WO2007025705A2

WO2007025705A2 - Method for the generation of antigen-specific t-cells and uses thereof

Info

Publication number: WO2007025705A2
Application number: PCT/EP2006/008421
Authority: WO
Inventors: Tim Greten; Firouzeh Korangy
Original assignee: Medizinische Hochschule Hannover
Priority date: 2005-08-29
Filing date: 2006-08-29
Publication date: 2007-03-08
Also published as: WO2007025705A3; WO2007025554A1

Abstract

The present invention relates to the generation and expansion of antigen-specific T-cells. Furthermore, the present invention relates to the use of antigen-specific T-cells obtained with the method according to the present invention in adoptive cell transfer therapy and in the treatment of various disorders and diseases including infections and cancer. Moreover, the present invention provides for kits and its use for generating and expanding antigen-specific T-cells. The present invention allows for the generation of antigen-specific T-cells suitable for adoptive cell transfer. The methods for the generation and expansion of antigen-specific T-cells include both in vitro and in vivo approaches.

Description

Method for the generation of antigen-specific T-cells and uses thereof

The present invention relates to the generation and expansion of antigen-specific T- cells. Furthermore, the present invention relates to the use of antigen-specific T-cells obtained with the method according to the present invention in adoptive cell transfer therapy and in the treatment of various disorders and diseases including infections and cancer. Moreover, the present invention provides for kits and its use for generating and expanding antigen-specific T-cells. The present invention allows for the generation of antigen-specific T-cells suitable for adoptive cell transfer. The methods for the generation and expansion of antigen-specific T-cells include both in vitro and in vivo approaches.

Background of the invention

The immune system represents the defence system present in its most advanced form in higher vertebrates. The immune system provides rapid and specific responses on all kinds of foreign substances. These foreign substances, known as antigens, induce the release from the lymphocytes, mainly the B-cells and T-cells, signals for cellular or humoral immune responses. The T-cells are responsible for the cellular immune response while the antibody producing B-lymphocytes supported by specific T-cells are the main component of the humoral response. However, an errant immune response is also responsible for autoimmune diseases or transplant tissue rejection.

T-cells are one of the major regulatory cell types of the immune system. The regulatory functions of T-cells depend not only on expression of a unique T-cell receptor, but also on expression of a variety of accessory molecules and effector functions associated with an individual T-cell response, e.g. T-helper cells versus cytotoxic T-cells. Each individual T-cell expresses a unique T-cell receptor. After clonal expansion of this individual T-cell, a number of identical T-cells are obtained displaying the same individual T-cell receptor having a specific antigenicity for an antigenic structure. It is the regulatory function of T-cells that often goes awry in the development of autoimmune diseases, plays a large role in tissue graft rejection, and can be important in tumour rejection. As stated above, T cells express a unique T-cell receptor (TCR). The cognate ligands are antigen containing major histocompatibility complex molecules (MHC), the MHC complex. That is, the TCR which is expressed on a T-cell surface recognizes the antigen present in form of a small antigenic peptide presented by the MHC molecules on the surface of the so called antigen presenting cells (APC).

The MHC molecules can be divided mainly into the MHC class I and MHC class Il molecules. The MHC molecules are encoded by a large complex of multiple genes, many of which are highly polymorphic. Thus, the variability of the MHC system is achieved in each individual by a multigenic system. The class I molecules consist of a heavy peptide chain noncovalently linked to a smaller peptide called β2- microglobulin (β2m). The largest part of the heavy chain is organized into three globular domains (crt , α2, and α3) which protrude from the cell surface; a hydrophobic section anchors the molecule in the membrane and a short hydrophilic sequence carries the C-terminus into the cytoplasm.

The α1 and the α2 domains which are most distal to the membrane, form a structure having a cavity. This cavity or groove formed by these two domains contains the antigenic structure, e.g. the peptide derived from the foreign substance.

The class Il molecules are also transmembrane glycoproteins consisting of an α and a β polypeptide chain or a y and δ. polypeptide chain, respectively. The α1 and the β1 domains form the groove responsible for the uptake and representation of the antigenic structure.

Essentially all nucleated cells carry class I molecules. In contrast, MHC class Il molecules are expressed by antigen presenting cells, like dendritic cells, macrophages and B-cells. MHC class I molecules are responsible for an immune response involving cytotoxic T-cells. That is, the MHC class I molecules contain antigenic structures present in the cytosol of the cells, i.e. endogenous cytosolic molecules. In contrast, the MHC class Il molecules present exogenous molecules to the T-cells of the helper-type. In the most recent years the adoptive cell-transfer therapy comes into the focus as an instrument for the treatment of e.g. cancer and in transplantation. This type of therapy is based on the use of antigen-specific T-cells as the inducer of an immune response against tumours or infections. In particular, cytotoxic T-cells represent the ultimate effector mediating the rejection of tumour cells or cells infected with foreign invaders like bacteria, parasites or virus.

However, one major hurdle in adoptive cell-transfer therapy before administering cells to the patient is the generation of a sufficient number of antigen-specific cells. Typically, the generation and expansion of the antigen-specific cells is conducted in vitro, although in vivo expansion is also possible. The expansion of T-cells may be affected specifically or unspecifically. For the use in e.g. adoptive cell transfer therapy, antigen-specific T-cells specific for the antigenic structure have to be prepared. In particular, the specificity of the antigen-specific T-cells against the foreign infection or the tumour cells is essential for attaining a successful therapy.

As indicated above, one major problem is the efficient production of a sufficient number of antigen-specific T-cells for later transfer into the recipient.

In principle, there are four different possibilities to produce antigen-specific T-cells:

(i) The use of dendritic cells as the antigen presenting cell pulsed or loaded with the defined peptide(s) of interest.

(ii) The use of peripheral blood mononuclear cells (PBMC) which have been driven to lymphoblasts and pulsed with the peptides of interest.

(iii) The use of lymphoblastoid cell lines where the natural peptides are acid- stripped and loaded with the peptides of interest, (iv) The use of cells expressing empty MHC molecules which are subsequently loaded with the peptide of interest.

Dendritic cells are considered to be the most important antigen presenting cells in the immune system. They sample antigen at the body's environmental interface and, under appropriate conditions, mature and migrate to the lymphatic organs were they induce primary and enhance secondary immune responses.

That is, dendritic cells are regarded as the primary antigen presenting cell system in humans because of their wide application in presenting primary antigen to T-cells. Self or foreign proteins are processed within a dendritic cell (DC) including processing system comprising the TAP molecules. The resultant peptide epitopes are presented by MHC molecules, and are transported to the surface of the DC. However, using the DCs in an approach to generate antigen-specific T-cells does not necessarily result in the same specificity of the antigen-specific T-cells. That means, using the same DC and the same antigen does not lead to the same antigen-specific T-cells but different clones may be obtained recognizing different epitopes of the antigen. In addition, pulsing the DC with the desired antigen would not lead the DC presenting only epitopes of the desired antigen but also other molecules may be presented in the MHC molecules of the DC.

Depending on the conditions under which the dendritic cells are induced, the response of the T-helper cells may be of the T-helper-1 type or the T-helper-2 type. Further, under appropriate conditions an unpolarized Th-O response can be induced or the cultivation with dendritic cells may anergize the T-cells which results in tolerance. The mechanisms involved in this plasticity are affinity and duration of MHC II-TCR (T-cell receptor) interaction, the expression pattern of costimulatory molecules, and the availability of cytokines.

The application of so called antigen-pulsed dendritic cells (DC) is an extensively explored field. The purpose of producing antigen-pulsed DC, i.e. DC loaded with antigens, is to immunize patients in need thereof against tumours, infectious agents or other pathogens. Great efforts are made with respect to the treatment of tumours that have become resistant to conventional therapeutical means. Especially in tumour therapy it is highly desirable to induce a sufficient antigen-specific Th-1 response or a cytolytic response via cytotoxic T-cells. A possibility to generate activated and antigen specific T-cells emplying co-cultivation with dendritic cells is described in Marten et. al., Cancer Immunol Immunother, 2002,51 :25-32. The authors describe that they succeeded in generating cytotoxic T- cells in an amount of up to 2.8 % antigen specific T-cells after co-culture with antigen pulsed dendritic cells.

Further, constructs of e.g. peptide-β2m-alpha-1 , alpha-2, and alpha-3 MHC class I fusion peptides which may be present in soluble form or membrane bound form can be used to separate antigen specific T-cells. For example, in Greten et. al, J Immunol Methods, 271 , 2002, 125-135, transfected cells have been generated having said fusion peptides on their surface for interaction with cognate TCRs. However, the system described therein still suffers in providing antigen-specific T-cells in a high ratio after cultivation. The fusion peptide described therein have been used as a sensitve tool to identify and to sort antigen-specific T-cells in peripheral blood. However, the use of fusion peptides to generate or to expand antigen-specific T-cells specific solely for the peptide in the construct is not described or envisaged therein. The cell system used in Greten el. al. suffers in employing a MHC class positive cell line, namely COS cells, which express functional self MHC molecules which present endogenous peptides. Therefore, not only T-cells specific for the peptide in the fusion peptides will be activated but also T-cells whether of T-helper cell type or cytotoxic T- cells which recognize other antigenic structures presented by self MHC molecules.

Thus, one object of the present invention is to provide a method for the fast and reliable generation and/or expansion of antigen-specific T-cells. Another object of the invention is the provision of overcoming the laborious work of generating DC cells for the co-culture and stimulation of antigen-specific T-cells and the provision of a sufficient amount of APC for the generation of antigen-specific T-cells.

The above objects and other objects which will be become apparent from the following description are achieved by the present invention. Description of the invention

The present inventors now discovered that the use of cells which are phenotypically negative for a functional MHC complex being transformed or transfected with a construct leading to the expression of functional MHC class I and/or class Il complexes containing the antigenic structure of the peptide of interest (POI) which can also be referred to as an artificial MHC complex, allows for the production of antigen-specific T-cells of the cytotoxic T-cell type or T-helper T-cell type depending on the MHC class. These cells expressing one type of functional MHC class I and/or class Il complex will also be referred to as artificial Antigen Presenting Cells or aAPC. Thus, the above cells do present a single antigenic structure to the immune competent cells, in particular to the T-cells. Since these transformed or transfected cells do have only one specific antigen bound in the pocket of the appropriate MHC molecule of class I or II, respectively, thus, forming an artificial, functional MHC complex, only those T-cells will be activated which interact with this specific complex resulting in antigen-specific T-cells specific for the antigenic structure present in the complex.

Therefore, according to one aspect of the present invention there is provided a method for the generation and/or expansion of antigen-specific CD 8+ T-cells comprising the steps of: a. providing a cell or cell line being phenotypically negative for a functional MHC class I complex; b. introducing a first construct comprising (i) a peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class I molecule or parts of said MHC class I molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a.); c. obtaining a cell or cell line (aAPC) expressing a peptide construct as defined in b) comprising the antigenic structure presented by the MHC class

I complex on their surface; d. co-culturing the cell or cell line obtained in c.) with cells comprising T- cells; and e. expanding antigen-specific CD8+ T-cells specific for the antigenic structure of the POI present in the construct as defined in b.).

In a further embodiment, the present invention relates to a method for the generation and/or expansion of antigen-specific CD 4+ T-cells comprising the steps of: f. providing a cell or cell line being phenotypically negative for a functional MHC class Il complex; g. introducing a first construct comprising (i) a peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class Il molecule or parts of said MHC class Il molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a.); h. obtaining a cell or cell line (aAPC) expressing a peptide construct as defined in b) comprising the antigenic structure presented by the MHC class I complex on their surface; i. co-culturing the cell or cell line obtained in c.) with cells comprising T- cells; and j. expanding antigen-specific CD4+ T-cells specific for the antigenic structure of the POI present in the construct as defined in b.).

As mentioned above, the cells obtained in step c.) do express only one specific artificial MHC complex, either MHC class I complex and/or MHC class Il complex as a functional MHC complex. Self MHC molecules or parts thereof expressed by said cell are non-functional insofar that they do not allow cognate interaction with its ligand to provide signals for activating cells bearing said ligands. In this connection, functional complex means that the MHC complex allows for a cognate interaction with the ligands, namely e.g. the TCR or BCR on lymphocytes.

That is, the cells obtained in step c.) which are characterized in functionally expressing a peptide construct comprising a peptide of interest including an antigenic structure, a spacer in between the peptide of interest and an MHC molecule, are recombinantly obtained cells which are also referred to as artificial antigen presenting cells (aAPC), as POI presenting cells or peptide-MHC-APC. As used herein, the term "Th cells" refers to Helper T cells, or CD4 positive (CD4+) cells.

As used herein, the term "CTL" refers to cytotoxic T-cells, or CD8 positive (CD8+) cells.

As used herein, the term "major histocompatibility complex", MHC molecules or "MHC" is a generic designation meant to encompass the histo-compatibility antigen systems described in different species including the human leucocyte antigens (HLA). Further, the term MHC molecule as used herein refers to MHC molecules themselves or functional fragments thereof which molecules or functional fragments are able to induce an antigen specific activation of cells expressing receptor molecules for the MHC molecule, like cells expressing the B-cell receptor or the T- cell receptor when these molecule or fragments are expressed in combination with a molecule, preferably a peptide, containing an antigenic structure, said combination of MHC molecule and antigenic structure, the MHC complex, is specifically recognized by the B-cell receptor or T-cell receptor, respectively. If not otherwise indicated, the term MHC molecules or MHC or major histocompatibility complex encompass class I and class Il molecules. The same is true for MHC complexes. MHC complexes comprise the MHC molecules and the antigenic structure, which comprises typically a peptide and encompass MHC class I as well as MHC class Il complexes.

As use herein, the term "adoptive immunotherapy" or "adoptive cell transfer" refers the administration of donor or autologous T-lymphocytes for the treatment of a disease or disease condition, wherein the disease condition results in an insufficient or inadequate immune response that is normally associated with Class I or Class Il MHC molecules, in particular with Class I or Class Il HLA molecules. Adoptive immunotherapy is an appropriate treatment for any disease or disease condition where the elimination of infected or transformed cells has been demonstrated to be achieved by CTLs or T helper cells.

Disease or disease conditions as used herein include but are not limited to cancer and/or tumours, such as, melanoma, prostate, breast, colorectal, stomach, throat and neck, pancreatic, cervical, ovarian, bone, leukemia and lung cancer; viral infections, such as, hepatitis B, hepatitis C, human immunodeficiency virus; bacterial infections, such as tuberculosis, leprosy and listeriosis, and parasitic infections such as malaria. Furthermore, the adoptive immunotherapy or adoptive cell transfer is useful in transplantation, like organ graft rejection, e.g. graft versus host or host versus graft reactions, or diseases associated with an transplantation like EBV or CMV infection.

As used herein, the terms "epitope", "peptide epitope", "antigenic peptide" and "antigenic structure" refers to a structure, like a peptide, derived from an antigen capable of causing a cellular or humoral immune response in a mammal. Such structures, like peptides, may also be reactive with antibodies from an animal immunized with the structures. In case of peptides, such peptides may be about five to twenty amino acid in length preferably about eight to fifteen amino acids in length, and most preferably about nine to ten amino acids in length. In particular, the length of the peptide associated with the MHC class I groove is of 8 to 11 amino acids in size and of the peptide associated with the MHC class Il groove is of 9 to 22 amino acids in size.

The construct (the first and/or second construct) to be introduced in the cell or cell line may be a DNA construct or a fusion peptide.

The media for the co-culture of the transformed or transfected cells obtained in step c), the aAPC, with cells comprising T-cells according to step d.) may further contain growth factors, cytokines, and/or substances, in particular salts, nutrients, or other auxiliary agents and/or drugs. Co-culture is conducted in suitable containers, like culture dishes or flasks. In one embodiment, the medium is a AB-medium characterized in that the FCS usually present in fully supplemented medium is substituted with AB-sera. The culture conditions are preferably 37°C and 5% CO2 atmosphere. Of course, it is necessary that the cells obtained in step c), the aAPC, and the cells comprising T-cells to be cultured with the aAPC are cultured under conditions allowing for expansion of antigen-specific T-cells with appropriate co- molecules depending on which type of T-cells should be expanded. Another preferred embodiment comprises the step of pre-stimulation of the aAPC obtained in step c.) with appropriate factors to induce expression of co-stimulatory molecules. For example, in case of using B-cell lines like Daudi cells, a pre- stimulation of transfected Daudi cells is conducted by cultivating said cells in a medium containing IL-4 and CD40L. After pre-stimulation, the stimulated aAPC are co-cultured with cells comprising T-cells according to step d.) of the present invention.

In another embodiment of the present invention aAPC additionally express peptides that are associated with various desired functions that enhance the generation and expansion of antigen-specific T-cells and the treatment of the subject, respectively. For example, in addition to peptides associated with the disease or disease being treated, the aAPC can express proteins associated with accessory molecules such as, lymphocyte function antigens (LFA-1 , LFA-2 and LFA-3), intercellular adhesion molecules 1 and 2 (ICAM-1 , ICAM-2), T-cell co-stimulatory factors (CD40, CD70, B7, Ox40, 4-1 BBL, CD27L) to enhance cell-cell adhesion or transduce additional cell activation signals. In particular, accessory molecules useful for the activation and expansion of the antigen-specific T-cells are e.g. IL-2, IL-7, IL-18, IL-12, IL-3, IL-5, IL- 10.

Also, in a further preferred embodiment, the aAPC do not express or secrete any costimulatory molecules necessary for the generation, activation and/or expansion of antigen-specific effector T-cells, Ike cytotoxic T-cells or helper T-cells, like CD4+ T- cells. As known in the art, typically at least a second signal is necessary to achieve full activation of T-cells. When the aAPC do not express or secrete any costimulatory molecules, T-cells will be rendered into an anergic state. Said anergic T-cells will not be able to act as effector or helper cells in the immune response.

Preferably, the co-cultivation according to step d.) and the expansion step e.) of the method according to the present invention is repeated at least once, more preferably, twice or more. In addition, the aAPC obtained in step c) of the method described above, may be obtained by a selection step or an enrichment step. The selection step may be any kind of selection of the desired transfected cell based on selection markers known in the art. For example, in case a DNA-construct is used for the introduction of the construct allowing the expression of the peptide construct according to the present invention, suitable selection markers are, for example, resistances to antibiotics such as ampicillin, kanamycin, neomycin, puromycin, zeocin, blastomycin or metabolic defects. Alternatively, GFP/EGF expressing constructs for selection, e.g. for sorting transfected cells can be applied.

Thus, antigen-specific T-cells in large amounts and in high purity can be harvested from the culture which may include a further purification step. These antigen-specific T-cells may be used further for various applications. These further applications include the adoptive immunotherapy or the adoptive cell transfer, respectively. Additionally, the cells may be useful for the treatment of cancer, allergic disorders, infectious diseases, autoimmune disorders and host versus graft or graft versus host reactions in transplantation.

The origin of the T-cells is mostly the blood of donors. However, since the number of the desired cell type, the antigen-specific T-cells, is very low, an expansion step for theses cells is necessary. Preferably, the cells containing T-cells to be cultured with transformed or transfected cell or cell line, the aAPC, are derived from the recipient himself or may be derived from a healthy volunteer. Alternatively, frozen cells may be used. Said frozen cells may be derived from the recipient of the adoptive cell-transfer therapy. Preferably, blood mononuclear cells, in particular peripheral blood mononuclear cells or peripheral blood lymphocytes are used for cultivation.

Particularly, the cells are of human origin.

As stated above, the cell used for the preparation of the artificial antigen presenting cell is a cell or cell line being phenotypically negative for a functional MHC class I and/or class Il complex. As used herein, the term "being phenotypically negative for a functional MHC class I and/or MHC class Il complex" refers to a cell or cell line which do not express an MHC class I and class Il complex, respectively, on their cell surface containing an antigenic structure. In this connection, functional complex means a complex composed of the MHC molecule and the antigenic structure present in the groove of the MHC molecule and able to interact with its cognate ligand as explained above.

The use of MHC negative cells expressing the peptide-β2m-MHC sequence allows for stimulation only of those T cells specific for the peptide of interest, with a defined specificity. The use of this constructs also inhibits the stimulation of other nonspecific T cells, which could be observed when antigen presenting cells are used, which express MHC presenting endogenous peptides.

Thus, cells being phenotypically negative for a functional MHC complex and expressing an artificial MHC complex as described herein can be used to obtain either CD4+ or CD8+ T-cells being specific for a particular antigen presented by said artificial MHC molecule. In a particular preferred embodiment, said cells expressing the artificial MHC class I or class Il complex on its surface additionally express a second construct comprising (i) a peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class I or class Il molecule or parts of said MHC class I or class Il molecule, or sequences encoding said second construct comprising (i) to (iii) into the cell or cell line of a.);

Hence, the present invention relates to methods further comprising the steps of introducing a second construct comprising (i) a second peptide of interest which is different to the first peptide of interest comprising an antigenic structure, (ii) at least one spacer and (iii) MHC class Il molecule or parts of said MHC class Il molecule, or sequences encoding said constructs comprising (i) to (iii) into the cell or cell line of step a) containing the artificial MHC class I complex; and obtaining a cell or cell line expressing a peptide construct comprising the antigenic structure of the second peptide of interest as defined above presented by a functional MHC class Il complex on the surface. In another embodiment, the present invention relates to a method further comprising the steps of introducing a second construct comprising (i) a second peptide of interest comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class I molecule or parts of said MHC class I molecule, or sequences encoding said construct comprising (i) to (iii) into the cell or cell line of step a) containing the artificial MHC class Il complex; and obtaining a cell or cell line expressing a peptide construct comprising the antigenic structure of the second POI as defined above presented by a functional MHC class I complex on their surface.

The above method allows the generation of both T-cell types, antigen specific CD8+ T-cells and CD4+ T-cells.

That is, the cell or cell line may be defective in expressing any functional self MHC class I or class Il molecules, e.g. by a genetic defect. In particular, the cell or cell line is a cell or cell line which does not express any self MHC class I or MHC class Il at all.

In another embodiment, the expression of the self MHC class I and/or class Il molecule is suppressed by means known in the art. For example, suppression is effected by siRNA-techniques. In another embodiment, the TAP molecules involved in the processing of antigen may be inactivated or its expression may be suppressed. Further, in case of MHC class Il complexes, the expression of the invariant chain may be disrupted, inhibiting the formation of self MHC class Il complexes.

Alternatively, the cell or cell line do express an MHC class I and/or class Il molecule which does not contain an antigenic structure. This can be achieved for example by antigen stripping or by manipulating the self MHC class I and/or class Il proteins in a way that no antigenic structure can assemble in the groove.

Rendering the cell or cell being negative for a functional MHC class I and/or class Il complex may be effected after transfection with the construct comprising the POI, at least one spacer and MHC molecule or parts thereof. It is preferred that the aAPC and/or the T-cells are of human origin.

The antigenic structure present in the construct introduced into the cell or cell line is a part of or the complete peptide of interest. As used herein, the term "peptide of interest, POI" refers to the peptide the antigen-specific T-cells are raised against. That is, in case of foreign invaders, the peptide of interest contains an antigenic structure specific for the foreign invader which will elicit an immune response in the subject.

To be not bound by theory, in case of the treatment of cancer, the antigenic structure expressed in connection with the MHC complex on the cell or cell line, the aAPC, is derived from tumour associated antigens. When treating viral or bacterial infections, the antigenic structure is derived from viral or bacterial antigens, respectively.

In case of autoimmune diseases, the antigenic structure is an antigenic structure derived from the "self-antigen" causative of the autoimmune disease. The same is true for allergic disorders where for the treatment of the antigenic structure the antigenic structure of the causative allergen is used as the POI in the construct.

For example, the POI is the influenza M1 peptide as described in Greten et al., J. Immunol. Meth., 2002, 125-135. In addition, the POI may be derived from tumour antigens, like the NY-ESO-Epitope, SLLMWITQC, an Epitope well known in the art.

In one embodiment, the construct introduced into the cell or cell line is a DNA- construct. Suitable constructs are described for example in Greten TF, et al, J. Immunol. Meth., 2002, 125-135 which is enclosed herewith by reference in its entirety. In a preferred embodiment the construct is composed of a nucleic acid sequence encoding a peptide comprising an antigenic structure, spacer, optionally β2-microgloblin, and at least parts of the MHC class I molecule. In particular, the construct has the following order of components, starting from the N-terminus: Leader sequence of the β2 microglobulin, the POI, a spacer, β2 microglobulin core sequence, a spacer, MHC class I sequence. This preferred embodiment is also shown in figure 1. In another embodiment of the present invention, the construct is composed of a nucleic acid sequence encoding a peptide comprising an antigenic structure, spacer, and at least parts of the MHC class Il molecule.

Various companies supply commercially available expression vectors for various cell systems useful in the present invention, e.g. Invitrogen, Qiagen, Stratagene, Clontech, Novagen, New England Biolabs, Pharmingen, Promega, Pharmacia, etc. The expression vectors isolated in this way can then be introduced into suitable cells, e.g. by electroporation, calcium phosphate coprecipitation, liposome-mediated transfection, etc., in a manner known to the skilled worker. Alternatively, it is also possible to use recombinant viruses produced by methods of molecular biology, which then in turn infect cells and bring about the expression of the peptide molecules by the infected cells. Suitable viral expression systems are, for example, the bacculovirus system, e.g. BacculoGold (BD Bioscience Pharmingen, Palo Alto, CA, USA), adenoviral expression systems such as, for example, ViraPortTM (Stratagene, La JoIIa, CA, USA), retroviral expression systems such as, for example, AdEasy (Stratagene, La JoIIa, CA, USA) etc.

Preferably, the transfection is a stable transfection and not a transient transfection since it is necessary to culture the obtained transfected cells over a long period of time.

In addition, the construct introduced into the cell or cell line may be a peptide molecule. Introduction of the peptide can be achieved by known methods. For example, introduction of the peptide can be achieved by using a translocation or targeting sequence known in the art.

Numerous amino acid sequences, especially derived from viruses, e.g. HIV tat or the protein VP22 which is derived from herpes simplex virus, which promote the transport of proteins, peptides and other classes of substances, such as, for example, nucleic acids or pharmaceutically active substances, into the interior of cells are known from the literature. For this purpose, these so-called translocation sequences are linked to the molecules to be transported (also called cargo molecules) either via covalent or via noncovalent linkages. Extracellular addition of the resulting compounds of translocation sequence and cargo molecule to cells is then possible. The translocation sequence facilitates the entry of the cargo molecule into the interior of the cell. This principle has likewise been described in numerous studies, especially for the HIV Tat sequence.

Numerous suitable translocation sequences are described in the literature. These translocation sequences include viral sequences, homeoprotein sequences, leucine zipper sequences, arginine and lysine-rich sequences, and various other sequences of proteins which are secreted despite the absence of a secretion signal sequence, etc.

Viral peptide sequences suitable as translocation modules

The peptide sequences suitable as translocation modules for the purposes of this invention include inter alia viral proteins or partial sequences of viral proteins such as, for example, the protein HIV transcriptional activator protein (HIV tat). The suitable Tat proteins include besides the Tat protein of the HIV 1 virus also the Tat proteins of other lentiviruses. Numerous modified Tat peptides have been described as sequences able to bring about translocation. These include Tat peptides which represent only partial sequences of the Tat protein, e.g. aa 47 to 57, Tat peptides which comprise point mutations, Tat peptides in which the sequence is reversed (inverted), or Tat peptides which comprise unusual amino acids such as, for example, D isomers of amino acids, etc. All these variations of peptide sequences are therefore generally suitable as translocation modules. Peptides also suitable for the purposes of the present invention as translocation modules are those derived from other viruses such as, for example, VP22 (herpes simplex virus 1 VP22 tegument protein). At present, commercial expression vectors comprising a VP22 sequence suitable for translocation are also available. These expression vectors therefore permit VP22 fusion proteins to be prepared (VoyagerTM VP22 system, Invitrogen, Breda, the Netherlands). Other viruses, e.g. Marek's disease virus 1 , a virus which causes lymphoma in chickens, also express a protein which is related to VP22 and which is likewise suitable as translocation module. These proteins and partial sequences of these proteins are mentioned only as examples, and numerous further peptides are known at present, and will become known in future, which are suitable as translocation modules for the purposes of the invention.

Homeoproteins suitable as translocation modules A further group of translocation modules suitable for the purposes of the present invention are peptides derived from the drosophila homeotic protein antennapedia (ANTp). Among others, ANTp peptides suitable as translocation module are those comprising an inverted sequence of ANTp, comprising D isomers of amino acids, or comprising point mutations in their sequence. It is additionally expected that numerous further ANTp sequence modifications are also possible and will presumably make translocation possible. ANTp peptide variants are also referred to as transport peptides. Further homeoproteins such as, for example, engrailed 1 (En1 ), engrailed 2 (En2), Hoxa 5, Hoxc 8, Hoxb 4 and KNOTTED 1 likewise comprise sequences which can be used as translocation module for the purposes of the present invention. KNOTTED 1 is in fact a plant protein, but is likewise suitable as translocation module in animal cells. These peptides are mentioned only as examples, and numerous further homeoproteins which comprise peptide sequences which may be suitable as translocation module for the purposes of the invention are known. Further previously undisclosed homeoproteins may also comprise sequences suitable as translocation module.

Arginine- or lysine-rich peptides suitable as translocation module

Arginine-rich peptides, frequently derived from RNA and DNA-binding proteins, represent further peptide sequences which can be used as translocation modules for the purposes of the present invention. Examples of such sequences are HIV 1 rev- (34-50), flock house virus coat protein FHV coat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-15), CCMV Gag-(7-25), P22 N (14-30), lambda N (1-22), phi 21 N-(12-29) and PRP6-(129-144) from yeast. It is likewise possible to use for the purposes of the invention polyarginine peptides having 4 to 16 or else having more than 16 arginine residues. In addition to polyarginine peptides, peptides which can also be used as translocation modules are those which, besides arginine, also comprise further amino acids, e.g. the W/R peptide (RRW RRWW RRWW RRW RR) or the R9-Tat peptide in which the 9 central amino acid residues of the total of 11 amino acids of a Tat peptide have been replaced by arginine residues (GRRRRRRRRRQ). It has additionally been possible to show that peptides which for example consist of nine lysine residues also have the ability to act as translocation module for the purposes of the invention. These peptides are mentioned only as examples and numerous further arginine- or lysine-rich peptides are suitable to be used as translocation module for the purposes of the invention. Further arginine- or lysine-rich peptides currently already known or to become known in future are presumably also suitable as translocation module. Sequences comprising guanidino or amidino groups are also suitable as translocation module for the purposes of the present invention.

When the construct introduced into the cells is a peptide, the amino acids may be naturally occurring amino acids or may be modified amino acids, like chemical derivatives of the amino acids, analogs thereof or D-amino acids. The use of modified amino acids stabilizes the peptide construct and decreases the degradation rate thereof.

As used herein, the term "analog" includes any polypeptide having an amino acid residue sequence substantially identical to the sequences used according to the present invention in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the present invention as described herein. Examples of conservative substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophobilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamin acid or another. As used herein, the term "conservative substitution" also includes the use of a chemically dehvatized residue in place of a non-dehvatized residue.

As used herein, the term "chemical derivative" refers to a subject polypeptide having one or more residues chemically dehvatized by reaction of a functional side group. Examples of such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p- toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, choloracetyl groups of formyl groups. Free carboxyl groups may be derivatized to from salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to from O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine. Also included as chemical derivatives are those proteins or peptides, which contain one or more naturally-occurring amino acids derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylsine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

In a further aspect, the present invention relates to pharmaceutical compositions comprising the cell or cell line expressing a peptide construct according to the present invention, i.e. the aAPC and, optionally, a pharmaceutically acceptable carrier. In a different aspect, the present invention relates to pharmaceutical compositions comprising the antigen-specific T-cells obtainable with the methods according to the present invention. Such pharmaceutical compositions comprise a therapeutically effective amount of the cells and, optionally, a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered with a physiologically acceptable carrier to a subject, as described herein. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans, The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium, chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium, carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (18th ed., Mack Publishing Co., Easton, PA (1990)). Such compositions will contain a therapeutically effective amount of the aforementioned cells, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In another preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical composition for use in connection with the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol.histidine, procaine, etc.

"Pharmaceutically or therapeutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.

"Therapeutically- or pharmaceutically-effective amount" as applied to the compositions of the instant invention refers to the amount of composition sufficient to induce a desired biological result. That result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the present invention, the result will typically involve e.g. a decrease in the immunological and/or inflammatory responses to infection or tissue injury. In another embodiment, the results include the lysis of tumour cells or other cells expressing or harbouring foreign invaders.

In addition, the present invention relates to a method for the preparation of a pharmaceutical comprising the steps of generation and/or expansion of antigen- specific T-cells according to the present invention and formulating said cells into a pharmaceutical composition optionally containing pharmaceutically acceptable carriers as described above. The artisan is well aware of methods for the production of a pharmaceutical using the anting-specific T-cells as described herein.

In a preferred embodiment, the pharmaceutical composition is a vaccine comprising the cell or cell line expressing a peptide construct as defined herein. Particularly preferred is that the cell or cell line is of human origin. In one embodiment the vaccine is for the prevention of host versus graft or graft versus host reactions in transplantation. In another embodiment, the vaccines according to the present invention are useful in preventing or treating autoimmune diseases or allergic diseases.

For example, the pharmaceutical composition may be used for inducing anergy of antigen-specific T-cells. This is particularly useful in the treatment of autoimmune diseases and allergic diseases. Autoimmune diseases are characterized by self- reactive T cells. Silencing these autoreactive T-cells can be performed by anergizing these cells specifically using aAPCs which anergize these cells rather than stimulate them.

Further, the Antigen-specific T-cells may be used in adoptive cell transfer therapy, practised as an ex vivo therapy.

As used herein, the term "ex vivo" or "ex vivo therapy" refers to a therapy where biological materials, typically cells, are obtained from a patient or a suitable alternate source, such as, a suitable donor, and are modified, such that the modified cells can be used to treat a disease which will be improved by the therapeutic benefit produced by the modified cells. Treatment includes the re-introduction of the modified biological materials, obtained from either the patient or from the alternate source, into the patient. A benefit of ex vivo therapy is the ability to provide the patient the benefit of the treatment, without exposing the patient to undesired collateral effects from the treatment. For example, high doses of cytokines are often administered to patients with cancer or viral infections to stimulate expansion of the patient's CTLs. However, cytokines often cause the onset of flu like symptoms in the patients. In an ex vivo procedure, cytokines are used to stimulate expansion of the CTLs outside of the patient's body, and the patient is spared the exposure and the consequent side effects of the cytokines. Alternatively under suitable situations, or conditions, where appropriate and where the subject can derive benefit, the subject can be treated concurrently with low level dosage of Y interferon, α interferon and/or IL-2. The expected effect of the interferons is to possibly sensitize the tumour cells to lysis by antigen specific CTL, and the effect of the IL-2 is to possibly enhance antigen specific CTL persistence.

Further, the antigen-specific T-cells obtained by the method according to the present invention are useful for the preparation of a therapeutical composition for the treatment of malignancies, cancer, allergic disorders, infectious disorders including viral, bacterial, fungal and parasite infections, autoimmune disorders and host- versus-graft of graft-versus-host reactions in transplantation as well as in tumour therapy. Preferably, the antigen-specific T-cells are of human origin.

The same is true for the aAPC according to the present invention. These cells can also be used in therapeutical compositions for the treatment of malignancies, cancer, allergic disorders, infectious disorders including viral, bacterial, fungal and parasite disorders, autoimmune disorders and host-versus-graft or graft-versus-host reactions in transplantation, e.g. in tumour therapy.

When the aAPC are used in pharmaceutical compositions, as a vaccine or in the method for the generation of antigen-specific T-cells, the aAPC are preferably treated in advance to prevent further proliferation of said cells while maintaining their stimulating activity. The skilled person is well aware of suitable methods to do so. For example, the aAPC may be irradiated with a sub-lethal dose of radiation. Alternatively, the aAPC are treated with chemical compounds which interfere with the proliferation of the cells without killing them. Other possibilities include a mitomycin-C treatment or UV-irradiation.

In particular, a pre-treatment step of the aAPC might even enhance later stimulation of T cells. That is, aAPC wherein the cell death program, i.e. induction of apoptosis, has been initiated by a pre-treatment step, like a pre-treatment step as described above, are particularly useful for later administration for in vivo or in vitro co- cultivation with cells comprising T-cells.

In a further aspect, the present invention relates to methods for treating an infection in a subject comprising the steps of administering the aAPC according to the present invention or the antigen-specific T-cells obtainable according to the present invention to a subject in need of said treatment.

Of course, the method of treatment may be a vaccination of the subject. In a preferred embodiment, the method is an ex vivo adoptive cell transfer therapy of a subject comprising the steps of: a. providing a cell or cell line being phenotypically negative for a functional MHC class I complex; b. introducing a first construct comprising (i) a peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class I molecule or parts of sadi MHC class I molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a.); c. obtaining a cell or cell line (aAPC) expressing a peptide construct as defined in b) comprising the antigenic structure presented by the MHC class I complex on their surface; d. co-culturing the cell or cell line obtained in c.) with cells comprising T- cells; and e. expanding antigen-specific CD8+ T-cells specific for the antigenic structure of the POI present in the construct as defined in b.) f. inoculating said subject with the antigen-specific CD8+ T-cells .

In a further embodiment, the present invention relates to a method of an ex vivo adoptive cell transfer therapy of a subject comprising the steps of:: a. providing a cell or cell line being phenotypically negative for a functional MHC class Il complex; b. introducing a first construct comprising (i) a peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class Il molecule or parts of sadi MHC class Il molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a.); c. obtaining a cell or cell line (aAPC) expressing a peptide construct as defined in b) comprising the antigenic structure presented by the MHC class I complex on their surface; d. co-culturing the cell or cell line obtained in c.) with cells comprising T- cells; and e. expanding antigen-specific CD4+ T-cells specific for the antigenic structure of the POI present in the construct as defined in b.). f. inoculating said subject with the antigen-specific CD4+T-cells . Preferably, the way of administration of the cells is selected from the group consisting of oral, intradermal, subcutaneous, intravenous, inhalative, intranasal, intranodal, or by injection into or near the tumour in case of cancer

The term "administered" means administration of a therapeutically effective dose of the aforementioned pharmaceutical composition comprising the respective cells to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

The methods are applicable to both human therapy and veterinary applications. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt%. The agents may be administered alone or in combination with other treatments.

The administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intra-arterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, intrabronchial, transdermal^, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.

The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Finally, the present invention relates to a kit for the generation and/or expansion of antigen-specific T-cells comprising a) a cell or cell line obtained in step c) of a method according to the present invention, namely aAPC, or b) a cell or cell line being phenotypically negative for a functional MHC class I complex in case of generating and/or expanding antigen-specific CD8+ T-cells or being phenotypically negative for a functional MHC class Il complex in case of generation and/or expansion of antigen-specific CD4+ T-cells; and a construct as defined in step b) of a method according to the present invention, c) instructions of conducting the method according to any one of claims 1 to 23.

Preferably, the kit for the generation and expansion of antigen-specific T-cells comprises a. a construct as defined above, b. molecules suppressing the expression of functional self MHC class I and/or class Il complex, e.g. siRNA, and c. instructions how the use the kit for generating and expanding antigen-specific

T-cells.

Further, the present relation is directed to the cell or cell line expressing solely the artificial, functional MHC complex as defined herein being otherwise phenotypically negative for a functional (self) MHC complex.

Brief description of the figures

Figure 1 : (A) the design of a construct for transfection of cells being phenotypically negative for a functional MHC class I and/or class Il complex is exemplied. The sequence of the modules is as follows: human β2m signal peptide, POI, spacer 1 , human β2m, spacer 2, HLA- A2.1 , transmembrane region of the HLA-molecule, cytoplsamic region of the HLA-molecule. In addition, the sites for the restriction enzymes Hind III and Not I are shown. (B) A Scheme of the corresponding artificial MHC complex of the construct shown in (A) is provided on the right. On the left, a drawing of a natural membrane anchored MHC class I complex is shown.

Figure 2: Scheme of the ex vivo generation of antigen-specific T-cells according to the present invention. Blood is isolated from e.g. the individual to be treated. The PBMC are isolated via a Ficoll gradient. The obtained PBMC are co-cultured with aAPC obtained according to the present invention, preferably by repeating the co-culturing at least once to obtain a sufficient number of peptide specific T cells. The aAPC are obtained by transfecting MHC negative cells with the construct comprising the POI and MHC molecule which may contain additional spacer. Transfection may be achieved by electroporation or other suitable methods for introducing DNA or fusion proteins into a cell. After culture and selection of appropriate aAPC, these cells are pre-treated before co-cultivation, e.g. by irradiation. The analysis of the amount and the ratio of peptide specific T cells obtained after co-cultivation may be conducted by methods known in the art, e.g. by flow cytometry.

Figure 3: The amount of antigen (peptide)-specific T cells after several cycles of co-cultivation is shown. Antigen-specific T cells are detected by intracellular IFN-g staining and CD8 staining after peptide stimulation. Initially, the amount of antigen-specific T cells was about 0,07%. After 3 weeks the amount of antigen-specific T-cells increased to 22,38 % and after 7 weeks antigen-specific T cells represented about 83,83% of the cells.

Figure 4: Figure 4 is a diagram showing the cytotoxic activity of the generated antigen-specific T cells. Shown is the specific lysis of target cells loaded with the specific antigenic peptide, the antigen-specific cells were raised against said peptide according to the method of the present invention. Shown is specific lysis in percentage at varying amounts of peptide (antigen). In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

Examples

Example 1

Generation of construct for transfection of MHC negative cells

The construct was basically produced as described in Greten et al., J. Immunol. Meth., 2002, 125-135 which is herein incorporated by reference in its entirety. The construct was further modified by introducing a sequence coding for GFP at the 3'- end of the construct, thus, allowing easy detection and selection of transfected cells.

The construct was introduced into the MHC complex negative Daudi cells (ATCC NO. CCL 213) by electroporation with 10 μg of the plasmid containing the construct (Gene Pulser, BioRad) following manufacturers instructions.

Example 2 Selection of stabe transfected cells

After introduction of the construct into Daudi cells by electroporation, permanently transfected cells were grown in media containing 0,5 mg/ml G418 and selected on the basis of an expression of the GFP. Verification of transfection was conducted after several cycles of cultivation to obtain cells or cell lines which are stably transfected. Cultivation was conducted in supplemented RPMI 1640 medium well known in the art.

Thus, aAPC were obtained expressing the construct according to the present invention. Example 3

Co-cultivation of a AP C and PBMC

PBMC were obtained from a healthy HLA-A2 positive volunteer by known methods. That is, PBMC were isolated by Ficoll gradient separation, washed twice with PBS and resuspended in AB-media (see below) in a final concentration of 4x 10E6/ml.

Before co-cultivation, aAPCs obtained in Example 2, were pre-stimulated with Co- stimulatory compounds. In particular, 1 x 10E06 Daudi aAPC were cultured in AB- medium (fully supplemented RPMI 16340 medium wherein AB-sera is supplemented instead of FCS) supplemented with 500 IU/ml IL-4 and 1 x 10E05 NIH3t3 CD40L cells, which express the CD40L molecule on their surface, in 1 ml in a 24 well plate for 24 h.

After 24h, the aAPC were irradiated with 80 Gy, washed in PBS and resuspended to a final concentration of 4 x 10E05/ml. 0,5 ml PBMC and 0,5 ml aAPC were plated into one well of a 48 well plate. The cells were incubated at 37⁰C in humidified air with 5% CO2. After 24 h 10cll/ml human recombinant IL-2 was added and cultivation was continued.

After 3 to 4 day, when the media turned yellow and the cells started to proliferate, cells were splitted and fresh AB media and 5 cU/ml hlL-2 was added. After 7 to 8 day, cells containing T cells were counted and resuspended in AB medium in a final concentration of 4 x 10E06/ml. Freshly pre-stimulated and irradiated aAPC were prepared as described above and the next cycle of co-stimulation was initiated by plating 0,5 ml fresh aAPC and 0,5 ml cells containing T cells received after the first cycle of co-cultivation in a 48 well plate as described above.

After a total of 7 cycles of stimulation, the cells containing T cells, which are now mainly antigen-specific T cells were analysed for peptide-specific IFN-g secretion and CD8 expression by flow cytometry. In figure 3 the results are shown demonstrating an efficient generation and expansion of antigen-specific CD8 positive T cells. Example 4

Determination of antigen-specific cytotoxic activity

The cytotoxic activity of the generated antigen-specific T cells was determined with the well known Cr51 release assay. The effector/target ratio was 20:1 That is, 20 times more antigen-specific T-cells were co-cultured with peptide loaded target cells. The peptide specific lysis of the target cells was determined at varying amounts of peptides. The results are shown in figure 4. Figure 4 clearly demonstrates specific lysis of the target cells by the antigen-specific T cells (effector cells).

Example 5

Production of a construct containing a tumour derived antigen as POI

Another construct useful for the generation and expansion of antigen-specific T cells according to the present invention is a construct containing the well known NY-ESO-

Epitope SLLMWITQC derived from the germ cell antigen NY-ESO which is frequently expressed in cancer. This epitope is an HLA-A2 restricted peptide derived from the cancer testis antigen NY-ESO-1 , which is a tumour antigen known to be expressed in melanoma, esophageal cancer, liver cancer, neuroblastoma, testicular carcinoma, multiple myeloma, sarcoma, ovarian cancer, breast cancer, non.small cell lung cancer, prostate cancer, bladder cancer.

The construct containing the NY-ESO-Epitope is produced by substituting the influenza M1 peptide present in the construct described in Example 1 with a nucleic acid sequence encoding the NY-ESO Epitope.

After transfection of Daudi cells with the above construct and generation of stable transfected aAPC, co-cultivation of pre-stimulated aAPC and PBMC was conducted as described in Example 3. The results obtained demonstrate the generation of antigen-specific T cells being specific for the NY-ESO Epitope.

Claims

Claims:

1. A method for the generation and/or expansion of antigen-specific CD8+ T- cells comprising the steps of: a) providing a cell or cell line being phenotypically negative for a functional

MHC class I complex; b) introducing a first construct comprising (i) a first peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class I molecule or parts of said MHC class I molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a); c) obtaining a cell or cell line expressing a peptide construct as defined in b) comprising the antigenic structure presented by a functional MHC class I complex on the surface; d) co-culturing the cell or cell line obtained in c) with cells comprising T- cells; and e) expanding antigen-specific CD8+ T-cells specific for the antigenic structure of the first POI present in the construct as defined in b).

2. A method for the generation and/or expansion of antigen-specific CD4+ T- cells comprising the step of: a) providing a cell or cell line being phenotypically negative for a functional MHC class Il complex; b) introducing a first construct comprising (i) a first peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class Il molecule or parts of said MHC class Il molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a), c) obtaining a cell or cell line expressing a peptide construct as defined in b) comprising the antigen structure presented by a functional MHC class Il complex on the surface; d) co-culturing the cell or cell line obtained in c) with cells comprising T- cells; and e) expanding antigen-specific CD4+ T-cells specific for the antigenic structure of first POI present in the construct as defined in b).

3. The method according to claim 1 or 2 wherein step d) and e) are repeated at least once.

4. The method according to any one of claims 1 to 3 further comprising the step of f) harvesting antigen-specific T-cells specific for the antigenic structure of the POI present in the construct as defined in step b).

5. The method according to any one of claims 1 to 4 wherein the cell or cell line obtained in step c) are expressing further accessory molecules necessary for the activation and expansion of T-cells.

6. The method according to any one of claims 1 to 5 wherein the co-culturing according to step d) is conducted in a medium containing cytokines and/or T-cells costimulatory molecules.

7. The method according to any one of claims 1 to 6 wherein before co- culturing according to step d) a pre-stimulation step of the cell or cell line obtained in c) is conducted.

8. The method according to any one of claims 1 and 3 to 7 wherein the cell or cell line is phenotypically negative for a functional MHC class Il complex.

9. The method according to any one of claims 2 to 7 wherein the cell or cell line is phenotypically negative for a functional MHC class I molecule.

10. The method according to any one of claims 1 to 9 wherein the cell or cell line is a cell or cell line being defective in expressing functional self MHC molecules.

11. The method according to any one of claims 1 to 9 wherein the cell or cell line is a cell or cell line wherein the expression of the functional cells MHC molecules is suppressed.

12. The method according to any one of claims 1 to 9 wherein the self-MHC complex of a cell or cell line used in step a) do not contain an antigenic structure.

13. The method according to any one of claims 1 to 12 wherein the cell or .cell line and/or the antigen-specific T-cells are a human cell or cell line and/or human antigen-specific T-cells, respectively.

14. The method according to any one of claims 1 and 3 to 13 further comprising the step of da) introducing a second construct comprising (i) a second peptide of interest which is different to the first peptide of interest comprising an antigenic structure, (ii) at least one spacer and (iii) MHC class Il molecule or parts of said MHC class Il molecule, or sequences encoding said constructs comprising (i) to (iii) into the cell or cell line of step a); and c2a) obtaining a cell or cell line expressing a peptide construct comprising the antigenic structure of the second peptide of interest as defined above presented by a functional MHC class Il complex on the surface.

15. A method according to any one of claims 2 to 13 further comprising the steps of d b) introducing a second construct comprising (i) a second peptide of interest comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class I molecule or parts of said MHC class I molecule, or sequences encoding said construct comprising (i) to (iii) into the cell or cell line of step a); and c2b) obtaining a cell or cell line expressing a peptide construct comprising the antigenic structure of the second POI as defined above presented by a functional MHC class I complex on their surface.

16. The method according to any one of the preceding claims wherein the first and/or second construct containing the first and/or second POI introduced into the said cell or cell line is a DNA-construct.

17. The method according to any one of claims 1 to 15 wherein the first and/or second construct comprising the first and/or second POI introducing to said cell or cell line is a peptide molecule.

18. The method according to any one of the preceding claims wherein the first and/or second construct containing at least parts of the MHC class I molecule is composed of a peptide consisting of an antigenic structure, spacer, and at least of parts of the MHC class I molecule, or a nucleic acid sequence encoding the same.

19. The method according to claim 18 wherein the construct is composed of β2 microglobulin signal peptide, spacer, a peptide containing the antigenic structure, spacer, β2 microglobulin, alpha-1 , alpha-2 and alpha-3 chain and a transmembrane and cytoplasmic region of the MHC class I molecule.

20. The method according to any one of the preceding claims wherein the first and/or second construct is composed of a peptide of interest, comprising the antigenic structure, at least one spacer and at least parts of the MHC class Il molecule, or a nucleic acid sequence encoding the same.

21. The method according to any one of the preceding claims wherein the cell or cell line expressing said construct are obtained by a selection step or an enrichment step.

22. The method according to any one of the preceding claims wherein the cells co-cultured with the cell or cell line obtained in step c) are blood mononuclear cells, preferably peripheral blood mononuclear cells or peripheral blood lymphocytes.

23. The method according to any one of the preceding claims wherein the cell or cell line obtained in step c) are treated to prevent proliferation while maintaining or enhancing the stimulating capacity before using the same in step d).

24. A method for the preparation of a pharmaceutical comprising the steps of a method for generation and/or expansion of antigen-specific CD8 and/or

CD4+ T-cells according to any one of claims 1 to 23 and further processing the antigen-specific T-cells obtained into a pharmaceutical optionally containing pharmaceutically acceptable carriers.

25. The use of the pharmaceutical composition obtained according to claim 24 comprising antigen-specific T-cells in adoptive cell transfer therapy.

26. The use of the pharmaceutical composition obtained according to claim 24 comprising antigen-specific T-cells as a therapeutical composition for the treatment of malignancies, cancer, allergic disorders, infectious disorders including viral, bacterial, fungal and parasite infections, autoimmune disorders and host-versus-graft or graft-versus-host reactions in transplantation.

27. The use of the pharmaceutical composition according to claim 26 in tumor therapy.

28. The use according to any one of claims 25 to 27 wherein the antigen- specific T-cells are of human origin.

29. The use of the cell or cell line obtained in step c) of a method as defined in claims 1 to 23 as a vaccine for the treatment of malignancies, cancer, allergic disorders, infectious disorders including viral, bacterial, fungal and parasite disorders, autoimmune disorders and host-versus-graft or graft- versus-host reactions in transplantation.

30. The use of the cell or cell line obtained in step c) of a method as defined in claims 1 to 23 in tumor therapy.

31. A vaccine comprising a cell or cell line expressing a peptide construct obtained in step c) of a method as defined in claim 1 to 23.

32. The vaccine according to claim 31 wherein the cell or cell line is treated to prevent proliferation while maintaining the stimulating capacity.

33. A pharmaceutical composition comprising a cell or cell line expressing a peptide construct as defined in any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

34. A method for treating an infection in a subject comprising: a) obtaining a cell or cell line as defined in step c) of the method according to claims 1 to 23; optionally together with antigen-specific T-cells obtained according to claim 1 to 23; b) inoculating said subject with the cell or cell line according to a) optionally containing antigen-specific T-cells.

35. A method for vaccinating a subject comprising: a) obtaining a cell or cell line as defined in step c) of the method according to claims 1 to 23; and b) inoculating said subject with said cell or cell line.

36. A method for adoptive cell transfer therapy of a subject comprising the steps of: a) providing a cell or cell line being phenotypically negative for a functional

MHC class I complex; b) introducing a first construct comprising (i) a first peptide of interest

(POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class I molecule or parts of said MHC class I molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a); c) obtaining a cell or cell line expressing a peptide construct as defined in b) comprising the antigenic structure presented by a functional MHC class I complex on the surface; d) co-culturing the cell or cell line obtained in c) with cells comprising T- cells; and e) expanding antigen-specific CD8+ T-cells specific for the antigenic structure of the first POI present in the construct as defined in b); and f) inoculating said subject with the antigen-specific T-cells.

37. A method for adoptive cell transfer therapy of a subject comprising the steps of: a) providing a cell or cell line being phenotypically negative for a functional MHC class Il complex; b) introducing a first construct comprising (i) a first peptide of interest (POI) comprising an antigenic structure, (ii) at least one spacer and (iii) an MHC class Il molecule or parts of said MHC class Il molecule, or sequences encoding said first construct comprising (i) to (iii) into the cell or cell line of a), c) obtaining a cell or cell line expressing a peptide construct as defined in b) comprising the antigen structure presented by a functional MHC class Il complex on the surface; d) co-culturing the cell or cell line obtained in c) with cells comprising T- cells; and e) expanding antigen-specific CD4+ T-cells specific for the antigenic structure of first POI present in the construct as defined in b); and f) inoculating said subject with the antigen-specific T-cells.

38. A method according to any one of claims 35 to 37 wherein the way of administration of the cells is selected from the group consisting of oral, intradermal, subcutaneous, intravenous, inhalative, intranasal, intranodal, or by injection into or near the tumor in case of cancer.

39. A kit for the generation and/or expansion of antigen-specific T-cells comprising a) a cell or cell line obtained in step c) of a method as defined in claims 1 to 23 or b) a cell or cell line being phenotypically negative for a functional MHC class I complex in case of generating and/or expanding antigen-specific CD8+ T-cells or being phenotypically negative for a functional MHC class Il complex in case of generation and/or expansion of antigen- specific CD4+ T-cells; and a construct as defined in step b) of a method as defined in claims 1 to 23 and c) instructions of conducting the method according to any one of claims 1 to 23.

40. A kit for the generation and/or expansion of antigen-specific T-cells comprising a) a construct as defined in step b) of a method as defined in claims 1 to 23; b) molecules suppressing the expression of functional self MHC class I and/or class Il complex, e.g. siRNA, and c) instructions how to use the kit for generating and/or expanding antigen- specific T-cells.

41. Cell or cell line obtained in step c) of a method as defined in any one of claims 1 to 23.

42. A cell or cell line according to claim 41 said cell or cell line do not express any additional costimulatory molecules necessary for stimulating and expanding antigen-specific T-cells.

43. The use of a cell or cell line according to claim 42 for inducing anergy of antigen-specific T-cells.