WO2000020433A1 - Agents cytotoxiques pour eliminer de maniere selective le lymphome et les cellules de leucemie et procedes d'utilisation correspondants - Google Patents

Agents cytotoxiques pour eliminer de maniere selective le lymphome et les cellules de leucemie et procedes d'utilisation correspondants Download PDF

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WO2000020433A1
WO2000020433A1 PCT/US1999/022733 US9922733W WO0020433A1 WO 2000020433 A1 WO2000020433 A1 WO 2000020433A1 US 9922733 W US9922733 W US 9922733W WO 0020433 A1 WO0020433 A1 WO 0020433A1
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cells
adenovirus
infection
cytotoxicity
cll
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Roger Strair
Arnold Rabson
Daniel Medina
Eileen White
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University Of Medicine And Dentistry Of New Jersey
Rutgers, The State University Of New Jersey
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • This invention relates to the fields of virology and oncology. More specifically, this invention provides adenoviral-based agents which are selectively cytotoxic for malignant cells of lymphoid origin.
  • lymphoid malignancies Many patients with disseminated malignancies are treated with chemotherapy at some point during their illness. While response rates are generally high in some malignancies, such as lymphoid malignancies, only a subset of patients will be cured. Even amongst the lymphoid malignancies some (e.g. chronic lymphocytic leukemia (CLL) , stage III/IV follicular small cleaved cell lymphoma, stage III/IV marginal zone lymphoma, stage III/IV mantle cell lymphoma, and multiple myeloma) are currently incurable with standard dose chemotherapy.
  • CLL chronic lymphocytic leukemia
  • Novel therapeutics under study for patients with these diseases include: high dose chemotherapy +/- radiation therapy (HDC) with autologous or allogeneic hematopoietic stem cell rescue (HSCR) ; immunotherapeutics such as interferon and interleukin-2 ; vaccines; monoclonal antibodies (or other targeted therapeutics, such as peptides/peptide-conjugates) ; novel chemotherapeutic agents; and antisense oligonucleotides .
  • HDC high dose chemotherapy +/- radiation therapy
  • HSCR autologous or allogeneic hematopoietic stem cell rescue
  • immunotherapeutics such as interferon and interleukin-2
  • vaccines monoclonal antibodies (or other targeted therapeutics, such as peptides/peptide-conjugates)
  • novel chemotherapeutic agents include antisense oligonucleotides .
  • Adenoviruses are non-enveloped DNA viruses. The genome consists of a linear, double
  • Adenovirus mRNA is produced in the nucleus and transported to the cytoplasm.
  • the important processing event of splicing was discovered.
  • Initiation of eukaryotic DNA replication in vitro was first studied successfully in extracts of cells infected with adenovirus type 2. These studies have resulted in the definition of both viral and eukaryotic host-cell factors involved in the initiation of DNA synthesis. Additional studies of adenovirus infection have contributed to our understanding of transcriptional regulation, cell cycle control, and cell death. Productive infection of cells with adenovirus often results in cytotoxicity and the release of new viral progeny.
  • adenoviruses may contribute to the cytotoxic effects observed after infection by wild-type or mutant adenoviruses. For example, productive infection, abortive infection with the induction of apoptosis, or a combination of these processes may result in death of infected cells.
  • productive infection, abortive infection with the induction of apoptosis, or a combination of these processes may result in death of infected cells.
  • Each of these possible outcomes results from complex interactions between host and virus that impact on viral replication, host cell cycle regulation, viral and host cell gene expression, the induction of apoptosis and host cell survival.
  • New insights into adenovirus biology and basic cellular physiology have made it apparent that there are multiple biological genetic features of several malignancies which suggest that they may serve as ideal targets of cytotoxicity mediated by adenoviruses that have been attenuated by mutation.
  • the initial studies demonstrating specific cytotoxicity of mutant adenoviruses in selected epithelial malignancies utilized an adenovirus with a mutation in the EIB 55K gene product (1,2) .
  • the rationale for the specificity of the cytotoxicity induced by infection with that virus was based on the complementation of a genetic defect of the virus (EIB 55K mutation) by a genetic feature of the malignant cell (p53 mutation) which allows the mutant virus to replicate and induce cytotoxicity.
  • This selective cytotoxicity induced by this virus mutant has been presumed to be a consequence of the requisite inactivation of p53 by the EIB 55k polypeptide during a fully productive infection. In the absence of the EIB 55k polypeptide (and presence of wild-type p53), an abortive infection results and cytotoxicity is reduced.
  • the present invention provides novel compositions and methods of the treatment of malignancies using novel adenovirus mutants.
  • the specific mutant to be used will often be selected individually from a panel of viruses which are used to infect a primary sample of the malignancy.
  • a method is provided for identifying adenovirures which mediate selective elimination of malignant cells. The method comprises obtaining a biological sample from a patient suspected of containing malignant cells which is then incubated in separate culture vessels with a panel of mutant adenovirus strains under conditions suitable for viral growth and infection. Following a suitable time period the cells are then assessed for selective cytotoxic effecs mediated by a strain of adenovirus .
  • Adenoviruses which are selectively cytotoxic for the malignant cells are then utilized to eradicate the malignant cells from the patient.
  • the selected adenovirus in a suitable biological buffer medium is then delivered to the patient via intravenous infusion or direct tumor injection.
  • the malignant cells are of lymphoid origin. Any adenovirus which is selectively cytotoxic for malignant cells is contemplated for use in the present invention.
  • Adenovirus strains known in the art are also suitable for eradicating cancer cells from biological samples and/or patients.
  • adenovirus selected from the group consisting of wild-type adenovirus, dl309, dl337, tsl25, ts400, adenovirus containing mutations in E1A, adenovirus containing mutations in EIB, adenovirus containing mutations in E2A, and adenoviruses containing mutations in E1A, EIB and or E2A (6-11) .
  • a method for purging stem cell grafts of malignant cells prior to reinfusion of the graft into a patient is provided.
  • a stem cell sample suspected of containing malignant cells is isolated from a patient.
  • Adenovirus in a suitable pharmaceutical carrier or biological buffer, which is selectively cytotoxic for the malignant cells of the patient, are identified and incubated with the sample under conditions whereby productive infection ensues and cell death results. This results in effective purging of the sample of malignant cells.
  • the stem cells are reinfused into the patient.
  • FIG. 1 is a histogram showing that CLL cells can be infected with an adenovirus encoding GFP .
  • CLL cells were infected with AdGFP, containing the GFP gene under the control of a CMV promoter (Quantum Biotechnologies, Montreal, Canada) .
  • AdGFP AdGFP
  • CMV promoter Quantum Biotechnologies, Montreal, Canada
  • the heavy line represents the fluorescence of infected cells.
  • the light line represents the fluorescence of uninfected cells .
  • Figures 2A and 2B are a pair of graphs demonstrating that adenovirus infection of CLL cells induces cytotoxicity.
  • Primary CLL cells from 7 patients were infected with Ad5d2309 or heat-inactivated Ad5d2309 (mock infected) . At the indicated times cell viability was determined by cell counting and trypan blue staining and compared to the viability of uninfected and mock infected cells.
  • Figure 2A the degree of cytotoxicity seen 4 days after infection of primary CLL with Ad5d2309.
  • Figure 2B - the degree of cytotoxicity seen 20 days after infection of primary CLL with Ad5dl309. In both figures, control refers to uninfected cells and mock refers to cells infected with heat inactivated virus.
  • Figures 3A and 3B are a pair of graphs showing that attenuated adenovirus infection of CLL cells induces cytotoxicity.
  • Primary CLL cells from 7 patients were infected with Ad5dl309, heat-inactivated Ad5d2309 (mock infected) , an EIA deleted adenovirus (E1A-) , or an adenovirus with a deletion of EIA and EIB (Pac3) .
  • cell viability was determined by cell counting and trypan blue staining and compared to the viability of uninfected and mock infected cells.
  • Figure 3A the degree of cytotoxicity seen 4 days after infection of primary CLL with each of the adenoviruses.
  • Figure 3B the degree of cytotoxicity seen 20 days after infection of primary CLL cells with each of the adenoviruses.
  • control refers to uninfected cells and mock refers to cells infected with heat inactivated virus.
  • Figure 4 is a graph showing a time course of cytotoxicity induced by adenovirus infection of CLL cells.
  • CLL cells from patient 7 were infected with Ad5d2309, Pac3 or Ad5d2337 and the degree of cytotoxicity was monitored by cell counting and trypan blue staining every 3-4 days.
  • Control refers to uninfected cells and mock refers to cells infected with heat inactivated virus .
  • Figure 5 is a graph showing that Ad5dl337 induces cytotoxicity after infection of primary CLL cells at a low multiplicity of infection (moi) .
  • CLL cells from patient 8 were infected with Ad5dl309, Pac3 or Ad5dl337 at different moi. Cell viability was determined by cell counting and trypan blue staining 8 days after infection. Control refers to uninfected cells and mock refers to cells infected with heat inactivated virus.
  • Figure 6 is a graph showing that adenoviruses do not induce cytotoxicity after infection of normal B-cells.
  • Cells obtained from a non-malignant human tonsil were infected with Ad5dl309, EIA- , Pac3 and Ad5d2337.
  • Ad5dl309, EIA- , Pac3 and Ad5d2337 were infected with Ad5dl309, EIA- , Pac3 and Ad5d2337.
  • the number of viable B- cells was determined by isolating viable cells and assaying for CD19 expression by flow cytometry.
  • Figures 7A and 7B are a pair of graphs showing that adenoviruses do not alter the ability of committed hematopoietic progenitor cells to form colonies in methylcellulose.
  • Blood mononuclear cells obtained after treatment of patients undergoing hematopoietic stem cell mobilization with G-CSF were infected with Ad5d2309 at an moi of 100 for the indicated periods of time and plated in methylcellulose (in the presence of IL-3, GM-CSF and epo) using commercially available media (Stem Cell
  • Colonies CFU-e, BFU-e, CFU-GM and CFU- GEMM were counted at 14 days. (-) indicates uninfected ; (+) indicates infected with Ad5d2309.
  • Figure 8 is a graph showing that temperature- sensitive viruses induce cytotoxicity after infection of CLL cells.
  • CLL cells from patient 7 were infected with a temperature sensitive viruses containing mutations in the E2A gene (Ad5tsl25) at the restrictive (37°) and permissive (33°) temperatures. Cytotoxicity was determined by cell counting and trypan blue staining at various times after infection. Control refers to uninfected cells and mock refers to cells infected with heat inactivated virus. Closed box - mock infection; open box - infection with Ad5d2309; triangle - infection with Ad5tsl25 at 33°; diamond - infection with Ad5tsl25 at 37°.
  • the present invention is directed to methods for killing lymphoma and leukemia cells.
  • the present inventors have discovered that infection of malignant cells obtained from patients with chronic lymphocytic leukemia (CLL) with adenovirus or mutants thereof results in measurable cytotoxicity and cell death.
  • Adenoviruses can successfully infect primary CLL cells and a replication-competent strain of human adenovirus 5 (Ad5dl309) results in cytotoxicity.
  • Ad5dl309 human adenovirus 5
  • Adenovirus-mediated cytotoxicity was also seen after infection of CLL cells with a variety of viruses attenuated by mutations in the adenovirus early region 1 (El) or early region 2 (E2) .
  • HeLa cells and 293 cells were grown in L modified Eagle's medium supplemented with 2mM L- glutamine, 10% fetal bovine serum (FBS) and antibiotics.
  • CLL cells were maintained in RPMI-1640 medium supplemented with 20% FBS, L-glutamine, antibiotics and 50 u/ml of IL-4 (Boehringer Mannheim) .
  • Ad5dl309 The viruses used in the experiments, Ad5dl309, EIA- , Pac3, EIB- and Ad5dl337 were described previously 14"16 .
  • the temperature sensitive (ts) virus Ad5tsl25 was generously provided by Daniel Klessig (Rutgers University, Piscataway, NJ) 20 . All viruses, with the exception of the Ad5tsl25, were grown and titered on 293 cells at 37°C.
  • the Ad5tsl25 virus was grown at 33°C and was titered on 293 cells at 33°C and 37°C.
  • Ad5dl309 was heat inactivated at 65° for 30 minutes.
  • the recombinant adenovirus containing the green fluorescent protein (GFP) gene was purchased from Quantum Biotechnologies, Montreal, Canada. That virus
  • AdGFP contains the GFP gene, under the control of a CMV promoter, in the EIA region of an adenovirus 5 containing a deletion in the E3 region.
  • Infections with Ad5tsl25 were conducted at both the permissive temperature (33° C) and the nonpermissive temperature (37°C) . After infection, the cells were washed three times with Dulbecco's phosphate buffered saline to remove non- adsorbed virus.
  • the cells were then pelleted by centrifugation at 500xg for 10 min, resuspended in 2 ml of medium and cultured in 24-well plates. At the indicated times, samples were removed for determination of cell viability by trypan blue staining, virus yield, and apoptosis. Mock infections utilized heat inactivated Ad5dl309.
  • Cell Viability At the indicated times, cells in the various replicate wells were resuspended and 100 ⁇ l were removed and analyzed for cell viability by trypan blue staining, and cell count. Cytotoxicity was calculated by a comparison of viable cell numbers in the infected samples as compared to uninfected controls.
  • Virus yield assay To assess virus replication, cell free supernatants were collected at the indicated times. The virus titer was determined by plaque assay on either 293 cells or Hela cells. Briefly, 1 ml of serial 10-fold dilutions of the cell free supernatant were added to 60 mm dishes containing a confluent layer of either 293 cells or Hela cells followed by incubation for 1 hr at 37°C. After incubation, the virus was removed and the plates were overlaid with agar as described 32 . Viral plaques were counted on day 14 and the viral titer was determined.
  • the following examples are provided to describe the invention in further detail. These examples, which set forth the preferred mode presently contemplated for carrying out the invention, are intended to illustrate and not to limit the invention
  • adenoviruses to infect primary CLL cells was established by infection of CLL cells with a recombinant adenovirus expressing the green fluorescence protein under the control of a CMV promoter (AdGFP, Quantum Biotechnologies, Montreal, Canada). Three days after infection, green fluorescence was measured by flow cytometry. A marked shift in fluorescence was seen after infection with AdGFP ( Figure 1) , indicating that primary CLL cells could be infected by adenoviruses.
  • AdGFP Quantum Biotechnologies, Montreal, Canada
  • adenoviruses induce cytotoxicity in CLL cells
  • cells were obtained from the blood of a series of patients. Each patient had a lymphocytosis that was morphologically and immunophenotypically characteristic of B-cell CLL.
  • populations of malignant cells at 90-99.9% purity were infected with Ad5dl309, a replication- competent strain of human adenovirus 5 containing a deletion in E3B 14 , and cell viability was monitored over the course of 20 days.
  • the Ad5dl309 virus was chosen since it has served as the parental strain for generation of well -characterized, mutant adenoviruses.
  • CLL cells from patient 7 persist in culture for > 22 days, allowing a time course of full cytotoxicity for this patient's cells to be undertaken (Figure 4) .
  • the parental virus (Ad5d2309) the highly attenuated virus containing a deletion of EIA and EIB (Pac3) , and the virus containing an EIB 19K deletion (Ad5dl337) were used to infect the CLL cells from patient 7.
  • Ad5dl337 was studied because it has been demonstrated to be highly cytotoxic to other cell types, resulting in cell death by apoptosis 2,3,16,25 .
  • cells from an eighth CLL patient were infected with Ad5dl309, the EIA deleted adenovirus (E1A-) , Pac3 or Ad5dl337 at different multiplicities of infection (moi) .
  • Ad5dl337 was the only virus that exhibited cell killing at day 8 ( Figure 5) .
  • Ad5dl309, the EIA- virus, and Pac3 all exhibited cytotoxicity.
  • Ad5dl337 was the most efficacious in inducing cell death ( Figure 5) .
  • Pac3 is listed twice in Table II as the supernatant from the infection on day 10 was titered on both 293 cells and HeLa cells.
  • the absence of detectable virus when the supernatant was titered on HeLa cells reflects the inability of the Pac3 virus, which contains a deletion in EIA and EIB, to generate plaques on HeLa cells (Table II) .
  • the Ad5tsl25 is listed four times in Table II, as the supernatants from infections at 33° and 37° were each titered at both 33° and 37°.
  • Table III demonstrates viral yields after a series of infections with a different patient's CLL cells.
  • the virus deleted of EIA and EIB (Pac3) and Ad5dl337 result in comparatively high virus yields in culture (>10 5 pfu/ml) .
  • infection of HeLa cells and 293 cells with the viruses produced during infection demonstrated the restricted host range and temperature sensitivity anticipated for infection by the temperature sensitive and Pac3 virus mutants.
  • Table III Comparable levels of viral production occur after infection of primary CLL cells with Ad5dl309, Ad5d2337 or Pac3.
  • CLL cells from patient 8 were infected with Ad5dI309, Ad5dl337, or Pac3.
  • Apoptosis as measured by subdiploid DNA content
  • Ad5dl337 a virus which readily induces apoptosis in other cell types 2,3, 15,l ⁇ , only resulted in a small increment in the number of cells with a subdiploid DNA content (as compared to mock infected cells) and an insignificant alteration of Hoechst 33258 staining or Annexin V staining. For example, at 6 days after infection 12% of CLL sample 7 cells infected with
  • Ad5dl337 had a sub-diploid DNA content compared to 7% of control or "mock" infected cells. These results imply that some of the cytotoxicity induced by infection may be a consequence of apoptosis, but the degree of apoptosis, as measured by the above studies, was far less than anticipated if apoptosis was the sole or major mechanism of cytotoxicity. Presumably, adenovirus gene expression and virus production in CLL cells results in cytotoxicity that is only partially a consequence of apoptosis.
  • B-cell CLL cells and even highly attenuated viruses were capable of inducing cytotoxicity.
  • p53 mutation is not common in CLL, with only 6% of standard CLL samples harboring mutations of p53 8-11 .
  • a broad spectrum of genetic aberrations have been described and many of these mutations may impact on cell cycle control or the regulation of apoptosis, and therefore render the malignant cell an excellent target for cytotoxicity after infection with an attenuated adenovirus.
  • EIA function is essential for productive infection, thus cellular mutations that activate downstream targets of EIA (for example, mutations or deletions of pRb) may be predicted to enhance replication of an EIA- adenovirus.
  • EIA itself has, under certain conditions, been demonstrated to mediate apoptosis 3,15,16,21"26 and infection with viruses containing EIA in the absence of the anti- apoptotoic functions of EIB enhances apoptosis in many cells.
  • Both the pRb and p300 binding domains may be important for this induction of apoptosis in certain cells, whereas pRb binding may be dispensable in other cell types 16,21"24 .
  • specific EIA mutations may be predicted to facilitate apoptosis and cytotoxicity in some cell types and the pro-apoptotic functions of EIA may be offset by EIB.
  • EIB 19K is of critical importance in preventing ElA-mediated apoptosis, and viruses that do not encode EIB 19K may facilitate cytotoxicity mediated by EIA and other viral genes, perhaps in conjunction with cellular changes that result in alterations of p300, pRb, Bcl-2 family members, or other cellular proteins which impact on the development of apoptosis 2,3,15,16,21"24 . Therefore, viruses with mutations in EIA, EIB or both may induce variable amounts of cytotoxicity, depending upon the specific genotype of the host .
  • Ad5dl337 consistently induced cytotoxicity in some samples of CLL to a greater extent than the parental Ad5dl309 (containing wildtype EIA and EIB) .
  • the biological functions of EIB 19K inhibit adenovirus cytotoxicity 2,3,15,16,21 , and viruses with a mutation of EIB 19K may be more cytotoxic than the parental virus in the context of low level virus production.
  • the cytotoxicity of the Pac3 virus is also interesting, considering the loss of both functional EIA and EIB, making Pac3 as highly attenuated as many of the adenovirus vectors commonly used for gene transfer.
  • E4 open reading frame 4 (E4orf4)
  • E4orf4 E4 open reading frame 4
  • the specific cellular genotype and phenot pe may complement adenoviruses with mutations necessary for productive infection, establish a milieu that fosters adenovirus-mediated apoptosis, or be cytotoxic as a consequence of other mechanisms, such as toxicity induced by adenovirus gene products.
  • the cytotoxicity of the Pac3 virus also demonstrates the capacity of a highly attenuated virus, similar to those used as vectors for gene transfer in clinical trials of human gene therapy, to replicate and induce cytotoxicity under certain conditions.
  • malignant cells from a patient with mantle cell lymphoma demonstrated cytotoxicity after infection with Ad5dl309, but no cytotoxicity was seen after infection with the Pac3 or EIA- viruses (unpublished) .
  • Understanding the nuances of the patterns of cytotoxicity seen after infections with different mutated viruses may lead to a better understanding of the functional state of the malignant cell, particularly as it relates to the cell cycle regulatory control and the pro-apoptotic/anti-apoptotic functions of the adenovirus El region.
  • EIA- and Pac3 viruses used in our studies and demonstrated to be cytotoxic to CLL cells contain the same degree of attenuation as many of the adenovirus vectors used for gene transfer/therapy 33"40 . Therefore, selectively cytotoxic adenoviruses might ultimately be developed for use ex vivo to "purge" hematopoietic stem cell grafts of CLL cells.
  • the concept of inducing cytotoxicity with Ad5tsl25 may also be relevant to this potential application of adenovirus- mediated cytotoxicity, as temperature-sensitivity may ultimately be incorporated into the viruses as an additional safety feature.
  • the phenotypic pattern of cytotoxicity induced by different members of the panel of viruses will provide the framework to correlate cytotoxicity with specific genetic features of the malignant cells, and provide additional information about the viral -host cell relationships that mediate cell death and productive infection.
  • Example II Screening primary malignant cells for selective adenovirus induced cytotoxity
  • Primary malignant cells from a patient are obtained from blood or a tissue biopsy and placed in culture.
  • the cells are infected with the panel of viruses and cytotoxicity is determined over the course of several days as described in Example I .
  • the virus that has induced the greatest degree of cytotoxicity is selected for either in vivo or ex vivo use as indicated by the condition of the patient.
  • One use entails selection of the specific virus for ex vivo purging of malignant cells contaminating hematopoietic stem cells.
  • a second use entails selection of a virus for injection into a tumor mass.

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Abstract

L'invention concerne des procédés d'identification d'adénovirus qui possèdent une cytotoxicité sélective vis-à-vis des cellules malignes d'origine lymphoïde. L'invention concerne aussi des procédés pour nettoyer les greffes de cellules souches des cellules malignes au moyen des virus cytotoxiques de la présente invention.
PCT/US1999/022733 1998-10-02 1999-09-30 Agents cytotoxiques pour eliminer de maniere selective le lymphome et les cellules de leucemie et procedes d'utilisation correspondants WO2000020433A1 (fr)

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AU64064/99A AU6406499A (en) 1998-10-02 1999-09-30 Cytotoxic agents for the selective killing of lymphoma and leukemia cells and methods of use thereof

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US10274298P 1998-10-02 1998-10-02
US60/102,742 1998-10-02

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WO2000020433A1 true WO2000020433A1 (fr) 2000-04-13

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5824544A (en) * 1995-03-24 1998-10-20 Genzyme Corporation Adenovirus vectors for gene therapy

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5824544A (en) * 1995-03-24 1998-10-20 Genzyme Corporation Adenovirus vectors for gene therapy

Non-Patent Citations (7)

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Title
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GAIDANO ET AL.: "Analysis of alterations of oncogenes and tumor suppressor genes in chronic lymphocytic leukemia", AM. J. PATHOL.,, vol. 144, no. 6, 1994, pages 1312 - 1319, XP002926238 *
LUCHER L.R.: "Abortive adenovirus infection and host range determinants", CURR. TOP MICROBIOL. IMMUNOL., vol. 199, 1995, pages 119 - 152, XP002926241 *
QUERIDO ET AL.: "Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the induction of DNA synthesis", J. VIROL., vol. 71, no. 5, May 1997 (1997-05-01), pages 3526 - 3533, XP002926240 *
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WHITE E.: "Regulation of p53-dependent apoptosis by E1A and E1B", CURR. TOP MICROBIOL. IMMUNOL., vol. 199, 1995, pages 33 - 58, XP002926242 *

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