WO1999063050A2

WO1999063050A2 - Method for preparation and in vivo administration of antigen presenting cell composition

Info

Publication number: WO1999063050A2
Application number: PCT/US1999/012142
Authority: WO
Inventors: Peter Van Vlasselaer
Original assignee: Dendreon Corporation
Priority date: 1998-06-02
Filing date: 1999-06-01
Publication date: 1999-12-09
Also published as: WO1999063050A3; NZ508936A; EP1082411A2; AU4226999A; JP2002517409A; CA2330027A1

Abstract

A method of obtaining a blood-cell fraction enriched for antigen presenting cells is disclosed. The method includes treating a patient with an agent effective to mobilize stem cells, obtaining a blood sample from a patient, subjecting it to density centrifugation over a medium with a defined density, harvesting the cell fractions enriched in precursor antigen-presenting cells, and culturing the harvested cells under conditions effective to induce cells having the morphology, phenotype, and function of dendritic cells. A method of using such antigen presenting cell for in vivo therapy and pharmaceutical compositions comprising such antigen presenting cells are also disclosed.

Description

METHOD FOR PREPARATION AND IN VIVO ADMINISTRATION OF ANTIGEN PRESENTING CELL COMPOSITION

Field of the Invention

The present invention relates to methods for obtaining antigen-presenting cells (APC) and precursors thereof from a patient and use of such cells for therapy in the patient. The methods of the present invention include a multi-step therapeutic approach comprising in vivo therapy with APC, and in particular, a method of preparing a composition containing APC useful for in vivo immunotherapy and compositions comprising such APC.

Background of the Invention

Numerous different therapeutic regimes are presently employed for cancer therapy. Such regimes may include one or more of: surgery, chemotherapy, x- irradiation, immunotherapy, stem cell isolation and replacement, and in vitro simulation of the total stem cell fraction or sub-fractions derived therefrom, followed by return of the stimulated cells to the patient. (Vlasselaer, U. S. Pat No. 5,474, 687, issued December 12, 1995). Not only are there various types of therapeutic protocols, but numerous variations of such protocols exist for mobilizing, identifying, and isolating the stem cells, fractionating the stem cells and for culturing them in vitro. [Kennedy, Pharmacotherapy, 18(1 Pt 2) p3S-8S, (1998)]. Most relevantly, this is true with respect to the dendritic cells (DC) found within the stem cell population, which can be stimulated to become particularly effective antigen presenting cells (APC). Such APC are capable of presenting antigen to any T cells, even T cells that have never encountered the subject antigen ("naive T cells").

Past and present cancer treatment regimes have obtained some success with surgery, x- irradiation. and one or both of chemotherapy and immunotherapy. Chemotherapy/ immunotherapy has been shown to result in mobilization of stem cells from the bone marrow into the peripheral blood. In vivo administration of cvtokines, which is gaining wide use either as an independent treatment, or as part of a treatment in combination with other cancer therapies, has been demonstrated to stimulate stem cell mobilization. (Heinzinger et al., Leukemia (ENGLAND), 12 (3) p333-9, 1998).

More recently, cancer treatment regimes have included stem cell replacement therapy following in particular, one or more of x-irradiation, chemotherapy, and immunotherapy with cytokines. Such stem cell replacement therapy generally involves collection of patient cells, stem cell isolation, and return of the stem cells to the patient.

Each of the regimens outlined above has met with some success; however, recurrence of disease may occur for a number of reasons such as the presence of residual tumor cells that remain after such treatment or the recurrence of disease.

Various forms of cellular immunotherapy which have gained wider acceptance recently include the in vivo administration of antigen as a means to vaccinate the patient against disease or cellular therapy with cells which have been in vitro stimulated by a particular antigen and are subsequently returned to the patient.

Vaccination with viable antigen-pulsed APCs can accommodate this clinical need. The invention described herein is directed to a means of collecting APCs from cancer patients and their use in immunotherapy.

Summary of the Invention

It is therefore a general object of the invention to provide methods for immunotherapy and cancer therapy that include the step of administering antigen presenting cells to a patient. It is another object of the invention to provide methods for obtaining such APC and compositions comprising such cells for use in immunotherapy.

The invention includes, in one aspect, a method for obtaining antigen presenting cells from a human patient. The method includes; ( 1 ) treating a patient with an agent effective to mobilize stem cells from the bone marrow into the peripheral blood, (2) obtaining from the patient a blood cell fraction enriched in peripheral blood mononuclear cells, (3) subjecting the blood cell fraction to density centrifugation,(4) harvesting the cells at the interphase to obtain a cell fraction enriched in precursor antigen-presenting cells, and (5) culturing the harvested cells under conditions effective to induce cells having the morphology, phenotype, and function of dendritic cells. The density centrifugation step is preferably carried out by layering the product of (2) over a separation medium having a density of 1.0605 ± 0.0005 gr/ml, an osmolality of 280 mOsm/kg H₂0, and a pH of 7.4.

The invention is based, in part, on the discovery that a separation medium such as a colloidal silica solution having a density of 1.0605 ± 0.0005 gm/ml, an osmolality of 280 mOsm/kg H₂0, and pH 7.4 effectively separates APCs or CD34⁺ stem cells from the majority of blood cells when apheresed blood or a bone marrow buffy coat is overlaid on the separation medium, and that this cell fraction is particularly enriched in APC.

In one aspect, the agent effective to mobilize stem cells from the bone marrow into the peripheral blood, is one or more chemotherapeutic agents or x-irradiation. In a further aspect, the mobilization is accomplished by administering to the patient one or more chemotherapeutic agents alone or in combination with a cytokine. The immunotherapy and cancer therapy methods of the present invention comprise the steps of (1) obtaining a cell fraction enriched in precursor antigen-presenting cells [as described in steps (l)-(4) above], (2) either returning a portion of the fraction of cells enriched in precursor antigen presenting cells to the patient prior to culturing, or culturing the entire fraction of cells enriched in precursor antigen presenting cells under conditions effective to induce cells having the morphology, phenotype, and function of dendritic cells followed by (3) administering the exposed cells to the patient. The cells of the present invention may be induced to have the morphology, phenotype, and function of dendritic cells by exposing the harvested cells to an antigen precursor such as a polypeptide containing a peptide antigen or other antigen which is effective to induce in the cultured cells, or by cell-surface presentation of one or more peptide antigens against which an immune response is desired.

In one aspect, the antigen precursor includes DNA encoding a polypeptide containing the peptide antigen or other antigen, and exposing includes introducing the DNA into the cells that have the morphology, phenotype, and function of dendritic cells.

In a related aspect, the invention includes a cell composition for use in immunotherapy containing a mixture of stem cells and precursor antigen presenting cells cultured under conditions effective to induce the morphology, phenotype, and function of dendritic cells of the type described above. The precursor APC of the present invention are further exposed to an antigen precursor effective to induce in the changed cells, cell-surface presentation of one or more peptide antigens against which an immune response is desired. These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

Brief Description of the Drawings Figure 1 depicts the antigen presenting capability of dendritic cells derived from the interphase versus the pellet of a blood cell fraction, from a mobilized patient, enriched in peripheral blood mononuclear cells, following density centrifugation on a medium having density of 1.0605 ± 0.0005 gm/ml, an osmolality of 280 mOsm/kg H20. and a pH of 7.4, harvest and culture for a time sufficient for induction to cells that have the morphology, phenotype, and function of dendritic cells. The results of 3H-TdR incorporation are presented following incubation of cells derived from the interphase and pellet fractions with allogeneic cells.

Figure 2 depicts the antigen presenting capability of dendritic cells derived from the interphase versus the pellet of a blood cell fraction, from a mobilized patient, enriched in peripheral blood mononuclear cells, following density centrifugation on a medium having a density of 1.0605 ± 0.0005 gm/ml, an osmolality of 280 mOsm/kg H20, and pH 7.4, harvest, and culture for a time sufficient for induction to cells that have the morphology, phenotype, and function of dendritic cells in the presence of Keyhole Limpet Hemocyanin (KLH). The results of 3H-TdR incorporation are presented following incubation of cells derived from the interphase and pellet fractions with allogeneic cells. Detailed Description of the Invention I. Definitions

Unless otherwise indicated, the terms below have the following meanings:

"Dendritic-precursor cells", or "DPC", are peripheral blood cells which can mature into dendritic cells under suitable conditions. DPC typically have a non-dendritic morphology and are not competent to elicit a primary immune response as antigen presenting cells.

"Dendritic cells", or "DC" are matured DPC, which typically have a dendritic cell morphology, that is, they are large veiled cells which extend dendrites when cultured in vitro. When pulsed with antigen or peptide, such DC are capable of presenting antigen to naive T cells. "Precursor antigen presenting cells" are cells that when exposed to an antigen or peptide are capable of becoming APC and presenting antigen to T cells.

"Antigen presenting cells" (APC) are cells exposed to an antigen or peptide, can activate CD8⁺ cytotoxic T-lymphocytes (CTL) or CD4^T helper T-lymphocytes in an immune response.

II. Mobilization of stem cells from the bone marrow into the peripheral blood

Transplantation of bone marrow and peripheral blood is performed in the clinic for the treatment of cancer and hematopoietic disorders. The bone marrow and peripheral blood products are processed before being reinfused in the patients.

According to the methods of the present invention, a patient may be treated with one or more chemotherapeutic agents or by x-irradiation as a part of the treatment regime for cancer therapy. Such treatment regimes have been demonstrated to result in mobilization of stem cells. [Heinzinger et al., Leukemia (ENGLAND) 12 (3) p333-9, (1998)]. Such mobilization may also be accomplished by other means including, but not limited to. administering to the patient one or more chemotherapeutic agents, which may or may not be followed by administration of a cytokine. Other treatment regimes may involve cytokine administration alone.

Methods of administration of the therapeutic agent or cvtokines of the present invention may include, but are not limited to, intravenous (IV) infusion through a central venous catheter, subcutaneous (SC) injection, oral, intranasal, transdermal, intraperitoneal (IP), intramuscular (IM), or intrapulmonary administration. Preferred chemotherapeutic agents for mobilization include, but are not limited to, one or more of, cyclophosphamide, etoposide or carmustine. Other chemotherapeutic agents include those commonly used in the relevant art. [Moskowitz et al., Clin. Cancer Res., 4(2)31 1-6, (1998)].

Preferred cytokines for mobilization include, but are not limited to, one or more of, granulocyte colony stimulating factor (GCSF) or granulocyte macrophage colony stimulating factor (GMCSF), stem-cell factor (SCF), fetal liver kinase-3 protein ligand (FLT3-L), interleukin 3 (IL-3), and thrombopoietin. Cytokines may also be administered to the patient in the form of fusion proteins. According to the present invention, the fraction of stem cells obtained from patients that have been treated by one or more of chemotherapy, x- irradiation and therapy with cytokines is enriched in precursor APC that can be isolated by the methods of the present invention, as set forth below.

III. Isolation of precursor APC

A. Density gradient separation

According to the methods of the present invention, a fraction enriched in APC may be obtained by (1 ) obtaining, from a human blood sample, a monocyte-depleted cell fraction containing peripheral blood lymphocytes and dendritic-precursor cells, (2) enriching the portion of dendritic cells in the harvested cells by density centrifugation, to obtain a fraction enriched in APC, (3) culturing the cell fraction in a serum-free medium for a period sufficient to produce a morphological and biological change in dendritic-precursor cells to cells having the morphology and function of dendritic cells, and (4) harvesting non-adherent cells produced by the culturing.

Any tubes suitable for use in centrifugation may be used for the practice of the invention. Although the exemplified method achieves step (2) by density centrifugation, as detailed below, it will be understood that other approaches may be used to obtain such a monocyte-depleted cell fraction. For example, counterflow elutriation centrifugation [Kabel, P.J., et al., Immunobiology

179:395-41 (1989)] may be employed to enrich for dendritic cells.

By way of example, apheresed blood from a cancer patient previously treated with G-CSF is directly loaded into a cell-trap centrifugation tube containing, e.g., a colloidal silica solution filled to a level above the constriction, which solution has been adjusted to the preferred density of 1.0605 ±

0.0005 gr/ml, osmolality of 280 mOsm/kg FLO and pH 7.4. The density of the gradient solution may be adjusted on a densitometer to precisely define its accuracy up to at least the fourth decimal place.

It should be noted that a variety of gradient materials may be used to achieve progenitor cell enrichment, and they include, but are not limited to, "FICOLL". "FICOLL-ITYPAQUE", cesium chloride. "PERCOLL" and equivalent colloidal silica solutions, any protein solution such as albumin and any sugar solution such as sucrose and dextran. However, the density gradient solution should be prepared and adjusted to the appropriate density, osmolality and pH according to that disclosed herein, prior to its use. The gradient solution should be added to a centrifugation tube in a volume sufficient to allow all the cells having a higher density to pass through the gradient during centrifugation.

Any tubes suitable for use in centrifugation may be used for the practice of the invention. It is preferred that a cell-trap tube which contains within it a constriction or a trap and a properly adjusted density gradient material for the density separation of CD34" cells be used in the centrifugation step of the present invention. (Van Vlasselaer, U. S. Pat No. 5,474, 687).

The present invention is based on the finding that the fraction present at the interphase following density centrifugation is enriched in DC, antigen presenting cells and precursors thereof. Accordingly, isolation of APC is dependent on the separation characteπstics of the density gradient used, which in turn depends on the physical attributes such as the density, osmolaπty and pH of the gradient material used It will be appreciated that any separation medium having a combination of these characteπstics such as presented above, is effective for obtaining a cell fraction enriched for APC and precursors thereof

The purity of DC in this fraction may be quantified using, for example, flow cytometry cell sorting (FACS) analysis, together with functional assays

B Antibody Staining And Flow Cvtometry In an exemplary assay, the cells are incubated with 10 L of an antibody and the DNA dye

LDS 751 (Exciton Inc , Dayton OH) per 10⁶ cells for 30 mm on ice in the presence of 5% rabbit serum Rabbit serum is used to reduce non-specific binding to the cells The cells are washed twice with PBS and subsequently fixed with 1 % paraformaldehyde Statistical analysis is performed on 10 flow events using a FACSScan flow cytometry system equipped with a LYSYS II program It will be understood that any of a number of immunoassay protocols routinely employed by those of skill in the art may be used to complete such analyses

C Culture of selected cell fractions

The separated cells from one or more of the interphase and pellet are harvested, washed, and resuspended in a suitable culture medium, inoculated into tissue culture vessels and cultured in a humidified incubator for at least 24 hours, preferably about 40 hours The culturing penod is sufficiently-long to produce a morphological and functional change in the dendritic-precursor cells

(DPC) to cells having the morphology and function of dendritic cells (DC)

This morphological change may be detected using, for example, photomicroscopy DC are large sized veiled cells which, when cultured in vitro, typically extend cytoplasmic processes from the cell surface

DC in the enriched cell fraction typically have a dendritic morphology when cultured in vitro According to the methods of the present invention, the culture medium used in the DC isolation procedure, and particularly in the culturing step descπbed in the above paragraph, is preferably serum-free

Serum-free media which resulted in improved purity of subsequently-harvested DC cells included DMEM/F-12, Enriched Monocyte SFM, AIM-V and Enriched AIM-V All of these are available from Gibco/BRL Life Technologies, Gaithersburg, MD Other serum-free media may also be employed in the practice of the present invention Examples include Hybπdoma Serum-Free Medium (Gibco), Protein-Free Hybπdoma Medium (Gibco), Iscove's Modified Dulbecco's Medium (IMDM, Sigma), and MCBD medium (Sigma) If additional purification is desired, the cell fraction enriched in DC, antigen presenting cells and precursors thereof may be subjected to additional purification steps. For example, antibodies directed against various antigens not expressed on DC, may be immobilized on a solid support and used to remove, or "negatively deplete", contaminating cells. Such additional purification can result in further enrichment of DC cells, without appreciable loss of APC.

IV. Biological activity of APC

A. Response to Allogeneic T Cell Stimulation

APC in the fraction have the characteristics of the DC cells stated above, as well as the ability to stimulate a primary immune response mediated by MHC class I restricted CTL. This functional competence is assessed by measuring the proliferative response in an allogeneic T-cell stimulation setting as detected by tritiated thymidine incorporation.

A further characteristic of APC is the ability to activate naive CD8" cytotoxic T-lymphocytes

(CTL) in a primary immune response, after being pulsed with an antigen. The degree of enrichment of APC in the final fraction may be determined using, for example, limiting dilution analysis in a

CTL-activating assay by mixing serial dilutions of allogeneic cells with a selected cell fraction and evaluating the expansion of T-cells, e.g., as detailed in Example II. The relative ability of the cells to present antigen in association with an MHC and capable of activating T-cells can be estimated based on the relative extent of cell proliferation following such stimulation.

B. Response to Stimulation by Keyhole Limpet Hemocyanin (KLH)

True antigen presenting cells can activate naive CD8^T cytotoxic T-lymphocytes (CTL) in a primary immune response, after being pulsed with an antigen. As confirmation that the response to allogeneic T cells is not a recall response, KLH, an antigen not normally encountered by such cells may be employed by those of skill in the art to determine the ability of APC to activate naive T cells. The respective ability of each cell fraction to present antigen in association with MHC and activate T- cells may be estimated based on the relative extent of cell proliferation following such stimulation.

In an exemplary method, fractionated cells are either ( 1 ) cultured for 40 hours, then mixed with allogeneic "stimulators" or cells derived from buffy coats that had received 3000 rads of irradiation, or (2) cultured for 40 hours in the presence of Keyhole Limpet Hemocyanin (KLH) and then mixed with allogeneic "stimulators" or cells derived from buffy coats that had received 3000 rads, in order to evaluate the ability of the cells to generate an allogeneic response or to present antigen to naive T cells, respectively. V. In vitro stimulation of precursor APC

A. Induction to APC

The cells of the present invention may be induced to have the morphology, phenotype, and function of dendritic cells by exposing the harvested cells to an antigen precursor such as a polypeptide comprising a peptide antigen or antigen(s) which is effective to induce in the cultured cells, cell-surface presentation of one or more peptide antigens against which an immune response is desired.

Several methods may be used to pulse APC with antigen, to make them effective or competent to activate a desired subset of CTL. For example, experiments detailed herein demonstrate that the cells may be exposed to antigenic peptides, and that these peptides can be processed through the "endogenous" class I pathway such that they are presented in association with MHC class I molecules, and accordingly are able to activate CD8+ CTL.

It had been demonstrated that, in addition to peptides, certain proteins may be introduced to

APC such that the proteins are processed through the MHC class I, as opposed to class II, pathway (see, for example, Mehta-Damani, et al). In particular, the incorporation of antigens into liposomes has been used to accomplish such targeting [e.g..Nair, S., et al., J. Immunol. Meth. 152:237 (1992),

Reddy, R., et al., J. Immunol. 148: 1585 (1992), Zhou. F., et al. J. Immunol. 149: 1599 ( 1992)].

Selected antigens can be introduced to APC by transfecting the cells with expression vectors containing genes encoding such antigens. DNA encoding a polypeptide containing the peptide antigen or other antigen, and exposing includes introducing said DNA into the morphologically changed cells. Transfection of APC with a gene encoding a desired antigen is an effective way to express the antigen in association with the class I MHC. Any of a variety of known methods [for example, Ausubel, F. M., et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. John Wiley and Sons, Inc., Media PA.. Mulligan, R.C. Science 260:926 (1993)], may be used for such transfections, including CaP0₄ precipitation, lipofection, naked DNA exposure, as well as viral vector-based approaches, such as retroviral, adenoviral, AAV, and vaccinia virus vectors.

The antigen may be any antigen against which it is desired to mount an immune response, such as a cancer-specific or viral antigen.

The density gradient/affinity cell separation method employed in the isolation of precursor APC may be used in a simple, closed device or kit. The APC or precursor thereof, isolated using the methods of the present invention may be used in a number of applications.

B. Antigen-Specific T-Lymphocvtes

The de ree of enrichment of APC in a cell fraction may be determined by measuring, for example, functional competence as assessed by the generation of peptide-specific CTL. The antigen presenting cell-enriched fraction is pulsed with an antigen, and serial dilutions of the pulsed fraction are made. The dilutions are then used to stimulate expansion of T-cells. The relative number of APC expressing the antigen in association with an MHC and capable of activating T-cells can be estimated based on the most diluted sample that results in T-cell expansion.

Antigen-specific cytotoxic T-lymphocytes are elicited essentially as described by Mehta- Damani, et al, 1994. By way of example, three HLA-A *0201 binding peptides may be used to elicit antigen-specific cytotoxic T-lymphocytes (CTL). For example, the first may correspond to amino acids 11-19 of the Tax gene product of human trophic leukemic virus 1 (HTLV-1; Elovaara, I., et al, J. Exp. Med. 177: 1567-1573 (1993); Kannagi, M., et al, J. Virol. 66:2928-2933 (1992); Zweerink, H.J., et al, J. Immunol. L50: 1763 -1771 (1993)], the second may correspond to amino acids 27-35 of the MART-1 antigen expressed on melanoma cells (Stevens, et al.) and the third may correspond to amino acids 464-472 of human immunodeficiency virus (HIV) reverse transcriptase in the polymerase gene. All peptides may be synthesized by Bachem Laboratories (Torrance, CA).

Stock solutions are prepared by dissolving the peptides in sterile filtered 1.0% acetic acid solution in LAL water (BioWhittaker. Walkersville, MD) at a concentration of about 1 g/ml. The isolated DC enriched cell fraction is resuspended in 1.0 mL of basal RPMI-1640 and incubated with 1 to 5 g/mL β2-microglobulin (Sigma Chemical Company, St. Louis, MO) and 1 to 5 g/mL peptide at 37°C for 1-2 hours. Following the incubation, peptide-pulsed DC are washed to remove excess peptide and mixed with autologous T-lymphocytes (14.5% metrizamide pellet cells) at a ratio of approximately 10: 1 to yield a cell concentration of 1.0 x 10⁶ cells/mL in AB Culture Medium supplemented with 4.0 U/mL of human recombinant IL-2 (Gibco Laboratories, Grand Island, NY). After 3 days of culture the IL-2 concentration is increased to 20.0 U/mL.

The T-lymphocytes are restimulated on a weekly schedule using autologous peptide-pulsed monocytes at a ratio of 10: 1. During restimulation, the IL-2 concentration is decreased to 4.0 U/mL and is subsequently increased to 20.0 U/mL after 3 days of culture following each restimulation. CTL cultures are typically expanded for 3-4 weeks before evaluation of antigen-specific target cell lysis.

C. Flow Cytometry

FACS analysis may be done using a FACSScan flow cytometer (Becton Dickinson, San Jose, CA) connected to a Hewlett-Packard HP-9000 computer (Hewlett-Packard, Palo Alto, CA) running "LYSIS II" software (Becton Dickinson). In such cases, the monoclonal antibodies used for analysis and their respective isotype controls may be purchased from Becton Dickinson. Briefly, approximately 100,000 cells are preincubated in each well of a 96-well plate with 50 μ\ of rabbit serum (Sigma Chemical Company, St. Louis, MO) in a final volume of 150 μL for 15-20 minutes at room temperature to block non-specific sites for antibody binding. 10 μL of desired FITC or PE- tagged monoclonal antibody are then added to the wells and the 96-well plate is incubated in the dark at 4°C for 30 minutes. The plate is then centrifuged to pellet cells and supernatant is aspirated off to remove unbound antibody. Pelleted cells are resuspended in 100 μL of D-PBS supplemented with 5% human AB serum, fixed and counterstained by addition of 100 μL of 1.0% paraformaldehyde (Sigma Chemical Company, St. Louis, MO) supplemented with 2.0 μg/mL of LDS-751 (Molecular Probes, Eugene, OR). LDS-751 fluoresces in the far-red spectrum (PerCP region-detected by FL3 fluorescence channel on the FACScan) and counterstains cells, allowing for distinction between non- nucleated cell (non-staining), nucleated viable cell (weakly staining) and nucleated non-viable cell (very bright staining) populations.

D. Mixed Lymphocyte Culture and Natural Suppressor Cell Activity

In an exemplary method, cells from two different buffy coats are mixed in a flat bottom 96 well multiwell plate at 10⁵ cells of each. One of the buffy coats is treated with 3000 rad of irradiation and referred to as the "stimulators". The other buffy coat is untreated and referred to as "responders." If there are differences in the MHC genes of the two individuals, the cells will proliferate over a period of from about 4 to 7 days. Unfractionated apheresed peripheral blood products (APBL) or cells from the different density fractions are added to these co-cultures at lO⁵ cells per well. These cells, referred to as "suppressors", are treated with 1500 rad prior to being added to the MLR. The cells are cultured for 5 days, then pulsed with [³H]-thymidine (1 Ci/well). 18 hours later, the cells are harvested and the amount of thymidine incorporated is determined in a scintillation counter. The percent suppression induced by the suppressor cells is determined by the formula:

% Suppression = 100 x cpm control - cpm experiment cpm experiment

VI. Methods for immunotherapy and cancer therapy comprising precursor APC

One of the useful features of the APC isolated by the methods of the present invention is that they are able to present antigens for the induction of primary T-cell responses. CD8^T CTL-mediated as well as CD4⁺ T cell proliferative responses in cases where the donor of the T-cells had been previously exposed to the antigen. As such, the APC are universally-useful antigen-presenting cells and can be employed in a wide range of immunotherapeutic, immunoprophyiactic and cancer therapeutic applications involving generation of primary and secondary immune responses.

Cells or cell membranes can be used, for example, in direct in vivo administration, ex vivo somatic therapy, in vivo implantable devices and ex vivo extracorporeal devices. They can also be employed in the screening of antigenicity and immunogenicity of peptide epitopes from tumor- and virus-specific antigens. APC treated or pulsed with appropriate antigens can be used as potent vaccine compositions, for example against pathogenic viruses or cancerous tumors. In certain cases, it may be advantageous to use cells obtained from one individual to treat a condition in a second individual (i.e., "allogeneic" cells). For example, HIV-infected individuals with

AIDS are often not able to mount anti-viral T-cell responses. In such cases, CTL can be isolated from healthy HLA-matched individuals, such as siblings, be stimulated or primed with antigen-pulsed DC in vitro, expanded, and administered back to the HIV-infected individuals.

The isolated cells may also be used, for example, in gene therapy applications, such as transfection of the cells so that they constitutively express desired antigens/gene products for therapeutic applications. Steps include obtaining a cell fraction enriched in precursor antigen- presenting cells [as described in steps above], and either returning a portion of the enriched fraction to the patient prior to culturing, or culturing the entire fraction of cells enriched in precursor antigen presenting cells under conditions effective to induce cells having the morphology, phenotype, and function of dendritic cells followed by administering the exposed cells to the patient.

In this context, apheresed blood samples are collected from a patient treated with one or more cytokines. By way of example, a patient is treated with, a cytokine or fusion protein thereof, such as one or more of GCSF, GMCSF, SCF, FLT3-L, IL-3, and thrombopoietin.

The apheresed samples are subjected to fractionation on a density gradient to form a cell layer at the gradient interphase between a first and a second separation medium each having different densities morphology, phenotype, and function of dendritic cells. For example, a first separation medium may have a density of 1.0605 ± 0.0005 gr/ml, an osmolality of 280 mOsm/Kg H₂0, and a pH of 7.4 and a second separation medium may have a lighter-density. The cells from the interphase are harvested to obtain the stem cell fraction which is enriched in precursor antigen-presenting cells as set forth in Example I.

A fraction of the harvested cells, "stem cells", APCs and precursors thereof, may be immediately returned to the patient. Alternatively, all of the harvested cells are cultured under conditions effective to induce cells having the morphology, phenotype, and function of dendritic cells. The induction is accomplished by exposing the harvested cells to an antigen precursor such as a polypeptide containing a peptide antigen or other antigen which is effective to induce cell-surface presentation of one or more peptide antigens by the cultured cells, against which an immune response is desired, as described above. Antigen precursors may be DNA encoding a polypeptide comprising the peptide antigen or other antigen, and exposing may include introducing said DNA into the morphologically changed cells.

Following in vitro induction of the dendritic cell fraction to APC, the exposed cells are administered to the patient. VII. Compositions comprising precursor APC for use in immunotherapy

In a related aspect, the invention includes a cell composition for use in immunotherapy containing a mixture of stem cells and precursor antigen presenting cells which have been exposed to the appropriate antigens, as set forth above. The precursor antigen presenting cell component of such compositions are cells that have been cultured under conditions effective to induce the morphology, phenotype, and function of dendritic cells of the type described above, and which are further exposed to an antigen precursor effective to induce cell-surface presentation of one or more peptide antigens against which an immune response is desired.

In some cases, cells derived from a tumor, other cancer-specific antigen(s) or viral antigens are used to induce the dendritic cells. In other cases, an APC fused to a tumor cell or virally-infected cell, is used to induce the dendritic cell fraction.

By way of example, antigen precursors may be DNA encoding a cancer-specific polypeptide comprising the peptide antigen or other antigen of interest, and exposing may include introducing the DNA into the morphologically changed cells. Following in vitro induction of the dendritic cell fraction to APC, the exposed cells comprise a pharmaceutical composition that may be administered to the patient.

From the foregoing, it will be appreciated that the invention provides for a rapid and high yield procedure to enrich for APC. Most importantly, the enriched APC cell population is characterized as having the function of true APCs, namely the ability to present antigen to naϊve T cells. A continuing debate exists among those of skill in the art regarding the phenotype of true APCs, in terms of cell surface markers and the expression levels thereof. However, a uniformly accepted criteria for true APCs is the functional ability to present antigen to naive T cells.

The methods described herein further provide a procedure for APC enrichment from a large blood volume, which enhances the use of such cells in both in vitro applications and in vivo therapy. Additionally, the procedure is rapid, convenient and cost effective. Processing of a complete sample requires no specialized instrumentation and can be performed by one person in a time frame of a few hours.

EXAMPLE I Mobilization and collection of Cells from a Patient

A. Mobilization

An apheresed peripheral blood sample from a mobilized patient was applied directly onto the density gradient. However, complete blood and bone marrow aspirates may be processed to a buffy coat (removal of red cells) before they are applied onto the density gradient. Patients were hydrated and treated ("mobilized") with G-CSF (Neupogen, Amgen,

Thousand Oaks, CA) administered by subcutaneous (SC) injection at a dose of approximately 10 g/kg/d. Apheresis was initiated upon recovery of the white blood cell count (WBC) to equal or more than 1 x 10⁹/L. Apheresis was performed using a Cobe Spectra Cell Separator (Lakewood, Colorado) at a rate of 80 ml/min for 200 min (total volume of 16 L).

B. Fractionation of Apheresed Blood The apheresed blood product was further fractionated by applying to an organosilanized colloidal silica density gradient material (U.S. Patent 4,927,750) adjusted to a density 1.0605 ± 0.0005 gr/ml ("BDS"), with an osmolality of 280 mOsm/Kg H₂0, and a pH of 7.4 and centrifuging for 30 minutes at 850 x g at room temperature. The cells lodged at the interphase of the gradient; i.e., on top of separation medium (BDS) were collected by transferring the entire content of the upper compartment of the tube into another 50 ml tube. The cell pellet in the region below the constriction was prevented from pouring off when the tube was inverted.

EXAMPLE II Evaluation of the function of APC A. Presentation of allogeneic T cell antigens

Following administration of GCSF, apheresed blood obtained from a cancer patient, was layered onto BDS, with a density of 1.0605 ± 0.0005 gr/ml, an osmolality of 280 mOsm/Kg H₂0, and a pH of 7.4, then centrifuged, as described above. The cells lodged at the interphase of the gradient and the cells in the pellet were evaluated for their respective ability to present antigen. The cells from the interphase of the gradient and the pellet were separately cultured for 40 hours and then mixed with allogeneic "stimulators" or cells derived from buffy coats that had received 3000 rads of irradiation. 10⁵ cells derived from either of the interphase, or pellet fraction, respectively, were mixed in flat bottom 96 well multiwell plates at with various numbers of stimulators per well in triplicate, and pulsed with [³H]-thymidine (1 ^Ci/well). 18 hours later, the cells were harvested and the amount of thymidine incorporated determined in a scintillation counter.

As depicted in Figure 1, while both cell populations appear to have antigen presenting capabilities, the cells from the interphase elicit a much stronger allo response than those derived from the pellet. This indicates that the APCs and precursors thereof reside within the interphase following the BDS density centrifugation procedure.

B. Presentation of naive antigen (KLH)

Cells were prepared as described in "A" above. The cells were cultured for 40 hours in the presence of 10 g/ml Keyhole Limpet Hemocyanin (KLH) and then mixed with allogeneic "stimulators" or cells derived from buffy coats that had received 3000 rads. 10⁵ cells derived from either of the interphase, or pellet fraction, respectively, were mixed in flat bottom 96 well multiwell plates at with various numbers of stimulators per well in triplicate. 18 hours later, the cells were harvested and the amount of thymidine incorporated determined in a scintillation counter.

As depicted in Figure 2, only the cells derived from the inteφhase appear to have the ability to present antigen to naive T cells, and consequently only the cells at the inteφhase have the functional characteristics of APCs.

The invention described herein represents a novel method which provides the combination of mobilized APCs and precursors thereof, together with a method for enrichment of an APC population having the function of APCs, commonly accepted as the definitive characterization of the APCs.

Claims

WHAT IS CLAIMED IS:

1. A method for obtaining antigen presenting cells from a human patient who is under treatment with an agent effective to mobilize stem cells, antigen presenting cells (APCs) and precursors thereof, from bone marrow into the peripheral blood, comprising:

(i) obtaining from the patient, a blood cell fraction containing peripheral blood mononuclear cells,

(ii) subjecting the blood cell fraction to density centrifugation,

(iii) harvesting the cells at the inte╧åhase, to obtain a cell fraction enriched in precursor antigen presenting cells, and

(iv) culturing the harvested cells under conditions effective to induce cells having the mo╧åhology, phenotype, and function of dendritic cells.

2. The method of claim 1, wherein said separation medium has a density of 1.0605 ┬▒ 0.0005 gr/ml.

3. The method of claim 1, wherein the patient is under treatment for cancer, and the treatment agent is a chemotherapeutic agent or x-irradiation.

4. The method of claim 1, wherein the patient is under treatment with a cytokine or fusion proteins thereof selected from the group consisting of colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), stem-cell factor (SCF), fetal liver kinase-3 protein ligand (FLT3-L) and thrombopoietin.

5. The method of claim 1, wherein said culturing is carried out in serum or protein free medium.

6. An immunotherapy method for treating a patient, comprising

(i) treating the patient with an agent effective to mobilize stem cells, antigen presenting cells (APCs) and precursors thereof, from bone marrow, over a period sufficient to cause cell mobilization from bone marrow into the peripheral blood,

(ii) obtaining from the patient, a blood cell fraction enriched in peripheral blood mononuclear cells,

(iii) subjecting the blood cell fraction to density centrifugation, (iv) harvesting the cells at the inte╧åhase, to obtain a cell fraction enriched in precursor antigen-presenting cells, (v) culturing the harvested cells under conditions effective to induce cells having the mo╧åhology, phenotype, and function of dendritic cells, and

(vi) administering the cultured, induced cells to the patient.

7. The method of claim 6, wherein said first separation medium has a density of 1.0605 ┬▒

0.0005 gr/ml.

8. The method of claim 6, wherein said culturing includes exposing the harvested cells to an antigen effective to induce in the cultured cells, cell-surface presentation of one or more peptide or protein antigens against which an immune response is desired.

9. The method of claim 6, wherein said culturing includes exposing the harvested cells to an antigen effective to induce cell surface presentation of a self antigen to na╧ève T cells.

10. The method of claim 6, wherein said culturing includes exposing the harvested cells to an antigen effective to induce cell surface presentation of an allogeneic antigen to na╧ève T cells.

11. The method of claim 6, wherein said culturing includes exposing the harvested cells to an antigen effective to induce cell surface presentation of a xenogeneic antigen to na╧ève T cells.

12. The method of claim 8, wherein said antigen includes a polypeptide containing such peptide antigen or other antigen.

13. The method of claim 8, wherein said antigen includes a tumor or viral lysate.

14. The method of claim 8, for use in treating cancer or infectious disease by immunotherapy, wherein the antigen(s) are cancer- or viral-specific antigen(s), respectively.

15. The method of claim 8, wherein said antigens includes DNA or RNA encoding a polypeptide comprising such peptide antigen or other antigen, and said exposing includes introducing said DNA or RNA into the mo╧åhologically and functionally changed cells.

16. The method of claim 6, wherein the mobilizing agent administered to the patient is a cytokine or fusion protein thereof selected from the group consisting of colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), stem-cell factor (SCF), fetal liver kinase-3 protein ligand (FLT3-L) and thrombopoietin.

17. The method of claim 6, wherein the patient is under treatment for cancer or an infectious disease, and the treatment agent is a chemotherapeutic agent.

18. The method of claim 17, wherein the patient is also under treatment with granulocyte colony stimulating factor (GCSF) or granulocyte macrophage colony stimulating factor (GMCSF).

19. The method of claim 17, which further includes administering a portion of the harvested cells to the patient, prior to said culturing, to replace stem cells in the patient following chemotherapy.

20. A human blood cell composition for use in immunotherapy, containing a mixture of stem cells and precursor antigen presenting cells, prepared by the steps of:

(i) treating a human subject with an agent effective to mobilize stem cells from bone marrow, over a period sufficient to cause stem-cell mobilization from bone marrow into the peripheral blood, (ii) obtaining from the subject, a blood cell fraction containing peripheral blood mononuclear cells,

(iii) subjecting the blood cell fraction to density centrifugation; and (iv) harvesting the cells at the inte╧åhase.

21. The cell composition of claim 21, wherein said separation medium has a density of 1.0605 ┬▒ 0.0005 gr/ml.

22. The cell composition of claim 21 , which is further cultured under conditions effective to induce cells having the mo╧åhology, phenotype, and function of dendritic cells.

23. The cell composition of claim 22, wherein said culturing includes exposing the harvested cells to an antigen precursor effective to induce in the cultured cells, cell-surface presentation of one or more peptide or protein antigens against which an immune response is desired.

24. The cell composition of claim 17, wherein said culturing includes exposing the harvested cells to an antigen precursor effective to induce cell surface presentation of a self antigen, to na╧ève T cells.

25. The cell composition of claim 17, wherein said culturing includes exposing the harvested cells to an antigen precursor effective to induce cell surface presentation of an allogeneic antigen, to na╧ève T cells.

26. The cell composition of claim 17, wherein said culturing includes exposing the harvested cells to an antigen precursor effective to induce cell surface presentation of an antigen, to na╧ève T cells.