EA041619B1 - METHOD FOR PRODUCING HUMAN ANTIGEN-SPECIFIC T-LYMPHOCYTES - Google Patents

Description

The field to which the invention relates

The present invention contemplates methods for producing human antigen-specific T-lymphocytes. These methods use signals targeted to MHC class II and associated with an antigen or fragment thereof to produce class I and II MHCs presenting proteins encoded by the RNA. Accordingly, the present invention relates to expression vectors containing MHC class II targeting signals and at least one antigen or fragment thereof, and their use for in vitro production of antigen-specific T-lymphocytes. T-cell clones and T-cell receptors (TCRs) specific for tumor antigens or viral antigens are also described.

Prerequisites for the creation of the invention

To combat chronic viral infections and tumors, adoptive T-cell transfer is carried out, which leads to T-cell cytotoxic responses. T cells that are naturally or recombinantly reactive to the patient's cancer are produced in vitro and then transferred back to the patient. Adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) has been successfully used to treat patients with advanced tumors. The main disadvantages of TIL therapy for widespread use in clinics are often the low immunogenicity of tumors, as well as the appearance in the thymus of negative T-cell selection mechanisms that actively remove T-cells with autoantigen specificity. Therefore, in many cases, T cells with high avidity for tumor-specific antigens and T cells with the desired specificity cannot be isolated from the patient's blood or from a tumor removed from the patient. To eliminate these limitations, a method was proposed for the transfer of genetically retargeted peripheral blood lymphocytes (PBLs).

In addition, during cancer and chronic infections, T-lymphocytes lose their function and become depleted.

With the help of gene therapy and using the T cell receptor (TCR), millions of tumor-specific T cells can be rapidly produced from the patient's blood. Gene therapy for the basic TCR paves the way for the development of a universal way to transfer tumor specificity to proliferating and functionally promising subpopulations of T cells. This gene therapy involves the transfer of the TCR genes of certain antigen-specific T cell clones isolated from donors compatible for the human leukocyte antigen HLA into the T lymphocytes of the recipients to confer on them the desired specificity for the antigen.

Adoptive T cell therapy using cytotoxic CD8 ⁺ -T lymphocytes (CTL) is a promising immunotherapy for the treatment of cancer or viral diseases. To enhance clinical responses, complementary transfer of CD4 ⁺ -T helper cells is being considered as a possibility to enhance CD8 ⁺ -CTL responses. It is known that CD4 ⁺ -T-lymphocytes enhance the main functions of CD8 + -CTL, and also have a great influence on the process of generating long-lived CD8 ⁺ -T memory cells. Therefore, rapid and efficient isolation and characterization of tumor antigen-specific CD4+ and CD8 ⁺ T lymphocytes is essential.

Many tumors express MHC class II molecules, and therefore TCRs isolated from CD4 ⁺ T cells are particularly valuable for directly attacking these tumors.

In order to expand the use of adoptive cell therapy (ACT) for the treatment of patients with rapidly growing tumors or chronic viral diseases, the transfer of enriched peptide-specific effector T cells (CD4 ⁺ T-helper cells and cytotoxic T-lymphocytes), which were selected for their ligand specificity to effectively attack viruses or tumor cells while not causing severe effects on normal tissues. These cells must be rapidly expanded to obtain a large number of cells ex vivo and their subsequent use in ACT. Alternatively, the T cell receptors (TCRs) of such ligand-specific T cells can be cloned and expressed as TCR transgenes in activated lymphocytes using either recipient peripheral blood lymphocytes or activated T cell clones with specific specificity that proliferate well and do not capable of attacking normal host tissues.

Therefore, the need to obtain antigen-specific TCRs and develop effective methods for the isolation of these TCRs is obvious.

Purposes and essence of the invention

Therefore, the aim of the present invention is to develop efficient methods for isolating T cells with antigen-specific TCRs. It would be desirable to develop a method for producing CD4-TCR and/or CD8-TCR. Another object of the present invention is to obtain TCRs or their functional parts, such as CDR3 regions. In this case, it is preferable to obtain TCRs that would have a high and/or optimal affinity for tumor antigens.

Therefore, in its first aspect, the present invention relates to a method for the production of human antigen-specific T-lymphocytes, comprising the following steps:

A) expression of at least one fusion protein containing

- 1 041619 at least one antigen or fragment thereof, an endoplasmic reticulum (ER) translocation signal sequence upstream of the N-terminus of the antigen, and a transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence downstream of the C-terminus of the antigen, in antigen-presenting cells; And

B) treating a cell population containing T-lymphocytes with antigen-presenting cells of step A) in vitro to activate antigen-specific T-lymphocytes specific for the antigen expressed by the antigen-presenting cell.

Attaching a targeting sequence, particularly in combination with an ER translocation signal sequence, to the desired antigen or fragment thereof allows for efficient loading of MHC class II complexes as well as MHC class I complexes that normally present cellular proteins. Loading of MHC class I complexes is also achieved in the manner described above. Therefore, this method allows the production of CD4+-TCR as well as CD8+-TCR.

The expression of at least one fusion protein in step A) may be transient expression or stable expression, and preferably transient expression, achieved, for example, by introducing ivt-RNA encoding at least one fusion protein. Expression of ivt-RNA has the advantage that ivt-RNA serving as a quality control can be rapidly produced and does not contain immunogenic protein contaminants.

In specific embodiments of the invention, the hybrid protein may contain at least two antigens or fragments thereof.

In some embodiments, the method may also include the step of enriching activated T lymphocytes. This enrichment step typically includes the following steps:

(a) contacting a cell population containing activated antigen-specific T lymphocytes with at least one binding molecule that specifically binds to at least one marker protein specifically expressed by activated T lymphocytes;

(b) isolating T lymphocytes to which at least one binding molecule is associated.

In preferred embodiments, at least one marker protein specifically expressed by activated T lymphocytes is selected from the group consisting of Ox40, CD137, CD40L, PD-1, IL-2 receptor, interferon y, IL-2, GM-CSF, and TNF-α. Furthermore, in step (a), the cells may also be contacted with a binding molecule that specifically binds to CD4 and/or a binding molecule that specifically binds to CD8.

In specific embodiments of the invention, the selection of activated CD4 T cells includes the following steps:

(a1) contacting the cell population of step B) with an anti-CD40 antibody to block the interaction between CD40-CD40L antigen-presenting cells and antigen-specific T-lymphocytes and to accumulate CD40L on the surface of T-lymphocytes;

(a2) contacting a cell population containing activated antigen-specific T-lymphocytes with an anti-CD40L antibody;

(b) isolation of T-lymphocytes labeled with anti-CD40L antibody and anti-CD4 antibody.

Another embodiment of the invention relates to a method according to any one of the preceding claims, wherein said method also includes the step

C2) identification of antigen-specific T-lymphocytes, including the following steps:

a) incubating expanded cell clones of a cell population containing activated antigen-specific T-lymphocytes (i) with antigen-presenting cells as defined in step A), and (ii) with control antigen-presenting cells;

b) comparing the activation profile during incubation (i) and (ii) for each cell clone;

c) identifying antigen-specific cell clones by comparison b);

where activation in stage (i), but not in stage (ii), indicates that the cell clone is antigen-specific; activation in steps (i) and (ii) indicates that the cell clone is not specific for the antigen; and the absence of activation in (i) and (ii) indicates that the cell clone is not activated.

In step B), antigen-presenting cells are added to the cell population containing T-lymphocytes at least once, or at least twice, or at least three times, or three times. The time interval between repeated additions of antigen-presenting cells can be 7-21 days, 12-16 days, or 13-15 days, or 14 days.

The ER translocation signal sequence is derived from an endosome/lysosome associated protein. The endosome/lysosome associated protein may be selected from the group consisting of LAMP1, LAMP2, DC-LAMP, CD68 or CD1b, and preferably the endosome/lysosome associated protein is LAMP1. Preferably, the ER translocation signal sequence is a human sequence. In specific embodiments, the ER translocation signal sequence comprises SEQ ID NO: 33 or a fragment thereof. In more specific embodiments, the ER translocation signal sequence consists of the following sequence of SEQ ID NO: 33.

The transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence may be derived from LAMP1 or DC-LAMP, and preferably from DC-LAMP. Preferably, the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence is a human domain.

Generally, the antigen presenting cells are selected from dendritic cells, activated B cells, monocytes, macrophages, EBV-transformed lymphoblastoid cell lines, preferably from dendritic cells, and more preferably from monocyte-derived dendritic cells.

Typically, the cell population containing T-lymphocytes is a population of peripheral blood lymphocytes. The cell population containing T-lymphocytes may be a population of undivided peripheral blood lymphocytes. The cell population may be enriched in T lymphocytes, and preferably in CD8+ and/or CD4 ⁺ T lymphocytes, by methods known to those skilled in the art.

In another aspect, the present invention relates to a method for the production of antigen-specific TCR, comprising the steps of the methods described above, as well as the step of isolating TCR from activated antigen-specific lymphocytes obtained by the methods described here.

Description of drawings

In FIG. 1 shows targeted presentation of MHC class II for activation and isolation of antigen-specific CD4 ⁺ T cells.

(a) Schematic representation of various EBNA-3C constructs used to validate functionality and the need for CrossTAg signaling. Shown are ORFs (from ATG to STOP), various components of the CrossTAg signal (LAMP1 and DC-LAMP), an HLA-DR11-restricted EBNA-3C epitope (3H10), and a polyA120 fragment.

(b) CrossTAg signal facilitates efficient cross-presentation of MHC class-II. IFN-γ secretion by EBNA-3C-specific CD4 ⁺ -T cell clone 3H10 was shown after 16-hour co-cultivation with ivt-RNA-transfected APCs (3-day-old monocytic DCs (3d-mDCs) or virus-transformed minilymphoblastoid cell lines Epstein-Barr (EBV) (mLKL)). Values are presented as mean+avg. sq. off for three repetitions.

(c) Expression of the activation marker CD40L is suitable for the isolation of antigen-specific CD4 ⁺ T cells. 3H10 cells labeled with purple CellTrace™ were mixed with PBL at decreasing concentrations and cultured together with autologous donor EBNA-3C-CrossTAg-ivt-RNA-transfected mDCs (APCs). CD40L expression induced by activation on CD4 ⁺ T cells was assessed after 6 hours of co-culture.

In FIG. 2 shows the induction and enrichment of CD4 ⁺ T cells specific for the C/T antigen and isolated from intact PBL.

(a) Activation-induced expression of surface CD40L and CD137 on CD4 ⁺ T cells isolated from PBL primed with C/T antigen. Activation marker expression was measured 6 h after specific re-stimulation of the in vitro PBL culture on days 13 and 27, respectively.

(b) Direct comparison of the proliferative activity of all CD40L ^positlve and CD40L ^negatlve CD4+ T cell lines. On day 28, CD4 ⁺ T cells were separated from the stimulated PBL culture by their expression of CD40L. The total number of cells was counted after FACS separation (day 0) and at the end of day 14 after specific restimulation (day 14). X-fold proliferation compared to proliferation on day 0 is shown.

(c) Comparison of induced cytokine secretion after antigen-specific restimulation. The secretion of IFN-γ by all CD40L ^positIve and CD40L ^negatIve CD4 ⁺ -T cell lines was measured after 16 hours co-culture with mDCs transfected with the CrossTAgantigen-ivt-RNA construct. Values are presented as mean+avg. sq. off for three repetitions.

In FIG. 3 shows a screening for CD4 ⁺ T cell clones specific for the C/T antigen.

(a) Representative screening data for clones derived from PBL culture stimulated with C/T antigen. IFN-γ secretion was assessed after 16 hours of co-cultivation with mDCs transfected with the CrossTAg-antigen-ivt-RNA construct (mixture of 4 antigens). Bars correspond to individual values.

(b) Confirmation of the specificity of the selected T-cell clones for the C/T antigen. The secretion of cytokines (IFN-γ and GM-CSF) was assessed after co-cultivation with ivt-RNA transfected mLAL (mixture of 4 antigens). Data are presented as individual values.

(c) Confirmation of CD4 co-receptor expression. Representative clones are shown.

In FIG. 4 shows the antigen specificity score.

Representative data for CD4 ⁺ T cell clones found to have

- 3 041619 specificity to one C/T antigen. The secretion of IFN-γ was determined after co-cultivation with ivt-RNA-transfected mlLKL. Values are presented as mean+avg. sq. off for duplicates or individual experimental data shown (*).

In FIG. 5 shows the need for recognition of the CrossTAg signal and protein.

(a) The need to use a CrossTAg signal to activate a CD4 ⁺ T cell clone based on ivt-RNA.

(b) Recognition of physiologically processed exogenous recombinant proteins.

The stimulatory ability of APCs transfected with CrossTAg-ivt-RNA (mLCL) antigen was directly compared with mLALs transfected with antigen-ivt-RNA without CrossTAg signal (a) or with recombinant protein-loaded mLALs (b), and such comparison was carried out according to secretion of IFN-γ isolated CD4 ⁺ T-cell clones carrying unique TCR. Mock transfected APCs and T cells alone served as controls. Values are presented as mean+avg. sq. off for three repetitions.

In FIG. 6 shows the definition of core sequences of an MHC class II epitope.

Peptide fragments detected by direct MHC class II epitope identification (DEPI) (boxed with dotted lines) were verified using short CrossTAg-ivt-RNA constructs. GAGE-1-TCR-2 transgenic 3H10 T cells (a) or XAGE-1-TCR-1- and -TCR-2-T cells (b) were co-cultured with APCs transfected with either an overlapping CrossTAg-ivt-RNA short construct (shown in black bars) or full-length antigen-CrossTAg-ivt-RNA (mLKL) construct. IFN-γ secretion was assessed after 16 hours of co-cultivation, and this figure shows the recognizable epitope core sequences (boxed with a solid line). Co-cultures of mock-transfected APCs or T cells alone served as controls. Values are presented as mean+avg. sq. off for three repetitions.

In FIG. 7 shows transgenic expression of CD4 ⁺ T cell receptors specific for the C/T antigen.

3H10 T cells were transfected with ivt-RNA encoding the corresponding TCR-α and -β chains of CD4+ T cell clones GAGE-1-TCR-1, XAGE-1-TCR-1 or -TCR-2. 3H10 TCR transgenic cells were cultured together with antigen-loaded APCs (mLCL) and IFN-γ secretion was detected after 16 hours of co-culture. Co-cultures containing mock transfected APCs and T cells alone served as controls. Values are presented as mean+avg. sq. off for three repetitions.

In FIG. 8 shows how the CrossTAg signal facilitates class II MHC loading via internal [endogenous] cell presentation pathways.

HLA-DRB1*11:01-positive and HLA-DRB1*11:01-negative DCs were transfected with EBNA-3CCrossTAg-ivt-RNA. Transfected and non-transfected DCs were co-incubated in all possible combinations and after 12 h were cultured together with the EBNA-3C-specific CD4 ⁺ -T cell clone 3H10. The secretion of IFN-γ by 3H10 cells was assessed after 16 hours of co-culture with DC. Values are presented as mean+avg. sq. off for three repetitions.

In FIG. 9 shows the characterization of 3-day mDCs for PBL priming.

(a) Surface marker expression was detected by staining with monoclonal antibodies (open curves) and control antibodies of the respective isotypes (gray hatched curves).

(b) Antigenic mRNA levels in ivt-RNA-transfected mDCs are shown. An x-fold increase in copy number of antigenic mRNA compared to non-transfected mDCs is shown. The number of copies of the antigenic mRNA was estimated using qRT-PCR using antigen-specific primers.

In FIG. 10 shows a discrimination strategy for sorting C/T antigen-specific CD4 ⁺ -T cells based on CD40L.

Lymphocytes were selected for their forward scatter (FSC; FSC-A: forward scatter area; FSC-H: forward scatter height) and side scatter (SSC-A). DAPI was used to exclude dead cells. CD4-positive cells of the monocellular fraction (FSC-A/FSC-H) were sorted by the level of CD40L expression and presented as common CD40L ^p ° ^sitlve and CD40L ^negatlve -CD4 ⁺ -T cell lines. In addition, CD40L ^posltlve -CD4 ⁺ -T cells were distributed in 96-well plates on a single cell basis.

In FIG. 11 shows alternative ER translocation signal sequences.

(a) Schematic representation of the vector constructs used to produce ivt-RNA. The target antigen of the characterized CD4 ⁺ T cell clone 3H10 (EBNA-3C-specific, HLA-DRB1*11:01-restricted) was cloned into the pGEM vector system containing combinations of ER translocation signals for human LAMP1, LAMP2, DC-LAMP , CD68 or CD1b (from 5' to antigenic sequence) and endosomal/lysosomal targeting sequence of human

- 4 041619 human DC-LAMP (from 3' to antigenic sequence), human DC-LAMP endosomal/lysosomal targeting sequence alone or without translocation and targeting sequences. In addition, the amino acid sequences of the signal peptides used (translocation signals in the ER) are shown, indicating the presence of the predicted peptidase cleavage sites.

(b) Cells of the CD4 ⁺ -T cell clone 3H10 were cultured together with individual ivt-RNA transfected APCs (mLKL). Cultures together with mock-transfected APCs served as controls. IFN-γ secretion was detected using a standard IFN-γ ELISA assay 16 hours after the start of co-cultivation. Values are presented as mean+avg. sq. off for three repetitions.

In FIG. 12 shows the simultaneous presentation of MHC class II and MHC class I using a CrossTAg-targeted signal.

(a) Schematic representation of the vector constructs used to produce ivt-RNA. Target antigens of the characterized CD4 ⁺ T-cell clone 3H10 (EBNA-3C-specific, HLADRB1*11:01-restricted) and the characterized CD8 ⁺ T-cell clone IVSB (tyrosinase-specific, HLA-A2*01:01- restricted) were cloned into the pGEM vector system in the presence or absence of sequences targeting CrossTAg. EBNA-3C was cloned as a 1 kb fragment (a.k. 421-780) of the full length gene sequence and contained the epitope for clone 3H10. Minigenes containing epitopes of clone 3H10 (EBNA-3C, a.k. 628-641, WRMFMRERQLPQS; SEQ ID NO: 36) and clone IVSB (tyrosinase, a .k.369-377, YMDGTMSQV; SEQ ID NO: 37), which were sequentially cloned into the backbone of the pGEM-CrossTAg vector. To facilitate stabilization of transcribed ivt-RNA molecules in the cytoplasm, all pGEM vector constructs contained a poly-A tail comprising 120 adenine base pairs (poly-A120) from the 3' end to the open reading frame (ORF).

(b) Separate fractions of mature DCs (mDCs) from an HLA-A2*01:01, HLADRB1*11:01 positive donor were transfected with the individual ivt-RNA constructs listed in (a). Seven hours after transfection, 1x105 cells of individual populations of mDC were cultured together with 1x105 cells of CD4 ⁺ T cell clone 3H10 or CD8 ⁺ T cell clone IVSB (1:1 ratio). Co-cultures together with mock-transfected mDCs (H ₂ O) or T cells alone served as controls. IFN-γ secretion was detected using a standard IFN-γ ELISA assay 16 hours after the start of co-cultivation. Values are presented as mean+avg. sq. off for three repetitions.

Detailed description of the invention

Before a detailed description of the invention, presented with reference to some preferred options for its implementation, the following general definitions.

The present invention illustrated below can be appropriately practiced in the absence of any element or elements, limitation or limitations not specifically disclosed in this application.

The present invention has been described with specific embodiments and with reference to certain drawings, but the present invention is not limited to these embodiments, but is limited only by the claims.

Used in the present description and the claims, the term containing is not considered to be excluding other elements. In accordance with the present invention, the term consisting of is considered as a preferred version of the term including. If it has been determined that a group includes at least a number of embodiments of the invention, then this should also be understood as that such a group preferably consists of only these embodiments of the invention.

The term expression as used herein refers to a process in which a polypeptide is produced based on the nucleic acid sequence of a gene. Accordingly, the term expressed protein or polypeptide includes, but is not limited to, intracellular, transmembrane, and secreted proteins or polypeptides.

Technical terms are used here in their conventional sense. If some terms have a specific meaning, then the definition of these terms will be given in the context in which these terms are used.

In one of its aspects, the present invention relates to a method for the production of human antigen-specific T-lymphocytes, comprising the following steps:

A) expressing at least one fusion protein comprising at least one antigen or fragment thereof, an endoplasmic reticulum (ER) translocation signal sequence upstream of the N-terminus of the antigen, and

- 5 041619 a transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence behind the C-terminus of the antigen in antigen-presenting cells; And

B) treating a cell population containing T-lymphocytes with antigen-presenting cells of step A) to activate in vitro antigen-specific T-lymphocytes specific for the antigen expressed by the antigen-presenting cell.

A fragment may be an antigen sequence that is specific for that antigen, ie. not present in any other protein or peptide of a mammal, and in particular a human. The fragment may be shorter, for example, at least 5%, at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, than antigen sequence. The fragment may be at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more amino acids long.

In one embodiment, the fusion protein contains at least two antigens or fragments thereof. The fusion protein may contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 antigens or fragments thereof. The fusion protein may contain less than 100, less than 50, less than 40, less than 30, less than 20, less than 10 antigens or fragments thereof.

The term activate antigen-specific T lymphocytes refers to the activation of naive T cells (induced de novo) and to the activation of memory T lymphocytes (reactivation).

Typically, antigen-presenting cells and a cell population containing T-lymphocytes come from the same donor. Also contemplated are the allorestricted constructs described in EP1910521, in which a nucleic acid encoding an MHC molecule is expressed in antigen-presenting cells of a donor that lacks an MHC gene corresponding to said transferred MHC molecule.

Expression of at least one fusion protein in step A) may be transient expression or stable expression. In preferred embodiments of the invention, expression is transient expression, achieved, for example, by introducing an ivt-RNA encoding at least one fusion protein. Expression of ivt-RNA has the advantage that ivt-RNA serving as a quality control can be rapidly produced and does not contain immunogenic protein contaminants.

In vitro treatment of T-lymphocytes, also called priming, produces a number of different activated populations of lymphocytes. Typically, the treatment in step B) is a co-cultivation of antigen-presenting cells with a cell population containing T-lymphocytes. T cells that recognize MHC:epitope complexes in antigen-presenting cells are activated, and some of these cells are specific for complexes of MHC molecules presenting the epitope of the antigen expressed in step A). These T cells of interest must be separated from T cells that recognize MHC molecules, independent of the peptide or MHC molecules that present epitopes not derived from the antigen expressed in step A).

For treatment of antigen-presenting cells in step B), antigen-presenting cells are added to the population containing T-lymphocytes at least once. The first addition of antigen-presenting cells is also referred to as priming. The antigen presenting cells may be added multiple times, such as at least two or at least three times. The second and each subsequent addition of antigen-presenting cells is also called restimulation, since at these stages, already activated T-lymphocytes receive an additional stimulus for further proliferation. In some embodiments, antigen presenting cells are added once. In other embodiments, the antigen-presenting cells are added twice. In another embodiment, the antigen presenting cells are added three times. Other embodiments relate to methods in which antigen-presenting cells are added three or more times. New APCs can be added to T cell cultures every 7-21 days, or every 12-16 days, or every 13-15 days, or every 14 days. One skilled in the art will appreciate that the cells are cultured in fresh culture medium on a regular basis, which contains additional cytokines.

Treatment of a cell population containing T-lymphocytes with antigen-presenting cells in vitro means that the treatment is not carried out in an organism such as a mammal, but in cell culture in vitro. Cell culture conditions are known to those skilled in the art and include the addition of cytokines such as IL-2, IL-4, IL-7 and/or IL-15 and the like, depending on the type of T cells produced (Schendel, D.J. et al., Human CD8+ T lymphocytes, 1997, in: The Immunology Methods Manual (I. Lefkovits, ed.), p. 670-690 Regn, S. et al., 2001, The generation of monospecific and bispecific anti-viral cytotoxic T lymphocytes (CTL) for the prophylaxis of patients receiving an allogeneic bone marrow transplant, Bone Marrow Transplant, 27:53-64; Su, Z. et al., Antigen presenting cells transfected with LMP2a RNA induce CD4+ LMP2a cytotoxic T lymphocytes which kill via a Fas-independent mechanism, Leuk Lymphoma, 43(8):1651-62).

- 6 041619

In some embodiments, the method may also include the step of enriching activated and/or antigen-specific T-lymphocytes. This enrichment step typically includes the following steps:

(a) contacting a cell population containing activated antigen-specific T lymphocytes with at least one binding molecule that specifically binds to a marker protein specifically expressed by activated T lymphocytes or at least one MHC molecule presenting an epitope of the desired antigen;

(b) isolating T lymphocytes to which at least one binding molecule or at least one MHC molecule is associated, presenting an epitope of the desired antigen.

In specific embodiments of the invention, this method may include the step of enriching activated T-lymphocytes. This enrichment step typically includes the following steps:

(a) contacting a cell population containing activated antigen-specific T lymphocytes with at least one binding molecule that specifically binds to a marker protein specifically expressed by activated T lymphocytes;

(b) isolating T lymphocytes to which at least one binding molecule is associated.

In other specific embodiments, the method may also include the step of enriching T-lymphocytes that are specific for the desired antigen. This enrichment step typically includes the following steps:

(a) contacting a cell population containing activated antigen-specific T-lymphocytes with an MHC molecule presenting an epitope of the desired antigen;

(b) isolating T lymphocytes to which at least one MHC molecule is associated presenting an epitope of the desired antigen.

Enrichment of activated T lymphocytes based on marker proteins specifically expressed by activated T lymphocytes makes it possible to enrich a wide range of activated T cells, regardless of restriction and specific epitope.

A binding molecule that specifically binds to a marker protein may be, but is not limited to, an antibody, an antibody derivative, an antibody fragment, or a conjugate of the aforementioned molecules with another molecule. The binding protein may be labeled, for example, to facilitate sorting procedures such as FACS or MACS.

A marker protein specifically expressed by activated T lymphocytes can be any surface protein or secreted protein that is expressed by activated T lymphocytes and is not generally expressed by non-activated T lymphocytes. In preferred embodiments, at least one marker protein specifically expressed by activated T lymphocytes is selected from the group consisting of OX40, CD137, CD40L, PD-1, IL-2 receptor, interferon-γ, IL-2, GM-CSF, and TNF-α. With these markers, activated T-lymphocytes can be enriched regardless of the specific epitope presented by the TCR. This method facilitates the isolation of T cells that recognize all potential immunogenic epitopes of the selected antigen and is, for example, particularly suitable for poorly characterized antigens.

In the enrichment step, selected cells can be pooled into subpopulations or directly isolated as single cell clones. In specific embodiments of the invention, the cells in the first stage of enrichment are combined, and in the subsequent stage of enrichment they are separated into separate clones. Separation into individual clones can be carried out without any restrictions by limited dilution or by automated single cell sorting by FACS or MACS. Preferably, single cell sorting is carried out using FACS.

In specific embodiments of the invention, the selection of activated CD4 T cells includes the following steps:

(a1) contacting antigen-presenting cells expressing at least one fusion protein of step A) with an anti-CD40 antibody to block interaction between CD40-CD40L antigen-presenting cells and antigen-specific T lymphocytes and to accumulate CD40L on the surface of T lymphocytes;

(a2) contacting a cell population containing activated antigen-specific T-lymphocytes with an anti-CD40L antibody;

(b) isolation of T-lymphocytes labeled with anti-CD40L antibody and anti-CD4 antibody.

To select activated T cells with secreted proteins, e.g., cytokines such as interferon-γ, bispecific molecules recognizing T cell surface markers and target cytokines are immobilized with the secreted cytokine on the cell surface, and then they can be detected with using a labeled detection antibody as described by Becker et al. (Becker, C. et al., 2001, Adoptive tumor therapy with T lymphocytes enriched through an IFN capture assay, Nature Med., 7(10):1159-1162).

- 7 041619

In addition, in step (a), the cells may be further contacted with a binding molecule that specifically binds to CD4 and/or a binding molecule that specifically binds to CD8.

Alternatively, enrichment can be performed using MHC molecules presenting the epitope of the antigen of interest (ie, the antigen exogenously expressed by the antigen-presenting cells in step A). MHC molecules can be labeled, for example, to facilitate sorting procedures such as FACS or MACS. The low affinity interaction between the TCR and the corresponding peptide:MHC complexes can be prevented by assembling soluble peptide:MHC multimers as described by Wilde et al. functional avidity, Blood, 114(10):21312139; 2009), such as, but not limited to, dimers, trimers, tetramers, pentamers, hexamers, heptamers, or octamers. In addition, so-called peptide:MHC streptamers can be used, which reversibly bind to TCRs, allowing high-affinity TCRs to be isolated without the risk of inducing functional modifications or activation-induced cell death (Knabel et al. (2002), Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer, Nature Medicine, 8(6), 631-7). The reversible properties of T-cell interaction with the streptamer are based on a modified form of streptavidin (streptactin) that acts as the backbone of the streptamer.

This approach allows targeted enrichment of epitope-specific TCRs with a specific restriction.

Isolation of activated and/or antigen-specific T-lymphocytes can be carried out using fluorescence activated cell sorting (FACS) as well as magnetic activated cell sorting (MACS). For FACS, a binding molecule that specifically binds to a marker protein or to an MHC molecule presenting an epitope of the desired antigen is labeled with a fluorescent dye. FACS is particularly suitable for isolating small amounts of molecules in high purity. For MACS, a binding molecule that specifically binds to a marker protein or to an MHC molecule presenting an epitope of the desired antigen is labeled with a magnetic particle, such as a magnetic sphere. MACS is particularly suitable for quick sorting of all crops.

In addition, in step (a), the cells may be further contacted with a binding molecule that specifically binds to CD4 for further enrichment and/or with a binding molecule that specifically binds to CD8.

Another embodiment of the invention relates to a method according to any one of the preceding claims, wherein said method also includes the step

C2) identification of antigen-specific T-lymphocytes, including the following steps:

a) incubating expanded cell clones of a cell population containing activated antigen-specific T-lymphocytes (i) with antigen-presenting cells as defined in step A), and (ii) with control antigen-presenting cells;

b) comparing the activation profile during incubation (i) and (ii) for each cell clone;

c) identifying antigen-specific cell clones by comparison with b);

where activation in step (i) but not in step (ii) indicates that the cell clone is antigen specific.

The step of identifying C2) antigen-specific T-lymphocytes can be carried out after the step of enrichment C1) of activated T-lymphocytes or, alternatively, after the step of treating the cell population containing T-lymphocytes B) with antigen-presenting cells without carrying out the step of enrichment C1).

Another embodiment of the invention relates to a method according to any one of the preceding claims, wherein said method also includes the step

C2) identification of antigen-specific T-lymphocytes, including the following stages:

a) incubating at least one cell fraction of the cell population containing activated antigen-specific T-lymphocytes (i) with antigen-presenting cells as determined in step A), and (ii) with control antigen-presenting cells or in the absence of antigen-presenting cells;

b) comparing the activation profile during incubation of (i) and (ii) for at least one cell fraction;

c) identifying the fraction of cells that are antigen specific by comparison b);

where activation in step (i) but not in step (ii) indicates that the cell fraction is antigen specific.

Another embodiment of the invention relates to a method according to any one of the preceding claims, wherein said method also includes the step

- 8 041619

C2) identification of antigen-specific T-lymphocytes, including the following steps:

a) incubating expanded T-cell clones of a cell population containing activated antigen-specific T-lymphocytes (i) with antigen-presenting cells as determined in step A), and (ii) with control antigen-presenting cells or in the absence of antigen-presenting cells;

b) comparing the activation profile during incubation (i) and (ii) for each cell clone;

c) identifying antigen-specific cell clones by comparison b);

where activation in step (i) but not in step (ii) indicates that the cell clone is antigen specific.

The activation profile of T lymphocytes can be determined, for example, by assessing activation-induced cytokine release or antigen-targeted cytolysis capacity.

To assess activation-induced cytokine secretion, T cells can be cultured with antigen-loaded APCs. In this case, various ratios of effector cells to target cells (E:T) can be used. T cells incubated with antigen-presenting control, ie. mock-transfected APCs, or in the absence of stimulating cells, can be used as a negative control. Culture supernatants are evaluated using a standard enzyme-linked immunosorbent assay (ELISA). Examples of markers include, but are not limited to, secretion of granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-y (IFN-γ), IL-2, and TNF-α. The secretion of IFN-γ, IL-2 and TNF-α after interaction with an antigen correlates with improved antitumor function, and therefore it is particularly suitable for assessing antigen-induced cytokine secretion by cytotoxic CD8 ⁺ -T cells. In addition, IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) are well-characterized cytokines for assessing antigen-specific T-cell clones polarized by CD4 ⁺ -T-helper-1 (Th1).

If many antigens are used simultaneously for the primary induction of T cells, then separate APC populations expressing each stimulating antigen can be mixed in equal proportions and used in T cell co-cultures. Thus, initial screening assays performed to assess specificity only predict overall antigenic reactivity for all of the target antigens presented here. To assess specificity to one antigen, subsequent co-cultivation of antigen-reactive T cells with APCs transfected with ivt-RNA of the same species is necessary.

In addition, the cytotoxic activity of individual T clones can be assessed, for example, using chromium release assays. In such assays, target cells are labeled with radioactive chromium and contacted with T cells. After cytolysis, radioactive chromium is released into the supernatant and detected 4 h after the start of co-cultivation. The specific release of chromium is normalized to spontaneous release, assessed by incubating the target cells in the absence of effector cells. Accordingly, a high amount of chromium in the supernatant correlates with excellent cytolytic T-cell activity. Chromium release assays are preferably performed to screen for tumor antigen-specific CD8 ⁺ T cells.

Suitable antigen-presenting cells for use in identifying antigen-specific T-lymphocytes may be, for example, tumor cell lines expressing the desired antigen and the desired MHC molecules; stabilized antigen-presenting cell lines expressing common MHC molecules; or antigen-presenting cells taken from the same donor from which the population containing T-lymphocytes was taken.

One example of a stabilized antigen-presenting cell line is the T2 human lymphoid cell line expressing the frequently occurring HLA-A*02:01 allele and having a defective natural epitope presentation, and such a cell line can be externally loaded with short peptides, e.g. synthetic peptides, i.e. . this cell line can be used to screen for HLA-A*02:01-restricted CD8 ⁺ T lymphocytes. Another example is the human K562 cell line, which does not express HLA class I and II and which can be stably or transiently introduced with any HLA molecule of interest, and therefore such a cell line can serve to screen for CD8 ⁺ -T-lymphocytes and CD4 ⁺ - T-lymphocytes.

Donor-derived antigen-presenting cells may be, for example, isolated monocytes that mature to form dendritic cells. Mature dendritic cells have an optimal ability to activate.

Another example of valuable APCs derived from a donor are B-lymphocytes immortalized by Epstein-Barr virus (EBV), i.e. so-called lymphoblastoid cell lines (LCL). Since LCLs are naturally derived from B cells, such APCs have an advantageous function such as processing and antigen presentation. In addition, LAL express co-stimulatory molecules such as B7.1 (CD80) and B7.2 (CD86) as well as the corresponding adhesion molecules, which enhance the stimulatory activity. Shortened EBV mutant strains (mini-EBV) can be used to produce mini-EBV-transformed B cells (mLCL), which lack most of the lytic cycle genes, resulting in reduced EBV-dependent T-lymphocyte activation (Kempkes, B. et al (1995), Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein Barr virus DNA, J. Virol, 69(1):231-238, 1995 Moosmann et al (2002), B cells immortalized by a mini-Epstein-Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells, Blood, 100(5):1755-1764).

Donor LCL/mLCL can be used to assess the specificity of T cells, in particular when a large number of isolated T cell clones need to be evaluated. Antigen loading of LAL/mLLL can be carried out, for example, by ivt-RNA transfection by retroviral transduction and delivery of an external peptide or protein.

Isolation of antigen-specific T-lymphocytes is based on a comparison of the activation profile of T-lymphocytes incubated (i) with antigen-presenting cells expressing the desired antigen and (ii) with control antigen-presenting cells or in the absence of antigen-presenting cells.

Activation by (i) antigen presenting cells expressing the desired antigen but not (ii) control antigen presenting cells or in the absence of antigen presenting cells indicates that the T cell clone is antigen specific. The term activated compared to non-activated means a reduction of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% of the level of secretion (ie secretion of IFN-γ) by T-lymphocytes incubated with antigen-presenting cells expressing the desired antigen.

The ER translocation signal sequence may be derived from an endosome/lysosome associated protein.

The ER translocation signal sequence used in the disclosed method may be a sequence obtained by sorting from a protein localized in endosomes/lysosomes. As used herein, endosome/lysosome localized proteins are proteins that are localized in the membrane or in the lumen of endosomes and/or lysosomes of a cell.

Examples of proteins localized in endosomes or lysosomes are glycosidases such as alpha-galactosidase A/GLA, endo-beta-N-acetylglucosamine αminidase H/Endo H, alpha-Nacetylgalactosaminidase/NAGA, galactosylceramidase/GALC, alpha-N-acetylglucosaminidase/ NAGLU, glucosylceramidase/GBA, alpha-galactosidase/a-Gal, heparanase/HPSE, alpha-L-fucosidase, heparinase I, tissue alpha-L-fucosidase α/FUCA1, heparinase II, beta-galactosidase-1/GLB1, heparinase III, betaglucuronidase/GUSB, hexosaminidase A/HEXA, beta(1-3)-galactosidase, hyaluronan-lyase, beta(1-4)galactosidase, hyaluronidase 1/HYAL1, chitinase-like protein 1, hyaluronidase 4/HYAL4, protein 2, chitinase-like 3, alpha-L-iduronidase/IDUA, chitinase-3-like protein/ECF-L, chitobiase/CTBS, chitotriosidase/CHIT1, lactase-like protein/LCTL, chondroitin B-lyase/chondroitinase B, lysosomal alpha-glucosidase , chondroitinase ABC, MBD4, chondroitinase AC, NEU-1/sialidase-1, cytosolic beta-glucosidase/GBA3, O-GlcNAcase/OGA, endo-beta- M-acetylglucosaminidase F1/Endo F1, PNGasa F, endo-beta-M-acetylglucosaminidase F3/Endo F3, SPAM1; lysosomal proteases such as AMSH/STAMBP, cathepsin H, cathepsin 3, cathepsin K, cathepsin 6, cathepsin L, cathepsin 7/cathepsin 1, cathepsin O, cathepsin A/lysosomal carboxypeptidase A, cathepsin S, cathepsin B, cathepsin V, cathepsin C/DPPI, cathepsin X/Z/P, cathepsin D, galactosylceramidase/GALC, cathepsin F, egumain/asparaginyl endopeptidase; sulfatases such as arylsulfatase A/ARSA, iduronate-2-sulfatase/IDS, arylsulfatase B/ARSB, N-acetylgalactosamine-6-sulfatase/GALNSv, arylsulfatase G/ARSG, sulfamidase/SGSH, glucosamine(N-acetyl)-6-sulfataseα /GNS, sulfatase-2/SULF2; or other lysosomal proteins such as BAD-LAMP/LAMPS; hyaluronidase 1/HYAL1; CD63; LAMP1/CD107a; CDM6PR; LAMP2/CD107b; clathrin heavy chain 1/CHC17; Rab27a; heavy chain clathrin 2/CHC22; UNC13D, CD68, CD1b or DC-LAMP.

The ER translocation signal sequence is derived from an endosome/lysosome associated protein. The endosome/lysosome associated protein may be LAMP1, LAMP2, DC-LAMP, CD68, CD1b, and preferably LAMP1. The preferred ER translocation signal sequence is a human sequence. The ER translocation signal sequence comprises at least one of SEQ ID NO: 33, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 46. In some embodiments, the signal sequence ER translocations may consist of a sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47. In specific embodiments of the invention, the sequence of the translocation signal in the ER contains the sequence of SEQ ID NO: 33 or a fragment thereof. In more specific embodiments of the invention, the ER translocation signal sequence consists of the following sequence

- 10 041619 SEQ ID NO: 34.

The endosomal/lysosomal targeting sequence may be derived from LAMP1 or DC-LAMP, and preferably from DC-LAMP. Endosomal/lysosomal targeting sequence is usually part of the transmembrane and cytoplasmic domain. Thus, the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence may be derived from LAMP1 or DC-LAMP, and preferably DC-LAMP. Preferably, the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence is a human domain. Typically, the endosomal/lysosomal targeting sequence contains a Y-XX motif followed by a hydrophobic amino acid. Preferably, the endosomal/lysosomal targeting signal sequence is YQRI. The transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence may include the sequence of SEQ ID NO: 54 or a fragment thereof. For example, a transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence may include SEQ ID NO: 35 or a fragment thereof.

The term hydrophobic amino acid is well known to those skilled in the art. Examples of hydrophobic amino acids are Ala, Ile, Leu, Phe, Val, Pro, Gly, Met, Trp, Tyr, Pro, Cys.

Typically, antigen presenting cells are selected from dendritic cells, activated B cells, monocytes, macrophages, EBV-transformed lymphoblastoid cell lines, preferably dendritic cells, and more preferably monocytic dendritic cells.

Antigen-presenting cells may contain different populations of antigen-presenting cells, where each population expresses different antigenic fusion proteins.

In some embodiments, the antigen-presenting cells are mature dendritic cells obtained by a method comprising the following steps:

i) obtaining monocytes;

ii) incubating the monocytes of step (i) with IL-4 and GM-CSF;

iii) incubating the monocytes of step (ii) with IL-4 and GM-CSF in combination with a maturation cocktail.

The maturation cocktail may contain at least one selected from the group consisting of IL-β, TNF-α, IFN-γ, TLR7/8 agonist, PGE2 and TLR3 agonist, or combinations thereof. For example, the TLR7/8 agonist may be R848 and/or the TLR3 agonist may be poly(1:C). The incubation step ii) may be continued for at least 2 days. Incubation step iii) may be continued for at least 12 hours and preferably 24 hours.

Typically, the cell population containing T-lymphocytes is a population of peripheral blood lymphocytes. The cell population containing T-lymphocytes may be a population of undivided peripheral blood lymphocytes. The cell population may be enriched in T lymphocytes, and preferably in CD8+ and/or CD4 ⁺ T lymphocytes.

Another aspect of the invention relates to an expression vector comprising a human endoplasmic reticulum (ER) translocation signal sequence and a human transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence.

The vector may contain a promoter for in vitro transcription of the mRNA. The ER translocation signal sequence is derived from an endosome/lysosome associated protein, eg LAMP1, LAMP2, DC-LAMP, CD68, CD1b, and most preferably LAMP1. Preferably, the ER translocation signal sequence is a human sequence. In specific embodiments, the ER translocation signal sequence comprises SEQ ID NO: 33 or a fragment thereof. In more specific embodiments, the ER translocation signal consists of the following sequence of SEQ ID NO: 34.

The endosomal/lysosomal targeting sequence may be derived from LAMP1 or DC-LAMP, and preferably from DC-LAMP. Endosomal/lysosomal targeting sequence is usually part of the transmembrane and cytoplasmic domain. Thus, the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence may be derived from LAMP1 or DC-LAMP, and preferably DC-LAMP. Preferably, the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence is a human domain. Typically, the endosomal/lysosomal targeting sequence contains a Y-XX motif followed by a hydrophobic amino acid (X means any naturally occurring amino acid). Preferably the endosomal/lysosomal targeting signal is YQRI. The transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence may include the sequence of SEQ ID NO: 54 or a fragment thereof. For example, a transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence may include SEQ ID NO: 35 or a fragment thereof.

- 11 041619

In some embodiments, the expression vector also includes restriction sites located between the ER translocation signal sequence and the human transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence. In other embodiments, the vector also includes at least one antigen or fragment thereof inserted between the human endoplasmic reticulum (ER) translocation signal sequence and the human transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence.

In specific embodiments of the invention, the vector contains a sequence encoding at least two antigens or fragments thereof. The vector may contain a sequence encoding at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 antigens or fragments thereof. The vector may contain a sequence encoding less than 100, less than 50, less than 40, less than 30, less than 20, less than 10 antigens or fragments thereof.

In some embodiments of the invention, the vector contains a nucleic acid sequence encoding the full amino acid sequence of the antigen. Alternatively, the vector contains a fragment of the nucleic acid sequence encoding the amino acid sequence of the antigen. As shown in FIG. 12, antigen-presenting cells that have been introduced with a vector containing fragments of two different antigens can induce the activation of different TCRs specific for the two antigens.

Typically, the antigen is a tumor antigen or a viral antigen. The tumor antigen may be selected from the group consisting of a viral tumor antigen, a tumor-specific antigen, a tumor-associated antigen, and an antigen that causes specific mutations in a patient and is expressed in the patient's tumor cells.

Viral tumor antigens, also referred to as oncogenic viral antigens, are antigens from oncogenic viruses such as oncogenic DNA viruses such as hepatitis B, herpes and papillomaviruses and oncogenic RNA viruses. Tumor-specific antigens cause tumor-associated mutations that are expressed exclusively by tumor cells. The group of tumor associated antigens includes, for example, tissue-specific cancer/testicular antigens or tissue differentiation antigens such as MART-1, tyrosinase or CD20. Preferably the tumor antigen is a tumor associated antigen, and more preferably the tumor associated antigen is a cancer antigen/testicular antigen (C/T antigen). The C/T antigen can be selected from the group consisting of members of the MAGE family, e.g. MAGE-A1, MAGE-A3, MAGE-A4, including but not limited to single point mutation tumor antigens, NY-ESO1, tumor antigen/ testicular antigen 1B, GAGE-1, SSX-4, XAGE-1, BAGE, GAGE, SCP-1, SSX-2, SSX-4, CTZ9, CT10, SAGE and CAGE. Preferably, the C/T antigen may be selected from the group consisting of GAGE-1, SSX-4 and XAGE-1. Preferably, an antigen that causes specific mutations in a patient and is expressed in tumor cells of the patient is not expressed in non-cancer cells of the patient.

In another aspect, the present invention relates to an antigen-presenting cell, and in particular to dendritic cells, expressing at least one fusion protein containing at least one antigen or fragment thereof, an endoplasmic reticulum (ER) translocation signal sequence before the N-terminus of the antigen , and a transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence beyond the C-terminus of the antigen.

In another aspect, the present invention relates to the use of the expression vector described herein for the in vitro production of antigen-specific T-lymphocytes.

In another aspect, the present invention relates to T-lymphocytes for use in a method for the prevention or treatment of cancer, including the introduction of a mammal T-lymphocytes obtained by the methods described here.

In another aspect, the present invention relates to a method for the production of antigen-specific TCR, including the steps of the methods described above, as well as the additional step of isolating TCR from activated antigen-specific lymphocytes obtained by the methods described here.

TCR isolation and subsequent sequence analysis are described, for example, in Steinle et al. (In vivo expansion of HLA-B35 alloreactive T cells sharing homologous T cell receptors: evidence for maintenance of an oligoclonally dominated allospecificity by persistent stimulation with an autologous MHC/peptide complex. The Journal of Experimental Medicine, 181(2), 503-13 ; 1995). Sequence analysis can be performed, for example, using PCR or next generation sequencing methods. Methods for identifying a nucleic acid sequence are well known to those skilled in the art.

The TCR is made up of two distinct protein chains, a and b. The TCR-α chain contains variable regions (V), junction regions (J), and constant regions (C). The TCR-α chain contains variable regions (V), divergent regions (D), junction regions (J), and constant regions (C). Rearranged areas

The V(D)J of both TCR-α and TCR-β chains contain hypervariable regions (CDRs, complementarity determining regions), among which the CDR3 region determines specific epitope recognition.

In one of its aspects, the present invention relates to TCR specific to GAGE-1. In one embodiment, the GAGE-1 specific TCR specifically recognizes at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 48 and SEQ ID NO: 49, or fragments thereof. The fragment may be at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more amino acids long. The TCR can specifically bind to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 48 and SEQ ID NO: 49, or fragments thereof.

A GAGE-1 specific TCR may comprise a TCR-α chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 7, and a TCR-β chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 8.

Some embodiments of the invention relate to a TCR receptor specific for GAGE-1 and comprising a TCR-α chain having the amino acid sequence of SEQ ID NO: 7 and a TCR-β chain having the amino acid sequence of SEQ ID NO: 8.

In addition, the present invention relates to a TCR receptor specific for GAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 7 , and CDR3 having the sequence of SEQ ID NO: 3;

the TCR-β chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 8 and a CDR3 having the sequence of SEQ ID NO: 4.

Some embodiments of the invention relate to a TCR receptor specific for GAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 7, and CDR3 having the sequence of SEQ ID NO: 3;

the TCR-β chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 8, and a CDR3 having the sequence of SEQ ID NO: 4.

Some embodiments of the invention relate to a TCR receptor specific for GAGE-1 and containing a TCR-α chain encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 5, and a TCR-β chain encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the SEQ sequence ID NO: 6.

Some embodiments of the invention relate to a TCR receptor specific for GAGE-1 and containing the TCR-α chain encoded by the nucleotide sequence of SEQ ID NO: 5 and the TCR-β chain encoded by the nucleotide sequence of SEQ ID NO: 6.

In addition, the present invention relates to a TCR receptor specific for GAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 5 and contains the CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 1;

the TCR-β chain is encoded by a nucleotide sequence that is at least 80%

- 13 041619 is identical to the sequence of SEQ ID NO: 6 and contains the CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 2.

Some embodiments of the invention relate to the TCR receptor specific to

GAGE-1 and comprising a TCR-α chain and a TCR-P chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % is identical to the sequence of SEQ ID NO: 5, and contains the CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 1;

the TCR-β chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 6, and contains the CDR3 region encoded by the nucleotide the sequence shown in SEQ ID NO: 2.

In another aspect, the present invention relates to a TCR specific to SSX-4 and containing a TCR-α chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 15, and a TCR-β chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identical SEQ ID NO: 16.

Some embodiments of the invention relate to a TCR receptor specific for SSX-4 and comprising a TCR-α chain having the amino acid sequence of SEQ ID NO: 15 and a TCR-β chain having the amino acid sequence of SEQ ID NO: 16.

In addition, the present invention relates to a TCR receptor specific for SSX-4 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 15 , and CDR3 having the sequence of SEQ ID NO: 11;

the TCR-β chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 16 and a CDR3 having the sequence of SEQ ID NO: 12.

Some embodiments of the invention relate to a TCR receptor specific for SSX-4 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 15, and CDR3 having the sequence of SEQ ID NO: 11;

the TCR-β chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 16, and a CDR3 having the sequence of SEQ ID NO: 12.

Some embodiments of the invention relate to a TCR receptor specific for SSX-4 and containing a TCR-α chain encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 13, and a TCR-β chain encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the SEQ sequence ID NO: 14.

Some embodiments of the invention relate to a TCR receptor specific for SSX-4 and comprising the TCR-α chain encoded by the nucleotide sequence of SEQ ID NO: 13 and the TCR-β chain encoded by the nucleotide sequence of SEQ ID NO: 14.

In addition, the present invention relates to a TCR receptor specific for SSX-4 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 15 and contains the CDR3 region encoded by the nucleotide

- 14 041619 the sequence shown in SEQ ID NO: 9;

the TCR-β chain is encoded by a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14 and contains a CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 10.

Some embodiments of the invention relate to a TCR receptor specific for SSX-4 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 13 and contains a CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 9;

the TCR-β chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 14 and contains the CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 10.

In one of its aspects, the present invention relates to TCR specific to XAGE-1. In one embodiment, the XAGE-1 specific TCR specifically recognizes at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 50 and SEQ ID NO: 51, or fragments thereof. The fragment may be at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more amino acids long. The TCR can specifically bind to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 50 and SEQ ID NO: 51, or fragments thereof.

A TCR specific for XAGE-1 may include a TCR-α chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 23, and a TCR-β chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 24.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain having the amino acid sequence of SEQ ID NO: 23 and a TCR-β chain having the amino acid sequence of SEQ ID NO: 24.

In addition, the present invention relates to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 23 and CDR3 having the sequence of SEQ ID NO: 19;

the TCR-β chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 24 and a CDR3 having the sequence of SEQ ID NO: 20.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 23, and CDR3 having the sequence of SEQ ID NO: 19;

the TCR-β chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 24, and a CDR3 having the sequence of SEQ ID NO: 20.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and containing a TCR-α chain encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 21, and the TCR-β chain encoded by a nucleotide sequence that is at least

- 15 041619 50%, at least 60%, at least 70%, at least 80%, at least

85%, at least 90%, at least 95%, at least 96%, at least

97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 22.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising the TCR-α chain encoded by the nucleotide sequence of SEQ ID NO: 21 and the TCR-β chain encoded by the nucleotide sequence of SEQ ID NO: 22.

In addition, the present invention relates to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 21 , and contains the CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 17;

the TCR-β chain is encoded by a nucleotide sequence that is at least 80% identical to SEQ ID NO: 22 and contains a CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 18.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 21, and contains a CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 17;

the TCR-β chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 22, and contains the CDR3 region encoded by the nucleotide the sequence shown in SEQ ID NO: 18.

In another aspect, the present invention provides a TCR specific for XAGE-1 and comprising a TCR-α chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 31, and a TCR-β chain having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identical SEQ ID NO: 32.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain having the amino acid sequence of SEQ ID NO: 31 and a TCR-β chain having the amino acid sequence of SEQ ID NO: 32.

In addition, the present invention relates to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 31 , and CDR3 having the sequence of SEQ ID NO: 27;

the TCR-β chain contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 32 and a CDR3 having the sequence of SEQ ID NO: 28.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 31, and CDR3 having the sequence of SEQ ID NO: 27;

the TCR-β chain contains an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 32, and a CDR3 having the sequence of SEQ ID NO: 28.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and containing a TCR-α chain encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least by 80%, by

- 16 041619 at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 29, and a TCR-β chain encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO : thirty.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising the TCR-α chain encoded by the nucleotide sequence of SEQ ID NO: 29 and the TCR-β chain encoded by the nucleotide sequence of SEQ ID NO: 30.

In addition, the present invention relates to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 29 , and contains the CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 25;

the TCR-β chain is encoded by a nucleotide sequence that is at least 80% identical to SEQ ID NO: 30 and contains a CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 26.

Some embodiments of the invention relate to a TCR receptor specific for XAGE-1 and comprising a TCR-α chain and a TCR-β chain, where the TCR-α chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 29, and contains a CDR3 region encoded by the nucleotide sequence shown in SEQ ID NO: 25;

the TCR-β chain is encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 30, and contains the CDR3 region encoded by the nucleotide the sequence shown in SEQ ID NO: 26.

The present invention also relates to nucleic acid molecules encoding TCRs as defined above.

Changes can be made to the overall nucleic acid sequence to optimize codons. These changes can lead to conservative substitutions in the expressed amino acid sequence. Such changes can be made in hypervariable and non-hypervariable regions of the amino acid sequence in the TCR chain that do not affect its function. Generally, additions and deletions should not be introduced into the CDR3 region.

The term conservative amino acid substitutions is known to those skilled in the art and preferably means that codons encoding positively charged residues (H, K and R) are replaced by codons encoding positively charged residues; codons encoding negatively charged residues (D and E) are replaced by codons encoding negatively charged residues; codons encoding neutral polar residues (C, G, N, Q, S, T and Y) are replaced by codons encoding neutral polar residues and codons encoding neutral non-polar residues (A, F, I, L, M, P, V, and W) are replaced by codons encoding neutral non-polar residues. These substitutions may occur spontaneously, or they may be introduced by random mutagenesis or site-directed mutagenesis. These substitutions can be made without compromising the essential characteristics of these polypeptides. One of skill in the art can easily routinely screen for amino acid and/or nucleic acid variants encoding those variants to determine, using known methods, whether these substitutions can significantly reduce or impair ligand-binding ability.

Epitope tags are short amino acid fragments against which a specific antibody can be produced, which in some embodiments of the invention allows the specific identification and detection of a labeled protein added to a living organism or cultured cells. Detection of the labeled molecule can be accomplished by any of a variety of methods. Examples of such methods are immunohistochemical analysis, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (western blot) and affinity chromatography. An epitope tag is attached to a known epitope (antigen binding site) on the protein of interest to allow binding of the known and often high affinity antibody and thereby to specifically identify and detect the tagged protein that has been added to a living organism or cultured cells.

In the context of the present invention, the term functional fusion protein containing a TCR-α and/or TCR-β chain means a TCR or a TCR variant, for example, modified by adding

- 17 041619 changes, deletions or substitutions of amino acids that retain at least the basic biological activity. In the case of the α- and/or β-chain of TCR, this term means that both chains retain their ability to form a T-cell receptor (either with the unmodified axis/or β-chain, or with another α- and/or β-chain of the hybrid protein according to the invention), which has a biological function, and in particular the ability to bind to a specific peptide-MHC complex of the specified TCR and/or transmit a functional signal after the specific interaction of the peptide with the MHC.

In specific embodiments, the TCR may be modified to be a functional T-cell receptor (TCR) α- and/or β-chain fusion protein, wherein said epitope tag is 6 to 15 amino acids in length, and preferably 9 up to 11 amino acids. In another embodiment, the TCR may be modified to be a functional T-cell receptor (TCR) α- and/or β-chain fusion protein, wherein said functional T-cell α- and/or β-chain fusion protein receptor (TCR) contains two or more epitope tags located either at a distance from each other or directly in tandem. Variants of the hybrid protein may contain 2, 3, 4, 5 or even more epitope marks, provided that the hybrid protein will retain its biological activity/function (functionality).

Preferred is a functional T-cell receptor (TCR) α- and/or β-chain fusion protein according to the invention, wherein said epitope tag is selected from tags, which are, but are not limited to, CD20 or HER2/neu tags or other standard tags, such as a ty-tag, FLAG-tag, T7-tag, HA (hemagglutinin)-tag, His-tag, S-tag, GST-tag or GFP-tag. The myc, T7, GST, GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed to be highly antigenic (see, for example, US Pat. Nos. 4,703,004 and 4,851,341). myc-Label may be preferred because high-quality reagents are available for its detection. Clearly, epitope tags may have one or more additional functions beyond antibody recognition. The sequences of these labels are described in the literature and are well known to those skilled in the art.

In another aspect, the present invention provides T cells expressing a TCR as defined above.

In addition, the present invention relates to a vector containing one or more nucleic acid sequences encoding a TCR as defined above. The preferred vector is a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector or particle, and/or a gene therapy vector.

A vector is any molecule or composition that is capable of inserting a nucleic acid sequence into a suitable host cell where the encoded polypeptide is synthesized. Typically and preferably, the vector is a nucleic acid that has been designed by recombinant DNA techniques known to those skilled in the art to include the desired nucleic acid sequence (eg, the nucleic acid of the invention). The vector may contain DNA or RNA and/or liposomes. The vector may be a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenovirus vector or particle and/or vector used in gene therapy. The vector may include nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements known to those skilled in the art. The vector is preferably an expression vector which includes a nucleic acid of the invention operably linked to sequences allowing expression of said nucleic acid.

In another aspect, the present invention relates to a cell into which the above nucleic acid sequence encoding a TCR has been introduced, as described above. The vector described above may be introduced into the T cell, containing a nucleic acid sequence encoding the TCR described above, or an in vitro transcribed RNA encoding said TCR. The cell may be a peripheral blood lymphocyte such as a T cell. A method for cloning and exogenous expression of TCR is described, for example, by Engels et al. (Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity, Cancer Cell, 23(4), 516-26, 2013).

In another aspect, the present invention relates to TCR or cells expressing TCR as defined above for use as a drug. Thus, the present application also contemplates a pharmaceutical composition comprising a TCR, or cells expressing a TCR, as described above, and a pharmaceutically acceptable carrier. Some embodiments of the invention relate to TCRs, or cells expressing TCRs as defined above, for use in the treatment of disease caused by cancer cells expressing GAGE-1, SSX-4, XAGE-1, or a mixture thereof. Thus, the present invention also relates to TCRs as defined above for use in the treatment of cancer. Accordingly, the present invention relates to a method for treating a patient in need of adoptive cell therapy, wherein said method comprises administering to said patient a pharmaceutical composition as defined herein. The patient may be suffering from diseases caused by cancer cells expressing GAGE-1, SSX-4, XAGE-1, or a mixture thereof.

These active ingredients according to the invention are preferably used in such a pharmaceutical composition in doses admixed with a suitable carrier or carrier material and suitable for the treatment of the disease or at least the amelioration of its symptoms. Such a composition may include (in addition to the active ingredient and carrier) excipients, salts, buffer, stabilizers, solubilizers, and other materials known to those skilled in the art.

The term pharmaceutically acceptable refers to a non-toxic material that does not adversely affect the efficacy of the biological activity of the active ingredient. The choice of carrier depends on the purpose of its application.

The pharmaceutical composition may contain additional components that enhance the effect of the active ingredient or enhance the effect of the treatment. Such additional components and/or factors may be part of the pharmaceutical composition and are introduced to achieve a synergistic effect or to minimize unwanted or side effects.

Methods for the preparation or preparation and use/administration of the active ingredients of the drug according to the invention are published in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, in the latest edition. Suitable administration is parenteral, eg intramuscular subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. For patient treatment, intravenous injection is preferred.

According to a preferred embodiment of the invention, the pharmaceutical composition is administered by infusion or injection.

An injectable composition is a pharmaceutically acceptable liquid composition containing at least one active ingredient, eg a propagated population of T cells (eg autologous or allogeneic for the patient being treated) expressing TCR. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition may additionally contain a small amount of one or more non-toxic excipients such as emulsifiers, preservatives and pH buffering agents and the like. Such injectable compositions suitable for use with the fusion proteins of the invention are standard and such compositions are well known to those skilled in the art.

Examples

Confirmation of effective cross-presentation of MHC class II by the CrossTAg vector.

For cross-presentation of MHC class II and RNA-encoded proteins, selected antigenic DNA sequences were interacted with a signal targeted to MHC class II (CrossTAg). For this purpose, the ER translocation signal of human lysosome-associated membrane protein 1 (LAMP-1) was fused at the 5' end to the transmembrane and cytoplasmic domains of human DC-LAMP. These two signaling components were separated by unique restriction sites, which allowed the integration of selected antigenic sequences in the same reading frame with CrossTAg. The LAMP-1 signal peptide was used to facilitate co-translational translocation of newly synthesized proteins into the ER. After translocation, a cytoplasmic signal targeted to DC-LAMP (ΥΧΧΦ motif; X means any naturally occurring amino acid; F means any hydrophobic amino acid; SEQ ID NO: 38) should communicate efficient shuttle delivery of the protein to the endosomal/lysosomal compartment.

The inventors integrated an incomplete Epstein-Barr virus (EBNA)-3C core antigen (EBNA)-3C coding sequence encoding a 1 kb fragment containing a known epitope for the EBNA-3C-specific CD4 ⁺ T cell clone 3H10 (described Xiaojun Yu, et al.: Antigen-armed antibodies targeting B lymphoma cells effectively activate antigen-specific CD4+ T-cells, Blood, 2015 Mar 5, 125(10):1601-10.; http://www.iedb.org /assayId/2445148), which is HLADRB1*11:01-restricted. Additional constructs containing only one signal or no signals were used to confirm the need for cross-presentation of each sequence of the desired MHC class II (Fig. 1a). The RNA was then transcribed in vitro from the linearized plasmids and transferred to other APCs expressing the desired HLA-DRB1*11:01 restriction element. Determination of the level of IFN-γ production after specific recognition of the antigen by clone 3H10 made it possible to effectively detect the cross-presentation of MHC class II and endogenously translated and processed protein in experiments on co-cultivation with DC, as well as with mini-EBV-transformed lymphoblastoid cell lines (mLCL) transfected EBNA-3C-CrossTAg-RNA (Fig. 1b). Detection of T-cell activation was only achieved using ivt-RNA containing a translocation signal in the ER

- 19 041619

LAMP-1. Compared to the explicit presentation of MHC class II achieved using

EBNA-3C-CrossTAg-PHK, the LAMP-1 signal peptide, taken alone, had little cross-presentation. These data were confirmed by the authors using one additional CD4 ⁺ T-cell clone specific for the HLA-DRB1*15:01-restricted epitope

EBV antigen BNRF1 (data not shown).

We then used 3H10 T cells to confirm whether the CrossTAg signal could communicate MHC class II presentation via 1) protein secretion and reuptake or 2) intracellular presentation pathways. For this, the authors transfected HLADRB1*11:01-positive or HLA-DRB1*11:01-negative DCs with the EBNA-3C-CrossTAg-PHK construct.

Transfected and non-transfected DCs were co-incubated in all possible combinations (HLA-DRB1*11:01-positive/negative) and then cultured together with 3H10 cells. APC recognition was only detected when HLA-DRB1*11:01-positive DCs were transfected with CrossTAg-PHK antigen. Recognition of non-transfected HLA-DRB1*11:01 positive DCs that could take up and process the antigen secreted by HLA-DRB 1*11:01 negative DCs was not detected (FIG. 8).

CD40L expression induced by CrossTAg antigen.

To develop a rapid method for the selective enrichment of antigen-specific CD4 ⁺ T cells, we evaluated the ability of antigen-CrossTAg-RNA-transfected DCs to induce CD40L expression in responsive CD4 ⁺ T cells. For this, 3H10 T cells were stained with a fluorescent detecting dye and mixed with autologous PBLs to final concentrations of 10, 5, 1, or 0.1% of all cells (Fig. 1c). These different fractions were cultured together with EBNA-3C-CrossTAg-ivtRNA-transfected autologous DCs and stained for CD40L expression. After a 6-hour co-cultivation, the authors detected overt expression of CD40L on EBNA-3C-specific 3H10 cells, but only very slight expression of CD40L was observed on autologous PBLs. Even at the lowest concentration of 3H10 cells (0.1%), subsequent sorting of CD40L-positive CD4 ⁺ T cells revealed a population of 66% 3H10 cells.

Alternative ER translocation signal sequences.

Assessing the level of IFN-γ production after specific antigen recognition by clone 3H10 allowed efficient detection of the cross-presentation of MHC class II and endogenously translated and processed protein in co-cultivation experiments with APCs transfected with various EBNA-3Cs labeled with CrossTAg-RNA and other molecules serving as as an alternative to CrossTAg-RNA (FIG. 11).

Effective presentation of MHC class II and MHC class I.

The authors have shown that the use of the CrossTAg signal facilitates not only the presentation of MHC class II, but also the presentation of MHC class I (Fig. 12).

Efficient presentation of multiple antigens encoded by the same ivt mRNA molecule.

The authors also developed an efficient method for the presentation of several epitopes derived from different antigens from the same ivt-mRNA. As shown in FIG. 12, antigen presenting cells express a construct comprising two epitopes derived from different antigens, and these cells activate the EBNA-3C-specific clone 3H10 as well as the tyrosinase-specific clone IVSB. Therefore, an ivt construct having two different epitopes facilitates the presentation of both epitopes.

PBL activation by DC cells subjected to pulsed RNA treatment.

In accordance with a large-scale approach, the authors have investigated the possibility of using multiple candidate antigens simultaneously for priming CD4 ⁺ -T cells. Thus, CD4 ⁺ T cells present in undivided PBLs were activated using DCs transfected with ivt-RNA molecules encoding four different C/T antigens (GAGE-1, MAGE-A4, SSX-4, and XAGE-1) that have been attached to the CrossTAg signal. DCs were transfected with each individual ivt-RNA construct by electroporation as described in the literature (Javorovic, M. et al. (2008), Inhibitory effect of RNA pool complexity on stimulatory capacity of RNA-pulsed dendritic cells, J. Immunother., 31( 1):52-62). The authors assessed the maturation status of transfected DCs by the levels of expression of costimulatory molecules using flow cytometry. The authors found that DC has a mature phenotype with a high level of expression of costimulatory molecules (CD80, CD83, CD86) and HLA class II (Fig. 9a).

To evaluate transfection efficiency, antigenic cDNA derived from mRNA extracted from transfected DCs was analyzed by quantitative RT-PCR. The data obtained indicated an increase in the copy number of the C/T antigen mRNA from 1.5x105 to 2x10' after electroporation (Fig. 9b). In addition, approximately 68% of DCs expressed green fluorescent protein (eGFP) at a level higher than that achieved using ivt-RNA eGFP, as determined by flow cytometry 12 h after transfection (data not shown) .

- 20 041619

After transfection with the CrossTAg-RNA antigen, four separate DC populations were pooled and used simultaneously to prime unseparated autologous PBLs from a healthy donor. The subsequent breeding procedure included two rounds of APC stimulation. Frozen aliquots of primary DC populations were thawed and used to restimulate the cultures.

Isolation of activated CD4 ⁺ -T cells.

At each 14 day co-culture interval, DC, primed autologous PBLs showed an overall 3-4-fold increase in cell numbers. Changes in CD4:CD8 ratios were assessed at different times to determine the degree of expansion of activated CD4+ T cells. In addition, PBL samples were incubated with antigen-loaded DCs in the presence of CD40 blocking antibody and then analyzed by flow cytometry using mAbs specific for CD4, CD137 and CD40L (Fig. 2a). Throughout the monitoring period, the CD4:CD8 ratio decreased from 1.7 on day 0 to 0.7 on day 27, indicating an increase in CD8 ⁺ T cell proliferation (data not shown). However, in the CD4 ⁺ T cell population, the percentage of CD40L-positive T cells increased from 4.6% on day 13 to over 30% on day 27. In contrast, the number of putative regulatory T cells (Tregs), which have been described as are CD4 ⁺ T cells positive for one CD137 (Schoenbrunn et al., A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg, J. Immunol., 2012, 189(12):5985-94) decreased by approximately 36%.

After the third cycle of stimulation, an enrichment of CD40L-positive CD4 ⁺ -T cells from the PBL culture was observed, and then these cells were directly cloned into 96-well plates and analyzed by FACS (Fig. 10). Cloned CD4 ⁺ -T cells were expanded. Cell surplus after the cloning procedure was defined as the surplus of all CD40L-positive (CD40Lpos) and CD40L-negative (CD40Lneg) CD4 ⁺ T cell lines. This assay indicated successful enrichment of C/T antigen-specific CD4 ⁺ -T lymphocytes, as CD4 ⁺ -T cells selected as CD40Lpos proliferated at a level that was approximately 4 times that of CD40Lneg-T cells within 14 days. after re-stimulation specific to the C/T antigen and released significantly more IFN-γ after culturing with RNA-transfected APCs (Fig. 2b, 2c).

Screening for CD4 ⁺ T-cell clones reactive with C/T antigen.

We tested T cell clones expanded in 96-well plates for reactivity with antigen in IFN-γ release assays 12 days after FACS cloning (FIG. 3a). Co-cultivation using DCs loaded with a mixture of all four CrossTAg-RNA antigens showed that along with non-reactive and non-specific T-cell clones, there are also T-cell clones that react with multiple antigens. Antigen-reactive T cell clones showed no background activation, with some exceptions, when they were co-cultured with mock-transfected DCs.

To confirm these observations, the authors retested individual clones one day later using ivt-RNA-transfected mLAL, which served as an alternative source of APC. IFNy and GM-CSF release assays confirmed the data previously obtained for DC (FIG. 3b). In addition, selected T cell clones were stained for the expression of CD4 and CD8 surface markers and all of these clones were found to express the CD4 co-receptor (FIG. 3c).

Molecular and functional characterization of antigen-specific CD4 ⁺ -T-cell clones.

To analyze the antigen specificity of individual antigen-reactive CD4 ⁺ T-cell clones, the authors cultured individual clones together with individual APC populations transfected with individual antigen-CrossTAg-RNA constructs. Responses were assessed by IFN-γ secretion using a standard ELISA (FIG. 4). The authors have detected antigen-specific CD4 ⁺ -T-cell clones that recognize each of the four C/T antigens used for priming. The T-cell clones showed no background activation by the mock-transfected APCs and no detectable cross-reactivity with other antigens they came into contact with during the priming process.

By analysis of the TCR repertoire, 4 unique T-cell receptor sequences were identified, derived from a variety of isolated clones. MHC restriction assays showed that different T cells recognized epitopes presented by different MHC class II allotypes (data not shown).

The authors demonstrated the important role of the antigen coupled to the CrossTAg signal, as evidenced by the fact that DCs supplied with ivt-RNA without this signal could not induce IFN-γ secretion from isolated CD4 ⁺ T cell clones (Fig. 5a). Activation-induced IFN-γ secretion was observed only when CD4 ⁺ T cell clones were cultured with APCs transfected with the CrossTAg-RNA construct. The exception was that

- 21 041619 a case where a T-cell clone expressed GAGE-1-TCR-2, which also recognized APCs transfected with RNA without corresponding sorting signals, albeit at significantly lower levels.

To confirm the specificity of the isolated CD4 ⁺ -T-cell clones according to the invention to the antigen, the authors loaded the APC with recombinant proteins. CD4 ⁺ T cell clones showed positive secretion of IFN-γ by protein-loaded APCs, and this secretion was comparable to the activation observed when cultured with antigen-CrossTAg-RNA-transfected APCs (Fig. 5b).

Direct identification of an MHC class II epitope.

For these 4 clones, the MHC class II direct epitope mapping (DEPI) method (Milosevic, S. et al. (2006), Identification of major histocompatibility complex class II-restricted antigens and epitopes of the Epstein-Barr virus) was applied to identify epitopes , which were recognized in association with class II MHC molecules. The authors confirmed the recognition of isolated antigenic fragments with short overlapping CrossTAg-RNA constructs, and these fragments were used to further confirm minimal epitope sequences (FIGS. 6a, 6b). As a result, GAGE _- 1-TCR-1 was found to recognize the GAGE-1 _{76_98} epitope presented by HLADRB5*01:01. Interestingly, two XAGE-1-specific CD4 ⁺ T cell clones recognize the same XAGE-1 epitope ₃₇ _ ₄₉ presented by two different class II MHC allotypes (HLADRB1*13:02 and HLA-DRB5*01: 01).

Transgene expression of C/T antigen-specific TCRs.

After analyzing the TCR repertoire, the authors re-engineered the isolated TCR sequences using TCR-containing expression vectors. CD4+-T cells of clone 3H10 (EBNA-3C-specific and HLA-DRB1*11:01-restricted) were transfected with the corresponding TCR-α and β-chains of CD4 ⁺ -T-cell clones GAGE-1-TCR-2, XAGE-1- TCR-1 or TCR-2. TCR-derived 3H10 cells were cultured together with APC loaded with C/T antigen (FIG. 7). Measurement of IFN-γ secretion showed that the specificity of all CD4+ T cell clones was successfully communicated to 3H10 cells without any negative effect on endogenous EBV-specific TCR. Thus, after TCR transfection, 3H10 cells recognized EBNA-3C-CrossTAg-ivt-PHK-transfected APCs, as well as APCs transfected with the corresponding C/T-antigen-CrossTAg-ivt-RNA constructs.

Methods.

genetic constructs.

The pGEM-eGFP-A120 vector was used as the initial construct to obtain the CrossTAg vector (S. Milosevic). This po1yA120 variant of the original pGEM vector imparts higher stability to the transcribed RNA and increases the level of protein expression. This plasmid also contains a unique Agel site at the 5' end of the eGFP cDNA, as well as a unique EcoRI site at the 3' end. Behind the poly-A tail is the Spel site, which provides linearization of the plasmid during the production of ivt-RNA.

Plasmid pGEM-CrossTAg-A120 was cloned by replacing eGFP with a cDNA encoding a signal targeting CrossTAg.

The CrossTAg sequence consists of a translocation signal in the ER of human lysosome-associated membrane protein-1 (LAMP-1, accession number: NP_005552, a.k. 1-28) and is 5' fused to the transmembrane and cytoplasmic domain of DC-LAMP ( registration number: NP 055213, a.k. 376-416). For insertion of the antigen-coding cDNA, individual CrossTAg sequences were separated by an 18 bp spacer containing the NheI, KpnI, and PstI restriction sites without disturbing the LAMP1 open reading frame (ORF). The codon-optimized CrossTAg sequence was actually designed using a cloning computer program and synthesized using GeneArt (Regensburg). The full-length CrossTAg sequence was then excised from plasmid DNA using the restriction sites AgeI (5' end) and EcoRI (3' end) and ligated into the pGEM-A120 MCS digested with both enzymes. For cloning various C/T-CrossTAg antigen constructs (pGEM-GAGE-1-CrossTAg-A120, pGEMMAGE-A4-CrossTAg-A120, pGEM-NY-ESO-1-CrossTAg-A120, pGEM-SSX-4-CrossTAg-A120 , pGEMXAGE-1-CrossTAg-A120), antigenic cDNA was amplified from plasmids by PCR (accession numbers: GAGE-1, U19142; MAGE-A4, NM_001011550; NY-ESO1, AJ003149; SSX-4, U90841; XAGE-1, AF251237) using forward and reverse gene-specific primers and ligated through the NheI and PstI/NotI restriction sites. Primers used for PCR reactions were supplied upon request. All antigenic sequences were inserted into the pGEM-CrossTAg-A120 cleaved CrossTAg signal without disturbing the original ORF. Complementary oligonucleotides (Metabion) were synthesized to confirm CD4 ⁺ T cell epitopes and then annealed. The sticky ends formed after annealing were used to directly ligate these short antigenic sequences into the CrossTAg vector.

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Production of ivt-RNA.

After SpeI linearization, the pGEM plasmids were used as templates for in vitro single molecule production of transcribed (ivt)-RNA using the mMESSAGE mMACHINE T7 kit (Ambion) according to the manufacturer's instructions. To evaluate quality control, the length of the ivt-RNA product was determined by agarose gel electrophoresis. Concentration and purity were determined on a Nanodrop ND-1000 spectrophotometer (Thermo Scientific).

Cell culture.

Monocytic 3 day old mature DCs (mDCs) were generated and transfected as described by Burdek et al. (Journal of Translational Medicine, 2010, 8:90). RNA transfection of mDCs and EBV-transformed lymphoblastoid minicell lines (mLKL) was achieved by electroporation as described by Burdek et al. (Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation, Journal of Translational Medicine, 2010, 8:90).

MLKL were cultured as suspension cultures in the medium for LKL described in the literature (Milosevic, S. et al. (2006), Identification of major histocompatibility complex class Il-restricted antigens and epitopes of the Epstein-Barr virus).

Protein loading into mlLCL was achieved by culturing 2x106 cells in 24-well plates in 2 ml LCL medium for 16 h in the presence of 25 µg of recombinant human protein GAGE-1, MAGE-A4, SSX-4 or XAGE-1 (enriched with bex -cHis-tags after expression in HEK-293T cells). At the end of the incubation, the cells were washed twice with RPMI 1640 medium and cultured together with specific CD4 ⁺ T cell clones.

Quantitative RT-PCR.

Cellular RNA of transfected and non-transfected DCs was isolated and the corresponding cDNA was synthesized using oligo-dT primers using the First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche). Differences in the amounts of antigen templates were determined by quantitative RT-PCR (qRT-PCR) using the LightCycler® 480 SYBR Green I Master Kit (Roche) according to the manufacturer's instructions. All gene-specific primers (α-enolase, GAGE-1, MAGE-A4, SSX-4 and XAGE-1) used for RT-PCR were supplied upon request. The results of measurements were normalized according to the data for α-enolase encoded by the housekeeping gene and analyzed by the AACP method.

T cell surface phenotyping and DC.

Surface markers expressed by T cells and DCs were detected using the following antibodies: PE-conjugated CCR7-specific antibody (3D12) (eBioscience), Hz450-conjugated CD4-specific antibody (RPA-T4), Hz500-conjugated CD8-specific antibody (RPA -T8), FITC-conjugated CD14-specific antibody (M5E2), PE-conjugated CD40-specific antibody (5C3), PE-conjugated CD40L-specific antibody (TRAP1), PE-conjugated CD80-specific antibody (L307.4), FITC-conjugated CD83 -specific antibody (HB15e), FITC-conjugated CD86-specific antibody (2331), APC-conjugated CD137-specific antibody (4B4-1), FITC-conjugated DC-SIGN-specific antibody (DCN46), PE-conjugated HLADR-specific antibody ( G46-6) (all from BD Biosciences). After washing, cells were stained for 30 min at 4°C and propidium iodide (2 μg/ml) was added to remove dead cells. Expression of all surface markers was analyzed by flow cytometry (LSRII, BD). The obtained data were analyzed using the computer program FlowJo 8 (TreeStar). Analysis of CD40L expression on the surface of T cells was performed as described in the literature (Frentsch, M. et al. (2005), Direct access to CD4+ T cells specific for defined antigens according to CD154 expression, Nat. Med., 11(10 ):1118-1124) using 2 μg/ml anti-CD40 antibody (clone G28.5, provided by M. Frentsch, Berlin-Brandenburg Center for Rehabilitation Therapy) and the results were evaluated 6 hours after the start of co-culturing T-cells: APC. De novo PBL priming with RNA-transfected DC 3-day-old healthy donor mDCs were transfected in separate populations with 2 single CrossTAg-RNA molecules encoding C/T antigens GAGE-1, MAGE-A4, SSX-4 and XAGE-1. After electroporation, the transfected mDCs were collected and mixed. The mDCs of this mixture were cultured in a 1:2 ratio with peripheral blood lymphocytes (PBLs) that did not adhere during culturing of PBMCs on a plastic plate during mDC production. Cells were cultured at 37°C in an atmosphere of high humidity. Interleukin-2 (IL-2, 20 U/ml; Chiron Behring) and 5 ng IL-7/ml (Promokine) were added 1 day later and then 2 days later. Mixed mDCs that were not co-cultivated with PBL were cryopreserved and thawed to re-stimulate the de novo induced PBL culture.

Isolation and propagation of antigen-specific CD4 ⁺ -T-cells.

Primed PBLs were co-cultured with CrossTAg-RNA-transfected mDCs (mixture of 4 antigens) at a ratio of 2:1 for 6 h in the presence of an anti-CD40 antibody as described in the literature (Frentsch, M. (2005), Direct access to CD4+ -T cells specific for defined antigens according to

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CD154 expression, Nat. Med., 11(10):1118-1124). After stimulation, cells were stained with aHTu-CD4- and anti-CD40L-specific antibodies (SK3 and TRAP1; BD Biosciences). DAPI was added to remove dead cells. Using FACSAria III (BD Biosciences), live CD40L-positive CD4 ⁺ T cells were distributed as single cells into the wells of round bottom 96 well plates. CD4+ T cell clones in 96-well plates were propagated using antigen-CrossTAg-ivt-RNA-transfected MLKL, feeder cells and IL-2.

Cytokine release assay.

To determine activation-induced cytokine secretion, 5 x 10 ⁴ T cells were cultured together with 1 x 105 ivt-RNA-loaded APCs (DC/mLCL) in 200 µl of T cell culture medium in round bottom 96-well plates at 37° C. in an elevated atmosphere. humidity. T cells with mock-transfected APCs or no stimulator cells were used as negative controls. After 16 hours of co-cultivation, supernatants were collected and evaluated by enzyme-linked immunosorbent assay (ELISA) using the human IFN-γ or GM-CSF OptEIA kit (both from BD Biosciences).

TCR gene transfer based on Ivt-RNA.

The rearrangements and sequences of TCR-α- and TCR-b-strands were determined by PCR using a panel of TCR-Va- and TCR-vp-specific primers as described in the literature (Steinle, A. et al. (1995), In vivo expansion of HLA-B35 alloreactive T cells sharing homologous T cell receptors: evidence for maintenance of an oligoclonally dominated allospecificity by persistent stimulation with an autologous MHC/peptide complex, J. Exp. Med., 181(2):503-513). After replacing the constant regions of both TCR chains with their murine counterparts, the codon-optimized sequences of the TCR-α and TCR-β chains were synthesized and cloned into an expression vector for RNA production. To confirm the specificity of these TCR sequences, cells of the 3H10 T cell clone (HLADRB1*11:01-restricted, EBNA-3C specific EBV) were co-transfected with TCR-α- and TCR-e-ivt-RNA and used in secretion assays. cytokines.

The present invention also includes the following embodiments.

Option 1.

A method for producing human antigen-specific T-lymphocytes, comprising the following steps:

A) expression of at least one fusion protein comprising at least one antigen or fragment thereof, an endoplasmic reticulum (ER) translocation signal sequence upstream of the N-terminus of the antigen, and a transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence for C- the end of the antigen, in antigen-presenting cells; And

B) treating a cell population containing T-lymphocytes with antigen-presenting cells of step (A) in vitro to activate antigen-specific T-lymphocytes specific for the antigen expressed by the antigen-presenting cell.

Option 2.

The method according to option 1, wherein the treatment in step (B) is culturing the antigen-presenting cells together with a cell population containing T-lymphocytes.

Option 3.

The method according to option 1 or 2, wherein the expression in step (A) is transient expression or stable expression, and preferably transient expression.

Option 4.

The method according to option 3, wherein transient expression is carried out by introducing ivt-RNA encoding at least one fusion protein.

Option 5.

A method according to any of the preceding embodiments, wherein said method also includes the step of

C1) enrichment of activated and/or antigen-specific T-lymphocytes.

Option 6.

The method according to option 5, where the enrichment of activated T-lymphocytes includes the following steps:

(a) contacting a cell population containing activated antigen-specific T lymphocytes with at least one binding molecule that specifically binds to a marker protein specifically expressed by activated T lymphocytes, or at least one MHC molecule presenting an epitope of the desired antigen;

(b) isolating T lymphocytes to which at least one binding molecule or at least one MHC molecule is associated, presenting an epitope of the desired antigen.

Option 7.

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The method according to option 6, wherein the binding molecule that specifically binds to the marker protein is an antibody, an antibody derivative, an antibody fragment, or a conjugate of the aforementioned antibodies with another molecule.

Option 8.

The method according to option 6 or 7, wherein at least one marker protein specifically expressed by activated T-lymphocytes is selected from the group consisting of: Ox40, CD137, CD40L, PD-1, IL-2 receptor, interferon-γ, IL- 2, GM-CSF and TNFa.

Option 9.

The method according to variant 8, wherein in step (a) the cells are also contacted with a binding molecule that specifically binds to CD4.

Option 10.

The method according to option 8 or 9, wherein in step (a) the cells are also contacted with a binding molecule that specifically binds to CD8.

Option 11.

The method according to option 6, where the selection of activated CD4-T cells includes the following steps:

(a1) contacting the cell population of step (B) with an anti-CD40 antibody to block the interaction between CD40-CD40L antigen-presenting cells and antigen-specific T lymphocytes and to accumulate CD40L on the surface of T lymphocytes;

(a2) contacting a cell population containing activated antigen-specific T-lymphocytes with an anti-CD40L antibody;

(b) isolation of T-lymphocytes labeled with anti-CD40L antibody and anti-CD4 antibody.

Option 12.

A method according to any of the preceding embodiments, wherein said method also includes the step of:

C2) identification of antigen-specific T-lymphocytes, including the following steps:

a) incubating expanded cell clones of a cell population containing activated antigen-specific T-lymphocytes (i) with antigen-presenting cells as determined in step (A), and (ii) with control antigen-presenting cells or in the absence of antigen-presenting cells;

b) comparing the activation profile during incubation (i) and (ii) for each cell clone;

c) identifying antigen-specific cell clones by comparison (b);

where activation in step (i) but not in step (ii) indicates that the cell clone is antigen specific.

Option 13.

The method according to any of the preceding embodiments, wherein in step (B), antigen-presenting cells are added to a cell population containing T-lymphocytes at least once, optionally at least twice, optionally at least three times, and optionally three times. times.

Option 14.

The method according to embodiment 12, wherein the time interval between repeated additions of antigen presenting cells is 7-21 days, preferably 12-16 days, more preferably 13-15 days, and even more preferably 14 days.

Option 15.

The method according to any of the preceding embodiments, wherein the ER translocation signal sequence is derived from an endosome/lysosome associated protein.

Option 16.

The method according to option 15, wherein the endosome/lysosome associated protein is selected from the group consisting of LAMP1, LAMP2, DC-LAMP, CD68 and CD1b, and preferably LAMP1.

Option 17.

The method according to any of the preceding embodiments, wherein the endosomal/lysosomal targeting sequence is from LAMP1 or DC-LAMP, and preferably from DC-LAMP.

Option 18.

The method according to any of the preceding embodiments, wherein the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence is derived from LAMP1 or DC-LAMP, and preferably from DC-LAMP.

Option 19.

The method according to any of the preceding embodiments, wherein the ER translocation signal sequence is a human sequence.

Option 20.

The method according to any of the preceding embodiments, wherein the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence is a human domain.

Option 21.

The method according to any of the preceding embodiments, wherein the ER translocation signal comprises SEQ ID NO: 33 or a fragment thereof.

Option 22.

The method according to option 20, wherein the ER translocation signal sequence consists of SEQ ID NO: 34.

Option 23.

The method according to any of the preceding embodiments wherein the antigen presenting cells are selected from dendritic cells, activated B cells, monocytes, macrophages, EBV-transformed lymphoblastoid cell lines, preferably from dendritic cells, and more preferably from monocyte-derived dendritic cells.

Option 24.

The method according to any of the preceding embodiments, wherein the antigen-presenting cells comprise different populations of antigen-presenting cells, wherein each population expresses a different antigenic fusion protein.

Option 25.

The method according to any of the preceding embodiments, wherein the antigen presenting cells are mature dendritic cells obtained by a method comprising the following steps:

i) obtaining monocytes;

ii) incubating the monocytes of step (i) with IL-4 and GM-CSF;

iii) incubating the monocytes of step (ii) with IL-4 and GM-CSF in combination with a maturation cocktail.

Option 26.

The method according to embodiment 25 wherein the maturation cocktail comprises a combination of IL-β, TNFa, IFNy, TLR7/8 agonist, PGE2 agonist and TLR3.

Option 27.

The method according to option 25 or 26, wherein the incubation step (ii) lasts at least 2 days.

Option 28.

The method according to embodiments 25-27 wherein the incubation step (iii) lasts at least 12 hours and preferably 24 hours.

Option 29.

The method according to option 28 wherein the TLR7/8 agonist is R848 and wherein the TLR3 agonist is poly(1:C).

Option 30.

The method according to any of the preceding embodiments, wherein the cell population containing T-lymphocytes is a population of peripheral blood lymphocytes.

Option 31.

The method according to any of the preceding embodiments, wherein the cell population containing T-lymphocytes is a population of undivided peripheral blood lymphocytes.

Option 32.

The method according to any of the preceding embodiments, wherein the cell population is enriched in T lymphocytes, and preferably in CD8+ and/or CD4 ⁺ T lymphocytes.

Option 33.

The method according to any of the preceding embodiments, wherein the fusion protein contains at least two antigens or fragments thereof.

Option 34.

T-lymphocyte obtained by the method in accordance with options 1-33.

Option 35.

An expression vector comprising a human endoplasmic reticulum (ER) translocation signal sequence and a human transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence.

Option 36.

Expression vector according to option 1, where the vector contains a promoter for in vitro mRNA transcription.

Option 37.

An expression vector according to option 35 or 36, wherein the ER translocation signal sequence is derived from an endosome/lysosome associated protein.

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Option 38.

An expression vector according to any one of options 35-37, wherein the endosome/lysosome associated protein is selected from the group consisting of LAMP1, LAMP2, DC-LAMP, CD68, CD1b.

Option 39.

An expression vector according to any one of options 35-38, wherein the endosomal/lysosomal targeting sequence is from LAMP1 or DC-LAMP, and preferably from DCLAMP.

Option 40.

An expression vector according to any one of options 35-39, wherein the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence is from LAMP1 or DC-LAMP, and preferably from DC-LAMP.

Option 41.

An expression vector according to any of embodiments 35-40, wherein the ER translocation signal sequence is a human sequence.

Option 42.

An expression vector according to any one of options 35-41, wherein the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence is a human domain.

Option 43.

An expression vector according to any one of options 35-42, wherein the ER translocation signal sequence contains SEQ ID NO: 33 or a fragment thereof.

Option 44.

An expression vector according to any one of options 35-43, wherein the ER translocation signal consists of the sequence of SEQ ID NO: 34.

Option 45.

An expression vector according to any one of options 35-44, wherein the endosomal/lysosomal targeting sequence contains the motif of SEQ ID NO: 38.

Option 46.

An expression vector according to any one of options 35-45, wherein the endosomal/lysosomal targeting sequence is SEQ ID NO: 39.

Option 47.

An expression vector according to any one of options 35-46, wherein the transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence comprises SEQ ID NO: 54 or a fragment thereof, such as SEQ ID NO: 35 or a fragment thereof.

Option 48.

An expression vector according to any of the options 35-47, also containing a restriction site located between the ER translocation signal sequence and the human transmembrane and cytoplasmic domain containing the endosomal/lysosomal targeting sequence.

Option 49.

An expression vector according to any one of embodiments 35-48, wherein the vector also includes at least one antigen or fragment thereof that is inserted between a human endoplasmic reticulum (ER) translocation signal sequence and a human transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting subsequence.

Option 50.

An expression vector according to option 49, wherein the vector includes at least two antigens or fragments thereof that are inserted between a human endoplasmic reticulum (ER) translocation signal sequence and a human transmembrane and cytoplasmic domain containing an endosomal/lysosomal targeting sequence.

Option 51.

An expression vector according to any of embodiments 35-50, wherein the vector includes a nucleic acid sequence encoding the full length amino acid sequence of the antigen.

Option 52.

An expression vector according to any one of options 35-51, wherein the vector includes a fragment of a nucleic acid sequence encoding the amino acid sequence of the antigen.

Option 53.

An expression vector according to option 52, wherein the antigen is a tumor antigen or a viral antigen.

Option 54.

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An expression vector according to option 53, wherein the tumor antigen is selected from the group consisting of a viral tumor antigen, a tumor-specific antigen, a tumor-associated antigen, and an antigen that causes specific mutations in a patient and is expressed in the patient's tumor cells.

Option 55.

An expression vector according to any of embodiments 35-54, wherein the tumor antigen is a tumor associated antigen.

Option 56.

An expression vector according to any one of options 35-55, wherein the tumor associated antigen is a cancer antigen/testicular antigen (C/T antigen).

Option 57.

An expression vector according to any one of embodiments 35-56 wherein the C/T antigen is selected from the group consisting of MAGE-A1, MAGE-A3, MAGE-A4, NY-ESO1, IB tumor/testis antigen, GAGE-1, SSX -4, XAGE-1, BAGE, GAGE, SCP-1, SSX-2, SSX-4, CTZ9, CT10, SAGE and CAGE.

Option 58.

An expression vector according to any one of options 35-57 wherein the C/T antigen is selected from the group consisting of GAGE-1, SSX-4 and XAGE-1.

Option 59.

The use of an expression vector according to any of the options 35-58 for in vitro production of antigen-specific T-lymphocytes.

Option 60.

T lymphocytes for use in a method for preventing or treating cancer comprising administering T lymphocytes to a mammal according to embodiment 34.

Option 61.

A method for producing an antigen-specific TCR, comprising the steps of a method according to any of embodiments 1-33, as well as the step of isolating a TCR from activated antigen-specific lymphocytes.

Option 62.

TCR isolated from lymphocyte according to variant 34.

Option 63.

TCR specific for GAGE-1 and containing a TCR α-chain comprising a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 5, a TCR β-strand comprising a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO : 6.

Option 64.

A TCR specific for GAGE-1 according to variant 63 and containing the α-chain of the TCR, which is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 5, and which contains a CDR 3 region encoded by the nucleotide sequence represented by in SEQ ID NO: 1, a TCR β-strand that is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 6 and that contains a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 2.

Option 65.

TCR specific to SSX-4 and containing a TCR α-chain comprising a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 13, a TCR β-strand comprising a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO : 14.

Option 66.

TCR specific for SSX-4 according to variant 65 and containing the TCR α-strand, which is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 13, and which contains a CDR 3 region encoded by the nucleotide sequence presented in SEQ ID NO: 9, TCR β-strand encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 14 and which contains a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 10.

Option 67.

TCR specific to XAGE-1 and containing the α-chain of TCR, including the CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 21,

-

Claims (4)

A TCR β chain comprising a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 22. Option 68. A TCR specific for XAGE-1 according to variant 66 and containing a TCR α-strand encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 21 and which contains a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 17, a TCR β-strand which is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 22 and which contains a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 18. Option 69. TCR specific to XAGE-1 and containing a TCR α-chain comprising a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 29, a TCR β-chain comprising a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO : thirty. Option 70. TCR specific for XAGE-1 according to variant 68 and containing the TCR α-strand, which is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 29, and which contains a CDR 3 region encoded by the nucleotide sequence presented in SEQ ID NO: 25, a TCR β-strand which is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 30 and which contains a CDR 3 region encoded by the nucleotide sequence shown in SEQ ID NO: 26. Option 71. A TCR according to any of embodiments 62-70 for use in a method for preventing or treating cancer. Option 72. A method for preventing or treating cancer, comprising the step of administering a TCR to a mammal according to any one of embodiments 62-70. CLAIM 1. A method for producing human antigen-specific T-lymphocytes, comprising the following steps: A) expression of at least one fusion protein comprising at least two antigens or fragments thereof, an endoplasmic reticulum (ER) translocation signal sequence upstream of the N-terminus of the N-terminal antigen of said at least two antigens, and a transmembrane and cytoplasmic domain containing endosomal/lysosomal targeting sequence at the C-terminus of the C-terminal antigen of the specified at least two antigens in antigen-presenting cells; And B) treating a cell population containing T-lymphocytes with antigen-presenting cells of step (A) in vitro to activate antigen-specific T-lymphocytes specific for the antigen expressed by the antigen-presenting cell. 2. The method of claim 1, wherein said method further comprises the step of C1) enrichment of activated and/or antigen-specific T-lymphocytes, including the following steps: (a) contacting a cell population containing activated antigen-specific T lymphocytes with a binding molecule that specifically binds to a marker protein specifically expressed by activated T lymphocytes or to MHC molecules presenting an epitope of the desired antigen; (b) isolating T lymphocytes to which a binding molecule or an MHC molecule is bound, presenting an epitope of the desired antigen. 3. The method of claim 2, wherein the marker protein specifically expressed by activated T lymphocytes is selected from the group consisting of Ox40, CD137, CD40L, PD-1, IL-2 receptor, interferon γ, IL-2, GM-CSF and TNF-α. 4. A method according to any one of the preceding claims, wherein said method further comprises the step of C2) identification of antigen-specific T-lymphocytes, including the following steps: a) incubating propagated cell clones of a cell population containing activated antigen-specific T-lymphocytes, -