ex, since you seem to have a deep understanding of the basic science, then perhaps you can explain what exactly separates the dendritic cells of -L and -Direct. As someone who clearly doesn’t possess your superior knowledge of the basic science, I’ve wondered about that myself. We all know that the administration, source of antigen, (lysate vs. tumor tissue in vivo) activation method, and maturity of the cells are different, but what specifically in the cell characteristics and critical quality attributes of the actual dendritic cells are different?
Please feel free to use these examples and claims made in Northwest Bio’s patents for their assays:
The following examples are provided merely as illustrative of various aspects of the invention and shall not be construed to limit the invention in any way.
Examples Example 1: Co-Stimulation Assay In this example, an antigen-independent co-stimulation assay is used to measure the quality of preparations of dendritic cells. Dendritic cells preparations were made from 26 different human subjects, as follows: PBMC were isolated from leukophereses blood from each patient and cultured for 6 days in OptiMEM media (Gibco-BRL) supplemented with 5% autologous plasma, followed by another day of culture in the presence of BCG, a dendritic cell maturation agent.
Peripheral blood mononuclear cells (PBMC's) were prepared as follows: Leukopheresed blood was diluted with buffered saline, overlaid upon FICOLL solution and spun for 20 minutes at 2000 rpm. The white cells at the interface were isolated. The co-stimulatory function was removed from PBMC using magnetic bead selection. Briefly, antibodies for MCH class II were coupled to magnetic beads (Dynal Corp., New York). The magnetic beads were added to PBMC to remove cells having MHC class II molecules as follows: Beads were added to PBMC at 2-10 beads per cell, and incubated for one hour. Following this incubation, bead-bound cells (APC) were removed using a magnetic device. The resulting population of PBMC were largely APC-free and contained >50% T cells.
The proliferation assay was performed as follows: 1×104 dendritic cells were added to each well of a 96-well culture plate and contacted with 1 ng of anti-CD3 antibody (BD Pharmingen, San Diego, Calif.). Then 1×105 enriched T cells (supra) were added, resulting in a final volume of 0.2 ml per well. The plate was incubated for 26 hours, and then pulsed with 3H-thymidine. The plate was further incubated for 18 hours before harvesting and determination of incorporated label.
T cell proliferation (delta cpm) was measured as the difference between 3H-thymidine incorporation by T cells stimulated with a sample of the dendritic cell preparation in the presence of anti-CD3 antibody minus 3H-thymidine incorporation by T cells stimulated with the sample of the dendritic cell preparation alone. The mean delta cpm for each dendritic cell preparation was calculated as the mean of triplicate samples.
The results of the assay are shown in the following Table 1.
T cells incubated with anti-CD3 antibody alone exhibited a mean cpm of about 370. This low level of 3H-thymidine incorporation establishes that anti-CD3 antibody was added at suboptimal concentrations. T cells co-cultured with a sample of the dendritic cell preparation alone exhibited an average cpm of about 2281 cpm. In contrast, the mean delta cpm for T cells co-cultured with anti-CD3 antibody and the dendritic cells was 39,747 cpm, with a standard deviation of 14,972 cpm. The distribution of the delta cpm values was normal, but with significant skewing to the higher end of the range of delta cpm values.
A reference sample of a dendritic cell preparation from a normal human donor had a mean delta cpm of about 51,260, with a standard deviation of 12,911 cpm. Based on these data, dendritic cell preparations exhibiting proliferation of 15,000 delta cpm or greater were found to be of acceptable quality.
Example 2: Specificity of the Antigen Independent Co-Stimulation Assay The co-stimulation assay is based on the ability of certain types of APCs to stimulate antigen-independent T cell proliferation. The following studies were performed to establish the specificity of the assay.
The non-dendritic cell types most commonly found in dendritic cells preparations were prepared and used in the co-stimulatory assay alone and spiked into a characterized (reference) dendritic cell preparations. T cells, B cells and monocytes were purified from peripheral blood mononuclear cells (PBMC) by magnetic bead separation with negative-selection using antibodies. For T cells, antibodies to HLA-DR, CD19 and CD56 were used; for B cells, antibodies to CD2, CD3 and CD14 were used. For monocytes, antibodies to CD3, CD19 and CD56 were used.
The assays were performed as follows: T cells, B cells and monocytes were used instead of dendritic cells in the proliferation assay, as described in Example 1. T cells, used in place of dendritic cells, were irradiated to prevent proliferation. Then allogenic indicator T cells were added and proliferation determined 40 hours later, as described supra.
T and B lymphocytes, when used in place of dendritic cells, were unable to stimulate T cells in the co-stimulatory assay at any of the concentrations tested. As shown in the following Table 2, monocytes isolated from PBMC were able to stimulate T cell proliferation (3H-thymidine incorporation) when added at 2.5 times the cell number of dendritic cells. However, the proliferation was much lower than that obtained using an equal number of dendritic cells.
CD14 positive, CD11c positive cells and CD14 negative, CD11c positive cells in dendritic cell preparations were found to have equivalent co-stimulatory activity and were both considered to be dendritic cells.
Example 3: Characterization of Dendritic Cells The co-stimulatory activity of CD11c positive, CD14 positive cells and CD11c positive, CD14 negative were separated from a preparation of dendritic cells by fluorescent activated cell sorting (FACS) using labeled antibody against CD14 (Pharmingen).
In these assays, CD11c positive, CD14 positive cells and CD11c positive, CD14 negative cells from the dendritic cell preparation appeared to have equivalent co-stimulatory activity. Thus, both cell types were collectively referred to as dendritic cells.
Example 4: Effect of Dendritic Cell Viability on the Co-Stimulatory Assay The possible effect of dead cells on an assay according to the present invention was determined. Briefly, dendritic cells were killed by treatment with 1% formaldehyde for 30 minutes or by heating to 56° C. for 1 hour. These dead (killed) cell suspensions were tested in a co-stimulatory assay. The dead cells were mixed with live dendritic cells at defined ratios. The assays were performed as described above in Example 1, except as otherwise described below.
As shown in the following Tables 3 and 4, heat-killed dendritic cells retained essentially no activity in a co-stimulatory assay according to the present invention. Formaldehyde-treated dendritic cells still retain about 20% live dendritic cells, as determined by propidium iodide staining; these cells retain co-stimulatory activity at reduced levels. In a third experiment, the addition of killed cells did not interfere with the assay.
TABLE 3 Effect of Cell Viability on Co-Stimulatory Assay Cells Used Per Well Viability Delta cpm 104 live DC 100% 27482 104 total DC 63% 15791 104 formaldehyde-fixed DC 20% 6957 104 heat-killed DC 4% -42
TABLE 4 Effect of Dead Cells on Co-Stimulatory Assay Number or Dead Cells Kill Delta Added per Well Method cpm 1000 Formaldehyde 27,076 2000 Formaldehyde 27,336 3000 Formaldehyde 27,661 5000 Formaldehyde 26,478 1000 Heat 25,391 2000 Heat 24,270 3000 Heat 23,560
Example 5: Linearity of the Antigen-Independent Co-Stimulation Assay Increasing numbers of dendritic cells were added to fixed numbers of indicator T cells to determine the relationship between dendritic cell number and 3H-thymidine assay. Zero, 2000, 6000 or 10,000 dendritic cells were placed in wells. The following culture conditions were used, as described in Example 1 (e.g., 1 ng of anti-CD3 antibody per well with 105 T cells). The total incubation time was 40 hours; the last 18 hours of incubation was performed in the presence of 3H-thymidine.
3H-thymidine uptake of indicator T cells increased substantially linearly as the number of dendritic cells increased In particular, the delta cpms observed were 0, about 7,000 cpm, about 15,000 cpm and about 27,000 cpm, respectively. The formula y=2.7183-134.13 (R2=0.9879) approximated this linear relationship. These results demonstrated that co-stimulatory activity can be linearly dependent on the number of DC.
Example 6: Precision The precision of a co-stimulation assay according to Example 1 was determined by having three operators test the same lot of dendritic cells. Each operator tested the lot three times, once a day on three consecutive days. The data were analyzed for duplicability (intra-assay variance), repeatability (inter-assay variance), and reproducibility (inter-operator variance). The raw data are shown in the following Table 5. The coefficient of variation (CV) ranged from 1.25 to 16.18, with higher CV observed at lower levels of 3H-thymidine incorporation.
TABLE 5 Precision Of The Co-Stimulation Assay - Raw Data Indicator T Cells With Dendritic Cells With Antigen Without Anti-CD3 Antibody CPM CPM CPM Mean SD CV Oper- Day 1 856 947 940 914 50.6 5.54 ator 1 Day 2 3161 3769 3190 3373 343.0 10.17 Day 3 1126 1113 1226 1155 61.8 5.35 Oper- Day 1 870 1180 946 999 161.6 16.18 ator 2 Day 2 1092 1297 1379 1256 147.8 11.77 Day 3 3853 4249 4599 4234 373.2 8.82 Oper- Day 1 977 1223 1132 1111 124.4 11.20 ator 3 Day 2 913 1011 1218 1047 155.7 14.87 Day 3 1556 1835 2118 1836 281.0 15.30 Indicator T Cells With Dendritic Cells With Antigen Without Anti-CD3 Antibody % CPM CPM CPM Mean SD CV Oper- Day 1 24248 23523 26526 24765.67 1567.00 6.33 ator 1 Day 2 69711 73655 64396 69254 4646.39 6.71 Day 3 29232 31084 30453 30256.33 941.53 3.11 Oper- Day 1 26383 25821 26390 26198 326.51 1.25 ator 2 Day 2 35386 34414 31738 33846 1889.16 5.58 Day 3 36821 35714 38678 37071 1497.73 4.04 Oper- Day 1 31390 31968 32644 32000.67 627.64 1.96 ator 3 Day 2 28011 31085 28443 29179.67 1664.14 5.70 Day 3 42181 40188 44625 42331.33 2222.32 5.25
All conditions were run in triplicate, so triplicate cpm values were examined as a measure of duplicabilty. Repeatability and reproducibility were analyzed using delta cpm. The mean delta cpm, standard deviation and inter-assay Coefficient of Variation (CV) for each operator are depicted in the following Table 6. The CV of Operator #1 was 57.8%, for Operator, 2 14.4%, and for Operator 3, 19.6%. Reproducibility is represented by the CV of the mean delta cpm for all three operators and is 14%.
TABLE 6 Repeatability And Reproducibility Mean Coeffi- Delta Delta Delta Delta Standard cient of cpm cpm cpm cpm Deviation Variation Operator 1 23852 65881 29101 39611 2901 57.8 Operator 2 25199 32590 32837 30209 4340 14.4 Operator 3 30890 28133 40495 33173 6489 19.6 MEAN 34331 4807 14.0
Example 6 PSMA-loaded dendritic cells are assayed as follows: The loaded dendritic cells are lysed using a detergent, and the lysate equivalent of 5×105 cells is electrophoresed in each lane of a 7.5 percent SDS PAGE gel. After resolution of the lysate proteins at 150 volts for about an hour, the proteins are transferred to a PVDF or nylon membrane. Western blotting is performed using a PSMA-specific monoclonal antibody, 4D8 (ATCC HB 12487; U.S. Pat. No. 6,150,508). The binding of antibody is visualized by chemiluminescence and exposure to film. The identity of PSMA is determined by co-localization of a standard PSMA protein run on the gel.
The previous examples are provided to illustrate, but not to limit, the scope of the claimed inventions. Other variants of the inventions will be readily apparent to those of ordinary skill in the art and encompassed by the appended claims. All publications, patents, patent applications and other references cited herein are hereby incorporated by reference.
Claims 1. A method for determining the quality of the antigen-independent, co-stimulatory activity of a population of antigen presenting cells (APCs), comprising: providing allogeneic T cells having a known functional activity and being substantially free of co-stimulatory activity; providing a sample of APCs of unknown co-stimulatory activity; contacting the T cells with a sub-optimal concentration of an antigen-mimetic agent, wherein the antigen-mimetic consists of a CD3 binding agent that is an antibody, a plant lectin, or a mitogen; contacting the T cells with the sample of APCs of unknown co-stimulatory activity; determining the activation of the T cells contacted with the antigen-mimetic agent and the sample of APCs; and comparing the determined activation of the T cells with a standard activation index for the T cells to determine the quality of the co-stimulatory activity of the population of APCs. 2. The method of claim 1, wherein the T cells and the APCs are syngeneic, or allogeneic. 3. (canceled) 4. The method of claim 1, wherein the CD3 binding agent is anti-CD3 antibody, or an antigen binding fragment thereof. 5. The method of claim 1, wherein the APCs are dendritic cells. 6. The method of claim 5, wherein the dendritic cells are mature dendritic cells derived from immature dendritic cells by contacting ex vivo with a dendritic cell maturation agent, or the dendritic cells are immature dendritic cells. 7. (canceled) 8. The method of claim 1, wherein the T cells have been substantially depleted of peripheral blood mononuclear cells expressing CD14, CD54, CD80, CD83 or CD86 molecules on their cell surface, or the T cells have been substantially depleted of peripheral blood mononuclear cells expressing MHC class II molecules on their cell surface. 9. (canceled) 10. The method of claim 1, wherein the activation of the T cells is determined by 3H-thymidine uptake assay, by assaying T cell cytokine production, or is determined by detecting the modulation of expression of a T cell activation marker. 11. (canceled) 12. The method of claim 10, wherein the assayed T cell cytokine production is IFN? or Interleukin 2 production, the assayed T cell cytokine production is extracellular cytokine production, or the assayed T cell cytokine production is intracellular cytokine production. 13-15. (canceled) 16. The method of claim 10, wherein the T cell activation marker is CD25, CD69, CD44 or CD125. 17. The method of claim 16, wherein the T cell activation marker is detected using labeled antibody capable of binding to the T cell activation marker. 18. The method of claim 1, wherein comparing the determined activation with the standard activation index includes comparing the determined T cell activation with activation of the T cells contacted with the sample of dendritic cells alone to determine the quality of the dendritic cells. 19. The method of claim 1, wherein the standard activation index is a threshold value, or is a range of values, each value associated with a predetermined quality of dendritic cells. 20. (canceled) 21. The method of claim 1, further comprising determining presentation of a predetermined antigen by the APCs. 22. The method of claim 21, wherein presentation of the predetermined antigen is determined by Western blotting, flow cytometry or activation of antigen-specific T cells. 23. A method for determining the quality of the antigen-independent co-stimulatory activity of a preparation of dendritic cells, comprising: contacting a first quantity of T cells, which are substantially free of co-stimulatory activity and have a known functional activity, with a suboptimal quantity of an antigen-mimetic agent, wherein the antigen-mimetic consists of a CD3 binding agent that is an anti-CD3 antibody, a plant lectin, or a mitogen, and with a first sample of a dendritic cell preparation of unknown co-stimulatory activity; determining a first activation value for the first quantity of T cells; contacting a second quantity of T cells with a second sample of the dendritic cell preparation or the suboptimal quantity of the antigen-mimetic agent; determining a second activation value for the second quantity of T cells; and comparing the first and second activation values to determine the quality of the co-stimulatory activity of the dendritic cell preparation. 24. The method of claim 23, wherein the T cells are allogeneic with respect to the dendritic cell preparation, or wherein the T cells are syngeneic with respect to the dendritic cell preparation. 25. (canceled) 26. The method of claim 23, further comprising determining presentation of a predetermined antigen by the dendritic cells. 27. The method of claim 26, wherein presentation of the predetermined antigen is determined by Western blotting, flow cytometry or activation of antigen-specific T cells. 28. A method for determining the quality of a preparation of dendritic cells, comprising: (1) providing a dendritic cell preparation of unknown co-stimulatory activity and unknown antigen presenting ability for a predetermined antigen; (2) determining the co-stimulatory activity of the dendritic cell preparation, said determination of co-stimulatory activity comprising (a) providing allogeneic T cells of known functional activity and substantially free of co-stimulatory activity; (b) contacting the T cells with a suboptimal quantity of an antigen-mimetic agent, wherein the antigen-mimetic is a CD3 binding agent consisting of an anti-CD3 antibody, or an antigen binding fragment thereof, a plant lectin, or a mitogen, and with a first sample of the dendritic cell preparation; (c) determining the activation of the contacted allogeneic T cells; and (d) comparing the determined activity of the contacted T cells with the standard activation index for the T cells to the determined co-stimulatory activity of the dendritic cell preparation; (3) determining presentation of the predetermined antigen by the preparation of dendritic cells, said determination of presentation comprising: (a) contacting a second sample of the dendritic cell preparation with the predetermined antigen; and (b) determining the amount of predetermined antigen presented by the dendritic cells; and (4) determining the quality of the dendritic cell preparation based on the determined co-stimulatory activity and determined antigen-specific presentation of the predetermined antigen. 29. The method of claim 28, wherein the antigen-mimetic agent is a CD3 binding agent, a plant lectin or a mitogen. 30. The method of claim 28, wherein the dendritic cells are mature dendritic cells derived from immature dendritic cells by contacting ex vivo with a maturation agent, wherein the dendritic cells are immature dendritic cells, or wherein the T cells have been substantially depleted of peripheral blood mononuclear cells expressing MHC Class II, CD14, CD54, CD80, CD83 or CD86 molecules on their cell surface. 31-32. (canceled) 33. The method of claim 28, wherein the activation of the T cells is determined by 3H-thymidine proliferation assay, by assaying T cell cytokine production, or by expression of at least one T cell activation marker. 34. The method of claim 28, wherein the activation of the T cells is determined by assaying T cell cytokine production. 35. The method of claim 34, wherein the T cell cytokine production is IFN? or Interleukin 2 production, wherein the T cell cytokine production is extracellular cytokine production, or wherein the T cell cytokine production is intracellular cytokine production. 36-38. (canceled) 39. The method of claim 33, wherein the T cell activation marker is CD25, CD69, CD44 or CD125. 40. The method of claim 39, wherein the T cell activation marker is detected using labeled antibody capable of binding to the T cell activation marker. 41. The method of claim 28, wherein determining the co-stimulatory activity includes comparison of the determined T cell activation with a standard activation index for the T cells. 42. The method of claim 41, wherein the standard activation index is a threshold value, or is range of values, the values associated with different predetermined co-stimulatory activities. 43. (canceled) 44. The method of claim 28, wherein presentation of the predetermined antigen is determined by Western blotting, flow cytometry or activation of antigen-specific T cells.
