WO2001039594A2

WO2001039594A2 - Cryopreservation of antigen-loaded dendritic cells and their precursors in serum-free media

Info

Publication number: WO2001039594A2
Application number: PCT/US2000/032722
Authority: WO
Inventors: Tatsue Monji; Madhusudan V. Peshwa
Original assignee: Dendreon Corporation
Priority date: 1999-12-03
Filing date: 2000-12-01
Publication date: 2001-06-07
Also published as: AU1811801A; WO2001039594A3

Abstract

A serum-free cryopreserved dendritic cell (DC) precursor and antigen (Ag)-loaded DC composition and a method of obtaining such a composition is disclosed. The method includes cryopreserving the cells in a manner effective to result in good recovery, viability, maintenance of the ability to differentiate into mature DC for DC precursors and maintenance of DC phenotype and Ag-specific immune function for mature Ag-loaded DC.

Description

CRYOPRESERVATION OF ANTIGEN-LOADED DENDRITIC CELLS AND THEIR PRECURSORS IN SERUM-FREE MEDIA

Field of the Invention The present invention relates to compositions and methods relating to cryopreservation of dendritic cell (DC) precursors, and DC products.

Background of the Invention DC are effective antigen presenting cells (APC) . They are potent allostimulators and the only APC capable of priming naive helper and cytotoxic T lymphocytes. In T cell-mediated cancer immunotherapy, induction of tumor-specific immune response depends on effective antigen (Ag) presentation. Because of their unique ability to prime naive T lymphocytes, DC have been found to be promising in the treatment of various cancers such as B cell lymphoma, melanoma, prostate cancer, and breast cancer. In a general exemplary treatment protocol, DC precursors collected from either the bone marrow or peripheral blood of a patient are cultured ex vivo in the presence of tumor Ag . During the culture period, DC precursors process tumor Ag and differentiate into mature, Ag-loaded DC. The Ag-loaded DC are then reinfused into patients, where the APC induce immune responses directed to cancer Ag. In methods currently employed for immunotherapy using ex vivo stimulated APC, DC precursors are cultured immediately after collection from the patient, e.g. a cancer patient, and Ag-loaded mature DC reinfused into the patient as soon as they are harvested. Such time constraints not only limit the potential therapeutic uses for DC, but also necessitate leukapheresis products from a patient at multiple time points during the course of immunotherapy.

Ag-loaded DC have already processed the Ag and have the ability to present Ag to the immune cells and can therefore quickly generate Ag-specific immune responses. Cryopreservation of Ag-loaded mature DC in a functionally potent state has not been demonstrated to date.

Standard freezing medium for cultured cells includes about 5-10% dimethyl sulfoxide (D SO) or glycerol and 10-50% serum (Voetman AA, et al . , Blood 63(l):234-7, 1984; Utsugi T et al., Biotherapy 5(4):301-8, 1992). Some procedures utilize controlled-rate freezing while others include an insulated container in which vials of cells m freezing medium are placed in a freezer (i.e., at -70 to -80°C) .

Studies have shown that DC are more sensitive to freezing injury than either lymphocytes or macrophages. (See, e.g., Makino M and Baba M, Scand J Immunol 45 ( 6) : 618-22, 1997; Taylor MJ et al., Cryobiology 27 (3) : 269-78 , 1990; Delfini C et al . , Ric Clin Lab 9(1) :61-6, 1979.)

The inclusion of serum in cryopreservation medium containing cells to be used for in vivo therapy in human subjects presents the disadvantages that cellular compositions exposed to serum may contain foreign serum proteins, antibodies, infectious agents, etc. Other problems associated with the use of serum- containing cryomedia are the significant lot-to-lot variation in the content and quality of serum, and that cells preserved in serum-containing cryomedia cannot be directly reinfused into patients after thawing.

Accordingly, research efforts are under way to develop a method to preserve DC and DC precursors in order to further facilitate clinical application of the cells. Additionally, the ability to cryopreserve DC or their precursors makes the use of allogeneic DC practical in raising an Ag-specific immune response in allogeneic DC therapy (Peshwa et al . , Cell Transplantation, 7: 1-9, 1998) .

Summary Of The Invention

The present invention provides methods and compositions for cryopreservation of APC precursors and APC.

A cryopreservation composition comprising APC precursors or APC is prepared by obtaining a blood sample from a subject, treating the blood sample in a manner effective to obtain a population of APC precursors or APC and suspending the APC precursors or APC in serum-free cryopreservation medium containing DMSO, such that APC precursors or APC frozen to a temperature of -70°C or lower for at least 24 hours in the cryopreservation composition maintain viability with a recovery of at least 70%, upon thawing the cells.

In one aspect, the cryopreservation method includes bouyant density cell separation to obtain from a blood sample, a cell fraction containing peripheral blood lymphocytes and DC precursors. In some cases, this fraction is cultured in a serum- free or protein-free medium for a period sufficient to produce morphological, phenotypic and functional changes in DC precursors to cells having the morphology, phenotype and function of DC. In another aspect, the APC precursors are DC precursors which maintain the phenotype and function of DC precursors upon thawing.

In still another aspect, the APC are Ag-loaded DC, which maintain the phenotype and Ag-specific function of Ag-loaded DC upon thawing the cells.

The invention further provides a serum-free cryoprotective medium containing from about 5 to 20% DMSO (volume percent) , for cryopreservation of APC precursors and APC, such that APC precursors or APC frozen to a temperature of -70°C or lower in the cryopreservation medium for at least 24 hours maintain a viability and recovery of at least 70% upon thawing the cells. In some cases, the cryoprotective medium is protein-free and may contain from about 1 to 30% human serum albumin (HSA) .

In other cases, the cryoprotective medium contains a commercially available serum-free medium, exemplified by AIM-V, XVIVO 10, XVIVO 15, XVIVO 20 and StemPro. In still another aspect, the invention provides a method for cryopreserving and cryoprotecting APC precursors and APC by obtaining a blood sample from a subject; treating the blood sample in a manner effective to obtain a population of APC precursors or APC; and suspending the APC precursors or APC in a serum-free cryopreservation medium containing DMSO, such that APC precursors or APC frozen to a temperature of -70°C or lower for at least 24 hours in the cryopreservation medium maintain a viability and recovery of at least 70% upon freezing and thawing.

In the cryopreservation method of the invention, the APC precursors may be DC precursors which maintain the phenotype and function of DC precursors upon thawing and the APC may be mature Ag-loaded DC, which maintain the phenotype and Ag-specific function of DC upon thawing the cells.

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 Figures 1A-Q depict the results of 3-color FACS analysis for expression of the DC-associated markers: CDllc (Fig. IB, 1G, 1M) , CD40 (Fig. 1C, 1H, IN), CD54 (Fig. ID, 1J, IP), CD86 (Fig. IE, IK, 1Q) , and HLA-DR on the lineage-negative population, where the expression on fresh DC (Fig. 1A-E ), DC generated from cryopreserved precursors (Fig. 1F-K) and DC cryopreserved as DC products (Fig. 1L-Q) , was evaluated.

Figures 2A-C depict the results of allogeneic and autologous T cell proliferation assays. Fig. 2A presents the results of an analysis of the stimulation of allogeneic T cells

(allostimulation) by fresh DC (Fresh PA2024), DC cryopreserved as precursors (Precursor Cryo PA2024), and DC cryopreserved as Ag- loaded DC (Product Cryo PA2024) . All the DC were cultured in the presence of recombinant PA2024, and their allostimulatory ability was measured based on ³H-thymidine incorporation by the T cells. Figs. 2B and 2C present the results of an evaluation of the antigen presenting ability of fresh DC (Fresh w/o Ag, Fresh PA2024 or Fresh HER500), DC cryopreserved as DC precursors (Precursor Cryo w/o Ag) , then cultured in the presence of Ag (Precursor Cryo PA2024; Fig. 2B or Precursor Cryo HER500; Fig. 2C) , versus DC cryopreserved as Ag-loaded DC with the PA2024 (Product Cryo PA2024; Fig. 2B) or the HER500 (Product Cryo HER500; Fig. 2C) Ag, based on T cell proliferation as indicated by ³H-thymidine incorporation. Figures 3A-D and 3E-H depict the results of FACS analysis for expression of CD54 on DC generated from cryopreserved precursors (Fig. 3A-D) and DC cryopreserved as DC products (Fig. 3E-H) following 0 (3A, 3E) , 4 (3B, 3F) , 8 (3C, 3G) and 24 (3D, 3H) hours in lactated Ringer's solution at 4°C. Figures 4A and 4B depict the results of an analysis of the stimulation of allogeneic T cells by DC cryopreserved as DC precursors, then cultured in the presence of recombinant PA2024 (Fig. 4A) , versus DC cryopreserved as PA2024-loaded DC (DC products; Fig. 4B) . The results are based on T cell proliferation as indicated by ³H-thymidine incorporation at 0, 4, 8 and 24 hour time points in lactated Ringer's solution at 4°C.

Detailed Description of the Invention

I . Definitions Unless otherwise indicated, the terms below have the following meanings:

"Dendritic cell precursors", or "DC precursors", are peripheral blood cells which can mature into DC under suitable conditions. DC precursors may comprise a mixture of peripheral blood cells and typically have a non-dendritic morphology and are not competent to elicit a primary immune response as APC. "Dendritic cells", or "DC" are mature DC, which typically have a DC morphology, that is, they are large veiled cells which extend dendrites when cultured ex vivo . When pulsed with Ag or peptide, such DC are capable of presenting Ag to naive T cells. The term "DC products" as used herein refers to Ag-loaded DC generated by ex vivo culture in serum-free or protein-free medium in the presence of antigen, for a time sufficient for the cells to acquire the morphology, phenotype and function of mature DC. The term "mature DC" as used herein refers to DC generated by ex vivo culture in serum-free medium for a time sufficient for the cells to acquire the phenotype, morphology and function of mature DC.

"Antigen presenting cell precursors" or "APC precursors" are cells that when exposed to differentiation stimulating conditions, e.g., environmental stimuli, cell culture, or cytokines, are capable of becoming mature APC and presenting Ag or peptide to T cells .

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

As used herein, the term "lin -" refers to a cell population that is negative for cell surface expression of the lineage markers expressed on T cells, B cells, monocytes/macrophages, NK cells, eosinophils, and neutrophils: for example, CD3, CD14, CD16, CD19, CD20, and CD56.

As used herein, the term "allostimulatory" means capable of stimulating allogeneic T cells due to differences in MHC molecules expressed on the cell surface.

As used herein, the term "Ag-loaded mature DC" refers to peripheral blood mononuclear cell (PBMC) -derived cells enriched for DC precursors and cultured ex vivo in the presence of Ag, e.g., a tumor Ag . Such "Ag-loaded mature DC" include DC and various types of PBMC including professional APC, monocytes/macrophages, which are positive for cell surface expression of CD14, and B lymphocytes, which are positive for cell surface expression of CD19.

As used herein, "immunogen" refers to a substance that is able to stimulate or induce a humoral antibody and/or cell- mediated immune response. "Antigen" or "Ag" refers to a substance that reacts alone or in the context of MHC molecules with the products of an immune response (e.g., antibodies, T-cell receptors) which have been stimulated by a specific immunogen. Ag therefore include the specific immunogens giving rise to the response (e.g., antigenic peptides, proteins or polysaccharides) as well as the entities containing or expressing the specific immunogens (e.g., viruses, bacteria, etc. ) .

"Tumor antigens" refer to tumor-associated Ag and tumor- specific Ag . Examples of tumor Ag include HER-2/neu, prostatic acid phosphate (PAP) and any of a number of proteins expressed on tumor cells.

As used herein, the term "cryopreserved" refers to cells that have been resuspended in a cryomedium, frozen at a temperature of -70°C or lower, and preserved at that temperature for a minimum of 24 hours.

II . Isolation and Characterization of DC precursors and DC PBMC were collected from healthy donors by standard leukapheresis and DC precursors isolated by either a one-step or a successive two-step buoyant density centrifugation procedure, as further described below.

DC may be generated from such DC precursors by culture ex vivo in serum-free or protein-free medium for 40 hours, in the absence of exogenously added cytokines, as detailed in co-owned

USSN 60/158,618.

The purity of DC in this fraction may be quantified using, for example, flow cytometry (i.e., FACS) analysis, together with functional assays. In some cases, during the 40-hour culture period DC precursors were pulsed with Ag, e.g., recombinant PA2024 or

HER500 in a manner effective to yield Ag-loaded DC.

A. Phenotypic characterization Historically DC have been characterized as negative for the cell surface markers CD3 (T-cells) , CD14 (monocytes), CD19/20 (B- cells), CD56 (NK cells) and positive for HLA class II expression. (Macatonia, SE et al . , Immunology 74 : 399-406, 1991; Markowicz S and Engleman EG, J. Clin . Invest . 85:955-961, 1990; Young JW and Steinman RM, Cell . Immunol . 111:167-182, 1987).

The DC population is characterized phenotypically as lineage negative, i . e . , negative for expression of the cell- surface lineage markers CD3, CD14, CD16, CD19, CD20, and CD56; strongly positive for the cell-surface marker CD86 ("CD86- bright"); and positive for cell surface expression of MHC class II molecules such as HLA DR (Lin-/86++/DR+) . They also express DC-associated markers such as CDllc, CD40 and CD54. This Lin-

/86++/DR+ population is not detected in freshly isolated PBMC or in cell fractions enriched for DC precursors.

It has recently been demonstrated that DC precursors acquire a mature DC phenotype during the period of ex vivo culture. The cells upregulate expression of CDllc, CD40, CD54, CD86, and MHC class II molecules, as further described in co- owned USSN 09/684,308.

The FACS profile of DC generated from fresh precursors, those generated from cryopreserved precursors and those cryopreserved as DC products was compared. Although the DC culture contains various types of cells such as T lymphocytes, B lymphocytes and monocytes/ macrophages, FACS analysis facilitates the analysis of DC lineage cells. DC products were stained with FITC-Lin, PE-CDllc, -CD40, -CD54, or -CD86 and Per-CP-HLA-DR, as detailed in Example 3. The results indicate that: (1) cryopreserved DC precursors maintain the ability of the cells to acquire a mature DC phenotype, and (2) cryopreserved DC products maintain their mature DC phenotype.

B. Functional characterization

The acquisition of the mature DC phenotype correlates with the functional maturation of DC, in that DC precursors become APC which are not only allostimulatory but also capable of presenting Ag to autologous T cells.

III . Cryopreservation of DC precursors and Ag-loaded DC

Given the various utilities for DC precursors and Ag-loaded DC, the ability to provide a ready supply of cryopreserved cells of either type, particularly cells which can elicit Ag-specific immune responses, represents a significant advantage that can facilitate various therapeutic uses of the cells. For example, a large scale culture of DC precursor cells or Ag-loaded DC may be cryopreserved in aliquots of the appropriate size for individual doses of cells for use in a particular immunotherapeutic protocol .

Numerous cryopreservation protocols have been reported in the literature for primary cells and cultured cell lines. Examples include: the cryopreservation of enucleated human neutrophils (PMN cytoplasts) in a medium containing 10% (v/v) fetal calf serum and 10% (v/v) DMSO, and stored at -70 °C, resulting in a recovery of 75% (Voetman AA, et al . , 1984); the cryopreservation of human monocytes separated from buffy coats of healthy donors in 70% medium, 20% fetal bovine serum and 10% DMSO, frozen in a stepdown freezer, and stored at -180 °C, resulting in a viability of greater than 90% (Utsugi T et al . , 1992); effective cryopreservation of blood cell transplants with medium containing 55% oxypolygelatine, a plasma expander; 6% hydroxyethylstarch; and 5% DMSO (Di Nicola M et al . , Bone Marrow Transplant 18 (3) : 619-23, 1996); and the cryopreservation of fresh PBMC from which DC were recovered and established DC in freezing medium containing 12% DMSO and 25-30% fetal calf serum, with the viability of established DC maintained at greater than 90% (Makino M and Baba M, Scand J Immunol 45 ( 6) : 618-22, 1997).

The present invention is directed to the specific effects of cryopreservation in liquid nitrogen on DC precursors collected from peripheral blood, as well as on mature Ag-loaded DC generated from the DC precursors, with an emphasis on serum-free and protein-free cryomedium and large scale cryopreservation. The effects of cryopreservation on DC precursors may be evaluated based on factors including, but not limited to: (1) recovery and viability after cryopreservation and thawing; and (2) the ability to differentiate ex vivo into mature DC.

The effects of cryopreservation on DC products (i.e., Ag- loaded DC) may be evaluated based on factors including, but not limited to: (1) recovery and viability after cryopreservation and thawing; and (2) changes in phenotype and function due to cryopreservation.

A. Small Scale Cryopreservation

The effect of small scale cryopreservation of DC precursors and DC products in serum-free medium containing 5% human serum albumin (HSA) and 10% DMSO, is described in Examples 1 and 2, respectively. In the small scale cryopreservation studies described herein, about 10-100 x 10⁶ cells per ml were frozen at 1 ml/vial. The recovery and viability of the cells, summarized in Tables IA and IIIA, respectively, suggest that small scale cryopreservation of DC precursors enriched by buoyant density centrifugation was effective under the serum-free conditions described herein. While the examples provided herein include specific reference to a particular concentration of HSA, it will be understood that the amount of HSA in the freezing medium may vary, but is generally in the range of 1% to 30% (v/v or volume percent) . However, any concentration of HSA that results in a cell viability of at least 50% and a cell recovery of at least 50%, and preferably a cell viability and recovery of at least 70 or 80% may be used in the DC precursor and Ag-loaded DC compositions, as well as the cryopreservation methods described herein.

Similarly, although the examples refer to 10% DMSO, those of skill in the art will recognize that DMSO concentrations of from about 5% to as high as 20% (v/v or volume percent) may be included in the cryopreservation methods described herein. Generally, lower concentrations of DMSO are preferred, e.g., about 5% to 10%, however, any concentration of DMSO that results in a cell viability of at least 50% and a cell recovery of at least 50%, and preferably a cell viability and recovery of at least 70 or 80% may be used in the DC precursor and Ag-loaded DC compositions, as well as the cryopreservation methods described herein.

B. Large Scale Cryopreservation

A cryopreservation process which is practical on a clinical scale must be applicable to cryopreservation of large numbers of cells. The effect of large scale cryopreservation on the recovery and viability of DC precursors and DC products is described in Examples 1 and 2, respectively. In such large scale cryopreservation, about 15-300 x 10⁶ cells per ml were frozen in a volume of 20 ml/bag. The recovery and viability of DC precursor cells and DC products is summarized in Tables IB and IIIB, respectively.

The results suggest that DC precursors and DC products enriched by buoyant density centrifugation may be cryopreserved on a large scale under the serum-free conditions described herein.

The exemplary procedures described herein make reference to AIM-V serum-free medium containing 10% DMSO with 15-300 x lOV l in a volume of 20 ml/bag using a rate-controlled freezing system (Forma) for large scale crypopreservation of DC precursors and Ag-loaded DC. While the examples provided herein include specific reference to a rate-controlled freezing and a particular type of serum-free medium, it will be understood that numerous methods of freezing in a rate-controlled or non-rate-controlled manner are routinely employed by those of skill in the art. Similarly, various serum-free media are commercially available and may be used in carrying out the invention. Examples include XVIVO 10, XVIVO 15, XVIVO 20, StemPro and any commercially available serum- free media. In addition, although the large scale cryopreservation examples refer to 10% DMSO, those of skill in the art will recognize that DMSO concentrations of from about 5% to as high as 20% may be included in the cryopreservation methods described herein. Generally, lower concentrations of DMSO are preferred, e . g. , about 5% to 10%.

It will be understood that any freezing procedure, type of serum-free medium and concentration of DMSO that results in a cell viability of at least 50% and a cell recovery of at least 50%, and preferably a cell viability and recovery of at least 70 or 80% may be used in the DC precursor and Ag-loaded DC compositions, as well as the cryopreservation methods described herein .

C. Effects of cryopreservation on the recovery and viability of DC products

Cryopreserved DC precursors were cultured ex vivo under conditions effective to generate mature DC and the percent (%) recovery and % viability of the cells compared to DC generated from fresh precursors, as described in Example 2. Mature DC were also generated ex vivo from fresh DC precursors in the presence or absence of tumor Ag. They were then cryopreserved on either a small or large scale under the same conditions as DC precursors, as described in Example 2.

The overall recovery of mature DC following ex vivo culture and cryopreservation was good in all cases irrespective of the stage at which cells were cryopreserved (i.e., DC cryopreserved as DC precursors versus mature DC) . These results confirm that DC, either as precursors or as mature DC, cryopreserved well in serum-free medium under the conditions described herein with cryopreservation accomplished on a clinical scale. D. Effects Of Cryopreservation On The Phenotype And Function Of DC Phenotypic analysis

The effect of cryopreservation on the ability of DC precursors to mature was assessed by comparing the cell surface expression of molecules typically expressed on mature DC ("DC- associated markers") following ex vivo culture for 40 hours, as further described in Example 3. The effect of cryopreservation on the phenotype of DC products is also described in Example 3. The results of 3-color FACS analysis are summarized in Fig. 1A-E for fresh DC products, Fig. 1F-K for DC generated from cryopreserved precursors and Fig. 1L-Q for cryopreserved DC products. The results demonstrate that there is no marked difference in the expression of DC-associated markers among DC generated from fresh precursors, DC generated from cryopreserved precursors and DC cryopreserved as DC products, suggesting that cryopreservation of DC, either at the stage of precursors or DC products, does not affect their phenotype.

E . Functional analysis During ex vivo culture DC precursors differentiate into mature, competent DC with allostimulatory and Ag-presenting abilities. DC generated from both fresh and cryopreserved DC precursors were allostimulatory, with no significant difference in the observed allostimulatory activity between DC derived from fresh versus cryopreserved precursors.

The results of Ag-specific T cell proliferation assays indicate that DC generated in the presence of the recombinant tumor Ag PA2024 or HER500, stimulated autologous T cells, while DC cultured in the absence of Ag were not stimulatory to the T cells. There was no difference in T cell stimulatory activity between Ag-loaded DC derived from fresh versus cryopreserved precursors. Together, these results suggest that cryopreservation procedures described herein do not affect the ability of DC precursors to differentiate into mature, competent DC, that are not only allostimulatory but also capable of stimulating autologous T cells in an Ag-specific manner.

Similarly, cryopreservation of DC products using the methods of the invention did not affect their allostimulatory or Ag-presenting ability. F. Stability of DC products

The stability of DC products, generated from either cryopreserved precursors or cryopreserved as mature DC products, was assessed as further described in Example 4. DC products, generated from either cryopreserved precursors (Table VA) or cryopreserved as DC products (Table VB) , were stable in lactated Ringer's solution, with a high percentage recovery and viability for up to 24 hours. The stability of DC products was also demonstrated in phenotypic analysis and functional assays, which indicate that DC products, generated from either cryopreserved precursor (Table VIA, Fig. 4A) or cryopreserved as mature DC products (Table VIB, Fig. 4B) , maintain their phenotype and allostimulatory activity for up to 24 hours at 4°C in lactated Ringer's solution.

G. Characteristics of cryopreserved DC precursors, and Ag- loaded DC

The results described herein indicate that (1) DC precursors enriched from peripheral blood may be cryopreserved on both a small and large scale in liquid nitrogen for a period of 2-24 weeks in serum-free cryomedia. At temperatures below -120°C, the duration of storage can be extended indefinitely beyond 24 weeks without impacting cell recovery, viability, phenotype or function. Good recovery and viability is observed upon thawing the cells and the cryopreservation conditions described herein do not affect the ability of DC precursors to differentiate into mature DC, as evidenced by both phenotype and function.

The recovery and viability of cryopreserved DC products is comparable to that of cryopreserved precursors, and cryopreservation of DC products did not affect their phenotype or function.

In addition, the stability of DC products, resuspended in lactated Ringer's solution at 4°C, indicates that DC products, generated from either cryopreserved precursors or cryopreserved as DC products are stable in lactated Ringer's solution for up to 24 hours with good recovery, high viability and no significant change in the phenotype or allostimulatory activity of the DC products over the 24 hour period. IV. Utility

Various utilities for cryopreserved DC precursors, DC and Ag-loaded DC (derived from bone marrow, PBMC, or other tissues and cell lines), include but are not limited to, one or more of:(l) cellular immunotherapy with a uniform cell population which may be administered at multiple time points; (2) utility as a base line sample for clinical monitoring following DC immunotherapy and other vaccination studies; (3) utility as a diagnostic and prognostic tool; (4) utility in ex vivo and in vivo generation of T cells specific to naive or weak Ag; (5) utility in generation of immune chimeras for allo- and xeno- transplantation; (6) utility in gene therapy; (7) utility in various research and development activities; and (8) utility in immunomodulatory therapies using autologous, allogeneic or xenogeneic DC for both stimulating Ag-specific immune responses and treating autoimmune disease.

Cells that have been cryopreserved under serum-free conditions provide the advantage of being free of infectious agents, foreign proteins (which may be antigenic) , antibodies, etc., that are typically found in serum. Accordingly, cryopreserved under serum-free conditions can be directly reinfused into patients after thawing. Additionally, serum-free conditions are more reproducible without the lot-to-lot variability that is inherent in any serum preparation, assuring better quality control of products prepared under such conditions .

Materials And Methods

Cells . PBMC were collected from healthy donors by standard leukapheresis . DC precursors were isolated by either a one-step or a successive two-step buoyant density centrifugation procedure using buoyant density solutions, BDS 77 and BDS 65 (Dendreon Corp.). Autologous T cells were purified from leukapheresis products by either a one-step buoyant density centrifugation procedure using BDS 77, or two-step buoyant density centrifugation using BDS 77 and BDS 65, followed by negative selection affinity chromatography (R&S Systems) . Allogeneic T cells were enriched from buffy coat preparations by the same method as autologous T cells.

Monoclonal antibodies (mAb) . Commercially available mAb were used to stain DC and DC precursors for flow cytometry including the following: (1) the fluorescein (FITC) conjugated antibodies, CDla (Biosource, BS), CD14 (Becton Dickinson, BD) , CD66b (Coulter/Immunotech, Cl), HLA-DR (BD) , Lin 1 (BD) and IgGl (BD) ; (2) the PE-conjugated antibodies, CD3 (BD) , CDllc (BD) , CD19 (BD), CD40 (BD) , CD54 (BD) , CD56 (BD) , CD86 (Pharmingen) and IgGl (BD) ; and (3) the PerCP-conjugated antibodies HLA-DR (BD) and IgG2a (BD) .

Ex vivo culture of DC precursors. DC precursors were cultured in Teflon bags (American Fluoroseal) at a density of lxl0⁷/ml in Aim-V medium supplemented with 2 mM glutamine in a humidified incubator at 37°C under 5% C0₂ for 40 hours. During the culture period DC precursors were pulsed with Ag, PA2024 at 10 μg/ml or HER500 at 20 μg/ml.

Flow cytometry. Cell surface phenotype analysis was carried out using samples consisting of approximately 1 x 10⁷ cells, which were incubated in 10% normal mouse serum in phosphate buffered saline (PBS) for 10 min., washed in PBS and resuspended in 300 μl PBS. The cell suspension was then dispensed at 30 μl/well into round-bottom 96-well plates. FITC-, PE-, and PerCP-conjugated mAb were added at 10 μl/well and incubated with the cells for 20 min. in the dark on ice. Cells were then washed with 200 μl/well of PBS and resuspended in 400 μl/well in PBS, then analyzed by FACScan (Becton Dickinson) using cells labeled with isotype-matched control Ab as a negative control .

Mixed Lymphocyte Reaction (MLR) . Cells were irradiated at 3,000 rad and used as stimulators at 800 - 8xl0⁵ cells/well in triplicate wells of round-bottom 96-well plates. As responders, 5xl0⁴ allogeneic T cells were added to each well. One μCi of ³H- thymidine was added to each well for the final 18 hours of a 6- day incubation period. At the end of incubation, cells were harvested and ³H-thymidine incorporation measured. Wells containing either stimulators alone or responders alone served as controls .

Ag presentation assays. DC precursors were cultured ex vivo for 40 hours in the presence or absence of Ag . Exemplary antigens include PA2024 and HER500, as further described below. Ag-loaded DC were then washed and used for assays either immediately following culture or following cryopreservation and thawing. The T cell stimulatory activity of the Ag-loaded DC was measured by incubating 800 - 8 xlO⁵ Ag-loaded DC with 1x10" autologous T cells per well in triplicate wells of 96-well round bottom plates. Ag was not added during the assay period. Proliferation of T cells was measured as above.

Antigens (Ag) . Two exemplary recombinant tumor Ag evaluated herein were: (1) recombinant PA2024, a fusion protein consisting of PAP and human granulocyte macrophage-colony stimulating factor (hGM-CSF) ; and (2) recombinant HER500, a fusion protein comprising 300 amino acids of the N-terminal extracellular domain and 200 amino acids of the C-terminal intracellular domain of HER-2/neu and hGM-CSF.

EXAMPLE 1 Cryopreservation Of DC Precursors In Serum-Free Media

Small scale cryopreservation. Cells were resuspended at 20-200 x lOVml in precooled 5% human serum albumin (HSA) (Swiss Red Cross) . An equal volume of 20% DMSO in the above HSA solution was then added dropwise. The mixture was aliquoted in cryovials at 1 ml/vial and frozen at -80°C in a cryochamber (Nalgene) overnight. The vials were transferred to a liquid nitrogen tank in the following morning.

Recovery and viability of DC precursors after small scale cryopreservation . DC precursors were isolated from PBMC by two- step buoyant density centrifugation. Cells were cryopreserved in a cryomedium containing 10% DMSO and 5% HSA using a cryochamber at -80°C overnight. Frozen vials were then transferred to a liquid nitrogen tank. After 2-24 weeks of cryopreservation, cells were thawed and their recovery and viability examined by trypan blue exclusion. As summarized in Table IA, the recovery of DC precursors was 80.1 ± 29.2% with a viability of 93.8 ± 1.4% (n=4) . These results suggest that DC precursors enriched by buoyant density centrifugation cryopreserved well under the above-described conditions. The results also suggest that a cryochamber is an effective means for freezing DC precursors. Table I . Recovery and Viability of Cryopreserved DC Precursors

A. Small Scale

Large scale cryopreservation

Cells were resuspended at 30-600 x 10⁶/ml in AIM-V. An equal volume of 20% DMSO in AIM-V was then added gradually. The mixture was frozen in freezing containers (Cryocyte, Baxter) at 20 ml/bag using a rate-controlled freezing system (Forma) .

Recovery and viability of DC precursors following large scale cryopreservation

The effect of large scale cryopreservation on recovery and viability of DC precursors was assessed by freezing DC precursors in AIM-V (Life Technologies) containing 10% DMSO at 15-300 x 10⁶ /ml in a volume of 20 ml/bag using a rate-controlled freezing system (Forma) . After 2-4 weeks of cryopreservation in liquid nitrogen, the cells were thawed and their recovery and viability examined.

The results of large scale cryopreservation of DC precursors summarized in Table IB, indicates that the recovery of DC precursors was 100.7 ± 8.4% with viability of 96.8 + 1.2% (n=4) . The results demonstrate that DC precursors enriched by buoyant density centrifugation tolerate the processes of freezing and thawing in serum-free medium on a clinical scale. The recovery of DC precursors after large scale cryopreservation was superior to those of small scale cryopreservation, while viability after large scale cryopreservation was as good as that of small scale cryopreservation. Table I . Recovery and Viability of Cryopreserved DC Precursors

B. Large Scale

Recovery and viability of DC products generated from cryopreserved precursors

Cryopreserved DC precursors were cultured ex vivo in AIM-V serum-free medium in the presence of PA2024. After 40 hours of culture, cells were harvested and their recovery and viability examined as above. The recovery of DC generated from cryopreserved precursors in the presence of PA2024 was 80.4 ±

5.6% with a viability of 96.4 ± 0.4% (n=4) (Table IIB) . These results are comparable to those obtained for DC generated from fresh precursors, for which a recovery of 101.9 ± 14.8% and a viability of 97.3 ± 3.1% was observed (Table IIA) . These results indicate that cryopreserved DC precursors survive through the subsequent ex vivo culture period.

Table II. Recovery and Viability of DC products after ex vivo Culture A. DC Products generated from Fresh Precursors

DC Products generated from Cryopreserved Precursors

EXAMPLE 2

Effects Of Cryopreservation On The Recovery

And Viability of DC Products.

Recovery and viability of cryopreserved DC products

DC products were generated ex vivo from fresh DC precursors in the presence or absence of tumor Ag . They were then cryopreserved on either a small or large scale under the same conditions as DC precursors. After 4-16 weeks of cryopreservation, DC products were thawed and their recovery and viability examined. As shown m Table IIIA, the mean recovery of PA2024-loaded DC cryopreserved on a small scale was 69.1 ± 16.0% with a viability of 80.0 ± 9.6%, while that of DC products cryopreserved on a large scale was 87.7 ± 2.4% with 98.2 ± 2.6% viability (Table IIIB) .

Table III. Recovery and Viability of Cryopreserved DC Products A. Small Scale

Table III. Recovery and Viability of Cryopreserved DC Products B . Large Scale

The overall recovery of DC, through ex vivo culture and cryopreservation either as DC precursors or as mature DC, is summarized in Table IV. The overall recovery of DC products generated ex vivo from cryopreserved DC precursors in the presence or absence of tumor Ag (Table IVA) , was compared to the overall recovery of DC products generated ex vivo from fresh DC precursors in the presence or absence of tumor Ag and cryopreserved as mature DC products (Table IVB) .

Both DC precursors and DC products were cryopreserved on a large scale under the conditions described above, and % recovery was evaluated after 4-16 weeks of cryopreservation.

The overall recovery of mature DC following ex vivo culture and cryopreservation was good in all cases irrespective of the stage at which cells were cryopreserved (i.e., DC cryopreserved as DC precursors versus mature DC) . These results confirm that DC, either as precursors or as mature DC, cryopreserved well m serum-free medium in clinical scale cryopreservation.

Table IV. Overall Recovery of DC through Ex vivo Culture and Cryopreservation

A. Cryopreserved as DC Precursors

Table IV. Overall Recovery of DC through Ex vivo Culture and Cryopreservation

B. Cryopreserved as DC Products

EXAMPLE 3 Effects Of Cryopreservation On The Phenotype And Function

Of DC products

Phenotypic analysis

The effect of cryopreservation on the ability of DC precursors to mature was assessed by comparing the cell surface expression of molecules typically expressed on mature DC following ex vivo culture for 40 hours, as described above.

The FACS profile of DC generated from fresh precursors and those generated from cryopreserved precursors was compared by gating on the Lin-negative/HLA-DR-positive population with analysis focused on evaluating the expression of CDllc, CD40, CD54 and CD86 on DC lineage cells. The effect of cryopreservation on DC cryopreserved at the stage of mature Ag- loaded DC was also examined.

The results of 3-color FACS analysis are summarized in Fig. 1A-E for fresh DC products, Fig. 1F-K for DC generated from cryopreserved precursors and Fig. 1L-Q for cryopreserved DC products. DC products generated from cryopreserved precursors or DC cryopreserved as DC products expressed all of the above DC- associated markers. The results demonstrate that there is no marked difference in their expression of DC-associated markers among fresh and cryopreserved DC products as well as DC generated from cryopreserved precursors suggesting that cryopreservation of DC, either at the stage of precursors or at the stage of DC products, does not affect their phenotype. Functional analysis

During ex vivo culture DC precursors differentiate into mature, competent DC with allostimulatory and Ag-presenting abilities. The Ag-presenting ability of DC generated from fresh precursors versus DC derived from cryopreserved precursors was compared in a MLR. Ag-specific T cell stimulatory activity of the DC was also examined in T cell proliferation assays using autologous T cells and Ag-loaded DC generated in the presence of the recombinant tumor Ag, PA2024 or HER500. DC generated from both fresh and cryopreserved precursors were allostimulatory as shown in Fig. 2A. There was no significant difference in the observed allostimulatory activity between DC derived from fresh and cryopreserved precursors.

The results of Ag-specific T cell proliferation assays are summarized in Fig. 2B and 2C. DC generated in the presence of PA2024 stimulated autologous T cells, while DC cultured in the absence of Ag were not stimulatory to the T cells. There was no difference in T cell stimulatory activity between PA2024-loaded DC derived from fresh versus cryopreserved precursors (Fig. 2B) . Similar results were obtained with DC cultured in the presence of HER500 (Fig. 2C) .

Together, these results suggest that cryopreservation does not affect the ability of DC precursors to differentiate into mature, competent DC, that are not only allostimulatory but also capable of stimulating autologous T cells in an Ag-specific manner. Similarly, cryopreservation of DC products does not affect their allostimulatory and Ag-presenting abilities. DC products cryopreserved at the stage of mature Ag-loaded DC were as stimulatorynm as fresh DC to allogeneic (Fig. 2A) and autologous (Fig. 2B-C) T cells.

EXAMPLE 4.

Stability of DC products

The stability of DC products, generated either from cryopreserved precursors or DC cryopreserved as mature DC products was assessed using the small scale cryopreservation methods described herein. DC products generated from cryopreserved precursors were resuspended immediately after their harvest at lxl0⁷/ml in lactated Ringer's solution, while DC cryopreserved as DC products were thawed and resuspended as above. Cells were kept at 4°C for up to 24 hours. At T=0, 4, 8 and 24 hours, aliquots of the cell suspensions were removed and the recovery, viability, phenotype and allostimulatory activity of the cells examined.

The recovery and viability of DC products at T=0, 4, 8 and 24 hours are summarized in Table V. DC products, generated either from cryopreserved precursors or cryopreserved as DC products, were stable in lactated Ringer's solution up to 24 hours with a recovery of 88.7 ± 11.8% and a viability of 87.1 ± 4.7% (Table VA) , while DC cryopreserved as DC products had a recovery of 86.9 ± 22.1% and a viability of 75.4 ± 17.2% (Table VB) .

Table V. Stability of DC Products — Recovery and Viability A. DC Generated from Cryopreserved Precursors

DC Cryopreserved as DC Products

The results of FACS analysis are summarized as percent positive among the total live cells for CD3, CD14, CD19, CD56, CD66b, HLA-DR, and CDla/CD54 for both DC generated from cryopreserved precursors (Table VI A) and DC cryopreserved as DC products (Table VI B) .

The representative results of CD54 analysis of the DC/monocytes population are shown in Fig. 3A-H. There was no significant change in the phenotype of DC products either as percent positive for the above markers or as fluorescence intensity for CD54 during the 24-hour period. These results indicate that cells in DC products are stable in their phenotype for up to 24 hours in the above conditions.

Table VI. Stability of DC Products-Phenotype¹

A. DC Generated from Cryopreserved Precursors

B. DC Cryopreserved as DC Products

The stability of DC products was also demonstrated in functional assays. DC products, either generated from cryopreserved precursors (Fig. 4A) or cryopreserved as mature DC products (Fig. 4B) , maintained their allostimulatory activity for up to 24 hours at 4°C in lactated Ringer's solution. Together, these results indicate that DC products, either generated from cryopreserved precursors or cryopreserved as DC products, are stable under the above conditions for up to 24 hours.

Donor: 9610-TR-08

Claims

WHAT IS CLAIMED:

1. A cryopreservation composition comprising antigen presenting cell (APC) precursors or APC, prepared by a process comprising the steps of:

(a) obtaining a blood sample from a subject;

(b) treating the blood sample in a manner effective to obtain a population of APC precursors or APC; and

(c) suspending said APC precursors or APC in serum-free cryopreservation medium containing dimethyl sulfoxide (DMSO);

(d) freezing said APC precursor or APC suspension at a temperature of -70°C or lower, wherein APC precursors or APC within said cryopreservation composition maintain a cell viability and cell recovery of at least 70% upon freezing and thawing said cells.

2. The cryopreservation composition according to claim 1, wherein said APC precursors are dendritic cell (DC) precursors.

3. The cryopreservation composition according to claim 1, wherein said APC are antigen-loaded (Ag-loaded) mature DC.

4. The cryopreservation composition according to claim 2, wherein said DC precursors frozen for at least 24 hours maintain the phenotype and function of DC precursors upon thawing the cells .

5. The cryopreservation composition according to claim 3, wherein said Ag-loaded DC frozen for at least 24 hours maintain the phenotype and Ag-specific function of Ag-loaded DC upon thawing the cells.

6. A cryoprotective medium for cryopreservation of APC precursors and APC, comprising: serum-free cryopreservation medium and DMSO, wherein APC precursors and APC frozen to a temperature of -70°C or lower in said cryopreservation medium for at least 24 hours maintain a cell viability and cell recovery of at least 70% upon freezing and thawing said cells.

7. The cryoprotective medium according to claim 6, wherein said serum-free cryopreservation medium is protein-free.

8. The cryoprotective medium according to claim 7, wherein said protein-free medium is supplemented with from about 1 to 30% human serum albumin (HSA) .

9. The cryoprotective medium according to claim 6, wherein said serum-free cryopreservation medium is selected from the group consisting of AIM-V, XVIVO 10, XVIVO 15, XVIVO 20 and StemPro .

10. The cryoprotective medium according to claim 6, wherein the medium comprises DMSO at a concentration of between about 5% and 20% (volume percent) .

11. A method for cryopreserving and cryoprotecting APC precursors and APC, comprising:

(a) obtaining a blood sample from a subject;

(b) treating said blood sample in a manner effective to obtain a population of APC precursors or APC;

(c) suspending said APC precursors or APC in serum-free cryopreservation medium containing DMSO;

(d) freezing said APC precursor or APC suspension at a temperature of -70°C or lower for at least 24 hours; and

(e) thawing said APC precursor or APC, wherein APC precursors or APC within said cryopreservation medium maintain a cell viability and cell recovery of at least 70% upon freezing and thawing

12. The method according to claim 11, wherein said serum- free medium is protein-free.

13. The method according to claim 12, wherein said protein-free medium is supplemented with from about 1% to 30% HSA.

14. The method according to claim 11, wherein said serum- free cryopreservation medium is selected from the group consisting of AIM-V, XVIVO 10, XVIVO 15, XVIVO 20 and StemPro.

15. The method according to claim 11, wherein said APC precursors are DC precursors.

16. The method according to claim 11, wherein said APC are Ag-loaded mature DC.

17. The method according to claim 15, wherein said DC precursors maintain the phenotype and function of DC precursors upon thawing said cells.

18. The method according to claim 16, wherein said Ag- loaded DC maintain the phenotype and Ag-specific function of DC precursors upon thawing said cells.