JP2006517184A5 - - Google Patents

Description

Abbreviations Table A549: Human lung epithelial tumor cell line AcNPV: Autographa californica nuclear polyhedrosis virus BMDC: Bone marrow-derived dendritic cells BV422: CCL21 expressing recombinant baculovirus BV762: Raf expressing recombinant baculovirus CCL21: CC motif ligand 21 chemokine Secondary lymphoid tissue chemokine CD86: marker for dendritic cell maturation CR: complete response CTL: cytotoxic T lytic DC: dendritic cell FACS: fluorescence activated cell sorting GM-CSF: granulocyte-macrophage colony stimulating factor GV : Granulosis virus HIV: Human immunodeficiency virus i. t. : Intratumoral mCCL21: Mouse CCL21
MHC: Major histocompatibility complex MHCII: MHC class II
MLA-DR: MHC class I antigen MOI: Multiplicity of infection NPV: Nuclear polyhedrosis virus PBMC: Peripheral blood mononuclear cell PFU: Plaque-forming unit qd: Quaque Die (daily administration)
rhCCL21: Recombinant human CCL21
s. c. : Subcutaneous Sf (Sf9): Spodoptera frugiperda
Tn (Tn5): Trichoplusia ni
UV: UV VLP: virus-like particles.

Despite the advances described above, the success of immunological methods has been (1) tumor-specific antigenicity in which treatment is limited to specific cancer types, (2) poor antigen presentation by tumor cells, and ( 3) There were limitations due to the ability of tumor cells to produce immunosuppressive factors to avoid immune surveillance. Thus, there remains a need in the art for an effective and widely applicable cancer therapy. To satisfy this need, the present invention provides a new immunostimulatory method for the treatment and prevention of cancer.

That is, the present invention is particularly, through the administration of a non-pathogenic virus to a subject, inhibiting the growth of cancer, provides the necessary subject treatment method anticancer therapy including inhibition and resistance of cancer metastasis To do. The cancer immunotherapy disclosed herein is significant in that it does not rely on the discovery of tumor-specific antigens. Rather, administration of non-pathogenic viruses is widely applied and effective in multiple tumor types. Reference may be made to Examples 2-6.

While not intending to be limited to a particular mechanism of action, we believe that the anti-cancer activity of a non-pathogenic virus is due, at least in part, to its immunostimulatory properties. For example, baculovirus activates dendritic cell maturation and cytolytic T cell (CTL) responses both in vivo and in vitro. Reference may be made to Examples 9-10. In addition, Gronowski et al. (1999) J Virol. 73: 9944-51.

The term “viral particle” refers to a virus that is composed or modified from its native form, and thus cannot replicate in a natural host cell . Methods for preparing virus particles are known in the art. The structure and functional integrity of virus-like particles can be assessed by electron microscopy, immunogenicity analysis and standard plaque tests. For example, Hamakubo et al. , WO 02/06305 discusses the generation of isolated baculovirus-like particles.

The term “viral closure” refers to a structure comprising a plurality of viral particles embedded within a virally encoded protein crystal . When the protein crystal is dissolved, a plurality of virus particles are released, and each virus particle can then infect host cells.

A representative method for baculovirus production is, for example , as described in Example 1, which uses Spodoptera frugiperda (Sf). Another representative host cell and amplification method is described in US Pat. Nos. 5,405,770 (Heliothis subflexa cell line) and 6,379,958 (Spodoptera frugiperda cell line, which show advanced baculovirus production). ing.

After incubation, the viral agent thus produced is subjected to techniques known in the art such as polyethylene glycol (PEG) precipitation, ultracentrifugation and chromatographic purification, such as the use of ion exchange resins, size exclusion chromatography. Recovered by chromatography, affinity chromatography or a combination thereof. See US patent application 2002/0015945 (chromatographic purification); US patent 6,194,192 (virus adsorption on sulfated fucose- containing polysaccharides).

Non-pathogenic viruses, such as baculoviruses, can also be transcriptionally silent in mammalian host cells. That is, in some embodiments, a non-pathogenic virus can be a type of virus that is specifically excluded from current gene therapy because no heterologous gene is expressed.

The term “non-pathogenic” further encompasses viruses that are pathogenic in their native form and have been modified to be non-pathogenic. Such modifications include genetic modifications (eg, disruption of genes essential for viral replication as described above for the baculovirus gp64 gene; and / or disruption of viral promoters for transcriptional inactivation in the host species). To do. For example, the species-specific pathogenicity of baculovirus is partly because the baculovirus promoter is silent in species other than Lepidoptera. When a heterologous promoter is inserted into the genome of a baculovirus, the modified virus is capable of gene expression in non-lepidopterous cell lines, such as various mammalian cell lines. Boyce & Bucher (1996) Proc Natl Acad Sci USA 93: 2348-52; Carbonell et al. , (1985) J Virol
56: 153-60; Carbonell & Miller (1987) Appl.
Environ Microbiol 53: 1412-7; Hofmann et al. (1995) Proc Natl Acad Sci USA 92: 10099-103. A viral promoter that is initially active in mammalian cells is similarly modified to have the opposite result, so that it is no longer pathogenic in mammalian species. Site-directed mutagenesis methods for base pair alterations, deletions or small-scale insertions are known in the art and are described, for example, in the references mentioned above.

In some embodiments, the non-pathogenic virus is inactivated as described below. Anti-cancerous non-pathogenic viruses can be subjected to any one of a variety of inactivation methods to prevent the virus from infecting its native host cells. Using the tests described herein, one skilled in the art can select an inactivation method that preserves the anticancer activity of the virus. In some embodiments, the inactivation method allows entry of the virus into the host cell and interferes with transcription and / or replication of the viral genome. In some embodiments, it can be genetically modified so that the virus can enter the cell but not normal transcription and / or replication.

While not intending to be limited to a particular mechanism of action, we believe that the anti-infectious disease activity of non-pathogenic viruses is due, at least in part, to its immunostimulatory properties. For example, baculovirus activates dendritic cell maturation and cytolytic T cell (CTL) responses both in vivo and in vitro. Reference may be made to Examples 9-10.

The term “cancer” generally refers to tumors including primary and metastatic tumors. In some embodiments, the tumor is a solid tumor. The term “tumor” includes solid tumors and carcinomas of any tissue in a subject, for example, breast, colon, rectum, lung, oral genital region, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder, bile duct Small intestine, ureters such as kidney, bladder and urothelium, female organs such as cervix, uterus, ovary (eg choriocarcinoma and gestational trophoblastic disease), male organs such as prostate, seminal vesicle, anticancer and reproduction Cell tumors, endocrine lines, eg thyroid, adrenal and pituitary, skin (eg hemangiomas and melanoma), bone and soft tissue, blood vessels (eg Kaposi's sarcoma), brain, nerve, eye and meninges (eg astrocytoma, Glioma, glioblastoma, retinoblastoma, neuroma, neuroblastoma, schwannoma and meningioma).

The term “systemic immune response” as used herein refers to an immune response in lymphoid tissues associated with the lymph nodes, spleen or intestine where cells such as B lymphocytes of the immune system develop. For example, a systemic immune response includes the production of serum immunoglobulin (IgG). Furthermore, the systemic immune response refers to antigen-specific antibodies circulating in the bloodstream and antigen-specific cells in lymphoid tissues in systemic compartments such as the spleen and lymph nodes.

The invention also relates to methods and compositions useful for inducing cytotoxic T cell mediated responses in mammalian subjects, including humans. In some embodiments, the present invention relates to the use of non-pathogenic viruses to induce cytotoxic T cell mediated responses. That is, the present invention provides a method for producing an antigen preparation containing a non-pathogenic virus and an antigen. The term “antigen” refers to a substance that activates (positive or negative) lymphocytes by interacting with receptors on T cells or B cells. Positive activation results in immune responsiveness and negative activation results in immune tolerance. Antigens can include proteins, carbohydrates, lipids, nucleic acids, or combinations thereof. Antigens can include heterologous (eg, antigens that are not typically present in the host subject) or autologous antigens (autoantigens).

(C1. Virus inactivation)
In some embodiments , live non-pathogenic viruses used in the methods of the invention are inactivated prior to administration to a subject. A non-pathogenic virus cannot replicate in a mammalian host as defined above. Inactivation that renders the virus non-replicating in its native host cell can be implemented as another safety measure.

Virus inactivation is any suitable means, such as disrupting the lipid or protein components of the viral envelope, modifying the virus so that it cannot recognize the target cell, destroying the viral nucleic acid, and / or making the virus incapable of replicating. This can be achieved. Representative methods of virus inactivation include, for example, surfactant treatment (eg, Triton-X100®), alkylation with secondary ethyleneimine (BEI), photochemical inactivation, UV photoinactivation, radiation Includes irradiation, physical disruption (eg sonication, electron beam), genetic inactivation and combinations thereof. Rueda et al. (2000) Vaccine 19: 726-34 and Henzler & Kaiser (1998) Nat Biotechnol 16: 1077-9. In some embodiments, inactivation does not significantly reduce the antigenicity and / or activity of the virus. Viral inactivity is tested using standard methods for measuring viral infectivity.

If the virus can withstand sufficient heat treatment for inactivation , pasteurization is a simple method for inactivation. In some embodiments, the heating is performed for a minimum amount of time to minimize viral protein damage. In some cases, viral damage can be minimized by using stabilizers and sodium citrate, saccharose and / or glycine.

Alternatively, chemical inactivation, such as mild pepsin treatment at low pH values or exposure to detergents , can be used to disrupt the lipid bilayer, so that they have enveloped viruses, including baculoviruses Can be used for the inactivation of See U.S. Pat. Nos. 4,820,805 and 4,764,369. Aziridine secondary ethyleneimine is a powerful alkylating agent that inactivates viruses by selectively interacting with nucleophilic groups of nucleic acids rather than proteins.

Other photosensitizers include psoralen halides, angelicins, kerins and coumarins, each having a halogen substituent and a water soluble moiety such as quaternary ammonium ions or phosphonium ions. Substitution with a halogen atom, particularly a bromine atom, on the psoralen molecule increases the binding constant of the sensitizer to DNA due to the hydrophobic nature of bromine. In some embodiments, but not only requires light quantum only one only to activate the brominated sensitizer, two photons to cause crosslinking of DNA using non-brominated psoralen Brominated photosensitizers are used because they are necessary. See for example US Pat. No. 5,418,130.

The carrier can be selected to allow sustained bioavailability of the non-pathogenic virus to the site in need of treatment. The term “sustained bioavailability” refers to prolonged release of non-pathogenic viruses from a carrier , metabolic stability of non-pathogenic viruses, systemic transport of compositions containing non-pathogenic viruses, and non-pathogenic Includes factors including, but not limited to, effective administration of sex viruses.

Non-pathogenic virus of the invention in the tumor, peritumoral, systemic, parenteral (e.g., intravenous injection, intramuscular injection, intraarterial injection and infusion techniques), oral, transdermal (topical), intranasal (inhalation) And can be administered to the subject intramucosally. The delivery method is selected based on considerations such as the type of carrier or vector, the therapeutic effect of the composition, the location of the target area and the condition to be treated.

In some embodiments where the cancer is non-neoplastic growth, the non-pathogenic virus is administered at or around the affected area. The term “surrounded area ” refers to less than about 15 cm from the outer edge of non-neoplastic growth, less than about 10 cm from the outer edge of non-neoplastic growth, and less than about 5 cm from the outer edge of non-neoplastic growth. Refers to a site less than about 1 cm from the outer edge of non-neoplastic growth or less than about 0.1 cm from the outer edge of non-neoplastic growth. The non-pathogenic virus of the present invention can be delivered to one or more of the affected area and / or surrounding area. In some embodiments, the non-pathogenic viruses of the invention are administered at multiple sites within and / or around non-neoplastic growth.

In some embodiments, the composition is tested in vitro or in vivo to determine an “effective amount”. For example, in the methods described herein for inducing cell death, suitable tests include, for example, in vitro cell viability tests, such as TUNEL tests or other fluorescent system tests, such as Cell-Titer Blue (Promega Corp). Tests to monitor DNA fragmentation; and cytochrome C release tests, soft agar growth tests, contact inhibition tests, and tumor growth in nude mice; tumors are injected into test animals and cancers are administered after administration of the composition of the invention. A test involving monitoring attenuation, and a transgenic mouse test in which the transgenic mouse has a tumor and monitoring cancer attenuation; an in vivo test disclosed herein; of a cell through a barrier such as the Matrigel barrier In vitro methods for measuring migration (eg Ca Cer Res.2003 Aug 1:63 (15): 4632-40; Am J Chin Med.2003; 31 (2): 235-46); Yang et al. reported in vitro invasive and in vivo metastasis studies (Cancer Res. 61, 5284-5288, July 1, 2001).

International patent application WO 98/50071 describes the use of VLPs as adjuvants to enhance the immune response of antigens administered with virus-like particles (VLPs). S. Clair et al. Describe the use of protein crystals to enhance humoral and cellular responses (St. Clair, N. et al., Applied Biol. Sci., 96: 9469-9474, 1999).

According to convention, the number of substances can be one or more. The expression “about” as used herein refers to a measurable numerical value, for example, a distance around a tumor or an affected area, ± 20% or ± 10% from a specified amount, ± 5%, ± Including variations of 1% or ± 0.1 %, such variations are appropriate when performing the disclosed method or otherwise practicing the present invention. In some embodiments, “about” is meant to include ± 10% variation from a particular amount.

(D1. Prediction of in vivo activity)
Predicting in vivo anti-tumor activity is often difficult and unreliable. The present invention provides a method for predicting in vivo anti-tumor activity. In some embodiments, the method comprises contacting the compound with tumor cells and peripheral blood mononuclear cells and measuring cell death of the tumor cells. In some embodiments, the compound is a non-pathogenic virus or a non-pathogenic insect-specific virus. For example, cell death can be measured using any method including measurement of apoptosis, necrosis, cell viability, and the like. Tumor cells that can be used include, for example, lung cancer cells (eg A549 cells, 3LL-HM cells), breast cancer cells (eg 4T1 cells; MT901 cells; MAT BIII cells), prostate cancer cells, colon cancer cells, skin cancer cells (eg B16 black). Seed cells), pancreatic cancer cells, liver cancer cells, brain cancer cells, bone cancer cells (for example, MG-63 cells), gastric cancer cells, or esophageal cancer cells.

The invention provides a process for the preparation of anticancer compositions and anti-infectious disease compositions for sale. In some embodiments, the composition comprises one or more non-pathogenic viruses. In some embodiments, the non-pathogenic virus is an Autographa californica nuclear polyhedrosis virus. In some embodiments, the non-pathogenic virus comprises an inactivated virus, virus particle, virosome, virus-like particle, virus closure or virus component. In some embodiments, the process includes conducting sales trials on the composition to ensure that the safety, toxicity, and efficacy of the composition meet certain guidelines.

In some embodiments, the process further comprises collecting any portion of the composition for one or more analyzes of safety, efficacy or toxicity. In some embodiments, the safety and / or efficacy and / or efficacy of any portion is compared to the safety and / or efficacy and / or efficacy of the second anti-cancer or anti-infectious disease composition. Comparison data is created for review prior to sale.

(Example 1. Preparation of recombinant baculovirus)
The full-length sequence encoding human CCL21 (Genbank accession number NM002989) was cloned into the baculovirus transfer vector pVL1392 (Pharmingen, San Diego, California), and BACULOGOLD® using the recommended method of origin. Co-transfected with WT genomic DNA (Pharmingen, San Diego, California). The recombinant baculovirus obtained by this procedure was subcloned by plaque purification on Sf9 insect cells to obtain several isolates expressing human CCL21. Those clones with exceptional expression characteristics when compared to the original virus were selected. This baculovirus isolate was amplified in a low multiplicity of infection (MOI) to obtain a high-titer, low-passage stock for protein production. The baculovirus expressing human CCL21 was named B V 422. Additional recombinant baculoviruses were made similarly. For example, a baculovirus expressing intracellular Raf protein was prepared and named BV762.

(Example 2. Inhibition of tumor growth in an animal model of lung cancer)
Tumor cells were inoculated after acclimating 9-11 week old C57BL / 6 mice for a minimum of 7 days. Mice were inoculated with 2 × 10 ⁵ early passages (less than 10 passages) of 3LL-HM tumor cells subcutaneously on the right flank. Tumor size was measured twice per week. When tumors reached 50-100 mm ³ (typically 7 days after tumor inoculation), mice were randomly grouped. Baculovirus-expressing CCL21 was administered intratumorally to tumor-bearing mice. Usage of dose and dose was varied in order to optimize the suppression of tumor. Mice were sacrificed when the tumor volume of either group reached 3000 mm ³ (typically 33-35 days after inoculation in the control group of mice).

As shown in FIG. 1, growth of 3LL tumors was delayed by intratumoral administration of baculovirus-expressing CCL21. Dose of CCL21 were optimized to complete inhibition of tumor growth is achieved. The administration method with 2 or 3 injections at a relatively high dose showed similar efficacy compared to the administration method with 6 injections at a relatively low dose. Some tumor suppression was also observed with a single dose.

(Example 3. Inhibition of tumor growth in an animal model of breast cancer)
9-11 week old Balb / c mice were acclimated for a minimum of 7 days before inoculating tumor cells. Mice were inoculated with 2 × 10 ⁵ 4T1 cells subcutaneously on the right flank. Tumor size was measured twice per week. When tumors reached 50-100 mm ³ (typically 7 days after tumor inoculation), mice were randomly grouped. Baculovirus was administered intratumorally to tumor-bearing mice. Dose was varied to determine the optimal effective dose. Mice were sacrificed when the tumor volume of either group reached 3000 mm ³ (typically 33-35 days after inoculation in the control group of mice). As shown in FIG. 2, growth of 4T1 tumor was delayed by intratumoral administration of CCL21.

(Example 5. Resistance to tumor re-attack)
As described above, mice bearing tumors were prepared and baculovirus was administered. Mice that showed complete tumor attenuation or mice that did not show complete tumor attenuation were surgically removed and subjected to tumor re-challenge two days after the last dose of baculovirus. Mice were anesthetized by intraperitoneal administration of 200 μl ketamine / xylazine mixture (4: 1 ketamine: xylazine diluted 10-fold in phosphate buffered saline), tumors were removed and wounds closed with staples. One to four days after tumor removal, mice were re-attacked by subcutaneous administration of 2 × 10 ⁵ 4T1 cells at sites other than the original tumor site. Re-attack tumor volume was measured twice per week. For mice that showed complete tumor attenuation, the mice were equally subdivided into two groups. One group was re-challenge with the same tumor cells, 1 × 10 ⁵ per mouse on the opposite side of the abdomen. The other group was challenged with different syngeneic tumor cells (B16F10) on the left abdomen at 1 × 10 ⁵ per mouse. Tumor growth at the re-attack site was monitored. Mice treated with baculovirus could tolerate re-attack by 3LL-HM tumor cells but were not resistant to different syngeneic tumor cells.

(Example 9. Activation of dendritic cell maturation by baculovirus)
When wild type baculovirus was added to dendritic cell (DC) cultures, maturation was induced, as evidenced by increased cell surface expression of activation markers. As shown in FIGS. 11A and 11B, baculovirus activates mouse bone marrow-derived DCs and human monocyte-derived DCs.

Mouse DCs were prepared from bone marrow according to standard methods. In other words, bone marrow was isolated from female 6-8 week old Balb / c or C57B1 / 6 mice (Charles River Laboratories, Hollister, Calif.) And heat inactivated with 10% cell culture grade dimethyl sulfoxide (DMSO) added. Frozen fetal bovine serum (−80 ° C.) at a concentration of 2 × 10 ⁷ cells / ml. A portion of frozen cells was thawed rapidly and washed to remove DMSO. Cells in a 150 mm suspension culture dish containing 20 ml of 20 ml of RPMI medium (Sigma-Aldrich, St. Louis, Missouri) containing 200 U / ml murine GM-CSF (PreproTech, Rocky Hill, New Jersey). I put it in. On day 3 of culture, murine GM-CSF was again added to the cells, and on day 5 half of the culture volume was centrifuged and replaced with fresh medium containing GM-CSF. BMDC was collected by gentle pipetting.

Example 10. Activation of in vivo CTL induction by baculovirus
Immunization of mice with baculovirus and soluble protein antigen (HIVp24) induced a robust antigen-specific CTL response. The spleens of the immunized mice were collected 2 weeks after the third immunization. Individual spleens were combined so that 5 spleens were included in each sample. Were cultured in dishes for 24 wells at a density splenocytes 5x10 ⁶ cells per well of immunized mice. Of these cells, 1 × 10 ⁶ cells were (1) a synthetic p7g peptide, ie an H-2Kd restricted CTL epitope corresponding to amino acids 199-208 of HIV-1 _SF2 p24gag; and (2) a pGagb peptide, ie HIV-1 _SF2 Sensitized with an H-2Db restricted CTL epitope corresponding to amino acids 390-398 of p55gag. The peptide was used at a concentration of 10 μM for 1 hour at 37 ° C. The splenocytes were then washed and cultured with the remaining 4 × 10 ⁶ untreated cells. Splenocytes were used as splenocyte medium: RPMI 1640 and α-Mem (1: 1) (10% heat inactivated fetal bovine serum (Hyclone, Logan, Utah: inactivated in 56 ° C. water bath for 30 minutes), 100 U / ml penicillin, 10 μg / ml streptomycin, 10 ml / L of 100 mM sodium pyruvate and 50 μM 2-mercaptoethanol were added (RPMI1640 (Sigma-Aldrich, St. Louis, Missouri)) was added to 100 mM L-glutamine (Giboko, Grand Isl , New York) and α-Mem was stimulated as a bulk culture in 2 ml of the minimal essential medium (Alpha Medium) supplemented with L-glutamine, deoxyribonucleosides or ribonucleosides. . Furthermore, 5% rat T-StimIL2 (rat T-Stim: Collaborative Biomedical Products, Bedford, Massachusetts) was used as an IL2 raw material and added to the medium immediately before culturing the cells.

(Initial sample preparation and serial dilution in transfer plate)
(Initial screening test)
In order to improve the chance of detecting PBMC cytotoxicity inducers, samples were first prepared at relatively high concentrations. Sample concentration was reduced in subsequent tests depending on the activity of the sample.

(Transfer of sample dilution to target cell (test) plate)
The contents of the dilution plate were transferred to the test (cell) plate. The final sample concentration was _^1/2 of the original dilution plate. A plate-to-plate transfer program propet was used for the transfer. The test plate was incubated while preparing PBMC.

(Toxicity control plate for direct cytotoxicity of test sample to target cells)
Two identical transfer (dilution) plates were established and the medium was transferred to a 2-cell plate. Plate 1 was added with 50 μl of PBMC preparation and subjected to induced cytotoxicity measurement. To the second plate, 50 μl of PBMC preparation medium was added to measure the direct cytotoxicity of the test sample.

(Isolation of peripheral blood mononuclear cells (PBMC) from human peripheral blood)
A 1:10 dilution (3 liters) of bleach was prepared. All test tubes and pipettes in contact with blood were bleached overnight. The beaker was drained and all items were disposed of in a sharp or biological hazardous material container. Blood was handled after removing the cap from the 50 ml test (to prevent contamination of the cap with blood). The blood was added to a 250 ml disposable polycarbonate flask and diluted with an equal volume of HBSS. About 17 ml of Ficoll plaque solution was added per 50 ml polystyrene test tube, and blood was added 30 minutes later. The blood was layered on top of the Ficoll plaque solution without disturbing the Ficoll layer. 4 ml of blood / HBSS was added to each 3 ml of Ficoll plaques (ie 17 ml Ficoll and 23 ml blood / HBSS per 50 ml tube).

It was centrifuged at 1800RPM tubes at room temperature for 30 minutes (400xg) (Sor v al GLC -2B centrifuge). As much of the upper layer as possible was aspirated using a 25 ml serological pipette, leaving about 5 ml of the top of the second layer. The remainder of the first layer was removed using a 5 ml serological pipette. The second layer containing B and T lymphocytes was collected using a sterile 5 ml serological pipette and placed in a new 50 ml test tube. The collection was diluted with 3 volumes of RPMI without additives. The resulting solution was centrifuged for 15-20 minutes at 900 rpm (100 × g) (Wash 1). The supernatant was removed with a serological pipette, the cells were resuspended in 40 ml RPMI, and centrifugation was repeated at 900 rpm (wash 2). The supernatant was removed with a serological pipette and the cells were resuspended in 40 ml PBMC prep medium (RPMI supplemented with 0.5% BSA). The total volume of resuspended cells was recorded strictly. Cells were counted with a Coulter counter to determine cell density, and a 1: 5 dilution of cell concentrate in PBMC conditioned medium was used for counting .

The resuspension volume required to obtain the appropriate cell density was determined. For the cytotoxicity test, the desired density was 2 × 10 ⁶ cells / ml and for PBMC freezing the desired density was 10 × 10 ⁶ cells / ml. The final resuspension volume was calculated by multiplying the cell density by the total volume and then dividing the product by the final desired cell density (2 or 10 × 10 ⁶ cells / ml).

The resuspension volume required to obtain the appropriate cell density was then determined. For the cytotoxicity test, the desired density was 2 × 10 ⁶ cells / ml. The final resuspension volume was calculated by multiplying the cell density by the total volume and then dividing the product by the final desired cell density (2 or 10 × 10 ⁶ cells / ml). Centrifugation was repeated at 900 rpm with the rest of the resuspended cells. The supernatant was removed and the cells were resuspended in the final resuspension volume.

Administering BV to PBMC in suspension with shaking, followed by centrifugation wash, significantly non-specific (well of 0% BV-treated cells) stimulation of cytotoxic response to MG-63 target cells However, it was considered different for A549 cells .

FIG. 1 is a line graph showing that baculovirus-expressed CCL21 suppresses tumor growth and induces complete tumor attenuation in a 3LL mouse model of lung cancer. As shown in Example 2, administration of CCL21 includes 6 intratumoral injections of CCL21 at the stated concentrations. Increasing the dose per injection also increased the frequency of tumor suppression and complete response. A one-way ANOVA analysis was performed using the data collected on day 24. Albumin versus administration group, p <0.001; 25 μg mCCL21 vs 6 μg / AlbmCCL21, P <0.01; 25 μgmCCL21 vs 25 μghrCCL21, P> 0.05. mCCL21, mouse CCL21; rhCCL21, recombinant human CCL21; B Gold, recombinant mouse CCL21 expressed from baculovirus was used as a control lot (Gold standard); Alb, albumin; qd, Quaque Die (daily administration), CR, complete A response. FIG. 2 is a line graph showing suppression of tumor growth in a 4T1 mouse model of breast cancer after administration of baculovirus-expressing CCL21 described in Example 3. rhCCL21 is recombinant human CCL21; qd is Quaque Die (administered daily). FIG. 3 is a bar graph showing suppression of spontaneous 4T1 lung metastasis after administration of baculovirus-expressing CCL21 shown in Example 6. Black are animals that have not undergone surgical resection of tumors; hatched lines are animals from which tumors have been surgically removed one day after the last dose of CCL21 (Surg). FIG. 4 is a line graph showing that administration of baculovirus-expressing CCL21 confers resistance to tumor re-attack, as shown in Example 5. In short, 4T1 tumors were established in Balb / c mice and baculovirus expressing CCL21 was administered by intratumoral administration to a subset of host mice. One day after the last dose of CCL21, the tumor was surgically removed. One day after tumor resection, mice were re-challenged by subcutaneous injection of 4 T1 cells on the opposite side of the original tumor. Naive is a mouse that has never had a tumor and has not received CCL21; Alb + Surg + Re-chlg has previously had a 4T1 tumor and has received albumin; hCCL21 + Surg + Re-chlg has previously The mice have a 4T1 tumor and have received CCL21 administration. In the Alb + Surg + Re-chlg group, 1 out of 10 mice showed complete resistance to tumor growth. In hCCL21 + Surg + Re-chg, 6 out of 10 mice showed complete resistance to tumor growth. Figures 5A-5B show baculovirus-induced resistance to tumor re-attack. In the 3LL tumor model, animals that have been successfully treated with baculovirus-induced CCL21 are resistant to re-attack by the same tumor for extended periods of time. FIG. 5A shows complete tumor attenuation after administration of baculovirus-derived mouse recombinant CCL21 (see, eg, FIG. 1), and the contralateral abdomen in the same tumor at 60, 70, and 80 days after the final administration of baculovirus-induced CCL21. This is a summary of experiments using animals that were re-attacked. Animals were resistant to re-challenge for at least 70 days. FIG. 5B is inoculated 30 days after the end of baculovirus CCL21 administration and induction of complete decay (“tumor boost”) and reversed with the same tumor at 80, 120, 160 and 200 days after the last dose of baculovirus-induced CCL21. This is a summary of experiments using animals that re-attacked the abdomen on the side. These results show that tumor boost can be used to extend resistance to tumor re-attack until at least 200 days. 6 shows yeast or E. coli. 2 is a line graph showing that CCL21 produced in E. coli does not result in tumor attenuation in a 3LL tumor model of lung cancer. hCCL21-B (HBPG1) is recombinant human CCL21 expressed in baculovirus lot number HBPG1; hCCL21-B1 / 2 (HBPG1) is recombinant human CCL21 expressed in baculovirus lot number HBPG1 at a concentration of hCCL21 (HBPG1) Recombinant human CCL21 expressed in baculovirus lot number HBPG1 derived from a concentrated (10 mg / ml) solution; hCCL21-Bconc (HBPG1) is hCCL21-Y (HYPG4) Recombinant human CCL21 expressed in yeast lot number HYPG4; hCCL21-E (HEDS4) Recombinant human CCL21 expressed in E. coli lot number HEDS4; qd, Quaque Die (administered daily). Inhibition of tumor growth in albumin-administered mice when compared to mice administered hCCL21-B or hCCL21-B novel, p <0.01; tumor growth in mice administered hCCL21-Bconc or hCCL21-B1 / 2 Inhibition, P <0.05. FIG. 7 is a line graph showing the in vitro chemotaxis activity of baculovirus-expressing CCL21 preparations that are inactive in vivo. The chemotaxis test can be carried out essentially as described in PCT International Application WO 00/38706. In vivo activity was tested as described in Examples 2-6. HBDS2. p is baculovirus lot number HBDS2. recombinant human CCL21 derived from p; MBDS2. c is baculovirus lot number MBDS2. a recombinant murine CCL21 derived from c; HBPG1 is a recombinant human CCL21 derived from a baculovirus lot number HBPG1; HBPG1 + HBDS2. vp is HBPG1 mixed with an equimolar ratio of HBDS2 administered with vinylpyridine; HYPG4 is recombinant human CCL21 derived from yeast lot number HYPG4; Recombinant human CCL21 derived from E. coli lot number HEDS4. FIG. 8 shows a Western blot prepared with the sample described and then probed with an anti-gp64 antibody. Lane 1 is purified baculovirus; lane 2 is conditioned medium from uninfected Tn5 cell culture; lane 3 is conditioned medium from Tn5 cells infected with wild-type baculovirus; lane 4 is recombinant. Conditioned medium from a Tn5 cell culture infected with BV422 encoding human CCL21; lane 5 is an uninfected Tn5 cell pellet; lane 6 is a cell pellet from a Tn5 culture infected with wild-type baculovirus Lane 7 is a cell pellet from a Tn5 culture infected with BV422; lane 8 is a human recombinant CCL21 derived from baculovirus lot number HBPG1, which is the filtrate after removal of contaminants greater than 50 kDa; lane 9 is greater than 50 kDa Miscellaneous Residue of the sample in lane 8 contains (protein 5 [mu] g); lane 10 remnants of the sample in lane 8 containing 50kDa than contaminants (proteins 10 [mu] g); lane 11 recombinant human baculovirus derived unfiltered 5 μg of CCL21 lot number HBPG1; lane 12 is 5 μg of unfiltered recombinant human baculovirus-derived CCL21 lot number HBPDS4; lane 13 is 5 μg of unfiltered recombinant human baculovirus-derived CCL21 lot number HBMC1; lane 14 is unfiltered recombinant 5 μg of human baculovirus-derived CCL21 lot number HBDS1. FIG. 9 is a line graph showing that the antitumor activity of baculovirus-expressed CCL21 is removed by filtration to remove high molecular weight contaminants from the preparation. The purified baculovirus, a contaminant of the CCL21 preparation (FIG. 8) effectively suppresses tumor growth in the same manner as the baculovirus-expressing CCL21 preparation. FIG. 10A is a line graph showing PBMC-mediated toxicity of tumor cells in vitro when exposed to the described composition, as shown in Example 8. The cell pellet sample induced a greater cytotoxic response than the supernatant sample. Cells infected with either CCL21 expressing baculovirus or control baculovirus BV762 showed significant cytotoxicity. GAM is growth medium not conditioned by cell culture (control); BV422 is a CCL21 expressing baculovirus; BV762 is a Raf expressing baculovirus. FIG. 10B is a line graph showing PBMC-mediated toxicity of tumor cells in vitro when exposed to the described composition after filtration through a 0.2 μm filter as described in Example 8. Cytotoxicity was significantly reduced by removal of high molecular weight material by filtration. Cell pellet samples showed residual mild to moderate cytotoxicity. FIG. 11A is a bar graph showing changes in dendritic cell expression of CD86 and MHCII in response to the stimulants described. Mouse bone marrow derived dendritic cells were prepared and analyzed as described in Example 9. Vaccinia virus (VLP) expressing HIV gag protein was used as a positive control. Like VLP, live baculovirus induced CD86 and MHCII expression, indicating DC maturation. Black indicates CD86 expression; gray indicates MHCII expression. FIG. 11B is a bar graph showing changes in dendritic cell expression of CD86 and MHCII in response to the stimulants described. Human monocyte-derived dendritic cells were prepared and analyzed as described in Example 9. Vaccinia virus expressing HIV gag protein (p55VLP) was used as a positive control. Like p55VLP, live baculovirus induced DC maturation. Black indicates CD86 expression; gray indicates HLA-DR expression. FIG. 12 shows the results of a chromium release test performed as described in Example 10. Vaccinia virus (VLP) expressing HIV gag protein was used as a positive control. Like VLP, live baculoviruses act as powerful adjuvants that induce cytotoxic T cell lysis. FIG. 13 shows the PBMC-mediated toxicity of tumor cells in vitro when exposed to the described composition as shown in Example 8. FIG. 14 shows the correlation of PBMC cytotoxicity studies with in vivo anti-tumor activity. FIG. 15 shows the block of baculovirus tumor cell killing by anti-gp64 monoclonal antibody. See Example 13. FIG. 16 shows in vitro PBMC-induced tumor cell killing induced by inactivated baculovirus. See Example 14. FIG. 17 shows that tumor cells are the primary target for baculovirus. See Example 15. FIG. 18 shows suppression of tumor growth in an animal model of lung cancer using baculovirus. See Example 16. FIG. 19 shows baculovirus-induced protection from pathogenic virus attack in vivo and in vitro. Cells were challenged in vitro and in vivo with vesicular stomatitis virus (VSV). See Example 17. FIG. 20 shows the bystander effect. The maximum killing effect is achieved even when only 20% of the tumor cell population has been exposed to baculovirus.