WO2008061047A2

WO2008061047A2 - Tissue specific gene therapy treatment

Info

Publication number: WO2008061047A2
Application number: PCT/US2007/084385
Authority: WO
Inventors: Jongming Li
Original assignee: Thomas Jefferson University
Priority date: 2006-11-10
Filing date: 2007-11-12
Publication date: 2008-05-22
Also published as: WO2008061047A3; CN101182509A

Abstract

The present invention relates to solid phase T cell selection for antigen specific T cell purification. Aspects of the present invention relate to methods for binding cells to a substrate, removing target T cells from a population of T cells, depleting alloreactive T cells from a sample such as bone-marrow or stem cell transplant cells, and measuring the frequency of antigen-specific T cells in a sample.

Description

e TISSUE SPECIFIC GENE THERAPY TREATMENT

CROSS REFERENCED APPLICATIONS

This application claims the benefit under 35 U. S. C. 1 1.9{e) of U.S. Provisional

Application Serial No: 60/858.287 filed on November 10, 2006, the contents of which are incorporated herein by reference

FIELD OF THE INVENTION

The field of the present invention includes the immobilization of eel is on a substrate and solid phase T cell selection for antigen specific T cell purification Aspects of the present invention reiate to methods for binding cells to a substrate, remov ing target T cells from a population of T cells, depleting ailoreacti e T cells from bone-marrow transplant cells, and measuring the frequency of antigen-specific T cells in a sample.

BACKGROIiND OF THE INVENTION

The adhesi e characteristics of adherent cells h

av e been utilized in v arious methods such as cell-based micro arrays, tissue engineering, and methods of isolating or expanding a population of specific cells The adhesive properties have been enhanced through the use of extracellular matrix proteins (e.g collagen or fibronectin) or their derivative peptides as well as polymer treated surfaces such as gelatin-oated glass slides. Frequently , such methods rely on hydrophobic interactions A major limitation of such techniques has been the inability of extending then- vise to non-adherent cells such as dendritic ceils or EBV transformed B ceils. Also, relying o

n non-covaent bond interactions can result in release of cells from the substrate. A variety of techniques hav e been dev eloped for selection and expansion or deletion of a population of antigen-specific T cells. The expansion of a specific population of cells can enhance their therapeutic immune effects in procedures such as stem cell transplantations wherein patients can benefit from, for example, increasing the population of lymphocytes that exhibit anti-tumor effects. Equally important are methods for depleting T cells from a population. For example, depletion of alloreactive T cells from donor lymphocytes to reduce occurrence or severity of the potentially lethal graft versus host disease. The methods may utilize differential density centrifugation, magnetic beads, or MHC tetramers associated with a specific peptide and bound to a fluorochrome. These methods are generally costly, time-consuming, and inefficient. Frequently, antigen specificity can be lost. Other methods utilize a membrane layer of elutriated antigen- presenting cells, such as monocytes, but these methods are limited to adherent cells, require knowledge of the MHC restricted epitope peptide in order to select antigen-specific cells, and may require special centrifuges (e.g. Elutra™).

[0005] A variety of methods are available for determining frequency of antigen- specific T cells in a given sample. The most common are flow cytometric intracellular cytokine assays, ELISPOT assays, and MHC-tetramer assays. These methods determine the amount (i.e. the concentration) of activated antigen-specific cytotoxic T-cells but require special equipment such as ELISPOT readers or flow cytometers. These methods are generally time and labor intensive and costly.

[0006] Adoptive T cell therapy is currently being investigated as an approach to treat malignant diseases in humans. Clinical studies in melanoma, nasopharyngeal cancer, Hodgkin's lymphoma, and leukemia have shown promising results. Clinically it involves the harvest of immunologically sensitized T cells, in vitro activation and expansion of these lymphocytes, and reintroduction of these cells to the host to effect the regression of tumors. There are numerous protocols for stimulating, isolating, and expanding effector cytotoxic T cells ex vivo that each uses a different combination of antigen presenting cells, cytokines, forms and concentrations of antigen, and durations of stimulation cycles. However there are several clinical bottlenecks that limit its clinical applications. These include both quantitative limitations (inability to generate sufficient number of antigen specific CD8 T cells) and qualitative limitations (generation of dysfunctional cytotoxic T cells because of excessive provision of cytokines, and costimulation).

[0007] For example, it usually takes several months (4 to 5 months) to engineer EBV- specific cytotoxic T cells in sufficient quantity and quality in clinical trials treating nasopharyngeal cancer, Hodgkin's lymphoma. Additionally, the cost of production of these effector T cells has been prohibitively high to allow widespread use. SUMMARY OF THE INVENTION

[0008] The present invention relates to solid phase T cell selection for antigen specific T cell purification. Aspects of the present invention relate to methods for binding cells to a substrate, removing target T cells from a population of T cells, depleting alloreactive T cells from a sample such as bone-marrow or stem cell transplant cells, and measuring the frequency of antigen-specific T cells in a sample.

[0009] One aspect of the present invention relates to a method for binding cells to a substrate. In this method the substrate is contacted with a 3-aminopropyltriethoxysilane linker (silane linker) such that the silane linker binds to the substrate. Next, the silane linker is contacted with a 2-O-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6-dichloro-s- triazine linker (PEG linker) such that the PEG linker binds to the silane linker. Next, the PEG linker is contacted with a cell under conditions to allow binding of the cell to the PEG linker. [0010] Another aspect of the present invention relates to an alternative method of binding cells to a substrate. In this method a protein-coated substrate is contacted with a 2-O- 4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6-dichloro-s-triazine linker such that the linker binds to the protein coated substrate. Next, the substrate and linker are contacted with a cell under conditions to allow binding of the cell to the 2-0-4,6-dichloro-s-triazine- polyethylene glycol-2-O-4,6-dichloro-s-triazine.

[0011] Those skilled in the art will recognize that substrates used in the various embodiments of the present invention can be glass, a polymer, metal, metal alloy, or any suitable substrate. Additionally, the substrate can be coated with a polymer, peptide, or protein and can be shaped as a slide, bead or other convenient form. [0012] In another embodiment the substrate is polyethylene terephthalate (PET) or glass coated with a protein such as bovine serum albumin (BSA) or a fragment thereof. [0013] A further aspect of the present invention relates to a method of removing target T cells from a population of T cells. In this method antigen-presenting cells are contacted with an antigen such that the antigen-presenting cells present the antigen. The antigen-presenting cells are then bound onto a substrate using any of the methods of the present invention discussed above. The population of T cells are contacted with the antigen- presenting cells that have been bound to the substrate such that T cells that are specific for the antigen bind to the antigen-presenting cells. The T cells that are non-specific for the antigen are then eluted. [0014] Another aspect of the present invention relates to a method of depleting alloreactive T cells from a sample of cells such as bone-marrow or stem cell transplant cells. In this method recipient-derived antigen-presenting cells are bound to a substrate using any of the methods of the present invention discussed above. Donor cells are passed over the substrate and contacted with recipient-derived antigen-presenting cells under conditions to allow any donor T cells that are specific for the antigens to bind to the antigen-presenting cells. The non-alloreactive cells are then eluted from the substrate. In one embodiment the substrate may be contained within a column or other suitable vessel or container. [0015] Yet another aspect of the present invention relates to a method of measuring the frequency of antigen-specific T cells in a sample. First, antigen-presenting cells are labeled with, for example, a fluorescent dye while T cells are labeled with, for example, a fluorescent dye that is distinct from the fluorescent dye used to label the antigen-presenting cells. Next, the antigen-presenting cells are immobilized on a surface using any of the methods of the present invention discussed above. Then the antigen-presenting cells are contacted with an antigen of interest causing the antigen-presenting cells to present the antigen. Next, lymphocytes are added to a second surface and the two surfaces are assembled such that the layer of the lymphocytes is formed in close contact to the layer of antigen- presenting cells. The assembly is then incubated for a sufficient time to allow immunologic synapse formation. Next, the immunologic synapses that form are detected and counted. The frequency of antigen-specific T cells is determined by dividing the total number of immunologic synapse formed by the total number of lymphocytes.

[0016] In another embodiment, antigen-presenting cells are labeled with, for example, a fluorescent dye while T cells are labeled with, for example, a fluorescent dye that is distinct from the fluorescent dye used to label the antigen-presenting cells as mentioned above. Antigen-presenting cells and T cells are then concentrated and mixed together. A single layer of cells can be formed with these cells on or in between, for example, glass slides. The assembly is then incubated for a sufficient time to allow immunologic synapse formation. Next, the immunologic synapses that form are detected and counted. The frequency of antigen-specific T cells is determined by dividing the total number of immunologic synapse formed by the total number of lymphocytes.

[0017] The methods of the present invention offer several advantages over previous approaches. Under previous methodologies for selecting antigen specific T cells, cells that spontaneously adhered to surfaces, such as monocytes, tended to float off the surface over time and are therefore not useful for selecting protein or tumor cell specific T cells. Furthermore, previous methods required special centrifuges for elutriation of monocytes or were limited to selecting antigen specific T cells only when MHC restricted epitope peptides were known. Additionally, antigen presenting cells that do not spontaneously adhere to surfaces such as dendritic cells, EBV transformed B cells (LCL), and others can now be can now be used to select antigen specific T cells. Those skilled in the art will recognize that the methods of the present invention can utilize a wide variety of cells including but not limited to those cells described herein.

[0018] Adoptive T cell therapy is currently being investigated as an approach to treat malignant diseases in humans. Clinical studies in melanoma, nasopharyngeal cancer, Hodgkin's lymphoma, and leukemia have shown promising results. Clinically it involves the harvest of immunologically sensitized T cells, in vitro activation and expansion of these lymphocytes, and reintroduction of these cells to the host to effect the regression of tumors. There are numerous protocols for stimulating, isolating, and expanding effector cytotoxic T cells ex vivo that each uses a different combination of antigen presenting cells, cytokines, forms and concentrations of antigen, and durations of stimulation cycles. However there are several clinical bottlenecks that limit its clinical applications. These include both quantitative limitations (inability to generate sufficient number of antigen specific CD8 T cells) and qualitative limitations (generation of dysfunctional cytotoxic T cells because of excessive provision of cytokines, and costimulation).

[0019] For example, it usually takes several months (4 to 5 months) to engineer EBV- specific cytotoxic T cells in sufficient quantity and quality in clinical trials treating nasopharyngeal cancer, Hodgkin's lymphoma. Additionally, the cost of production of these effector T cells has been prohibitively high to allow widespread use. To overcome these obstacles, we developed a novel solid phase T cell selection method to purify and expand antigen specific T cells. In this method, a fixing agent that developed in our laboratory fixes lymphoblastoid cell lines (LCL) on the surface of a solid support to select and purify T cells. The underlying hypothesis is that antigen-specific T cells recognize their cognate antigens and bind to them faster than non-antigen specific T cells. Indeed our data demonstrated that antigen-specific T cells are preferentially selected over non-antigen-specific T cells. After removing the floating cells, the antigen-specific T cells were subsequently "concentrated" on the surface because of immunological synapse formation. These activated T cells are then expanded with addition of low dose IL-2 (20 U/ml). This combination of selection and expansion enables us to generate LCL specific T cells with purity >60% and total cells >10⁸ in one week. More than 90% purity of LCL specific T cells can be achieved after 2^nd round T cell selection and expansion within 2 weeks. LMP2 specific T cells can be enriched to >30% when T cells are selected with LCL that are pulsed with LMP2 epitope peptides. This new T cell purification method is not only easy to perform, but also rapid and inexpensive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic showing EBV-specific T cells purification by solid phase T cell selection method.

[0021] Figure 2 is a graph showing EBV-specific T cell purification by solid phase T cell selection method.

[0022] Figure 3 is a graph showing the effect of selection time.

[0023] Figure 4 is a histogram showing cell count after T cell selection and expansion.

[0024] Figure 5 is a graph showing the results of cytotoxicity experiments.

[0025] Figure 6 is a schematic showing the general scheme of LMP2-specific T cells purification by solid phase T cell selection method.

[0026] Figure 7 is a graph showing CLG-specific T cells generated by CLG-pulsed-

LCL selection lyse T2 cell line pulsed with CLG and do not have significant killings of T2 cell line pulsed with CMV pp64 peptide P₄9_5-5o₃. Each point represents the mean of data from triplicate experiments.

[0027] Figure 8 is a graph showing Lymphocytes generated from various selection times lyse autologous LCL, whereas there is no significant percent lysis of K562 and LCL when MHC I antibody (w6/32) was added to the latter (cytotoxicity data from 5 minutes selection T cells were shown as representative). Each point represents the mean of data from triplicate experiments.

[0028] Figure 9 is a graph showing M27L-specific T cells generated by immobilized

LCL selection method recognized their targets and lysed melanoma cells preferentially over non-matched monocytes.

[0029] Figure 10 shows CEA CAPl-6D-specific T cell selection and expansion.

Before selection there was 0.2% of CAPl-όD-specific T cells. After first selection and expansion, the frequency of CAPl-6D-specific T cells was increased to 10%. After second selection and expansion, the frequency of CAPl-6D-specific T cells was increased to 35%. [0030] Figure 11 shows WTl- specific T cell selection and expansion. Before selection there was 0.2% of WTl -specific T cells. After first selection and expansion, the frequency of WTl- specific T cells was increased to 11%. After second selection and expansion, the frequency of WTl- specific T cells was increased to 40%.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention relates to methods for binding cells to a substrate, removing target T cells from a population of T cells, depleting alloreactive T cells from a sample, for example, bone-marrow transplant cells, and measuring the frequency of antigen- specific T cells in a sample.

[0032] In one embodiment, the present invention relates to a method for binding cells to a substrate comprising the steps: (i) providing a substrate; (ii) contacting the substrate with a 3-aminopropyltriethoxysilane linker such that the linker binds to the substrate; (iii) contacting the substrate and 3-aminopropyltriethoxysilane linker with 2-O-4,6-dichloro-s- triazine-polyethylene glycol-2-O-4,6-dichloro-s-triazine such that the 2-O-4,6-dichloro-s- triazine-polyethylene glycol-2-O-4,6-dichloro-s-triazine binds to the 3- aminopropyltriethoxysilane linker; and (iv) contacting a cell under conditions to allow binding of the cell to the 2-O-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6-dichloro-s- triazine.

[0033] In another embodiment, the present invention relates to a method of binding cells to a substrate comprising the steps: (i) providing a protein-coated substrate; (ii) contacting the protein-coated substrate with a 2-0-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6-dichloro-s-triazine linker such that the linker binds to the protein coated substrate; and (iii) contacting the substrate and linker with a cell under conditions to allow binding of the cell to the 2-O-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6-dichloro-s- triazine.

[0034] Substrates are any desired dimensionally stable solids which may consist of a ceramic substance, a glass, a metal, a crystalline material, a plastic, a polymer or copolymer, any combinations thereof, or a coating of one material on another. For example, but not limited to, (semi) noble metals such as gold or silver; glass materials such as soda glass, pyrex glass, vycor glass, quartz glass; metallic or non-metallic oxides; silicon, monoammonium phosphate, and other such crystalline materials; transition metals; plastics or polymers, including dendritic polymers, such as poly( vinyl chloride), poly( vinyl alcohol), poly(methyl methacrylate), poly( vinyl acetate-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, polystyrenes, polypropylene, polyethyleneimine; copolymers such as poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride), poly(ethylene- co-acrylic acid) or the like. In a preferred embodiment, the substrate may be modified to enable the immobilization of biological molecules, for example, but not limited to, coating with gold or silver, formation of a patterned surface, etc.

[0035] Another embodiment of the present invention relates to a method of removing target T cells from a population of T cells comprising the steps: (i) contacting antigen- presenting cells with an antigen such that the antigen-presenting cells present the antigen; (ii) binding the antigen-presenting cells onto a substrate using any of the methods of the present invention; (iii) contacting a population of T cells with the antigen-presenting cells that have been bound to the substrate such that T cells that are specific for the antigen bind to the antigen-presenting cells; and (iv) eluting the T cells that are non-specific for the antigen. [0036] Yet another embodiment of the present invention relates to a method of depleting alloreactive T cells from transplant cells comprising: (i) binding recipient-derived antigen-presenting cells to a substrate using any of the methods of the present invention; (ii) passing donor cells over the substrate thereby contacting the donor cells with the recipient- derived antigen-presenting cells under conditions to allow donor T cells that are specific for the antigens to bind to the antigen-presenting cells; and (iii) eluting the non-alloreactive cells from the substrate.

[0037] A further embodiment of the present invention relates to a method of measuring the frequency of antigen-specific T cells in a sample comprising: (i) labeling antigen-presenting cells (APCs) with a fluorescent dye; (ii) labeling T cells with a fluorescent dye that is distinct from the fluorescent dye used to label the APCs; (iii) immobilizing the APCs on a surface using any of the methods of the present invention; (iv) contacting the APCs with an antigen of interest causing the APCs to present the antigen; (v) adding lymphocytes to a second surface; (vi) assembling the two surfaces such that the layer of the lymphocytes is formed in close contact to the layer of APCs, (vii)incubating the assembly for a sufficient time to allow immunologic synapse formation; (viii) detecting the number of immunologic synapses formed; and (ix) determining the frequency of antigen-specific T cells by dividing the total number of immunologic synapse formed by the total number of lymphocytes. Alternatively, antigen-presenting cells are labeled with a fluorescent dye while T cells are labeled with a fluorescent dye that is distinct from the fluorescent dye used to label the antigen-presenting cells as mentioned above. Antigen-presenting cells and T cells are then concentrated and mixed together. A single layer of cells can be formed with these cells in between two glass slides. The assembly is then incubated for a sufficient time to allow immunologic synapse formation. Next, the immunologic synapses that form are detected and counted. The frequency of antigen-specific T cells is determined by dividing the total number of immunologic synapse formed by the total number of lymphocytes.

EXAMPLES

Example 1- Fixing cells to glass surface or polyethylene terephthalate (PET) surface

[0038] Materials Required:

Phosphate buffered saline (PBS) pH8.5

PBS with pH 2 (roughly adjusted with IN HCL based on pH stripes pH 0-14)

PBS pH strips pH 0-14 (EM Science, cat. 9590) pH strips pH 6.5-10 (EMD Chemicals Inc, cat. 9583)

Activated PEG polymer

Saturated sodium bicarbonate solution

5% BSA in PBS, sterile

[0039] Principle: BSA will adhere to glass or PET surface spontaneously. Activated

PEG polymer has an activated functional group at its each end, which will attach to BSA and cell surface protein respectively.

[0040] Procedure:

[0041] Add 3 ml of 5% BSA in a 100ml Pyrex glass beaker and allow it to stand overnight at room temperature. Decant BSA solution and wash with PBS three times to remove non-adhering BSA.

[0042] 5 ml PBS is added into a 50-ml conical tube containing 0.4 gram of activated

PEG polymer (pH will immediately changed to acidic). After vigorous agitation, add solution into 100ml Pyrex beaker which was pretreated with BSA. Monitor pH with pH strips to keep pH at 8 to 9. After 15 min at room temperature (2O⁰C), decant and rinse with PBS three times to remove residual un-reacted polymer. Add 3 ml PBS (pH 2). After addition of acidic PBS, cells can be fixed either right way or 24 hours later on the surface.

[0043] Prepare cells for fixation: if indicated, irradiate cells first before fixation. 15 x

10⁶ EBV transformed B cells (LCL) or other type of cells is washed twice with 50 ml PBS.

The protein-free cells are suspended in 2.5 ml PBS (pH8.5) and slowly introduced into the Pyrex Beaker along its inner edge. Move under the microscope for observation with a sterile cover on top. Do not disturb it for 2-3 minutes so that cells precipitate to the surface as soon as possible. Then move the beaker to cell culture incubator (37⁰C and 5% CO2). After 30 minutes, gently shake and decant the media. Wash gently with 10% FCS/RPMI for 3 times to remove floating cells. Allow the fixed cells to recover in 10%FCS/RPMI 1640 or 10%NABS/RPMI 1640 in culture incubator for 1 hour before further experiment. [0044] Results: 3.5 + 0.2 x 10⁶ LCL cells could be fixed on the surface of 100 ml

Pyrex^Tm beaker. 88 + 2 % of these cells are alive 24 hours after fixation when placed in cell culture incubator.

Example 2- Antigen-specific T cell selection

[0045] 50x10⁶ peripheral blood mononuclear cells (PBMC) in 50 ml complete medium (CM) consisting of RPMI 1640 medium containing 10% heat- inactivated normal AB serum were added to 5x10⁶ LCL in 75 cm2 culture flasks. On day 7, these primed lymphocytes were counted and subject to selection with LCL cells fixed on the solid support surface.

[0046] Solid phase T cell selection procedure:

[0047] 1^st round selection: After LCL (irradiated with 8000 rad) were fixed on the surface of 100 ml Pyrex beaker, primed lymphocytes (on day 7) at a concentration of 25 x 10⁶ ml^"1 in 3 ml warm CM were then added. Selection time was examined at 5, 10 and 15 minutes. Non-adherent cells were removed with a transfer pipette. The remaining adherent cells were washed gently three times with warm CM and again after 15-30 min incubation.

After 24 h, adherent cells were removed by gentle agitation with a pipette and transferred to a culture flask in 100 ml CM and rhIL-2 (final concentration, 2OU ml^"1). rhIL-2 was added every 2-3 days. On day 14, cultured cells were analyzed by flow-cytometric intracellular cytokine assay (ICC). Figure 1 illustrates the general scheme of T cell selection.

[0048] 2ⁿ round selection: T cells generated by 5 minutes selection on day 14 were used for a 2" round T cell selection and expansion. On day 21 cultured cells were analyzed by ICC.

Traditional EBV-specific T cell stimulation method

[0049] As a control, we also prepared polyclonal EBV-specific T cells with traditional stimulation method. 5OxIO⁶ PBMC were co-cultured with autologous LCLs (irradiated with 8000 rad) at a responder/stimulator ratio of 10:1 in culture flask. These were re- stimulated on day 7 and then again on day 14 at a responder/stimulator ratio of 4:1. IL-2 was added to a final concentration of 20 U ml^"1 starting on day 7 and then every 2-3 days. On day 21, cultured cells were analyzed by ICC.

[0050] Results: We have performed T cell selection experiments with PBMC from 3 donors and some representative data are shown below.

[0051] On day 7, IFN-γ producing CD8+ T cells (Donor 1, mean 2%) are measured by intracellular cytokine assay (ICC) in lymphocytes that are primed with LCL. See Figure 2. Subsequently these lymphocytes are selected (selection time 5 minutes) with LCL cells fixed on glass surface (■) and then expanded with IL-2. On day 14, IFN-γ producing CD8+ T cells are measured by ICC in cultured cells. These cultured cells are then selected again with LCL cells fixed on glass surface (■) and then expanded with IL-2. On day 21, IFN-γ producing CD8+ T cells are measured by ICC in cultured cells. As a control, the same primed lymphocytes are also stimulated with LCL without selection weekly twice (day 7 and 14) (A). After expanding T cells with IL-2, IFN-D producing CD8+ T cells are measured by ICC on day 14 (7 days after 1^st stimulation) and day 21(7 days after 2nd stimulation). [0052] The effect of selection time, see Figure 3. On Day 7, the same primed lymphocytes (Donor 1) shown in Figure 2 are selected by LCL cells fixed on the glass surface. The selection time is 5, 10 and 15 minutes. As a control, these same primed lymphocytes are also stimulated with LCL without selection at a responder/stimulator ratio of 4:1. On day 14, IFN-D producing CD8+ T cells are measured by intracellular cytokine assay (ICC).

[0053] Cell count after T cell selection and expansion, see Figure 4. Seven days after

1^st round T cell selection and expansion (donor 1) as shown in Figure 3, T cells were counted by Vi-CeIl Counter.

[0054] Cytotoxicity experiments, see Figure 5. T cells (Donor 1) generated by solid phase T cell selection method (selection time 5 minutes) can kill autologous LCL but not K562 cell line.

Example 3- LMP2-specific T cell selection

[0055] Procedure: Usually there are no detectable LMP2-specific T cells after PBMC are primed with LCL. The primed lymphocytes are then stimulated with LCL again and expanded with IL-2. [0056] After LCLs (irradiated with 8000 rad) are fixed on the surface of 100 ml Pyrex beaker, the adherent cells are pulsed with 10-20 DM selected LMP2 epitope peptides for 2 h at 37 ⁰C. The medium is removed and the adherent cells are washed three times in warm

PBS. Cultured lymphocytes at a concentration of 25 x 10⁶ ml^'1 in 3 ml warm CM are then added. Selection time is 5 minutes. Non-adherent cells are removed with a transfer pipette.

The remaining adherent cells are washed gently three times with warm CM and again after

15-30 min incubation. Next day, adherent cells are removed by gentle agitation with a pipette and transferred to a culture flask in 100 ml CM and rhIL-2 (final concentration, 2OU ml^"1).

IL-2 is added every 2-3 days. On day 7 after selection, cultured cells are analyzed by ICC.

Figure 6 illustrates the general scheme of T cell selection.

[0057] LMP2-specific T cells purification by Solid phase T cell selection method, see

Figure 6.

[0058] Results: 4 LMP2 peptides (all restricted to HLA-A*0201) are used in this experiment:

LLW: LLWTLWLL

CLG: CLGGLLTMV

FLY: FLYALALLI

TVC: TVCGGIMFL

[0059] Before selection, the frequencies (mean, Donor 5) for LMP2-specific T cells are:

LLW: 0.8%

CLG: 0.8%

FLY: 0.8%

TVC: 0.8%

Total LMP2-specific T cells: 3.2%%

Total EBV-specific T cells: 15%

[0060] After selection, the frequencies (mean) for LMP2-specific T cells are:

LLW: 1.5%

CLG: 4%

FLY: 10%

TVC: 7%

Total LMP2-specific T cells: 22% Total EBV-specific T cells: 70%

Example 4- Construction of a human cell column

[0061] We constructed a human cell column with this new activated PEG polymer and glass bead. The glass beads were coated with bovine serum albumin (BSA). Then activated PEG polymer was added in a condition described above so that PEG polymer is attached to BSA. LCLs were added following the conditions described above so that LCLs were bounded to the bead in the end.

[0062] This is for T cell depletion in human stem cell transplantation to treat malignancies. The purpose of T cell depletion is to reduce the acute GVHD (graft verse host disease). Currently, T cell depletion is through bead, photodepletion method which is costly and inefficient. The cell column is made of recipient antigen-presenting cells such as LCL or monocytes where these cells are fixed on the surface of glass beads by activated PEG polymer. Donors' lymphocytes will be allowed to pass down the column. Those alloreactive T cells (i.e. donor's T cells that can recognize recipients' antigen-presenting cells) will be selected and kept in the column. Cells that come out of column would be the ones that can not cause GVHD. This procedure should significantly reduce the cost and workload for T cell depletion.

Example 5- LMP2-specifϊc T cell selection with immobilized LCLs

[0063] 10Ox 10⁶ peripheral blood mononuclear cells (HLA* A0201 +) in 50ml culture medium consisting of RPMI 1640 medium containing 10% heat-inactivated normal AB serum were activated with 10x10⁶ monocytes loaded with EBV LMP2 peptide CLGGLLTMV (CLG) in 75 cm² culture flasks. On day 7, these were 0.8% of CLG-specific T cells.

[0064] After autologous irradiated (80Gy) LCLs, which were pulsed with 2μM peptide CLG for 4 hours prior to immobilization, were immobilized as described in Example I₁, they were loaded with peptide CLG again for 1 hour. Activated lymphocytes (90 x 10⁶ + 10 in 3 mL culture media, 37⁰C) were added onto immobilized antigen presenting cells in cell culture incubator. Various selection times (3, 5, 7.5, 10 and 15 minutes) were tested. Nonadherent cells were removed at the end of each selection time. The remaining adherent cells were gently washed with 37⁰C culture media. Washing was repeated after 30 minutes incubation at 37⁰C. IFN-γ producing cells were increased to 10%, 17%, 24%, 21% and 19% (mean) among cells selected at 3, 5, 7.5, 10 and 15 minutes, respectively on the support surface. Afterwards, these selected lymphocytes were expanded with addition of IL2 (20u/ml) the day after T cell selection and then every other day. On day 8, IFN-γ producing cells were increased to 55%, 70% and 65% (mean) among expanded cells of selection times 5, 7.5 and 15 minutes, respectively. There were 80 xlO⁶, lOOxlO⁶ and lOOxlO⁶ (mean) cells in final expanded cell population selected at 5, 7.5 and 15 minutes, respectively. More than 90% (mean) of these cell populations is CD3+CD8+. The cytotoxicity killings were shown in Figure 7.

Example 6- EBV-specifϊc T cell selection with immobilized LCLs

[0065J 100x 10⁶ peripheral blood mononuclear cells (HLA* A0201 +) in 50ml culture medium consisting of RPMI 1640 medium containing 10% heat-inactivated normal AB serum were activated with 10x10⁶ autologous irradiated (80Gy) LCLs in 75 cm² culture flasks. On day 7, these were 1.8% of EBV-specific T cells.

[0066] Autologous irradiated LCLs were immobilized as described in Example 1.

Activated lymphocytes (90 x 10⁶ + 10 in 3 mL culture media, 37⁰C) were added onto immobilized antigen presenting cells in cell culture incubator. Various selection times (1, 3, 5, 10 and 15 minutes) were tested. Non-adherent cells were removed at the end of each selection time. The remaining adherent cells were gently washed with 37⁰C culture media. Washing was repeated after 30 minutes incubation at 37⁰C. There were 22%, 44%, 20%, 12% and 8% (mean) IFN-γ producing cells among cells selected at 1, 3, 5, 10 and 15 minutes, respectively on support surface. Afterwards, these selected lymphocytes were expanded with addition of IL2 (20u/ml) the day after T cell selection and then every other day. On day 7, IFN-γ producing cells were increased to 80%, 65%, 50%, and 45% (mean) among the expanded cells of selection times 3, 5, 10 and 15 minutes, respectively. There were 30 xlO⁶, 100 xlO⁶, 12OxIO⁶ and 12OxIO⁶ (mean) cells in final expanded cell population selected at 3, 5, 10 and 15 minutes, respectively. When autologous monocytes were pulsed with EBV LMP2 peptides LLWTLWLL (LLW), CLG, and FLYALALLI (FLY) together and co- cultured with lymphocytes generated by selection and expansion, there were 17%, 19%, 9% and 7% (mean) IFN-γ producing cells in lymphocytes generated from 3, 5, 10 and 15 minutes selection. Lymphocytes generated here by solid phase selection method recognize and kill autologous LCL, whereas there are no significant killings toward K562 and autologous LCL when MHC I antibody (w6/32) was added to the LCL (Figure 8).

Example 7- Melanoma-specific T cell selection with immobilized LCLs

[0067] 100x10⁶ peripheral blood mononuclear cells (HLA*A0201+) in 50ml culture medium consisting of RPMI 1640 medium containing 10% heat-inactivated normal AB serum were activated with 5x10⁶ autologous irradiated (30Gy) dendritic cells loaded with M27L (Melan-A 26-35 variant ELAGIGILTV) in 75 cm² culture flasks. On day 7, these were 0.8% of M27L-specific T cells.

[0068] After autologous irradiated (80Gy) LCLs, which were pulsed with lOμM peptide M27L for 4 hours prior to immobilization, were immobilized as described in Example 1, they were loaded with peptide M27L again for 1 hour. Activated lymphocytes (90 x lO + 10 in 3 mL culture media, 37⁰C) were added onto immobilized antigen presenting cells in cell culture incubator. Various selection times (3, 5, 7.5 and 10 minutes) were tested. Non-adherent cells were removed at the end of each selection time. The remaining adherent cells were gently washed with 37⁰C culture media. Washing was repeated after 30 minutes incubation at 37⁰C. There were 6%, 7.4%, 8% and 8% (mean) IFN-γ producing cells among cells selected at 3, 5, 7.5 and 10 minutes, respectively on support surface. Afterwards, these selected lymphocytes of 10 minutes were expanded with addition of IL2 (20u/ml) the day after T cell selection and then every other day. On day 7, there were 65% (mean) IFN-γ producing cells among gated CD3+ cells (22OxIO⁶, >90% are CD3+ cells, mean). See Figure 9.

Example 8- CEA CAPl 6D-specific T cell selection with immobilized LCLs

[0069] After PBMCs (HL A* A0201 +) were activated by CEA CAP 1 6D-loaded-DCs

(lOμM) initially, followed by re-stimulation with monocytes pulsed with CEA CAPl 6D (lOμM), there was 0.2% (mean) of CAPl-6D-specific T cells. After 1^st selection with immobilized LCL loaded with CEA CAPl 6D (lOμM) (10 minutes selection time) followed by expansion with IL2 at 2OLVmI for 7 days, there were 10⁸ (mean) cells with 10% (mean) CAPl-6D-specific T cells (Figure 10). These lymphocytes were subject to 2^nd T cell selection with immobilized LCL loaded with CEA CAPl 6D (lOμM) (10 minutes selection time) followed by expansion of 7 days. There were 10⁸ (mean) cells with 35% (mean) CAP 1-6D- specific T cells (Figure 10). Example 9- WTl-specific T cell selection with immobilized LCLs

[0070] After PBMCs (HLA*A0201+) were activated by WTl -loaded-DCs (10μM) initially, followed by re-stimulation with monocytes pulsed with WTl (lOμM), there was 0.2% (mean) of WTl-specific T cells. After 1^st selection with immobilized LCL loaded with WTl (lOμM) (10 minutes selection time) followed by expansion with IL2 at 20U/ml for 7 days, there were 10⁸ (mean) cells with 11% (mean) WTl -specific T cells (Figure 11). These lymphocytes were subject to 2^nd T cell selection with immobilized LCL loaded with WTl

(lOμM) (10 minutes selection time) followed by expansion of 7 days. There were 10 (mean) cells with 40% (mean) WTl-specific T cells (Figure 11).

[0071] It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit and scope of the invention. Thus it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED:

1. A method of binding cells to a substrate comprising: a. providing a substrate, b. contacting the substrate with 3-aminopropyltriethoxysilane under conditions to allow binding of the 3-aminopropyltriethoxysilane to the substrate, c. contacting the substrate of step b with 2-O-4,6-dichloro-s-triazine- polyethylene glycol-2-O-4,6-dichloro-s-triazine under conditions to allow binding of the 2-O-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6- dichloro-s-triazine to 3-aminopropyltriethoxysilane; and d. contacting the substrate of step c with a cell under conditions to allow binding of the cell to the 2-0-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6- dichloro-s-triazine.

2. A method of binding cells to a substrate comprising: a. providing a protein-coated substrate, b. contacting the protein-coated substrate with 2-O-4,6-dichloro-s-triazine- polyethylene glycol-2-O-4,6-dichloro-s-triazine under conditions to allow binding of the 2-O-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6- dichloro-s-triazine to the protein coated substrate; and c. contacting the substrate of step b with a cell under conditions to allow binding of the cell to the 2-O-4,6-dichloro-s-triazine-polyethylene glycol-2-O-4,6- dichloro-s-triazine.

3. A method for removing target T cells from a population of T cells comprising: a. contacting antigen-presenting cells with an antigen such that the antigen- presenting cells present the antigen, b. binding the antigen-presenting cells onto a substrate using the method of claim 1 or 2, c. contacting a population of T cells with the antigen-presenting cells bound to the substrate of step b under conditions to allow the T cells that are specific for the antigen of step a to bind to the antigen-presenting cells of step b; and d. eluting the T cells that are non-specific for the antigen of step a.

4. A method of depleting alloreactive T cells from a sample comprising: a. binding recipient-derived antigen-presenting cells to a substrate using the method of claim 1 or 2, b. passing a sample comprising donor cells over the substrate of step a thereby contacting said sample with the recipient-derived antigen-presenting cells of step a under conditions to allow T cells from within said sample that are specific for the antigens of step a to bind to the antigen-presenting cells of step a; and c. eluting the non-alloreactive cells from the substrate.

5. A method of measuring the frequency of antigen-specific T cells in a sample comprising: a. labeling antigen-presenting cells (APCs) with a first label, b. labeling T cells with a second label distinct from the first label of step a, c. immobilizing the APCs of step a on a first surface using the method of claim 1 or 2, d. contacting the APCs of step c with an antigen of interest such that the APCs will present the antigen, e. adding lymphocytes to a second surface, f. contacting the two surfaces such that the layer of the lymphocytes of step e is formed in close contact to the layer of APCs of step d forming an assembly, g. incubating the assembly of step f for a sufficient time to allow immunologic synapse formation, h. detecting the number of immunologic synapses formed; and i. determining the frequency of antigen-specific T cells by dividing the total number of immunologic synapse formed by the total number of lymphocytes.

6. A method of measuring the frequency of antigen-specific T cells in a sample comprising: a. labeling antigen-presenting cells (APCs) with a first label, b. labeling T cells with a second label distinct from the first label of step a, c. mixing the labeled APCs of step a with the labeled T cells of step b, d. contacting the mixture of step c to a first surface, e. covering the surface of step e with a second surface forming an assembly, f. incubating the assembly of step e for a sufficient time to allow immunologic synapse formation, g. detecting the number of immunologic synapses formed; and h. determining the frequency of antigen-specific T cells by dividing the total number of immunologic synapse formed by the total number of lymphocytes.

7. The method of claims 1-6 wherein the substrate is selected from a group comprising glass, plastic, metal, metal alloy, ceramic, or a polymer.

8. The method of claims 1-6 wherein the substrate is coated with a polymer or protein.

9. The method of claims 1-6 wherein the substrate is a bead.

10. The method of claim 8 wherein the protein coating the substrate comprises BSA.

11. The method of claim 1 or 2 wherein the cells comprise antigen-presenting cells, tumor cells, and pathogen-infected cells.

12. The method of claim 1-6 wherein the cells are non-adherent cells.

13. The method of claim 1-6 wherein the substrate is a glass slide.

14. The method of claim 11 wherein the antigen-presenting cells are non-adherent cells.

15. The method of claims 1-6 wherein the substrate comprises Polyethylene terephthalate.

16. The method of claims 1-4 wherein the substrate is within a column.

17. The method of claim 3 wherein the donor cells comprise bone-marrow or peripheral stem cells.

18. The method of claim 4 wherein the donor cells comprise bone-marrow or peripheral stem cells.

19. The method of claim 5 wherein the first label of step a comprises a first fluorescent dye and the second label of step b comprises a second fluorescent dye.

20. The method of claim 6 wherein the first label of step a comprises a first fluorescent dye and the second label of step b comprises a second fluorescent dye.