glycerol, dilute ethanol and propylene glycol.

Lotions according to the invention include formulations suitable for application to the skin or eyes. Eye lotions may comprise sterile aqueous solutions, optionally containing an antiseptic, and may be prepared by methods similar to those described above for the preparation of drops. Lotions or liniments for the skin may also include skin drying and skin cooling agents such as alcohol or acetone, and/or humectants such as glycerin, or oils such as castor or peanut oil.

Creams, ointments or pastes according to the invention are semi-solid dosage forms of active ingredients for external use. They can be prepared by mixing the active ingredient in granular or powder form, alone or in solution or suspension in an aqueous or non-aqueous liquid, with an oily or non-oily base. The base may include hydrocarbons such as hard, soft or liquid paraffin, glycerin, beeswax, metallic soaps; viscose; oils of natural origin such as almond, corn, peanut, castor or olive oil; lanolin or its derivatives, Or fatty acids like stearic acid or oleic acid, and alcohols like propylene glycol or polyethylene glycol.

The composition may incorporate any suitable surfactant, such as anionic, cationic or nonionic surfactants, such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic substances such as silicaceous silicon dioxide, and other ingredients such as lanolin, may also be included.

When multiple administrations of picornaviruses are required, different viruses may be administered each time to avoid or minimize any immune response to previously administered viruses, and the course of treatment may be extended to one week to two weeks or longer, such as one to two weeks. Two months or more can be determined by the attending physician. More preferably, a virus to which the mammal has not previously been exposed or to which the mammal mounts a relatively small immune response can be administered, as can be determined by standard techniques.

A wide variety of routes of administration can be employed. For example, for hematological cancers, the route of administration can be intravenous, intratumoral, intraperitoneal, intramuscular, intraocular, subcutaneous, oral, topical, or ex vivo in autologous stem cell transplantation to purify malignant cells. cell.

The picornavirus is administered in an amount sufficient to treat hematological cancer (eg, an "effective amount"). A hematological cancer is "treated" when administration of the picornavirus to cancer cells causes lysis of at least some of the cancer cells, resulting in some shrinkage of the cancer, or preferably complete disappearance of the cancer. Some reduction in cancer, or preferably disappearance of cancer, is often caused by picornavirus-induced lysis of hematological cancer cells ("oncolysis").

Effective amounts will depend on the individual, and may also take into account, in part, the type of picornavirus: individual size, age, sex; and type, extent, and other characteristics of the hematologic cancer. For example, for human therapy, about ¹⁰² to about ¹⁰¹⁰ plaque forming units (PFU) of picornaviruses may be used, depending on the particular circumstances, such as the type, size and extent of the cancer present. For example, the inoculum may contain greater than about 10 ⁵ PFU, such as between about 10 ⁵ to about 10 ⁶ PFU, or between about 10 ⁶ to about 10 ⁷ PFU, or between about 10 ⁷ to about 10 ⁸ PFU. As a further example, the dose of virus may range from about 0.01 to about 1000 IVI/cell, such as about 0.01 to about 0.1 IVI/cell, or about 0.1 to about 1 IVI/cell, or about 1 to about 10 infectious viral units/cell, or from about 10 to about 100 infectious viral units/cell, or from about 100 to about 1000 infectious viral units/cell.

The picornavirus can be administered in a single dose or in multiple doses (ie, more than one dose). Multiple doses may be administered simultaneously or consecutively (eg, over a period of days or weeks). In therapeutic applications, treatment is generally continued for the duration of the disease condition, for example at least until the hematologic cancer is no longer detectable by conventional means. It is also contemplated that it may be necessary to continue treatment beyond cycles in which cancer is detectably present, such as when the treating physician suspects that an undetected tumor may be present. Picornaviruses can also be administered to more than one blood cancer in the same individual.

It is also to be understood that picornaviruses may be administered indirectly, by using the RNA genome or a complementary DNA copy of the genome, or a sufficient portion thereof, to induce a lytic response in virus-infected cancer cells. When administered, picornaviruses are still able to replicate in cells and cause the desired lytic infection and killing.

Typically patients are treated with an initial dose of virus and then monitored for an appropriate period of time before deciding to further administer virus to the patient, taking into account factors such as the patient's response to the initial dose of virus and the extent of viral infection and malignant cell death resulting from the initial treatment .

Ideally, the individual is treated with the virus over a period of time at predetermined intervals. The time interval may be daily or from 24 hours to 72 hours or more, such as weekly or monthly, and may be appropriately determined according to each case. The same or a different virus can be given each time, for example to avoid or minimize the effects of any immune response to a virus previously administered or exposed to, and the course of treatment can extend to one to two weeks or longer, as determined by the treating physician . Most preferably, a virus to which the individual has not been previously exposed, or to which the individual can be determined to mount a relatively small immune response using standard techniques, is administered. Also optionally and sometimes desirable, virus administration may be used in conjunction with immunomodulatory agents (eg, when it is desired to reduce the recipient's immune response to the administered virus).

While available and known viruses may be suitable for use in the methods of the invention, viruses modified or engineered using conventional techniques may also be used. For example, viruses can be modified to employ other cell adhesion molecules as cell receptors. For example, Coxsackievirus A21 can be modified using site-directed mutagenesis such that the peptide motif "RGD" is expressed on the surface of the viral capsid, as in the case of Coxsackievirus A9 (CVA-9). The RGD motif is recognized by all _av integrin heterodimers, for example, _this capsid modification allows the virus to bind integrin _αvβ3 , a cell adhesion molecule that has been shown in malignant Together with ICAM-1 is upregulated on the surface of melanoma lesions, resulting in enhanced uptake of the virus through interaction with integrin molecules or subsequent interaction with ICAM-1. Alternatively, viruses can be modified to recognize selectins such as E-selectin, or to target the NF-κB signaling pathway.

Viruses can be modified or selected to recognize alternative molecules for cell entry. Methods of such modification and selection are described in Applicant's co-pending application PCT/AU2005/000048, entitled "Modified oncolytic viruses", the contents of which are incorporated herein by reference. For example, PCT/AU2005/000048 discloses the isolation and selection of picornaviruses which are capable of infecting cells substantially lacking intercellular adhesion molecule-1 (ICAM-1), e.g. via decay accelerating factor (DAF) on the cells. , to lytically infect cells or induce apoptosis.

Example

The present invention will now be described in detail with reference to specific examples, but the scope of the invention should not be limited thereby.

Materials and methods

cells and viruses

The prototype strain of group A coxsackievirus CVA21 (Kuykendall strain) was obtained from Dr M. Kennett, Enterorespiratory Laboratory, Fairfield Hospital, Melbourne, Victoria, Australia. CVA21 was double plaque purified and grown in ICAM-1 expressing myeloma cells (SK-Mel-28). Viruses are laboratory-saved strains of CVA21, CVA18, CVA15, and CVA13. Prototype strains of these group A coxsackieviruses CVA13 (Flores), CVA15 (G-9), CVA18 (G-13) and CVA21 (Kuykendall) were also obtained from Dr M. Kennett. Initially, CVA13, CVA15 and CVA18 were propagated in HeLa-B cells, while CVA21Kuykendall was propagated in RD-ICAM-1 cells. Working laboratory strains of these viruses were then prepared from these preserved strains in SK-Mel-28 cells.

SK-Mel-28 cells were a kind gift from Dr P. Hersey, Oncology, Mater Hospital, Waratah, New South Wales, Australia. U266, RPMI-8226 and NCI-H929 multiple myeloma cell lines were obtained from Dr L. Lincz, Hematology, Mater Misericordiae Hospital (Hunter Hematology Research Group), Edith Street, Waratah, New South Wales, 2298, Australia. Other cell lines, B-cell lymphoma cell line SCOTT, B-prolymphocytic leukemia cell line JVM13, acute promyelocytic leukemia (APML) cell lines NB4 and HL-60, and monocytic leukemia cell line U937 were also obtained from Dr. Obtained from L. Lincz. SK-Mel-28 cells were maintained in DMEM containing 10% fetal calf serum (FCS), while U266, RPMI-8226, NCI-H929, SCOTT, JVM13, NB4, HL-60 and U937 cells were maintained in DMEM containing 10% FCS. cultured in PRMI. All cell lines were maintained at 37 °C in an environment of 5% _CO2 . Normal peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by layering whole blood samples on a gradient of Ficoll-Hypaque plus a 4:1 ratio, followed by centrifugation at 400 xg for 35 min. The intermediate layer was collected, resuspended in phosphate-buffered saline (PBS), and rinsed twice in PBS before use.

primary tumor cells

All methods were approved by the University of Newcastle Human Care and Ethics Committee (Newcastle, NSW, Australia) and the Hunter Area HealthService Human Care and Ethics Committee (Newcastle, NSW, Australia). Primary tumor cells were obtained from patients undergoing routine bone marrow examination at diagnosis of multiple myeloma. Bone marrow aspirates were collected from 19 patients as indicated in Table 1.

Table 1

patient

ID sample date gender age state 1 21/06/2004 m 55 recurrent 2 30/06/2004 m 76 recurrent 3 20/07/2004 f 44 Persistent 4 28/07/2004 f 69 MGUS 5 28/07/2004 m 80 MM diagnostic sample 6 11/08/2004 m 85 MGUS 7 10/09/2004 f 77 diagnosis 8 14/02/2005 m 44 diagnosis 9 16/02/2005 m 46 before transplant 10 23/02/2005 f 75 dry pumping 11 28/02/2005 m 55 PR 12 2/03/2005 m 70 recurrent 13 13/04/2005 m 57 PR 14 11/05/2005 f 80 MGUS 15 1/06/2005 f 72 Waldenstrom's macroglobulinemia 16 1/06/2005 f 60 MM diagnosis 17 03/08/2005 f 67 MGUS 18 10/08/2005 m 66 plasmacytoma 19 10/08/2005 f 72 MGUS

"PR" partial response; "dry pumping" refers to the absence of marrow (bone particles). All patients signed informed consent. Single-cell suspensions were obtained from bone marrow samples by overlaying the samples on Lymphoprep (Nycomed, Oslo, Norway) at a ratio of 2:1 and centrifuging at 400×g for 30 min. The mononuclear cell layer was collected, washed twice in PBS, and resuspended in RPMI containing 5% FCS before use in the next assay.

Antibody

A commercially available anti-CD54 antibody (Immunotech Coulter) directed against the N-terminal domain of ICAM-1 (Berendt AR et al., Cell. 1992; 68:71-81) cross-linked with fluorescein isothiocyanate (FITC) , Marseilles, France) for staining surface expressed ICAM-1. Anti-DAF IH4 mAb was obtained from Dr B. Loveland, Austin Research Institute, Heidelberg, Victoria, Australia. Anti-CD138-FITC mAb (Serotec, Oxford, UK) was specific for the plasma cell antigen Syndecan-1. Another commercially available anti-CD138 antibody cross-linked with phycoerythrin (PE) is from Miltenyi Biotec, CA, USA.

flow cytometry

Surface expression of ICAM-1 and DAF on multiple myeloma cell lines and other hematological cancer cell lines B-cell lymphoma, B prolymphocytic leukemia, acute promyelocytic leukemia (APML) and monocytic leukemia by dual color Analysis by flow cytometry. Briefly, dispersed cells (1×10 ⁶ ) were directly incubated with cross-linked anti-CD54-FITC mAb (diluted to 5 μg/ml in phosphate-buffered saline (PBS)) for 20 min on ice. Cells were then washed with PBS, pelleted at 1000 xg for 5 min, and then double-labeled with anti-DAF IH4 mAb (5 μg/ml PBS) for 20 min on ice. Cells were washed with PBS and resuspended in 100 μl of secondary antibody solution; R-phycoerythrin-cross-linked F(ab′) ₂ fragment of goat anti-mouse immunoglobulin diluted 1 in PBS (DAKO A/S, Denmark) :100, and incubated on ice for 20min. For each cell line, the appropriate cross-linking control antibody was also used for staining. Cells were washed and pelleted as above, resuspended in PBS, and analyzed for ICAM-1 and DAF expression using a FACStar analyzer (Becton Dickenson, Sydney, Australia).

Virus lytic infection and cell viability assays

Multiple myeloma cell lines RPMI-8226, U266 and NCI-H929 were cultured with or without CVA21 in 6-well culture plates for 48 hours. Normal human peripheral blood mononuclear cells were obtained from healthy volunteers as a control. The multiplicity of infection (MOI) for each culture was approximately 5 TCID ₅₀ /cell. Cytopathic effect (CPE) was assessed by trypan blue viability staining 48 h after CVA21 infection. Due to the affinity of trypan blue for proteins secreted into the culture supernatant by multiple myeloma cell lines, the culture supernatant was first removed by slow aspiration through a blunt 26 gauge needle prior to staining. The remaining cells were stained with 0.4% trypan blue solution for 5 min. Photographs were taken at a magnification of ×100.

In Vitro Growth Inhibition Assay

A modified microculture tetrazolium salt assay (Alley MC et al., Cancer Res. 1988;48:589-601) was used to study the effect of increasing concentrations of CVA21 on the growth of multiple myeloma cell lines. Briefly, multiple myeloma cells were harvested from exponential-phase maintenance cultures and distributed in a volume of 100 μl in multiple 96-well culture plates (1×10 ⁵ cells/well). 100 μl of medium, or medium containing virus (2×10 ⁻⁵ −20 TCID ₅₀ /cell) was dispensed into appropriate wells. To compare the growth inhibitory effects of other group A coxsackieviruses (CVA13, CVA15 and CVA18) with CVA21, 10-fold serial dilutions ranging from 1 x 10 ^-4 -100 TCID ₅₀ /cell were used. Wells containing no cells were used for blank "background" assays of media alone or virus solution alone.

Plates were incubated at 37°C for 2 days before adding MTT reagent (Sigma Chemicals, Sydney, New South Wales, Australia). Prepare MTT stock solution (5 mg MTT/ml PBS solution), sterile filter with 0.2 μm filter device, and store at 4 °C. To determine the level of growth inhibition, after 2 days of culture, 20 μl of MTT stock solution was added to each culture well, and culture was continued at 37°C for 4 h.

After incubation, the cell supernatant was removed from the wells by slow aspiration with a blunt 26-gauge needle and replaced with 150 μl DMSO. Gently shake at room temperature (RT) to completely dissolve the formazan crystals, and measure the absorbance of each well at 540 nm using an ELISA plate reader (Flow Laboratories, McLean, Virginia, USA). Cell line growth inhibition was then expressed as mean absorbance units as a percentage of control absorbance readings after subtraction of the mean "background" absorbance.

Virus yield and endpoint titration

After measuring CVA21CPE in three myeloma cell lines, experiments were performed to measure the yield of infectious virus particles produced during infection of U266, RPMI-8226, NCI-H929 cells and PBMC. Approximately 3 x ¹⁰⁶ cells of each cell line were infected with an MOI of 1 _TCID50 /cell. U266, RPMI-8226, NCI-H929 cells and PBMCs inoculated with CVA21 were washed twice with PBS and resuspended in 600 μl RPMI, after which each cell was divided into 3 equal portions of 200 μl each (~1×10 ⁶ cells/test tube ). The tubes were incubated at 37°C, and one aliquot of each cell line was collected and the cells were harvested by three consecutive freeze-thaw cycles at the time of 0, 12, 24 and 48 h. The virus yield in each cell lysate was determined by endpoint titration assay in SK-Mel-28 cells. The results are shown in Figure 4.

Briefly, for endpoint titration experiments, 10-fold serial virus dilutions (100 μl/well, 4 replicates of each dilution) were seeded in 96-well tissue culture plates in confluent monolayers of SK-Mel-28 cells, and Incubate for 48 h at 37°C in an environment of 5% CO ₂ . By incubating with crystal violet/methanol solution (0.1% crystal violet, 20% methanol, 4.0% formaldehyde/PBS) (100μl/well) for 24h, and then washing with distilled water three times, the cell survival rate was quantified, and the multiscan ELISA The relative absorbance of each well was read at 540 nm on an absorbance assay plate reader (Flow Laboratories, McLean, Virginia, USA). The 50% virus endpoint titer was calculated using the Spearman Karber method; a well was scored as positive if the absorbance value was less than the mean of negative three standard deviations (SD) of the control virus-free wells.

Experiments were also performed in selected hematological cancer cell lines, namely the MM cell line RPMI-8226, the monocytic leukemia cell line U937, and the acute promyelocytic leukemia (APML) cell line HL-60, to measure the Production of infectious viral particles (CVA21). The method was similar to that described above, although cells were infected with CVA21 at 10 _TCID50 /cell, but the virus was allowed to bind for 30 min at 37°C before being washed and placed in fresh medium (RPMI 10% FCS). Cells and supernatants were harvested at 0, 24 and 48 h after infection, and viral load was determined by endpoint titration on SK-MEL-28 cell monolayers. The results are shown in Figure 13.

virus growth rate

To study the replication kinetics of CVA21 in three myeloma cell lines, tubes containing 1×10 ⁶ U266, RPMI-8226, or NCI-H929 cells were used with approximately 3×10 ⁶ TCID ₅₀ CVA21 (MOI ~ 3 TCID ₅₀ /cell) Infect with 100 μl of virus sample. The test tube was shaken gently at room temperature for 30 min, and then the cells were washed 5 times with 5 ml RPMI each time, and the cells were resuspended in 1 ml RPMI containing 1% FCS. Each tube inoculated with CVA21 was then divided into 9 aliquots of 100 μl aliquots (~1×10 ⁵ cells/tube) for collection at appropriate time points. These tubes were incubated at 37°C during the experiments. Synchronized infections were interrupted at intervals of 0, 2, 4, 6, 8, 10, 12, 24, and 48 hours, and 1 sample of each cell line tested was collected at each time point by three consecutive Cells were lysed by freeze-thaw cycles, centrifuged at 10,000 x g for 5 min at room temperature, and virus yields in cell lysates were determined in an end-point titration assay.

DNA Fragmentation Detection

Apoptosis induced by CVA21 infection in MM cells was measured by detecting genomic DNA fragments. MM cell lines RPMI-8226, U266 and NCI-H929 were cultured in 6-well plates (5×10 ⁵ cells/well), and were infected with CVA21 (MOI～10TCID ₅₀ /cell) at 37°C for 24 hours before analysis, or not infected, Culture medium alone for 24h. The cells were pelleted by centrifugation at 800 g for 5 min, and then 500 μl of lysis buffer (5 mM Tris-HCl, 20 mM EDTA, 0.5% Triton X-100, pH 8.0) was added to the cell mass and incubated on ice for 20 min. Lysates were centrifuged at 12,000 g for 20 min, and DNA was extracted from the supernatant using phenol:chloroform. DNA was ethanol precipitated, washed in 70% ethanol, and resuspended in 30 μl TAE containing RNase (50 μg/ml). DNA samples were incubated at 37°C for 30 min and then analyzed by agarose gel electrophoresis. 15 microliters of extracted DNA was mixed with 3 microliters of loading buffer (0.25% OrangeG, 40% glycerol/TAE) and resolved on a 1.2% TAE agarose gel containing ethidium bromide. Gels were visualized under UV light and images were acquired digitally using the Gel Doc system (Bio-RAD, Regents Park, New South Wales, Australia).

Selective infection of myeloma cell lines co-cultured with normal peripheral blood mononuclear cells by CVA21

Each U266 and RPMI-8226 myeloma cells were mixed with PBMCs in RPMI medium supplemented with 10% FCS to give a final plasma cell concentration of 10%. The cell mixture was treated with CVA21 (1 MOI/total cell population) or left untreated for 72h. On day 3 of cleanup, samples were collected from each cell population mixture in PBS, stained first with anti-CD138-FITC (10 μl/ml) for 30 min, then with propidium iodide (PI) (5 μg/ml) After staining for 10 min, the number of untreated cancer cells was detected by flow cytometry. Cells were rinsed in 5 ml PBS, pelleted by centrifugation at 1000 g, and analyzed on a FACStar analyzer (Becton Dickenson, Sydney, Australia). No longer viable or untreated cells stained positive with propidium iodide (PI ⁺ ), while anti-CD138-FITC staining was used to identify multiple myeloma cells (CD138 ⁺ ) from normal PBMCs (CD138 ^- ) .

Ex vivo purification of patient bone marrow samples with CVA21

Primary tumor samples were obtained from two multiple myeloma patients who had given informed consent, and only the remaining cells from bone marrow aspirate collected during routine diagnostic procedures were used in this study. First, by double staining primary tumor cells (1 x 105 cells) with anti-CD138-PE (10 μg/ml) and anti-ICAM-1-FITC antibody ( ⁵ μg/ml), based on the standard surface receptor staining described above Raw data, ICAM-1 receptor levels were determined for each clinical sample. Three growth inhibition/MTT assays were then performed on clinical samples infected with CVA21. Oncolysis of CVA21 was then assayed by seeding approximately 1 x ¹⁰⁵ cells (in RPMI with 5% FCS) in 96-well plates and serial 10-fold dilutions ranging from 0 to 16 _TCID50 /cell. These cells were cultured at 37°C for 48h and then analyzed using the MTT assay.

After the growth inhibition assay, a single cell suspension (1×10 ⁶ cells in RPMI containing 5% FCS) from a clinical bone marrow sample was seeded in each well of a 6-well plate with 0, 2.75, 5.5 and CVA21 at a concentration of 11TCID ₅₀ /cell was cultured for 48 hours. Cells were then harvested by gentle pipetting, resuspended in 4 ml PBS, and rinsed once before performing cell viability assays. The selectivity of CVA21-mediated oncolysis for MM cells was determined by flow cytometry using the cell viability dye propidium iodide (5 μg/ml) and anti-CD138-FITC (10 μg/ml) antibody.

Other clinical samples (19 in total) as shown in Table 1 were similarly assayed.

result

MM cells express ICAM-1 and DAF

Flow cytometry was used to examine the relative levels of the CVA21 cellular entry receptor ICAM-1 and DAF on the surface of several MM cell lines. U266, RPMI-8226 and NCI-H929 cells were analyzed by flow cytometry after double staining with anti-ICAM-1 and anti-DAF antibodies. In the upper right quadrant of each dot plot (Fig. 1), stained cells were clearly evident compared to the crosslinker control, demonstrating elevated levels of ICAM-1 and DAF in each of the three cell lines. However, not all cells in PBMC samples showed surface expression of ICAM-1, and the staining of ICAM-1 in most cells was negative. However, PBMCs were positive for DAF expression, showing an upward migration of the population of PBMCs in the double stained samples compared to the cross-linked control.

Expression of ICAM-1 and DAF in Hematological Cancer Cell Lines

To further investigate the potential susceptibility of hematological cancers to picornavirus infection and lysis, expression of the entry receptors ICAM-1 and DAF was demonstrated on graphs of representative cell lines. After double staining with anti-ICAM-1 and anti-DAF antibodies, flow cytometry was used to measure the expression of CVA21 cellular entry receptors ICAM-1 and DAF in the B-cell lymphoma cell line SCOT and the B-prolymphocytic leukemia cell line JVM13 , relative levels on the surface of the acute promyelocytic leukemia (APML) cell lines NB4 and HL-60, the monocytic leukemia cell line U937 and the multiple myeloma cell line H929. The B-cell lymphoma cell line SCOTT, the B-prolymphocytic leukemia cell line JVM13, and the MM cell line H929 all demonstrated expression of ICAM-1 and DAF, compared to crosslinkers, in each dot plot (Fig. 11A and B). Stained cells are very evident in the upper right quadrant. The monocytic leukemia cell line U937 also demonstrated elevated levels of ICAM-1 and DAF expression, although to a lesser extent. However, in the acute promyelocytic leukemia (APML) cell lines NB4 and HL-60, most of the cells were positive for DAF expression and negative for ICAM-1.

MM cells are susceptible to lytic infection by CVA21

In order to test whether the multiple myeloma cells expressing ICAM-1 and DAF are susceptible to the oncolytic effect of CVA21, the cells were infected with CVA21 (MOI ~ 5 TCID ₅₀ /cell) at 37°C for 48h. Each multiple myeloma cell line showed initial signs of CVA21 infection at 24h. Between 24-48h post-infection, the greatest cytopathic effect was observed in the U266, RPMI-8226 and NCI-H929 cell lines, visualized by cell aggregation and uptake of trypan blue (Figure 2). Cytopathic effects were minimal in human peripheral blood mononuclear cells compared with three multiple myeloma cell lines.

Infection of CVA21 inhibits the growth of MM cells

The differential killing and growth inhibitory effects of CVA21 on multiple multiple myeloma cell lines were examined in more detail using the MTT cell viability assay. Inoculated U266, RPMI-8226 and NCI-H929 cells were exposed to increasing concentrations of CVA21 for 48 hours. Oncolysis of CVA21 was determined by MTT survival as a function of increasing doses of CVA21. Figure 3 shows growth inhibition curves and dose-dependent curves after exposure to CVA21. Each of the MM cell lines assayed was found to be sensitive, with the RPMI-8226 and U266 cell lines showing significant growth inhibitory responses to low concentrations of CVA21. Growth of normal PBMCs was inhibited after exposure to high concentrations of virus, but even at 20 MOI, 80% of PBMCs survived, compared to MM cell lines, each with a survival rate of less than 10%.

MM cells support the propagation of progeny CVA21

To determine the ability of CVA21-infected MM cells to produce infectious progeny for further oncolysis, the virus yield after CVA21 infection of U266, RPMI-8226, NCI-H929 and PBMC was measured. At 24 and 48 h post-infection, the virus production of the three myeloma cell lines showed a large increase in viral titers compared to the 0 h time point (Figure 4). However, PBMCs showed no increase in CVA21 production, providing evidence that CVA21 cannot replicate freely in normal human PBMCs. CVA21 proliferation in PBMCs continued up to 6 days without any increase in CVA21 replication or virus production (data not shown).

When assayed under similar but not identical conditions, the monocytic leukemia cell line U937 and the acute promyelocytic leukemia (APML) cell line HL-60 did not show increased CVA21 production at 24 or 48 h after infection, suggesting that CVA21 cannot Replicates freely within U937 or HL-60 (Figure 13).

CVA21 has a rapid growth rate in MM cells

One advantage of virotherapy of malignant cells is the ability to generate infectious progeny virus that can be further targeted to surrounding malignant cells at this site or even at a more distant site. Following simultaneous infection of the three multiple myeloma cell lines, whole cells and supernatants were collected at indicated time points (Figure 5). CVA21 was found to replicate very rapidly in cancer cell lines, and increased viral titers in infected multiple myeloma cells were observed as early as 4 hours after inoculation with CVA21 virus. One-step growth curve analysis of CVA21 in U266, RPMI-8226 and NCI-H929 cells demonstrated efficient viral replication to maximal titers between 8 and 12 h post infection.

CVA21-induced apoptosis in RPMI-8226 and NCI-H929 cells

To determine whether CVA21 induces apoptosis in multiple myeloma cells during infection, CVA21 was cultured with RPMI-8226, NCI-H929 or U266 cells at an MOI of approximately 10 TCID ₅₀ /cell, and passed DNA fragmentation at 24 h after infection. The assay measures apoptosis. CVA21 induced a DNA ladder characteristic of apoptosis (Figure 6) in infected RPMI-8226 and NCI-H929 cells (lanes 2 and 4, respectively), but not in infected U266 cells (lane 6). No DNA fragmentation was observed in U266 cells treated with CVA21, indicating that CVA21 oncolysis in this cell line did not induce apoptosis. The results indicated that CVA21 could induce apoptosis in RPMI-8226 and NCI-H929 cells, but might activate the anti-apoptotic signaling pathway during lysis of U266 myeloma cells. Lanes 1, 3 and 5 contain DNA extracted from pseudo-infected control cells.

Ex vivo purification of myeloma co-cultures containing malignant and non-malignant cells

The lysis of multiple myeloma cells but not normal cells by CVA21 was studied by mixing multiple myeloma cells with normal human peripheral blood mononuclear cells to generate approximately 10% tumor burden and purging with CVA21 for 3 days. Tumor specificity. Viable and non-viable populations of normal PBMC and multiple myeloma cells could be distinguished by staining with anti-CD138-FITC and PI. As seen in Figure 7 (“No virus”), viable RPMI-8226 or U266 cells (CD138 ⁺ /PI ⁻ ) were seen in the lower right quadrant of the flow cytometry dot plot prior to infection with CVA21 . After purging RPMI-8226 or U266 co-cultures with CVA21 at an MOI of 1 _TCID50 /cell for 3 days, less than 0.26% and 0.51% of viable myeloma cells remained, respectively. CVA21 was able to purify approximately 98% of RPMI-8226 and approximately 95.7% of U266 cells from co-cultures. A viable lymphocyte population is shown in the lower left quadrant, negative for CD138 and PI staining. Following infection with virus, these populations remained relatively unchanged even after 3 days of exposure to CVA21.

Purification of multiple myeloma cells from clinical bone marrow samples

After demonstrating potent oncolytic activity in vitro against several multiple myeloma cell lines, ex vivo purification of cancer cells from two primary multiple myeloma bone marrow samples was confirmed. Bone marrow aspirates from two routinely diagnosed patients were processed into single-cell suspensions. Cells were first stained with anti-CD138 and anti-ICAM-1 antibodies to determine surface expression of ICAM-1 on multiple myeloma cells. A clear correlation was found between myeloma status (CD138 ⁺ ) and ICAM-1 expression in two clinical samples, one of which is depicted in Figure 8A. In clinical sample #001, approximately 37% of all cells were multiple myeloma plasma cells and expressed the CD138 marker and ICAM-1. A similar proportion of myeloma cells was also found in the second patient, in which approximately 41% of the cells stained with CD138 and ICAM-1 (data not shown).

The results of the MTT assay on clinical samples from patients confirmed that treatment with CVA21 effectively inhibited the growth of myeloma cells in a dose-dependent manner after incubation with different concentrations of virus for 48 h. After challenging primary tumor cells (sample #001) with different concentrations of CVA21, the percentage of proliferating cells decreased steadily with increasing virus input (Figure 8B). Because primary tumor samples also contain a fraction of non-malignant cells, the specificity of CVA21-specific infection of MM cells still needs to be determined.

To test whether CVA21 selectively mediates the specific lysis of multiple myeloma cells but not normal cells, primary tumor samples were infected with different concentrations of CVA21 for 48 h, and then stained with anti-CD138 antibody and propidium iodide for analysis. The viability of CD138 ⁺ and CD138 ^- cells was measured by flow cytometry ( FIG. 9A ), and at 48 h, cytopathic effects were observed for three concentrations of CVA infection ( FIG. 9B ). Tumor cells from clinical sample #001 were cultured with CVA21 at concentrations of 0, 2.75, 5.5, and 11 TCID ₅₀ /cell for 48 h, and it was shown that with increasing virus input, viable myeloma cells (CD138 ⁺ /PI ^- ) decreased in percentage (Fig. 9A). Primary tumor cells infected with _11TCID50 /cell of CVA21, the percentage of detected malignant myeloma cells decreased by 74%. In this primary tumor sample, there was a steady increase in the number of non-viable myeloma cells (CD138 ⁺ /PI ⁺ ), corresponding to a decrease in viable myeloma cells (CD138 ⁺ /PI ⁻ ).

These results were mirrored in other clinical samples as well, with a maximum 90% reduction of viable tumor cells after longer incubation with CVA21 (data not shown). Even without virus treatment, low levels of non-viable cells were present in both clinical samples, and more importantly, the level of non-viable ^CD138- cells was basically not increased in the tested clinical samples after virus treatment. Another factor to consider is that ex vivo culture of these cells for 48 h also resulted in low levels of cell death, as observed in no virus controls. The percentage of viable multiple myeloma cells remaining in clinical sample #001 after treatment with different concentrations of CVA21, summarized from the flow cytometry data, is shown in Figure 9C.

To further evaluate the clinical utility of CVA21 as an ex vivo purging agent, additional BM aspirates were studied. The results of infection of BM aspirates from 5 MM patients and 2 MGUS patients with approximately 3 and 10 MOI concentrations of CVA21 are shown in Figure 10B(i). The combined results of a total of 19 clinical samples of different conditions (Table 1) are shown in Figure 10B(ii). CD138 ⁺ cell populations in all clinical samples examined expressed ICAM-1; a representative dot plot from patient #006 is shown (Figure 10A). After treatment with virus alone, CVA21 had potent oncolytic purging in all 7 clinical samples tested, as demonstrated by a substantial reduction in viable CD138 ⁺ cells and a depletion of MM cells in sample #008 The maximum ratio was 97.2% (Figures 10B(i) and 10E). Levels of viable CD138 ^- normal cells did not substantially decrease after virus treatment (FIG. 10D). More importantly, the progenitor cells in this fraction still retained the ability to support hematopoiesis. Figure 10C demonstrates that viable progenitor stem cells can be cultured after treatment of primary samples with CVA21; the average number of CFU-GM colonies from these three patient samples is shown. The number of CFU-GM colonies was 57-70% lower in virus-treated groups compared to sham-infected control samples.

Susceptibility of hematological cancer cell lines to CVA13, CVA15, and CVA18

Other group A coxsackieviruses that use ICAM-1 to infect and destroy cells, such as CVA13, CVA15, and CVA18, were also able to inhibit the growth of multiple myeloma cells. Using RPMI-8226 cells as a representative multiple myeloma cell line, treatment of these cells with CVA13, CVA15 and CVA18 demonstrated similar antitumor effects to CVA21 at concentrations ranging from 0.1 to 100 _TCID50 /ml (Fig. 12A). The effects of CVA13, CVA15, CVA18 and CVA21 were less consistent when tested on non-multiple myeloma cancer cell lines U937 and HL-60 (Figure 12B and C). Although some growth inhibitory effects were observed with each virus on U937 and HL-60 cells, further studies of the yield of CVA21 growth after infection of these cells showed that the cancer cell lines U937 and HL-60 were unable to support viral replication and thus could not Ideal candidate for viral therapy (Figure 13).

discuss

Coxsackievirus A21 is a new oncolytic agent that is beginning to be shown to be an effective antineoplastic agent against malignant melanoma. CVA21 is a common picornavirus associated with mild upper respiratory tract infections and has not been found to cause serious human disease (Rueckert RR. Picornaviridae: The Viruses and Their Replication (Picornaviridae: Viruses and their replication). In: Fields BN, Knipe DM, Howley PM eds. Fields Virology. Vol 1. Philadelphia: Lippincott-Raven; 1996: 609-645). In vitro assays on several MM cell lines demonstrated that CVA21 is a potent oncolytic agent against multiple myeloma. This conclusion is supported by the observed cytopathic effect of CVA21 on MM cell lines (Figure 2), and the apparent growth inhibition/cytotoxicity of CVA21 on MM cells as demonstrated by the MTT assay (Figure 3).

The data reported here also demonstrate that other picornaviruses (examples are CVA13, CVA15 and CVA18) are potent oncolytic agents against MM cells (Figure 12).

As demonstrated here for the reactivity of the monocytic leukemia cell line U937 to CVA13, CVA15, CVA18 and CVA21, this effect was not restricted to MM cells, although it was significantly more dose-dependent than MM cells (Fig. 12B).

Using flow cytometry, it was confirmed that the multiple myeloma cell lines U266, RPMI-8226, and NCI-H929 expressed high levels of the CVA21 viral entry receptors ICAM-1 and DAF (Fig. 1), which are lytic molecules of CVA21. determinants of tumor selectivity and efficacy.

Expression of ICAM-1 and DAF in cell lines established from other hematological cancers is also demonstrated here, highlighting the broad potential utility of using picornaviruses to treat hematological cancers. As shown in Figure 11, the B-cell lymphoma cell line SCOTT and the B-prolymphocytic leukemia cell line JVM13 both demonstrated expression of ICAM-1 and DAF. The monocytic leukemia cell line U937 also demonstrated elevated levels of ICAM-1 and DAF, albeit to a lesser extent. Consistent with the apparent lack of detectable susceptibility of the acute promyelocytic leukemia (APML) cell line HL-60 to CVA21, most HL-60 cells stained negative for ICAM-1.

The B-cell lymphoma cell line SCOTT and the B-prolymphocytic leukemia cell line JVM13 grew very slowly under the culture conditions used here. Therefore, it is not possible to directly demonstrate the oncolytic infection effect of CVA on any of these cell lines as it has been demonstrated for various other hematological cancer cell lines. However, given the demonstrated expression of ICAM-1 and DAF on each of these cell lines, applicants believe that each of B cell lymphoma and B prolymphocytic leukemia is susceptible to the methods of the invention of.

High levels of ICAM-1 expression in MM cells have been well described in the literature, leading to some proposals for anti-ICAM-1 mAb therapy as a potential treatment for MM (van de Stolpe A, van der Saag PT. Journal of Molecular Medicine 1996; 74:13-33; Huang YW et al., Cancer Res. 1995; 55:610-616). The reason for this high level of ICAM-1 expression in MM cells can be explained by the constitutive activation of the transcription factor NF-κB in MM cells (Hideshima T et al., J Biol Chem. 2002; 277:16639-16647).

NF-κB is a cell survival factor related to MM pathogenesis and drug resistance, and it is an important regulator of adhesion molecule expression in MM cells (Chauhan D et al., Blood. 1996; 87: 1104-1112). The 5' promoter region of the ICAM-1 gene contains several κB enhancer elements and represents one of the most important transcriptional regulatory elements in the regulation of ICAM-1 expression. Activation of NF-κB in MM cells and the resulting upregulation of adhesion molecules such as ICAM-1 have been shown to increase the binding of MM cells to bone marrow stromal cells (BMSCs) (HideshimaT et al., Oncogene. 2001; 20: 4519-4527). Adhesion of MM cells to BMSCs has been shown to induce transcriptional upregulation of NF-κB-dependent IL-6 (interleukin-6) by BMSCs, a growth and anti-apoptotic factor in MM (Chauhan D et al., Blood. 1997;89:227-234).

The MTT/growth inhibition assay showed that the highest level of cytotoxicity/growth inhibition was observed at an initial CVA21 input of ₂₀ TCID50/cell (Figure 3). Our results showed that CVA21 had potent cytotoxic and cytocidal effects on all three multiple myeloma cell lines tested and was less cytotoxic to normal human peripheral blood mononuclear cells (Fig. 3), confirming that Cytopathic effects observed after CVA21 infection (Fig. 2).

A desirable property of preferred oncolytic viruses is their ability to produce progeny virus, which can disseminate to surrounding uninfected tumor cells at the site or at a distant site to cause further oncolysis. The results showed that CVA21 was able to replicate efficiently in each of the multiple myeloma cell lines, supporting its use as a replication-competent oncolytic agent. In different myeloma cell lines, replication of CVA21 amplified the initial input dose by 100 to 1000-fold, whereas normal PBMCs infected with CVA21 under the same conditions were free of proliferative virus (Fig. 4).

Another important property of preferred viral oncolytics is an optimal level of replication efficiency. From the one-step virus growth curve analysis, it was found that CVA21 efficiently and rapidly replicated in MM cells, reaching the peak level of virus production between 8 and 12 h after infection (Fig. 5). This relatively rapid replication cycle facilitates the ability of progeny virus to shed and control MM disease progression. CVA21 inhibits the replicative capacity and cytotoxicity of MM cell growth, which is further evidence supporting the use of CVA21 virus in the treatment of MM, such as in vivo treatment of MM, and ex vivo selective purification of MM.

The results reported here demonstrate the generation of progeny virus, while preferentially for allowing dissemination to surrounding uninfected tumor cells at this site or at a distant site to cause further oncolysis, for oncolytic activity by picornaviruses. Infection-induced cell death in blood cancers is not required. The monocytic leukemia cell line U937 (Figure 12), which proved here to be susceptible to CVA, also did not demonstrate an observable ability to support the growth of CVA21 (Figure 13). Multiple administrations of picornaviruses may be of particular benefit in the treatment of these cancers.

Although the exact mechanism by which CVA21 causes MM cell death remains to be determined; the process is highly dependent on cellular transformation events, as well as genetic mutations present in different clinical samples and MM cell lines. CVA21 and other picornaviruses are thought to mediate cell death by a combination of halting host cell protein synthesis, inhibiting cellular glycoprotein transport, inducing apoptosis, and proteolytic digestion of transcription factors (Rueckert RR. Picornaviridae: The Viruses and Their Replication (Picoviridae: Viruses and their replication). In: Fields BN, Knipe DM, Howley PM, eds. Fields Virology. Vol. 1. Philadelphia: Lippincott-Raven; 1996:609-645). According to the DNA fragmentation assay performed on CVA21-infected RPMI-8226 and NCI-H929 cells, CVA21 was able to induce apoptosis in these two cell lines, but not in U266 cells (Fig. 6). The survival and growth of U266 cells depend on autocrine or paracrine IL-6 stimulation. Linked to cytokine-mediated activation of transcription factors such as signal transducer and activator of transcription (STAT)-3, the IL-6 signaling pathway may have some effect on the anti-apoptotic response of U266 cells after CVA infection.

A recent study (Bharti AC et al., Blood 2004; 103:3175-3184) demonstrated that U266 cells showed constitutively activated STAT3 in the nucleus, whereas RPMI-8226 cells were negative for constitutively activated STAT3. However, chemical inhibitors of NF-κB have been shown to induce apoptosis in U266 and RPMI-8226 cell lines, regardless of STAT3 activation, suggesting that STAT3 appears to have little significance in apoptosis triggered by chemical NF-κB inhibitors. But our results show that activation of STAT3 signaling pathway actually has an anti-apoptotic role in the oncolysis of CVA21-infected myeloma cells.

The role of NF-κB family transcription factors is indeed very complex, there is evidence that NF-κB may play a role in pre-apoptotic and anti-apoptotic signal transmission. New drugs for the treatment of MM, such as thalidomide (Keifer JA et al., JBiol.Chem.2001; 276:22382-22387), proteasome inhibitor PS-341 (Hideshima T et al., Cancer Res.2001; 61: 3071-3076), and arsenic trioxide As ₂ O ₃ (Kapahi P et al., J.Biol.Chem.2000; 275:36062-36066) have been shown to inhibit the activation of NF-κB, and help overcome the previous preclinical and early Drug resistance in common MM in clinical trials.

Data collected from clinical samples in this study suggest that the observed oncolytic capacity of CVA21 and other picornaviruses is not limited to multiple myeloma cell lines, but is also found in other hematological cancer cell lines and in primary tumors infected ex vivo. Also effective in tumor samples. Clinical samples had significant levels of CD138 plasma cells in the bone marrow, all expressing ICAM-1 when detected by flow cytometry (Fig. 8A). Upon infection with increasing doses of CVA21, substantial arrest of growth and purification of tumor cells was observed in all clinical samples, with a maximum of 98% inhibition achieved in one sample (#008). To evaluate the effect of CVA21 on normal and malignant cells in more detail, cells were stained with anti-CD138 and PI, and then analyzed using flow cytometry. In 14 of 17 measurable clinical samples, CVA21 effectively reduced the percentage of malignant cells by >80% (Fig. 10E). Analysis of additional clinical samples (reaching a total of 19) confirmed the generality of these observations (Figure 10). Increased viral input and spread of CVA21 infection increases the level of cancer cell purification. The clinical samples used in this study confirmed that CVA21 is beneficial for the purification of MM cells.

The substantial eradication of myeloma tumor cells from patient samples, together with the persistent viability of progenitor stem cells, clearly demonstrates that CVA21 specifically kills myeloma cells with relatively minimal effects on normal progenitor cells. Additionally, treatment with CVA21 was effective in both previously untreated as well as difficult-to-pretreat, chemotherapy-resistant tumor cells, suggesting that CVA21 may be a useful treatment option for MM. Also of interest is the role of CVA21 in clearing precancerous plasma cells from patients diagnosed with asymptomatic pre-MM, MGUS. Thirty percent of these patients go on to develop MM, but it is currently impossible to predict the prognosis for each individual, so this benign dyscrasia is usually left untreated. The potential use of CVA21 for prophylaxis in this subset of patients suggests a novel role for viral therapy in the prophylactic treatment of MM.

In addition to the direct cytotoxic effect on malignant cells, CVA21 may have other functions in the treatment of MM. Although most MM cells are initially sensitive to existing drug treatments, drug resistance is a common feature of MM in most cases. The combination of CVA21 as a chemosensitizer with drugs such as vincristine and doxorubicin gives better results than drugs alone, which is a further reason to study CVA21 in MM.

Currently, the avenues for treating multiple myeloma are limited and new therapeutic approaches are needed. Although the immunodeficiency status of patients with multiple myeloma is a real concern for virotherapy in patients with this malignancy, it is in these patients that the results obtained with virotherapy with CVA21 will be most successful due to lack of antiviral immunity. Additionally, MM is a disease characterized by the accumulation of slowly proliferating malignant plasma cells, thus presenting an ideal opportunity to effectively control tumor burden through oncolytic action of CVA21. Viral therapy is predicted to be most effective in slowly growing malignancies because rapidly growing tumors can escape viral oncolysis if dissemination of progeny virus is ineffective.

Should CVA21 have adverse consequences for patients, antiviral compounds such as pleconaril (Rogers JM et al., Adv Exp Med Biol. 1999; 458:69-76), or immune serum globulin (Rotbart HA. Pediatr Infect Dis J. 1999 ; 18:632-633) can control the infection of CVA21. Another option involves using CVA21 to purify MM and other hematological tumor cells ex vivo. Recently, a novel purification strategy using oncolytic reoviruses for autologous stem cell transplantation was evaluated in hematologic malignancies, but ex vivo purification of enriched multiple myeloma cells from a mixture of aphaeresis products was incomplete (Thirukkumaran CM et al., Blood. 2003; 102:377-387). CVA21 is a unique oncolytic virus that is selective for the cytotoxicity of multiple myeloma cells and may also have enhanced potential as an ex vivo purging agent.

Summarize

Our data demonstrate that CVA21 and other picornaviruses (such as CVA13, CVA15, and CVA18) are potent oncolytic agents against multiple myeloma cell lines and other hematological cancer cell lines in vitro, and direct oncolysis of MM and hematological cancer cells in vivo There are also potential applications in the role and ex vivo purification of autologous grafts of peripheral blood stem cells.

The present invention has been described in detail above, and those skilled in the art should understand that, as shown in the specific embodiments, various changes and modifications can be made to the present invention without departing from the spirit or scope of the present invention as broadly described. and/or modifications, including various omissions, substitutions and/or changes in form or details. Accordingly, the embodiments of the present invention are to be considered in all respects as illustrative and not restrictive.

references

Alain T et al., Reovirus therapy of lymphoid malignancies. Blood. 2002;100:4146-4153.

Alley MC et al., Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988;48:589-601.

Berendt AR et al., The binding site on ICAM-I for plasmodium falciparum-infected erythrocytes overlaps, but is distinct from the LFA-1-binding site. Cell. 1992;68:71-81.

BhartiAC et al., Nuclear factor-{kappa}B and STAT 3 are constitutively active in CD138 ⁺ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis. Blood. 2004;103:3175-3184.

ChauhanD et al., Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B.Blood.1996;87:1104-1112.

Chauhan D et al., Interleukin-6 inhibits Fas-induced apoptosis and stress-activated protein kinase activation in multiple myeloma cells. Blood. 1997; 89: 227-234.

Grote D et al., Live attenuated measles virus induces regression of humanlymphoma xenografts in immunodeficient mice. Blood. 2001; 97: 3746-3754.

Hideshima T et al., The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 2001; 61: 3071-3076.

Hideshima T et al., The role of tumor necrosis factor alpha in thepathophysiology of human multiple myeloma: therapeutic applications. Oncogene. 2001; 20: 4519-4527.

Hideshima T et al., NF-kappa B as a therapeutic target in multiple myeloma. J Biol Chem. 2002; 277: 16639-16647.

Huang YW et al., Anti-CD54(ICAM-1) has antitumor activity in SCID mice with human rnyeloma cells. CancerRes.1995;55:610-616.

Hughes et al., J. Gen Virol. 1989, 70:2943.

Kapahi P et al., Inhibition of NF-kappa B activation by arsenite through reaction with a critical cysteine in the activation loop of Ikappa Bkinase. J. Biol. Chem. 2000; 275: 36062-36066.

Keifer JA et al., Inhibition of NF-kappa B activity by thalidomidethrough suppression of IkappaB kinase activity. J Biol. Chem. 2001; 276: 22382-22387.

Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.

Rogers JM et al., Pleconaril. A broad spectrum antipicomaviral agent. Adv Exp Med Biol. 1999;458:69-76.

Rotbart HA. Antiviral therapy for enteroviral infections. Pediatr Infect Dis J. 1999; 18: 632-633.

Rueckert RR. Picornaviridae: The Viruses and Their Replication. In: Fields BN, Knipe DM, Howley PM, eds. Fields Virology. Vol.1. Philadelphia: Lippincott-Raven; 1996: 609-645.

Schirrmacher V et al., Antitumor effects of Newcastle Disease Virus invivo: local versus systemic effects, Int J Oncol. 2001;18:945-952.

Schmidt, NJ et al., Proc. Soc. Exp. Biol. Med., 1961, 107:63.

Schwab G et al., Characterization of an interleukin-6-mediated autocrine growth loop in the human multiple myeloma cell line, U266.Blood.1991;77:587-593.

Shafren D et al., J. Virol 1997, 71: 4736.

Shafren DR et al, Systemic therapy of malignant human melanomatumors by a common cold-producing enterovirus, coxsackievirus a21. Clin Cancer Res. 2004;10:53-60.

Sickles G.M., Proc. Soc. Exp. Biol. Med. 102:742.

The Merck Index, Thirteenth Edition, Merck & Co. Inc, Whitehouse Station, NJ, USA.

Thirukkumaran CM et al., Reovirus oncolysis as novel purging strategy for autologous stem cell transplantation. Blood. 2003; 102: 377-387.

PCT/AU00/01461 (WO01/37866), titled "A method of Treating a Malignancy in a Subject and a Pharmaceutical Composition For Use in Same (a method of treating a malignant tumor in a subject and a pharmaceutical composition used in the method)"

PCT/AU2003/001688 (WO2004/054613), titled "A method of treating a malignancy in a subject via direct Picomaviral-mediated oncolysis (method for treating malignant tumors in subjects via direct oncolysis mediated by picornaviruses)".

PCT/AU2005/000048, entitled "Modified oncolytic viruses".

Claims (7)

1. Use of a group A coxsackievirus selected from CVA13, CVA15, CVA18 and CVA21 in a therapeutically effective amount for the preparation of a medicament for treating multiple myeloma in a subject, wherein said virus can induce at least some multiple myeloma Myeloma cells undergo viral oncolysis. 2. The application according to claim 1, wherein the virus is administered intravenously, intratumorally, intraperitoneally, intramuscularly, and the malignant cells in the autologous graft are purified ex vivo before autologous stem cell transplantation, or in the Purification of malignant cells in autografts ex vivo prior to transplantation. 3. The use according to claim 2, wherein said autologous graft comprises hematopoietic stem cells. 4. The application according to claim 1, wherein the dose of the virus is in the range of 0.01 to 1000 virus infection units per cell. 5. The application according to claim 1, wherein the multiple myeloma cells are excessive to virus-cell entry receptor molecule intercellular adhesion molecule-1 (ICAM-1) and/or decay accelerating factor (DAF) Express. 6. The use according to claim 1, wherein the multiple myeloma cells constitutively express NF-κB. 7. The use according to claim 1, wherein said subject is human.

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