GENERATION AND EXPANSION OF LAK CELLS

Abstract: Process for generating and expanding cells having LAK cell activity comprises (a) treating human PBMC to deplete monocytes, (b) culturing the resulting peripheral blood lymphocytes in medium containing IL-2 and human plasma or serum to generate LAK activity and (c) expanding the cells by culturing at low cell density in medium containing IL-2 and human plasma or serum in a culture bag made from and organic polymeric, oxygen permeable film. Monocytes are depleted by treating the PBMC with and ester of an L-amino acid such as phenylalanine methyl ester.

Description: Title Generation and Expansion of LAK Cells Field of the Invention This invention relates to the generation and expansion of cells having lymphokine-activated killer (LAK) cell activity which are useful in adoptive immunotherapy. Background of the Invention Incubation of interleukin-2 (IL-2) with human peripheral blood mononuclear cells (PBMC) or mouse splenocytes induces a population of highly tumoricidal cells. This phenomenon has been referred to as lymphokine-activated killer (LAK) cell activity. The precursor of the LAK cells, also called effector cells, may be heterogeneous, but most of the activity apparently originates from large granular lymphocytes (LGL's) which comprise about 5% of peripheral blood lymphocytes (PBL's) and which have natural killer cell activity. Adoptive transfer of LAK cells to tumor bearing mice, with or without simultaneous administration of IL-2, has resulted in reduction in tumor burden in several animal models. These results encouraged Rosenberg and colleagues to develop clinical trials utilizing LAK cells alone, IL-2 alone, and finally an intensive treatment program utilizing both agents in patients with advanced solid tumors (Rosenberg et al., N. Engl. J. Med . 1987, 316:889-897). The toxicity of this combination regimen was considerable despite the fact that Rosenberg was able to deliver in man only 1 -10% of the equivalent dose compared to the effective murine dosees of LAK cells and IL-2 (based on weight). Nevertheless, a significant number of partial responses were seen and further trials of the combination of IL-2 and LAK cells are underway. Central to the problem of the utilization of LAK cells in man is the complexity of their generation (also referred to herein as induction or activation): patients are leukaphoresed, the leukaphoresis product is separated by Ficoll-Hypaque gradients, and the resultant mononuclear cells are then cultured at cell densities of l-3xl0 6 cells per ml for 3-5 days. Thus, the final culture volumes in roller bottles can reach 40 liters. Various changes and improvements have been made in this procedure. For example, European patent application 87107755.8, published December 2, 1987, and allowed U.S. application 07/038361, filed April 20, 1987, disclose that depletion of monocytes by exposure of mononuclear cells to phenylalanine methyl ester allows LAK cell induction at cell culture densities aboutlO-fold greater than the currently utilized LAK cell induction concentrations. European patent application 88101138.1, published Aug. 31, 1988, and U.S. application 7/008273, filed January 29, 1987, disclose that LAK cell induction can be carried out in bags made of organic polymeric, oxygen permeable film. European published patent application 88106566.8. published Nov. 9, 1988, and U.S. patent 4,808,151, issued February 28, 1989, disclose that the leukaphoresis product need not be separated on a Ficoll- Hypaque gradient prior to use in the activation process. International published application WO 88/00970 teaches that LAK cells can be expanded about 30-100 fold by culturing in 16 mm wells for at least 7 days at a cell concentration less than 10 6 cells per ml in medium containing IL-2 and 6% serum, and can be expanded about 1000 fold over the course of 21 days by continuing to culture in 16 mm wells in the presence of a T-cell mitogen and preferably an additional lymphokine. Cell concentration was maintained under 10 6 cells per ml by subculturing every 48 hours and adding fresh medium and IL- 2. Ya ada et al., Journal of Leukocyte Biology 41:505-517 (1987) and Yamada et al., Journal of Leukocyte Biology 42:263- 272 (1987) disclose separation of the LGL and T-cell subpopulations of PBL's and proliferation of each subpopulation by culturing in 24 well flat bottom culture plates in media containing IL-2. Initial cell concentration was 10 6 per ml. Cultured cells were subcultured every 3 or 4 days with fresh medium containing IL-2. The procedures of international application WO 88/00970 and the Yamada et al. articles are complex and inconvenient. There remains a need for a practical process to augment the number of LAK cells available for human LAK cell therapy. Summary of the Invention This invention is a process for generating and expanding cells having LAK cell activity which comprises (a) treating human peripheral blood mononuclear cells to deplete monocytes, (b) culturing the resulting peripheral blood lymphocytes in a culture medium containing IL-2 and human plasma or serum for about 2 to 7 days to generate LAK cell activity in the cells; and (c) expanding the cells by culturing them in culture medium containing IL-2 and human plasma or serum in amounts sufficient to sustain cell expansion, in a culture bag made from an organic polymeric, oxygen permeable film, at an initial cell concentration in the range of about 0.5x 1 0 6 to 1.5xl0 6 cells per ml, adding fresh medium from time to time, with or without removal of used medium, to bring the cell concentration back to within the range of about 0.5x10° to 1.5x10° cells per ml, and continuing to culture under these conditions for at least about 2 days. The total culture time for generation step (b) and expansion step (c) combined is at least about 9 days. Preferably, in step (a), the monocytes are depleted by treating the PBMC with a lower alkyl ester of an L- amino acid selected from alanine, aspartic acid, cysteine, glutamic acid, glutamine, phenylalanine, proline, tyrosine, tryptophan, and valine, or a hydrogen chloride salt thereof. Using preferred materials and methods in the process of this invention, as described in the examples below, we were able to generate cells with high LAK cell activity, and expand the cell number 5-10 fold with no loss of total LAK cell activity, using cells from healthy donors as well as cells from cancer patients. In our process plasma and serum were equivalent in their capacity to support LAK cell expansion but less than 10% plasma or serum supported suboptimal activation. Several serumless media failed to support LAK cell expansion or optimal LAK cell activation. Thus, we have developed a practical process to augment the number of LAK cells available for human LAK cell therapy and simultaneously reduce the complexity and volume of the induction system, so that murine equivalent doses of LAK cell therapy are now feasible in man. Detailed Description of the Invention Preferred organic polymeric, oxygen permeable bags for use in our process are those disclosed in U.S. patents 4,496,361 and 4,588,401 and European patent application 88101138.1, published Aug. 31, 1988, the disclosures of which are incorporated herein. As described in the European application, the bags are made from a copolymeric film material having an oxygen permeability of at least about 1.8xl0 5 urn 3 (STP)/(m 2 *sec«Pa), and a thickness of from about 0.04 mm to about 0.23 mm. The materials are selected from the group consisting of: (a) copolymers of ethylene and an alpha olefin of 4-10 carbon atoms having a density of from about 0.915-0.925 g/cm 3 ; (b) copolymers of ethylene and methacrylic acid; (c) ionomers; and (d) laminates or coextrudates of an ionomer/polyester elastomer and a linear low density polyethylene elastomer. The most preferred material is 4-5 mil thick film of a copolymer of ethylene (about 97%) and 1 -octene which is sold as Sclair® film by E.I. du Pont de Nemours and Company Bags made of Sclair® film are sold by DuPont for cell culture, including LAK cell generation, as SteriCell™ bags. Other materials which can be used to make bags for use in this invention are disclosed in U.S. patent 4,140,162, the disclosure of which is incorporated herein. The bag should have a sufficient volume to accomodate from about 0.2 to about 5 liters of cell suspension. SteriCell™ bags are available in three sizes: 50ml (44cm 2 bottom surface), 200 ml (178cm 2 bottom surface) and 1000 ml (711 cm 2 bottom surface). The bag is preferably equipped with suitable access ports or tubes which can be closed by means well known in the art, such as clamps, to maintain a reasonable level of protection from contamination. Preferably, the tubes are closed by heat sealing, using a sterile connection device such as that disclosed in U.S. Patent 4.369,779. After incubation, the LAK cells can be removed by using the sterile connection device to connect the access tube to the desired transfer chamber. In this manner, sterility of the system can be maintained. The bag can have a sterility preserving tip overfitting the end of the tubing, as disclosed in U.S. Patent 4,227,527. PBMC required for the process of the invention are obtained by repeated leukaphoresis using procedures well known in the art to collect up to about 5xl0 5 mononuclear cells. The PBMC are typically fractionated by Ficoll-Hypaque density gradient separation, although this step can be eliminated without unduly interfering with LAK cell induction, as taught in U.S. patent 4,808,151 , the disclosure of which is incorporated herein. The cells are then washed with a suitable salt solution, such as Seligmann's balanced salt solution (Gibco, Grand Island, N.Y.), and then resuspended in a suitable medium, such as RPMI 1640 (M.A. Bioproducts, Walkersville, MD) supplemented with at least about 10% by weight heat inactivated human serum or plasma. The resuspended PBMC preferably are treated with a lower alkyl ester of an L-amino acid selected from the list given above, or hydrogen chloride salt thereof. The lower alkyl group can be any such group containing 1 to about 4 carbon atoms; methyl and ethyl are preferred. The preferred ester is phenylalanine methyl ester (PME). Preferably, the HC1 salt of the ester is dissolved in RPMI 1640 and the pH of the resulting solution is adjusted to about 7.4 prior to adding the resulting solution to the suspension of PBMC. The amino acid alkyl ester is used at a concentration of about 1 mM to about 5 mM based on total volume of combined PBMC suspension and amino acid alkyl ester solution. Contacting of the PBMC and ester is preferably carried out at a temperature of about 20-25° C . Treatment of PBMC with amino acid alkyl ester causes depletion of monocytes from the PBMC and permits culturing at higher cell density during the LAK cell induction step of this invention. The treatment also confers on the ultimate LAK cell product enhanced activity and improves expansion. Monocytes can also be depleted from PBMC using conventional means such as passage of the PBMC over glass beads. The peripheral blood lymphocytes (PBL) obtained in this manner are preferably then treated with the amino acid lower alkyl ester to enhance LAK activition and expansion. To generate LAK cell activity, the resulting PBL in suspension are placed in the bag along with suitable medium and IL-2, the bag is closed to maintain sterility, and the suspension is incubated for about 2 to 7 days, preferably 3 to 5 days. Incubation is carried out at a temperature of about 35° to 39° C, preferably 37° C, preferably under an atmosphere containing about 3% to about 10% CO2, most preferably 5% CO2. Initial concentration of cells in suspension will generally be in the range of about 0.5x10 6 to 5xl0 7 cells per ml, preferably in the range of lxlO 6 to 1.5xl0 7 cells per ml, most preferably in the range of 5-20x10 6 cells per ml. Suitable media include RPMI 1640, supplemented with IL-2 and human serum or plasma or human serum albumin (HSA). The IL-2 concentration can be about 500 to 50,000 pM, preferably 1 ,000 to 10,000 pM. The IL-2 can be purified or recombinant IL-2 or a bioligically functional equivalent. During culture, the bag is maintained in a horizontal position, preferably on a rack that will allow gases to enter the side contacting the rack, and the cell suspension is maintained at a depth that will allow adequate oxygen to reach all cells. At the lowest cell density, the suspension depth can be up to about 4 cm, whereas at highest cell density the depth will be about 0.5 cm or less. For LAK cell expansion, fresh medium containing IL-2 and heat inactivated human serum or plasma is added to provide a cell suspension in which the cell concentration is in the range of about 0.5-2xl0 6 , preferably 0.5-1.5x10 6 cells per ml. Preferably, the cells are separated from the used medium before the fresh medium is added, by removing the cell suspension from the bag and spinning the cells down. For resuspension, the cells can be returned to the same bag, but will frequently be placed in a larger volume bag. For example, the generation step may be carried out at high cell density (concentration) in a 50 ml bag and the expansion step at low cell density in a 200 or 1 ,000 ml bag. The amount of serum or plasma in the medium is preferably at least 10% by weight based on total weight of all components of the supplemented medium. Less than 10% serum or plasma can be used, but supports suboptimal activation. Human plasma is preferred because it can be obtained in larger quantities at much less expense than human serum, because donors do not lose formed blood elements and because of recent advances in plasmapheresis technology. From time to time as needed to sustain cell expansion, fresh medium containing IL-2 and plasma or serum is added, preferably after removal of used medium, to return the cell concentration to a level within the range of about 0.5-2xl0 6 , preferably 0.5-1.5xl0 6 cells per ml. Depending on the number of cells initially and the extent of expansion, it may be necessary to split the culture into two or more bags. Replacement of used medium with fresh medium is referred to as passaging. Passaging and splitting the culture is referred to as subculturing. The frequency of adding fresh medium will depend upon the particular culture. Because passaging is labor intensive, it is expensive, so the frequency should be as low as possible to provide an acceptable level of expansion. In our experiments, we have found that passaging after 4 days gave acceptable results, but the cells can be passaged more frequently if desired. Except as indicated above, the materials and methods used in the expansion step are the same as those used in the generation step. The invention will be further described by the following examples, but is not limited thereby. Examples Materials and Methods Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque separation of EDTA anticoagulated venous blood drawn from healthy consenting donors or patients (see below). The cells were washed three times with Seligman's balanced salt solution (Gibco, Grand Island, NY) and resuspended in RPMI 1640 (M.A. Bioproducts, Walkersville, MD), supplemented with 1 mM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) (Gibco) (cRPMI) and 10% pooled heat inactivated human sera (HIS). Peripheral Blood Lymphocyte Preparation. Monocytes were depleted from PBMC preparation by treatment with phenylalanine methyl ester (PME). Briefly, PBMC were suspended to 5xl0°7ml in serumless RPMI 1640. The PME was dissolved in RPMI, the pH of the solution was readjusted to 7.4 with ImM NaOH and the solution was filtered through a 0.22- um filter. The PBMC were incubated with 5mM of PME in polypropylene 50-ml conical tubes (Falcon 2098: Falcon Labware) for 40 minutes at room temperature. The cells were washed with cold Seligman's balanced salt solution and resuspended in cRPMI-10% HIS. In comparative experiments human plasma (HIP) was obtained by anticoagulation of blood with 5 units of preservative free heparin/ml and then heat inactivated. Precipitated protein was then removed by eentrifugation (25,000 g x 1 hour) and in addition, in some cases, filtration through .22 micron filters. Cell Culture Peripheral blood lymphocytes (PBL) were suspended at a concentration of lxl 0 6 cells per ml in cRPMI containing 10% HIS and 7500 pM recombinant IL-2 (rIL-2). For the experiments reported in Tables 1, 3 and 4 below, 10 ml of cell suspension was added to a 50 ml SteriCell™ culture bag. For the experiment reported in Table 2 below, 100 ml of suspension was added to a 200 ml SteriCell™ culture bag. Both size bags had double entry ports. Cultures were incubated at 37° C under an atmosphere containing 5% CO2, for the times indicated in the tables. After the incubation period, the resultant nonadherent cells were harvested and resuspended in cRPMI supplemented with 20% FCS (Sterile Systems, Logan, UT) for cytotoxicity assays. Cytotoxicity Assays A 4 hour 5 1 Cr release assay was used to measure cytotoxicity of IL-2 treated cells (effector cells) to the Raji cell line (target cells), which is a natural killer cell-resistant, B-cell lymphoma cell line. Raji cells (2-10xl0 6 ) were incubated with 100 uCi of Na 2 51 Cr0 2 (DuPont NEN Products, Boston, MA) in 0.4 ml of Tris-phosphate buffered saline for 1 hour at 37°C. The cells were washed 4 times with RPMI-1640 containing 5% FCS and were resuspended to 10 5 cells/ml in cRPMI-20% FCS. The effector cells were suspended to various concentrations and 0.1 ml were added to round bottomed microtiter plates (Flow Laboratories, McLean, VA). The 5 I Cr labeled target cells were added to all wells in 0.1 ml and the plates were centrifuged at 200 x g for 5 minutes. After 4 hours of incubation at 37°C, the plates were centrifuged again and 0.1 ml of supernatant was removed from each well and counted in a gamma counter. Percentage of cytotoxicity was calculated from the formula (cpm, counts per minute): % of cytotoxicity = Experimental cpm-spontaneous cpm Total cpm - spontaneous cpm Each variable was tested in triplicate and the data are expressed as percentage of cytotoxicity or lytic units as indicated. One lytic unit is the number of tumor cells xlO 2 lysed by 8x10 3 effector cells. The lytic units are calculated from a plot of the percentage of cytotoxicity versus effector:target cell ratio from all ratios tested using a power curve formula. Thus, the reported lytic units reflect the activity measured at all effectoπtarget cell ratios. Results Table 1 shows results obtained using PBMC obtained from healthy donors. Since the starting cell number was lOxlO 6 , a 5-10 fold LAK-cell expansion was accomplished in relatively short term culture (10-14 days) utilizing normal lymphocytes. LAK activity fell by day 14 of culture to 63% of the peak activity seen on day 10. In subsequent experiments we used a similar system to determine if LAK-cell expansion could be accomplished with patient cells. Six patients with solid tumors were selected. All patients had received previous cytotoxic chemotherapy greater than one month prior to phlebotomy. The culture system differed from the one described in Table 1 only in that 200ml (100ml volume of cells) SteriCell™ culture bags were used and that passage times were decreased as seen in Table 2. Similar LAK-cell expansion and similar fall in lytic units were observed by day 14 of culture. Utilization of this culture system to expand LAK-cells for clinical use would require large volumes of human sera. We, therefore, attempted to solve this problem in 3 ways: a) use of lower serum concentrations; b) use of human plasma; c) use of serumless medias. We attempted to utilize the concentration of human sera for LAK-cell expansion (2%) currently used in clinical trials. The lower concentration of serum failed to support LAK cell expansion (Table 3) although LAK cell activity was observed at the lower serum concentration during the entire culture period. We next examined the use of human plasma prepared as described in Methods. In the representative experiments presented, pools of serum and heparin anticoagulated plasma were obtained from the same 5 normal donors and heat inactivated. Plasma (HIP) was then clarified by eentrifugation at 25,000 g x 1 hour. No statistically significant difference in LAK cell expansion or activation was observed when HIS or HIP were used. (Table 4). It appears that some plasma lots inhibited activation of LAK cells if not clarified by eentrifugation. Filtration of plasma was not helpful (data not shown). TABLE 1 Expansion of LAK cells In Vitro Utilizinα the SteriCell™ Culture Baα and Normal Donor Lvmphocvtes Dav £(a) 1Q 14 Cell No.( b ) LU Cell No. LU Cell No. LU Mean 13. 4 38 49 47 107 30 S.E. 0.6 7 8 8 22 5 ( a) Days of culture. At these time points lymphocytes were recultured in fresh media, r-IL-2 (7500 pM) at 0.5 x 10 6 /ml Starting cell number was 10x10 6 . ( b ) Data are expressed as cell number x 10 6 or as lytic units (mean ± standard error, N=6). Table 2 Expansion of LAK-cells in vitro utϋizinπ the SteriCell™ Culture Baα and Cancer Patient Lvmphocvtes Day 4 a -D 8 11 14 Cell No. LU Cell No. LU Cell No. LU Cell No. LU Mean 88 57 246 57 550 45 1306 34 S.E. 12.5 5 83 9 140 5 400 3 a) Days of culture. At these time points lymphocytes were recultured in fresh media as described in Table 1. Starting cell number (Day 0) was 100x10 6 lymphocytes. b ) Data are expressed as cell number (cell No.) x 10 6 or as lytic Units N=6 (LU) (mean ± standard error N=6). Table 3 Effect of Serum Concentration on LAK-cell Activity and Expansion in Normal Donors 10% Sera Day 4 a - b 8 12 1 5 Fold % Fold % Fold % Fold % Increas m Increase Kill Increase Kill Increase K i l l Mean 0.5 64 1.3 47 6.4 56 10 58 S.E. 0.1 2 .2 16 2.4 10 5 1 6 2.0% Sera Fold % Fold % Fold % Fold % Increase Kill Increase Kill Increase K il l Increase K i l l Mean 0.6 4 1 0.6 29 0.8 32 0.3 42 S.E. . 7 0.1 9 0.3 13 0.1 1 4 a) Days of Culture. At these time points, lymphocytes were recultured in fresh media as described in Table 1. Starting cell number was 10x10 6 lymphocytes. b ) Data are expressed as total fold increase in cell number on each day and as % kill at a 20:1 effector:target ratio (Mean ± standard error, N=3) 1 4 Table 4 Comparison of Serum and Plasma on LAK-cell Activity and Expansion in Normal Donors Serum Day a ' b 4 7 10 Fold % Fold % Fold % Increase Kill Increase Kill Increase Kill Mean 0.93 60 3 43 11 49 S.E. 0.04 3 0.4 8 2 8 Plasma Mean 1 .0 58 3.7 38 14 44 S.E. 0.6 5 0.5 7 3 7 a) Days of culture. At these time points lymphocytes were recultured in fresh media as described in Table 1. Starting cell number was 10x10 6 lymphocytes. b) Data are expressed as total fold increase in cell number on each day and as % kill at a 10:1 effector target ratio.

Claims: Claims 1. A process for generating and expanding cells having LAK cell activity which comprises (a) treating human peripheral blood mononuclear cells to deplete monocytes, (b) culturing the resulting peripheral blood lymphocytes in a culture medium containing IL-2 and human plasma or serum for about 2 to 7 days to generate LAK cell activity in the cells, and (c) expanding the cells by culturing in culture medium containing IL-2 and human plasma or serum in quantities sufficient to sustain cell expansion, in a culture bag made from an organic polymeric, oxygen permeable film, at an initial cell concentration in the range of about 0.5x10 6 to 1.5xl0 6 cells per ml, adding fresh medium from time to time as needed to sustain cell expansion, with or without removal of used medium, to bring the cell concentration back to within the range of about 0.5x10 6 to 1.5xl0 6 cells per ml, and continuing to culture under these conditions for at least about 2 days, the total culture time in steps (b) and (c) combined being at least about 9 days. 2. Process of claim 1 wherein: in step (a) the monocytes are depleted by treating the PBMC with a lower alkyl ester of an L-amino acid selected from alanine, aspartic acid, cysteine, glutamic acid, glutamine, phenylalanine, proline, tyrosine, tryptophan, and valine, or a hydrogen chloride salt thereof; in step (b) the cells are cultured for about 3 to 5 days; in step (c) the cells are cultured for at least about 4 days and used culture medium is removed before fresh culture medium is added; both steps (b) and (c) are carried out in a culture bag made from an organic polymeric, oxygen permeable film; and in steps (b) and (c) the culture medium contains at least about 10% human, heat inactivated serum or plasma, 3. Process of claim 2 wherein, in step (a) the lower alkyl ester is phenylalanine methyl ester and in steps (b) and (c) the medium contains plasma. 4. Process of claim 3 wherein, in steps (b) and (c), the bag is made from a copolymeric film material having an oxygen permeability of at least about 1.8xl0 5 um 3 (STP)/(m 2 «sec»Pa), and a thickness of from about 0.04 mm to about 0.23 mm., the materials being selected from the group consisting of: (a) copolymers of ethylene and an alpha olefin of 4-10 carbon atoms having a density of from about 0.915-0.925 g/cm 3 ; (b) copolymers of ethylene and methacrylic acid; (c) ionomers; and (d) laminates or coextrudates of an ionomer/polyester elastomer and a linear low density polyethylene elastomer. 5. Process of claim 4 wherein the bag is made from 4-5 mil thick film of a copolymer of ethylene and 1-octene. 6. Process of claim 1 wherein the LAK cells are derived from peripheral blood from a healthy donor. 7. Process of claim 1 wherein the LAK cells are derived from peripheral blood from a cancer patient. 8. Process of claim 5 wherein the cells are expanded about 5 to 10 fold compared to the number of peripheral blood lymphocytes initially used to generate the LAK cells. 9. Process of claim 8 wherein the cells after expansion exhibit cytotoxicity against Raji cells of about 30 to 60 lytic units in the standard four hour 51 Cr release assay.

Inventors: IRR JOSEPH DAVID, RINEHART JOHN JOSEPH, IRR, JOSEPH, DAVID, RINEHART, JOHN, JOSEPH
Applicants: DU PONT, UNIV OHIO STATE RES FOUND, E. I. DU PONT DE NEMOURS AND COMPANY, THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION
Application year: 1990
Main classification: C12N5
IPC codes: A61K35, A61P37, C08F10, C08F210, C12N5, C12R1