AU772703B2 - A method for the transfer of antigens to dendritic cells - Google Patents

Description

WO 00/73415 PCTIEP00/04712 A METHOD FOR THE TRANSFER OF ANTIGENS TO DENDRITIC CELLS Disclosure The present invention relates to a method for antigen transfer to dendritic cells, comprising cocultivation of dendritic cells with cells transfected with, or expressing the, relevant antigens.

Dendritic cells (DCs) are central players of the immune system. They present self-antigens during T cell development and foreign antigens for the induction of immune responses. Located in the majority of tissues, DCs capture and process antigens, and display at their surface MHC-peptide complexes with high efficiency. Moreover, they can upregulate costimulatory molecules and migrate to lymphoid organs, where they activate antigen-specific T cells.

Human DCs have a number of unique features and properties. They have been identified by their typical morphology, by the high membrane density of HLA class II and costimulatory molecules, and by the expression of a unique pattern of cell surface molecules. Functionally, DCs have the ability to endocytose and concentrate soluble proteins in the HLA class II compartment. They are strong stimulators for allogeneic T-lymphocytes in mixed lymphocyte reactions (MLR) and have the unique property of priming cord blood naive T-cells.

Human DCs can be generated in vitro from CD34+ cells in response to granulocyte/macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNFa). An alternative source of DCs is represented by the monocyte fraction of peripheral blood mononuclear cells (PBMCs) cultured in the presence of GM-CSF and interleukin 4 (IL- 4).

In the last few years, great attention has been given CONFIRMATION COPY WO00/73413 2 PCT/US00/14740 to the role of DCs in inducing an effective and long lasting antitumour immunity in various murine tumour systems by protecting the animal against an otherwise lethal tumour challenge. This has been obtained, for example, by pulsing DCs with a class I-restricted synthetic peptide, or with unfractionated peptides eluted from the tumour. The relevance of these animal models to the treatment of human cancer has been recently confirmed by a clinical study in which pulsed DCs have been utilized to induce the regression of established human lymphomas (Hsu et al., Nature Med. 2, 52-58, 1996).

In an effort to obtain in vitro or in vivo tumour antigen specific DCs, several different approaches have been proposed including gene transfer with viral vectors, with nucleic acids (plasmid DNA, mRNA) by means of liposomes, or loading dendritic cells with purified tumour antigens, with tumour lysates or peptides purified therefrom or chemically synthesized. DCs genetically engineered for Constitutive expression of a given antigen could provide an important advantage over antigen-pulsed DCs in terms of stable expression of the target gene product. This could overcome the limitation potentially represented by the rapid intracellular degradation of the HLA class 1-peptide complexes during the different phases of ex vivo isolation, manipulation, and in vivo administration. An additional advantage could be represented by the possibility of transducing entire tumour antigen gene(s), thus allowing presentation of yet unknown epitopes.

Direct transduction of DCs, however, involves difficult problems: DC from circulating monocytes cannot, for example, be infected by retroviral vectors, contrary to CD34+ marrow-derived dendritic cells. On the other hand, in immunologically compromised patients, mobilization of CD34+ WO 00/73415 PCT/EP00/04712 3 precursors is not advisable. Anyway, transfection of DCs is always as a rule poorly efficient.

Furthermore, the use of adenoviral vectors for transfer of the tumour antigens in DCs from monocytes mainly causes immunization to the vector viral components, making the subsequent uses of the vector less efficient.

It has now been found a particularly efficient method for the transfer of antigens to DCs which does not require transfection or transduction of the DCs themselves.

The method of the invention comprises cocultivation of DCs with "donor" cells expressing on their surface or within the cytoplasm the desired antigens, both of recombinant and natural origin. Examples of donor cells in which the expression of the antigen is obtained by transfection with plasmide vectors, or by transduction with viral or retroviral vectors, according to the invention, comprise, but are not limited to: primary cultures of tumour and non-tumour mammal cells or continue cell lines, fibroblasts or in general cells which can be efficiently transfected, such as HeLa, COS, NIH3T3, CHO or HEK-293 cells (ATCC No. CRL 1573), D cells or monocyte cultures.

Particularly preferred are NIH3T3 mouse fibroblasts. The transfection methods are based on the use of suitable plasmid expression vectors, viral or retroviral, which contain the concerned gene under control of a viral or cell-constitutive promoter (such as CMV, SV40, HSV TK, etc.) for high efficiency expression. Said vectors can also contain a gene for the selection of transduced or transformed cells (Neomycin, erbamycin, TK, etc.).

The methods for the transfection or transduction of the "donor cells" are conventional and comprise, for example, microinjection, electroporation, lipofection, precipitation of DNA with calcium phosphate or the use of DEAE dextran, the infection or other conventional WO 00/73415 PCT/EP00/04712 4 procedures as described for example in Sambrook et al.

(eds) (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York.

The method of the invention allows transfer in DCs of intact cytoplasmic and surface antigens, which can be either recombinant, such as those used for labeling and selecting the transfected /XLNGFr cells as described in Mavilio et al. (1994) Blood 83: 1988-1997, or natural, such as the histocompatibility antigens, or tumour antigens expressed by cells obtained from tumor biopsies.

Examples of antigens which can also be expressed in the recombinant form in the donor cells include, but are not limited to: "tumour antigens" or antigens specifically expressed by tumor cells, such as the members of the MAGE (Melanoma Associated Antigen) or BAGE family, Melan-A, gpl00, Mart-1, PSA (Prostate Specific Antigen), MUC-1, or foetal or embrionic antigens, such as AFP (a-Foetoprotein), or CEA (ChorioEmbrionic Antigen), or oncogens such as HER2/neu, or oncogens derived form "normal" gene mutation, such as p53, c-ras, or the idiotype of the V region of immunoglobulins hyperproduced in lymphomas, or still antigens coded by virus known as the etiological agents of neoplasias, such as EBV (Epstein-Barr Virus), HCV (Hepatitis C Virus), HPV (Human Papilloma Virus) or viral antigens such as HIV gpl60, HbsAg, etc.. As a rule, transfection can be performed using all the tumour and non tumour antigens known to date, particularly histocompatibility antigens, antigens of melanoma, carcinoma, sarcoma cells, or from lymphomas, epitheliomas, etc.

"Donor cells" expressing natural antigens are for example tumour cells from biopsies of patients, or cells expressing heterologous histocompatibility antigens, against which tolerance has to be induced, such as those of WO 00/73415 PCT/EP00/04712 an organ or bone marrow trasplant recipient, or the cells of the organ to be transplanted (liver, lung, pancreas, heart) for inducing tolerance in the host. The histocompatibility antigens are transferred to the cell surface of the DC and then they result co-expressed with those naturally coded by the DC. Dendritic cells obtainable according to the invention, loaded with the heterologous histocompatibility antigens and optionally treated with suitable cytokines, can be used for modulating GVHD (Graft Versus Host Disease), or in the control of the graft rejection of a transplanted organ, or for controlling autoimmmune reactions.

In case of cells transfected with intracellular antigens, the antigen can be transferred to DCs by subjecting the transfected or transduced cells to treatment with pro-apoptotic agents, such as radiations (UV, rays X), or cytokines such as TNF-a, or molecules such as actinomycin-D, or they are subjected to interaction with FasL or with antibodies capable of activating a proapoptotic cascade (anti-Fas, FasL antibodies, etc.). In this case, the intracellular antitumour antigen is uptaken by the DCs cells thanks to their ability to phagocyte apoptotic bodies. The phagocyted antigen material is then intracellularly processed by DCs and displayed on the cell surface in form of complexes with HLA class I and/or II peptides. In case of surface antigens, an efficient transfer of the intact molecule from the donor cells to DCs is obtained without inducing apoptosis. In this case the contact between DCs and donor cells is sufficient. DCs obtainable according to the method of the invention can be used for adoptive immunotherapy protocols, namely for the ex-vivo expansion of effector cells, usiog the manipulated DCs as cells presenting the antigen, or for active immunotherapy strategies, re-infusing the manipulated DCs WO 00/73415 PCT/EP00/04712 6 into the patient.

Administration of dendritic cells has been disclosed in W093/20185 and in EP-A-0 563 485.

The manipulated DCs according to the invention can further be used for the identification and the use, for example in tumour therapy, of novel epitopes directly generated by DCs processing of the complete antigen. The antigens can be expressed in the donor cells by transfection with cDNA coding for the complete antigen, or with cDNAs from "libraries" (for example tumour cells libraries), therefore allowing transfer to DCs, by the method of the invention, and their natural processing through sub-cellular compartments of DCs.

The dendritic cells obtainable according to the invention can therefore be used for activating HLA class I or II-restricted antigen-specific cytotoxic T cells.

The cocultivation conditions of DCs with cells transfected with, or expressing at their surface, the antigens, are conventional. Typically, cocultivation is mainteined from 12 to 48 hours, preferably about 24 hours, optionally in the presence of polybrene or of other agents promoting cell fusion. The methods for the stimulation of lymphocytes with the DCs loaded with the antigens according to the invention, are also conventional. For example, lymphocytes from neoplastic patients are cocultured with the irradiated DCs in IMDM medium (Iscove's Modified Dulbecco Medium) containing 10% human serum. Some days after the first stimulation, the medium is added with a concentration of 10 U/ml of interleukin 2.

The invention will be described in further detail in the following examples.

WO 00/73415 PCT/EP00/04712 7 Example 1: Transfer to DCs of the immunogenic fusion protein TN (Heroes Simolex thvmidine kinase and neomycin phosphotransferase) and of /\LNGFr surface marker Packaging cell line SFCMM2, already described (Bonini et al. Science 276, 1719-1724 (1997), produces a retroviral vector coding for the fusion protein containing the herpes simplex virus thymidine kinase (HSV-Tk) and the neomycin phosphotransferase (NeoR) and the /\LNGFr surface marker.

All the cell lines were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, antibiotics and foetal calf serum (FCS).

DCs were isolated from either heparinised fresh whole blood (5-6x10 7 or leukocyte-enriched buffy coats were allowed to adhere to T25 flasks. After 1 h at 37 0 C, the non-adherent cells were removed and the adherent cell layer was cultured in RPMI 10% FCS supplemented with LPS Ag/ml, GMCSF 800 U/ml, IL-4 100 U/ml, 2 mM L-glutamine, mM 21-ME.

At day 2 of culture, differentiating DCs were transduced using two different protocols: 1- by cocultivation with a monolayer of irradiated (100 Gys) packaging cells in the presence of polybrene (4 jug/ml).

After 72 hours, DCs were harvested, and seeded in fresh medium. 2- by substituting the culture medium-with the packaging line cell-free retrovirus-containing supernatant.

Three consecutive cycles of infection were performed every 24 hours.

The percentage of infected cells obtained with the two procedures was evaluated 48 hours after the last exposure to retrovirus, by flow cytometry for /LLNGFr expression performed with the mAb 20.4 (ATCC, Rockville, MD). The results, reported in la, show a very high surface marker DCs transduction efficiency, ranging from 50 to 90% and WO 00/73415 PCT/IEP00/04712 8 independent of the procedure utilized. The transduction procedure did not alter the immunophenotype, nor the stimulatory capacity of DCs, as shown in example 3 where, by measurement of the capability of DCs to induce a TNspecific cytotoxic response in peripheral blood lymphocytes, transgene TN transfer to CDs was monitored.

Example 2. Accuisition of the /\LNGFr surface marker The transfer mechanism of 1/LNGFr surface marker was evidenced by using the 3T3-TN//\LNGFr line for coculture of DCs, according to the protocol described in example 1, namely a cell line transduced using the transgene and expressing the cell surface marker /\LNGFr, but packaging defective and unable to produce any vector particle. The 3T3-TN//.LNGFr line was derived by transduction of NIH/3T3 fibroblasts (devoid of gag-pol env genes necessary for producing intact retroviral particles) with the retroviral vector produced by packaging cell lines SFCMM2.

In this vector-free system, uptake of the /1LNGFr marker was achieved at frequencies that were similar to those observed by the use of a vector-producing cell line (figure Ib). Furthermore, systematic microscopy examinations showed that only DCs in close and direct contact with the /LLNGFr-expressing cells were efficiently uptaking the marker and expressing it on their cell surface.

Example 3: Stimulation of specific PBL by DCs loaded with TN transqene Peripheral blood lymphocytes (PBLs) (2x10 6 from a Thymidino Kinase immune patient (TK-immune), were stimulated in vitro with autologous irradiated (50 Gys) DCs previously cocultered with the SFCMM2 vector irradiated as described in example 1. Lytic activity of the effector cells was tested after one round of stimulation. All the stimulations were performed in IMDM containing 10% human WO 00/73415 PCT/EP00/04712 9 serum. Three days after the 1st stimulation, a final concentration of 10 U/ml of IL2 was added to each culture.

Lytic activity of the effector cells was tested, eight days after the last restimulation, in a 4 hours cytolytic chromium release assay against appropriated target cells.

DCs transduced by cocultivation showed an ability to induce a strong immune response against the TN transgene (figure 2a), whereas DCs transduced by cell-free vectorcontaining supernatants failed to elicit any detectable response.

DCs cocultured with irradiated 3T3-TN//LNGFr cell line expressing the TN antigen in the cytoplasm, but unable to produce vector particles, induced a response comparable with that obtained with the vector-producing cells (Figure 2b).

Example 4: Transduction of MAGE-3 into DCs The same procedure as described in example 3 was substantially followed, utilizing packaging cell lines M3- CSM, coding for the melanoma-associated MAGE-3 tumorantigen, described in J. Immunol. 159, 2513-2521 (1997).

In order to measure phagocytosis of apoptotic bodies, M3-CSM packaging cells were labeled with the PKH-26 red fluorescent dye (Sigma), irradiated and added to a 2-days culture of DCs. Uptake of apoptotic cells by DCs was tested by flow cytometry and confocal microscopy analyses of the DCs population after 24 and 48 hours. Apoptotic cell death was assayed using Annexin and propidium iodide, according to the manufacturer's instructions. Negative controls were performed on the non-irradiated packaging cells.

The obtained results proved that about 30% of DCs took up apoptotic bodies within 24 hours, with the proportion of positive cells increasing over time. The presence of fluorescent bodies within the cytoplasm of DCs was confirmed by confocal microscopy. Appropriate control WO 00/73415 PCT/EP00/04712 experiments with unirradiated packaging cells confirmed the complete dependence of the phenomenon by irradiation Example 5: Stimulation of zumor infiltrating lymphocytes (TIL) with dendritic cells loaded with the MAGE-3 gene Tumour infiltrating lymphocytes (TILs) (2x10 6 were collected from a metastatic melanoma lesion and mixed with irradiated DCs (5x10 5 previously cocultured with irradiated M3-CSM packaging cells (Example The cultures were subjected to a first stimulation as described in example 3. The cultures were then restimulated after days in the same condition, while the third stimulation was performed using irradiated autologous T-blasts (2x10 6 transduced with the same vector. A first recognition was detected against vector-transduced but not against untransduced autologous T-blasts (figure Additionally, these effector cells recognized and killed the autologous melanoma cells (figure Taken together, these data indicate that genetically engineered DCs were able to induce a transgene product-specific immune response involving the tumour antigen MAGE-3.

Example 6: Acquisition of HLA-class I alloqeneic molecules by donor cells DCs obtained from a HLA-Bw4 negative donor were exposed to melanoma cells expressing the HLA-Bw4. After 24 hours, DCs were analyzed by flow cytometry for the expression of the relevant histocompatibility antigene HLA and were found to have acquired the HLA-Bw4 allele (figure 4a). Similar results were reproduced for the HLA-A2 system (figure 4b). To provide a direct image of the phenomenon a confocal microscopy analysis was performed. The fusion between cell membranes of DCs and donor cells was clearly evidenced under the microscope. Said analysis gives evidence that cell-to-cell contact is a prerequisite for transduction of surface molecules to DCs.

Claims (9)

1. A method for the transfer of intact antigens to dendritic cells, which comprises contacting donor cells expressing at their surface the desired antigen(s) with dendritic cells in culture, wherein the transfer is not mediated by apoptosis of donor cells.

2. A method as claimed in claim 1, wherein the antigen to be transferred is a tumor antigen.

3. A method as claimed in claim 1, wherein the antigen to be transferred is a recombinant antigen.

4. A method as claimed in claim 1, wherein the surface antigen is a HLA histocompatibility antigen.

A method as claimed in claim 1, wherein the surface antigen is a recombinant surface marker.

6. A method as claimed in claim 5, wherein the recombinant surface marker is ALNGFr.

7. A method as claimed in any one of claims 1 to 6, wherein transfected donor cells are fibroblasts.

8. Dendritic cells obtainable by the method of claims 1-7.

9. The use of the dendritic cells of claim 8 for the preparation of agents for use in active or passive immunotherapy protocols. The use of the dendritic cells of claim 8, loaded with the heterologous histocompatibility antigens and optionally treated with suitable cytokines for the preparation of agents for use in modulating GVHD (Graft Versus Host Disease) and in controlling the graft rejection of a transplanted organ. EMPFANGSZI AMENDED SHEET i l. J 8