Engineered immune cell targeting tenascin-c

Abstract

The present invention relates to a vector comprising at least one polynucleotide coding for an antibody complex and for a chimeric antigen receptor ("CAR") and a host cell comprising the vector. Moreover, the invention relates to the medical use of the vector or host cell and a method for engineering a host cell.

Description

[0001] Engineered immune cell

[0002] DESCRIPTION FIELD OF THE INVENTION

[0003] The present invention relates to a vector comprising at least one polynucleotide coding for an antibody complex and for a chimeric antigen receptor (“CAR”) directed against a tumour microenvironment (TME) protein.

[0004] STATE OF THE ART

[0005] The tumour microenvironment (TME) is highly heterogeneous and comprises cellular components such as fibroblasts, endothelial cells, adipocytes, immune and inflammatory cells and a complex non-cellular or extracellular matrix (ECM), which can promote tumour growth and spread into metastases. The extracellular matrix greatly influences an individual’s immune response towards tumour tissue, acting both as a physical barrier for immune cells and as a modulator of the signalling pathways involved in the immune response. Tenascin-C (TNC) is a glycoprotein of the extracellular matrix which is overexpressed in different types of cancer (i.e. glioblastoma, carcinoma, T-cell lymphoma). Recently, the TNC protein has aroused interest for precision medicine (target therapy) in view of its correlation with tumour progression and metastasis. In fact, antibodies directed against various domains of TNC or its isoforms have been generated which effectively permeate into tumour tissue that overexpresses them. Among them, the monoclonal antibody (MAB) ST2146 (tenatumomab), directed against the N-terminal domain “EGF-like repeats” of TNC, common to different isoforms of TNC, has reached the clinical trial phase I for the treatment of hepatocellular carcinoma as a radio conjugate. Various monoclonal antibodies conjugated with radioisotopes or cytokines acting against various TNC domains have also been developed and have demonstrated good tumour localization with respect to normal tissue.

[0006] The extracellular matrix is also one of the main obstacles to the use of CAR-T cells in solid tumours. T cells that express a chimeric antigen receptor (CAR-T cells) are T cells engineered to express a CAR directed against a specific tumour antigen. A limitation in the development of CAR-T therapy for non-haematological solid tumours is the selection of specific and / or overexpressed target molecules (tumour antigens),as well as the poor infiltration and persistence of immune cells in this type of tumours. Recently, it has been demonstrated that not only T cells, but also cytokine-induced killer (CIK) cells can be engineered with a CAR that recognizes CD19 in order to treat patients with acute lymphoblastic leukaemia (B-ALL). CIK cells are CD3+CD56+cells expanded in vitro, characterized by several phenotypic markers of natural killer (NK) cells and a cytotoxic activity independent of the major histocompatibility complex (MHC) against cancer cells.

[0007] Although CAR-T cells have demonstrated effectiveness in blood cancers, it is necessary to develop CAR technology so as to be able to apply it also in solid tumours, favouring permeability and persistence, and reduce the depletion of immune cells expressing this receptor.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention relates to a vector comprising at least one polynucleotide coding for an antibody complex and at least one polynucleotide coding for a chimeric antigen receptor (CAR).

[0010] In a preferred embodiment, the antibody complex is secretable.

[0011] Preferably, said CAR is directed against a protein of the extracellular matrix or of the tumour microenvironment. Preferably, the antibody complex recognizes and binds at least a portion of the human EGFR protein and / or at least a portion of the human CD3 protein.

[0012] Preferably, the CAR recognizes and binds at least a portion of the human TNC protein.

[0013] In one embodiment, the at least one polynucleotide coding for an antibody complex is complementary to a sequence having at least 80% identity with SEQ ID NO: 1, and / or the at least one polynucleotide coding for a CAR is complementary to a sequence having at least 80% identity with SEQ ID NO: 2 or 3.

[0014] Preferably, the at least one polynucleotide coding for an antibody complex is complementary to a sequence having at least 90% identity with SEQ ID NO: 1, and the at least one polynucleotide coding for a CAR is complementary to a sequence having at least 90% identity with SEQ ID NO: 2 or 3.

[0015] In one embodiment, the at least one polynucleotide coding for an antibody complexconsists of SEQ ID NO: 1, and / or the at least one polynucleotide coding for a CAR consists of SEQ ID NO: 2 or 3.

[0016] Preferably, the at least one polynucleotide coding for a CAR further includes at least one sequence coding for a domain selected from the group consisting of:

[0017] an lgG1 CH2-CH3 hinge domain; and / or

[0018] a CD28 transmembrane domain; and / or

[0019] an QX-40 co-stimulatory domain; and / or

[0020] a CD3 T-cell activating domain.

[0021] In one embodiment, the at least one polynucleotide coding for a CAR further includes at least one sequence coding for a domain selected from the group consisting of:

[0022] a CD8a hinge domain; and / or

[0023] a CD8a transmembrane domain; and / or

[0024] a 4-1 BB co-stimulatory domain; and / or

[0025] a CD3 T-cell activating domain.

[0026] Preferably, the antibody complex comprises or consists of a sequence having at least 80%, more preferably 90% identity with SEQ ID NO: 13 and the CAR comprises or consists of a sequence having at least 80%, more preferably 90% identity with SEQ ID NO: 14 or 15.

[0027] A second aspect of the present invention relates to a host cell which expresses an antibody complex and a chimeric antigen receptor (“CAR”). Preferably, the host cell is an immune cell, more preferably selected from the group consisting of a: peripheral blood mononuclear cell (PBMC), cytokine-induced killer (CIK) cell, T cell, and NK cell; preferably the immune cell is a CIK cell. Preferably, the host cell comprises the vector described above.

[0028] A third aspect of the present invention relates to the host cell described above or a composition comprising it, for use as a medicament.

[0029] A fourth aspect of the present invention relates to the host cell described above or a composition comprising it, for use in the treatment or prevention or follow-up of tumours. Preferably, the tumour expresses a larger amount of the tumour antigen which is the target of the antibody complex of the protein.

[0030] Preferably, the tumour expresses a larger amount of the EGFR protein compared to the corresponding healthy tissue.

[0031] A fifth aspect of the present invention relates to a method for engineering a host cellcomprising at least a step (I) of transferring into the host cell at least one vector as described above, preferably in combination with at least one vector encoding a transposase protein. Preferably, the host cell is an immune cell, more preferably selected from the group consisting of: PBMC, CIK cell, T cell, and NK cell.

[0032] BRIEF DESCRIPTION OF THE FIGURES

[0033] Figure 1 shows (A) the schematic structure of two transposon vectors, CAR-TNC4 and CAR-TNC5.

[0034] (B) The PBMCs transfected with the CAR-TNC4 or CAR-TNC5 plasmids and expanded to CIK cells for 21 days. The data show the total nucleated cells obtained starting from 10x106PBMCs.

[0035] (C-D) CAR expression analysed by flow cytometry at the end of CIK cell culture. The percentages of CAR-positive CIK cells (C) and the mean fluorescence intensity (MFI) of the CAR in these cells (D) are shown.

[0036] (E) Examples of flow cytometry histograms of purified or unpurified CARCIK-TNC4 or -5 cells.

[0037] (F-G) Immunophenotype at the end of culture of purified CARCIK-TNC cellular products, including CD4+, CD8+ and CD3+CD56+ populations (F) and effectormemory populations (G), analysed by flow cytometry. The results are the means and standard deviations of 3 experiments using different donors as the starting material. The statistics refers to the comparison between CARCIK-TNC4 and CARCIK-TNC5 with unmodified CIK cells (*p<0.05);

[0038] Figure 2 shows (A-B) the cytotoxicity in vitro of CIK cells, unmodified or modified with the two anti-TNC CAR vectors, measured against cell lines stably transfected with a vector called TNC-TM, bearing the “EGF-like repeat” domain of TNC bound to a transmembrane protein sequence (CEM-TNC-TM and HEK293-TNC-TM), with an effector: target (E:T) ratio of 5:1 for 4 hours (A) or against cancer cell lines naturally expressing TNC (HT-29, MDA-MB-231), with an E:T ratio of 1:1 for 24 hours (B). (C,D) The proliferation capacity in vitro measured with CFSE staining of the CIK effectors, unmodified or modified with CAR-TNC, and stimulation with CEM TNC-TM+ (C) or MDA-MB-231 (D), at different E:T ratios and analysis by flow cytometry. (E,F) The production of IFN-y (E) and IL-2 (F) by CIK cells, unmodified or modified with CAR-TNC, determined after 6 hours of co-culture with the targets CEM-TNC-TM+ or MDA-MB-231 at an E:T ratio of 1:1 and detected by means ofintracytoplasm ic staining of the cytokines. (*P<0.05, **P<0.01). (*P<0.05, **P<0.01, ***P<0.001);

[0039] Figure 3 shows a schematic representation of the various transposon vectors used, expressing the EGFRxCD3 BiTE alone, or placed upstream or downstream of the CAR-TNC5 and separated from the latter by a furin-T2A sequence, which enables cleaving of the polypeptide. All the vectors contain the LIR and RIR sequences recognized by transposase and the promoter pT-MNDU3.

[0040] (B) Expression of the anti-TNC CAR on CIK cells genetically modified with the various constructs specified.

[0041] (C) Cytotoxic activity in vitro of CIK cells, unmodified or modified with the CAR-TNC5, EGFRxCD3 or EGFRxCD3 / CAR-TNC5 constructs, against the cell line positive for TNC and EGFR MDA-MB-231 (N=3);

[0042] Figure 4 shows (A) a treatment scheme; (B) TNC expression and infiltration of CIK cells in tumour tissues were analysed by immunohistochemistry with human anti-CD3 and anti-TNC antibodies;

[0043] (C) survival curves of groups of 8 animals;

[0044] (D) mean weight of the groups of 8 treated animals.

[0045] Figure 5 shows: (A) the treatment scheme for NOD-SCID mice inoculated with MDA-MB-231 tumour cells and treated as schematically shown; (B) tumour growth curves in mice treated with CIK cells modified with CAR-TNC5 alone, EGFRxCD3 alone or with the double EGFR / CD3-CAR-TNC5 construct;

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] A first aspect of the present invention relates to a vector comprising at least one polynucleotide coding for an antibody complex and for at least one chimeric antigen receptor (“CAR”).

[0048] In a preferred embodiment, the antibody complex is a bispecific antibody or a bispecific antibody fragment.

[0049] In a preferred embodiment, the antibody complex comprises or consists of at least one single-chain variable fragment (scFv).

[0050] In a further embodiment, the antibody complex preferably comprises an scFv; even more preferably it comprises or consists of two scFvs able to recognize and bind at least two targets.In a particularly preferred embodiment, the antibody complex comprises two scFvs. Preferably, said two scFVs are linked together by means of a linker, more preferably a linker polypeptide.

[0051] In one embodiment, the antibody fragment is able to recognize and bind two different proteins; in other words, it is a bispecific antibody complex. Preferably, said antibody complex comprises or consists of a fusion protein comprising two single-chain variable fragments (scFvs) deriving from two monoclonal antibodies which recognize two different proteins. The antibody complex thus described is also defined as a “bispecific T cell engager” (BiTE).

[0052] It is general common knowledge that the scFv fragments of an antibody complex and / or a BiTE are linked by a linker peptide, such as, for example, the G4Sx3 (SEQ ID NO. 23) or G4Sx4 (SEQ ID NO. 24) peptide, consisting of a Gly-Gly-Gly-Gly-Ser (SEQ ID NO.

[0053] 25) amino acid sequence repeated 3 or 4 times. The skilled person is capable of adapting the linker according to need.

[0054] Preferably, the vector comprises at least one polynucleotide coding for an antibody complex and at least one polynucleotide coding for a CAR.

[0055] In a preferred embodiment, the antibody complex recognizes and binds at least a portion of a protein expressed by a tumour, preferably of a protein expressed on the membrane of a tumour cell. Preferably, the protein expressed by a tumour is a tumour membrane antigen.

[0056] In one embodiment, said protein expressed by the tumour is selected from the group consisting of: EGFR, HER2, HER3, GD2, CCR4, CD33, PD-L1, PD-1, GPC3, PSMA, CD19, CD20, CD22, CD37, CD38, and BCMA.

[0057] In one embodiment, said protein expressed by the tumour is the human epidermal growth factor receptor (EGFR).

[0058] In a preferred embodiment, the antibody complex recognizes and binds at least a portion of the human EGFR protein.

[0059] In another embodiment, the antibody complex recognizes and binds at least a portion of the EGFR protein of a species other than the human species.

[0060] In a preferred embodiment, the antibody complex recognizes and binds at least a portion of a transmembrane protein expressed by T lymphocytes. Preferably, said transmembrane protein is the human CD3 protein.In another embodiment, the antibody complex recognizes and binds at least a portion of the protein CD3 of a species other than the human species.

[0061] In a preferred embodiment, the antibody complex recognizes and binds at least a portion of protein expressed by a tumour and at least a portion of the transmembrane protein expressed by the T lymphocytes.

[0062] In one embodiment, the at least one polynucleotide coding for the antibody complex is complementary to a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 1.

[0063] In one embodiment, the at least one polynucleotide coding for the antibody complex is complementary to a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 1.

[0064] In one embodiment, the antibody complex comprises or consists of a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 13.

[0065] In one embodiment, the antibody complex comprises or consists of a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 13.

[0066] In a particularly preferred embodiment, the antibody complex recognizes and binds at least a portion of the human EGFR protein and at least a portion of the human CD3 protein.

[0067] Preferably, the antibody complex is a single-chain variable fragment (“anti-EGFR scFv”). Preferably, the antibody complex, preferably the anti-EGFR scFv, derives from the variable fragments of a monoclonal antibody; even more preferably, said monoclonal antibody is cetuximab.

[0068] In a further embodiment, the polynucleotide coding for the antibody complex is complementary to a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 4.

[0069] In another embodiment, the polynucleotide coding for the antibody complex is complementary to a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 4.

[0070] In one embodiment, the antibody complex comprises or consists of a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 16. Preferably, the antibody complex comprises or consists of a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 16.In a preferred embodiment, the antibody complex is a single-chain variable fragment (“anti-CD3 scFv”). Preferably, the anti-CD3 scFv derives from the variable fragments of a monoclonal antibody; even more preferably, said monoclonal antibody is blinatumomab.

[0071] In a further embodiment, the polynucleotide coding for the antibody complex is complementary to a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 5.

[0072] In another embodiment, the polynucleotide coding for the antibody complex is complementary to a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 5.

[0073] In a further embodiment, the antibody complex comprises or consists of a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 17.

[0074] In another embodiment, the antibody complex comprises or consists of a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 17.

[0075] In a particularly preferred embodiment, the at least one polynucleotide coding for an antibody complex comprises a fragment which codes for an scFv that recognizes and binds the human EGFR protein and a fragment which codes for an scFv that recognizes and binds the human CD3 protein.

[0076] In a particularly preferred embodiment, the antibody complex is secretable.

[0077] Preferably, the vector further includes a signal sequence, i.e. a sequence coding for a signal peptide suitable for enabling the secretion of the antibody complex from a cell. In one embodiment, the at least one polynucleotide coding for the antibody complex further includes a signal sequence. Preferably, said signal peptide is selected from: signal peptide of human serum albumin (HSA), signal peptide of the tissue-type plasminogen activator (tPA), signal peptide of the immunoglobulin K (IgK) chain, a signal peptide of an immunoglobulin heavy (IgH) chain; preferably, the signal peptide is a signal peptide of human serum albumin (HSA).

[0078] In a preferred embodiment, the signal peptide is complementary to a sequence that is 90%, preferably 95%, even more preferably 100% identical with SEQ ID NO: 10.

[0079] In a preferred embodiment, the signal peptide comprises or consists of a sequence that is 90% identical, preferably 95%, even more preferably 100% identical with SEQ ID NO: 22.In a preferred embodiment, the CAR recognizes and binds at least a portion of a protein of the extracellular matrix. Preferably, the extracellular matrix protein is a protein that is overexpressed in a tumour compared to its expression in a healthy tissue, i.e. not affected by the tumour.

[0080] Preferably, said extracellular matrix protein is human tenascin-C (TNC) or an isoform thereof.

[0081] In another embodiment, the CAR recognizes and binds at least a portion of the TNC of a species other than the human species.

[0082] CAR, i.e. chimeric antigen receptor, means an engineered receptor which, when expressed by cells of the immune system, for example by T cells (“CAR-T cells”), confers on them the ability to recognize a specific antigen, reprogramming their effector capacity towards specific targets. A CAR has an extracellular domain which consists of an scFv able to recognize and bind a specific antigen. This specific antigen can be, for example, expressed on the membrane of the cells to be targeted, or they can be present in the tumour microenvironment (TME), as in the context of the present invention, in which the CAR preferably recognizes TNC. This makes it possible to direct the effector and cytotoxic activity of the cells that express it where the antigen is present.

[0083] In a preferred embodiment, the CAR comprises or consists of a single-chain variable fragment (“anti-TNC scFv”). Preferably, the anti-TNC scFv derives from the variable fragments of a monoclonal antibody; even more preferably, said monoclonal antibody is tenatumomab.

[0084] In a preferred embodiment, the anti-TNC scFv comprises a G4Sx3 or G4Sx4 linker peptide. It is general common knowledge that the scFv fragments constituting the extracellular portion of a CAR are linked by a linker peptide, such as, for example, the G4Sx3 or G4Sx4 peptide, consisting of a Gly-Gly-Gly-Gly-Ser amino acid sequence repeated 3 or 4 times. The skilled person is able to adapt the linker according to need. In a further embodiment, the fragment that recognizes and binds at least a portion of the human TNC protein is complementary to a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 8.

[0085] In another embodiment, the fragment that recognizes and binds at least a portion of the human TNC protein is complementary to a sequence having 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 8.In a further embodiment, the at least a portion of the human TNC protein comprises or consists of a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 20.

[0086] In another embodiment, the portion of the human TNC protein comprises or consists of a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 20.

[0087] In a further embodiment, the fragment that recognizes and binds at least a portion of the human TNC protein is complementary to a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 9.

[0088] In another embodiment, the fragment that recognizes and binds at least a portion of the human TNC protein is complementary to a sequence having 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 9.

[0089] In a further embodiment, the at least a portion of the human TNC protein comprises or consists of a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 21.

[0090] In another embodiment, the portion of the human TNC protein comprises or consists of a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 21.

[0091] In a preferred embodiment, the at least one polynucleotide coding for the CAR is complementary to a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 2 or 3.

[0092] In a further embodiment, the at least one polynucleotide coding for the CAR is complementary to a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 2 or 3.

[0093] In a preferred embodiment, the CAR comprises or consists of a sequence having at least 70%, preferably at least 80%, even more preferably at least 90% identity with SEQ ID NO: 14 or 15.

[0094] In a further embodiment, the CAR comprises or consists of a sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 14 or 15.

[0095] In a particularly preferred embodiment, the vector further includes a polynucleotide coding for at least one cleavage site. Preferably, said polynucleotide coding for at least one cleavage site is positioned between the at least one polynucleotide coding for the antibody complex and the at least one polynucleotide coding for a CAR.The polynucleotides coding for cleavage sites enable the translation of solid polypeptides originating from a single polynucleotide and are known to the skilled person. For example, the cleavage sites can be of the “self-cleaving” type (i.e. T2A, P2A, E2A, F2A) and function during the protein translation process, or else they can be oligopeptides recognized by proteases or endonucleases (for example furin).

[0096] In a preferred embodiment, the polynucleotide coding for at least one cleavage site codes for the T2A peptide with or without a cleavage site for furin.

[0097] In a further embodiment, the previously described vector is a bicistronic vector, that is, it comprises at least one polynucleotide coding for a messenger RNA comprising at least two coding sequences, defined as cistrons, which are translated independently of each other, thus producing two distinct polypeptides.

[0098] In a preferred embodiment, the polynucleotide coding for at least one cleavage site is 90% complementary, preferably 95%, even more preferably 100% complementary to SEQ ID NO: 6 or 7.

[0099] In a preferred embodiment, the at least one cleavage site comprises or consists of a sequence having 90%, preferably 95%, even more preferably 100% identity with SEQ ID NO: 18 or 19.

[0100] In a particularly preferred embodiment, the vector comprises the at least one polynucleotide coding for the antibody complex followed by a polynucleotide coding for a cleavage site further followed by at least one polynucleotide coding for the CAR.

[0101] In a further embodiment, the vector comprises the at least one polynucleotide coding for the CAR, followed by a polynucleotide coding for a cleavage site, further followed by at least one polynucleotide coding for the antibody complex.

[0102] Without wishing to be bound by any theory, it is well known that CARs consist of various domains making it possible to reprogram the effector capacity of the T cells expressing them towards specific targets. CARs are formed by the extracellular antigen-binding domain, consisting of scFv fragments which bind a specific target, a hinge domain, a transmembrane domain, one or more co-stimulatory domains that confer survival and activation signals on the T cells, and, finally, a T-cell activating domain that enables the propagation of the signalling pathway associated with the CAR.

[0103] In a preferred embodiment, the at least one polynucleotide coding for the CAR further includes at least one sequence coding for a domain selected from the group consisting of:- a hinge domain selected from lgG1 CH2-CH3, CD8a, and CD28; and / or

[0104] - a transmembrane domain selected from CD8a and CD28; and / or

[0105] - one or two co-stimulatory domains selected from OX-40, 4-1 BB, CD27, CD28, and ICOS; and / or

[0106] - a CD3 T-cell activating domain.

[0107] In a particularly preferred embodiment, the at least one polynucleotide coding for the CAR further includes at least one sequence coding for a domain selected from the group consisting of:

[0108] - an lgG1 CH2-CH3 hinge domain; and / or

[0109] - a CD28 transmembrane domain; and / or

[0110] - an OX-40 co-stimulatory domain; and / or

[0111] - a CD3 T-cell activating domain.

[0112] In the context of the present invention, the preceding embodiment represents the polynucleotide coding for the CAR and the protein deriving therefrom and is defined as CAR-TNC5. Preferably, said polynucleotide coding for CAR-TNC5 consists of SEQ ID NO: 2.

[0113] In a further embodiment, the at least one polynucleotide coding for the CAR further includes at least one sequence coding for a domain selected from the group consisting of:

[0114] - a CD8a hinge domain; and / or

[0115] - a CD8a transmembrane domain; and / or

[0116] - a 4-1 BB co-stimulatory domain; and / or

[0117] - a CD3 T-cell activating domain.

[0118] In the context of the present invention, the preceding embodiment represents a polynucleotide coding for the CAR and the protein deriving therefrom and is defined as CAR-TNC4. Preferably, said polynucleotide coding for CAR-TNC4 consists of SEQ ID NO: 3.

[0119] A second aspect of the present invention relates to a host cell which expresses an antibody complex and a chimeric antigen receptor (“CAR”).

[0120] In a preferred embodiment, said host cell expresses the antibody complex and CAR previously described in detail. Preferably, said antibody complex is secretable.

[0121] In one embodiment, the host cell is an immune cell and is preferably selected from the group consisting of a: peripheral blood mononuclear cell (PBMC), cytokine-induced killer (CIK) cell, T cell, and NK cell.In a particularly preferred embodiment, the host cell is a CIK cell.

[0122] It is known in the prior art that cytokine-induced killer (CIK) cells constitute a group of heterogenous cells expanded ex vivo starting from PMBCs. They are in fact produced by adding, to the PBMCs, IFNy, IL-2 and anti-CD3antibodies, molecules which enable the cells to take on characteristics typical of both NK cells and T cells, expressing CD56 and CD3 respectively. The potentiality of CIK cells lies in their cytotoxic capacity, which is independent of the major histocompatibility complex (MHC), thus broadening their potential application while reducing the risk of GvHD.

[0123] In some embodiments, the host cell is autologous. Preferably, the autologous host cell derives from PBMCs or T or NK lymphocytes of the patient, and is collected, treated in such a way as to include and express said vector, expanded in vitro and subsequently reinfused into the patient.

[0124] In other embodiments, the host cell is an allogenic cell. Preferably, the allogenic immune cells derive from PBMCs, T cells, or NK cells of a healthy donor, which are collected and treated in such a way as to include and / or express said vector, expanded in vitro and subsequently infused into the patient.

[0125] In other embodiments, the host cell derives from induced pluripotent stem cells (iPSCs). Preferably, the cells derived from iPSCs are generated by reprogramming adult cells in a pluripotent state, which are then differentiated into T, CIK or NK cells, then treated in such a way as to include and express the vector previously described in detail and are subsequently expanded in vitro.

[0126] Therefore, in the context of the present invention, the host cell is a cell that can be used as immunotherapy, preferably in a context of adoptive cell therapy (adoptive cell transfer -ACT).

[0127] In one embodiment, the host cell comprises the vector previously described in detail. The TNC protein is one of the proteins making up the extracellular matrix (ECM) which is overexpressed in various tumours and constitutes a potential target for increasing the infiltration and persistence of CAR T cells in the tumour microenvironment.

[0128] Therefore, without wishing to be bound by any scientific theory, the host cell expressing the CAR that recognizes the protein TNC can permeate and persist to a greater degree within the tumour mass that overexpresses TNC. Moreover, the bispecific antibody complex or fragment enables both the cell expressing the CAR and the endogenous immune cells expressing CD3, as well as non-engineered CIK CD3+ cells, to be brought closer to the tumour cells to be eliminated, which in turn express the antigenrecognized by the CAR. In fact, since CD3 is a marker common to various immune populations, this technology would also increase the persistence and activation of nonengineered endogenous lymphocytes in the tumour microenvironment. Preferably, the expression of the CAR and of the bispecific antibody complex or fragment described will make it possible to increase the persistence of the immune cells or engineered cells in the solid tumour, and to deliver them and activate them against the tumour cells.

[0129] A third aspect of the present invention relates to a composition comprising the vector and / or the host cell described above.

[0130] In some embodiments, the composition is formulated for enteral or parenteral administration or, preferably, for parenteral administration.

[0131] Preferably, the composition is formulated for parenteral administration, more preferably as: a solution, a suspension, an injectable preparation, an infusion, a concentrate for injectable preparations, or a gel, with or without preliminary cryopreservation.

[0132] A fourth aspect of the present invention relates to the vector or host cell or composition comprising them as previously described, for use as a medicament.

[0133] A fifth aspect of the present invention relates to the vector or host cell, or the composition as previously described, for use in the treatment or prevention or follow-up of tumours.

[0134] Preferably, the vector or host cell or composition comprising them is administered to an individual who is in need thereof, more preferably to an individual who has been diagnosed with cancer.

[0135] In some embodiments, the vector or host cell or composition is administered in a single dose or in multiple doses over time, according to the patient’s needs and the type of cancer to be treated.

[0136] In some embodiments, the host cell or the composition comprising it is used in immunotherapy, preferably in cancer immunotherapy. Preferably, the host cell is used to improve the persistence and / or the effector activity of immune cells within the tumour microenvironment; said immune cells are preferably PBMCs, or CIK, T, or NK cells; more preferably said immune cells are cells which express a CAR.

[0137] Preferably, the treatment comprises the administration of the vector or host cell or of a composition containing said vector or host cell to an individual who has been diagnosed with a tumour, with the aim of destroying the tumour cells and reducing the size of the tumour.In other embodiments, the vector or host cell or a composition containing the vector or host cell is administered in combination with at least one other treatment, preferably with at least one other anti-cancer treatment.

[0138] Preferably, the prevention comprises the administration of the vector or host cell or composition containing said vector or immune cell to an individual at risk of developing a tumour, with the aim of enhancing the immune response of the patient and reducing the probability of developing cancer.

[0139] Preferably, the follow-up comprises the administration of the vector or host cell or the composition containing the vector or host cell to a patient who has received a cancer treatment with the aim of monitoring the patient’s response to the treatment and detecting any signs of tumour relapse. The follow-up can be conducted at regular intervals, such as every few months or once a year, depending on the type of tumour and the patient’s risk of relapse. The follow-up can include various tests and procedures, such as blood tests, imaging studies and biopsies. The optimization of the parameters of administration and the selection of the follow-up tests best suited to the patient represent activities that are well known and well-established for the skilled person.

[0140] In some preferred embodiments, the vector or host cell or composition is used for the treatment of a tumour, where said tumour is a solid tumour or a haematological tumour. Preferably, the solid tumour is selected from the group consisting of: a carcinoma, a sarcoma, a lipoma, a myelofibroma, a melanoma, a glioma, a meningioma, and a tumour of the nervous system. Preferably, the haematological tumour is a B- or T-cell lymphoma or a multiple myeloma.

[0141] In one embodiment, the tumour has an extracellular matrix (ECM) with a larger amount of tenascin-C (TNC) compared to healthy tissue, i.e. not affected by the corresponding tumour. Preferably, the tumour has cells which express higher levels of TNC than the corresponding healthy tissue and thus overexpress TNC.

[0142] In one embodiment, the tumour has an extracellular matrix and / or a tumour microenvironment (TME) with higher levels of TNC than the corresponding healthy tissue.

[0143] In a further embodiment, the tumour preferably expresses at least one antigen selected from the group consisting of: EGFR, HER2, HER3, GD2, CCR4, CD33, PD-L1, PD-1, MUC1, GPC3, PSMA, CD19, CD20, CD22, CD37, CD38, and BCMA.In a further embodiment, the tumour expresses a larger amount of the EGFR protein compared to the corresponding healthy tissue. Preferably, the solid tumour overexpresses EGFR.

[0144] In some embodiments, the tumour is a solid tumour that overexpresses a mutated form of the EGFR protein, preferably a mutation of the “gain-of-function” (GOF) type.

[0145] In a particularly preferred embodiment, the tumour has an extracellular matrix (ECM) with a larger amount of tenascin-C (TNC) and expresses a larger amount of the at least one tumour antigen previously described compared to the corresponding healthy tissue. Preferably, said tumour antigen is the EGFR protein. Optionally, the EGFR protein has a mutation of the “gain-of-function” (GOF) type.

[0146] A sixth aspect of the present invention relates to a method of treatment or prevention or follow-up comprising at least one step of administering the vector or host cell or composition comprising them as previously described to a patient in need thereof, wherein said patient has preferably been diagnosed with a tumour.

[0147] A seventh aspect of the present invention relates to a method for engineering a host cell, comprising at least a step (I) of transferring into the host cell at least one copy of the vector described in detail in the first aspect of the present invention.

[0148] Preferably, the step of transferring into the host cell at least one copy of the vector is carried out with a technique selected from: viral transduction (i.e. lentivirus, adenovirus, adeno-associated virus, retrovirus), electroporation, transfection, nucleofection, and lipofection.

[0149] In a preferred embodiment, the step of transferring into the host cell at least one copy of the vector takes place by nucleofection in combination with at least a second vector, preferably coding for a transposase protein.

[0150] In a preferred embodiment, the host cell is an immune cell and is preferably selected from the group consisting of a: PBMC, CIK cell, T cell, and NK cell. Preferably the immune cell to be engineered is a PBMC.

[0151] In a preferred embodiment, after the step of transferring into the PBMC at least one copy of the vector, there follows at least a step (II) of cellular expansion and / or a step (III) of treating said immune PBMCs with IFNYand / or IL-2 and / or an anti-CD3 antibody. The treatment of a PBMC with IFNY, IL-2 and an anti-CD3 antibody enables the differentiation of the PBMCs into cytokine-induced killer (CIK) cells, known for expressing membrane markers common to T cells (CD3), and NK cells (CD56). In a further preferred embodiment, after the treatment step (III), there is a step (IV) ofselecting the cells which express a CAR that recognizes and binds the TNC protein; said step (IV) preferably takes place by means of immunoselection techniques. The various types of techniques for selecting cells which express a CAR are activities known to the skilled person and are adapted based on needs and the context. The present invention would thus make it possible to overcome the current limitations of CAR technology regarding, for example, reduced permeability and persistence in solid tumours. In fact, the expression, by the immune cell, both of a CAR that recognizes a target of the extracellular matrix tumour and of the bispecific antibody fragment, which enables it to be brought close to the tumour cells, would increase the persistence of the immune cells in the tumour microenvironment, while specifically activating their cytotoxic capacity against cancer cells.

[0152] SEQUENCES

[0153] • Nucleotide sequence of the bispecific antibody complex (EGFRxCD3) - SEQ ID NO: 1 atgaagtgggtcaccttcatctctctgctcttcctgttttcgagcgcctactcccgcggcgtgttccgccgtgacattctgctgac ccagagccccgtgatcctgagcgtttcaccaggtgagcgcgtctccttctcctgccgagcatcccagtccataggcacaaa catccactggtaccagcagcgcaccaatggctccccgcgcctgctcatcaagtacgcgtccgagagcatttccggcatcc cctctcgcttcagcgggagtggatctggcaccgacttcactctgtctattaattctgtggagtctgaagacatcgcggactact attgtcagcagaacaacaactggcctaccacgtttggtgccggcactaagctcgagctgaaaggaggcggaggcagcg gcgggggcggatctggcggcggtggttctcaagtgcagttgaagcagtccggacctggactggtgcagccgtcccagag tctttccatcacctgcaccgtgtccggcttctcgctgaccaactacggcgtccactgggtccgccagtcgcccggcaaggg cctggagtggctcggcgtgatttggtctggaggcaacactgattacaacacgcccttcacctcccgcttgagcattaacaag gacaactccaagagccaggtctttttcaagatgaattccttgcagagcaacgacaccgccatctactattgtgcccgcgcc ctcacttactacgactacgagttcgcctattggggccagggaacattggtcaccgtgtccgctggcgggggcggttccgac atcaaactgcagcagtcgggggcggagcttgctcgcccgggtgcttccgtgaaaatgtcctgtaagaccagcggctatac gttcacacgctacaccatgcattgggtgaagcagaggcctggacagggcctcgaatggatcggttacatcaacccctctc ggggctacaccaactacaaccagaagtttaaagacaaggccacgctgacaaccgacaaaagctcgtccactgcctac atgcagctgtcctcactgacctccgaagattctgccgtgtactactgcgctcgttactacgatgaccattactgcctggactatt ggggccagggcacaacactgaccgtctcctccgtggagggcggcagtggaggttctggtggatccggagggtcaggtg gggtggatgacatccagcttacccagagtcccgctatcatgtctgcctccccaggggagaaggttactatgacctgtcgtgcttccagctccgtatcttacatgaactggtaccaacagaagagcggtacttctccgaagcgttggatctatgatacttccaagg tggcgtccggcgtcccctatcgttttagcgggagtggctccggcaccagctactcgctgaccatctcttctatggaggcgga ggacgcagccacctactactgtcaacagtggtctagcaaccccctgaccttcggtgctggcaccaaactggagctgaag caccaccaccaccaccattaa

[0154] • Nucleotide sequence of the receptor CAR-TNC5 - SEQ ID NO: 2 atggagaccgacaccctgctgctgtgggtgctgctgctgtgggtgccaggcagcaccggcgacatcgtgatgacacaag ctgctcccagcgtgccagtgacacctggcgagtctgtgtccatctcttgccggagcagcaagagcctgctgcacagcaac ggcaatacctacctgtactggttcctgcagaggcccggacagtctcctcagctgctgatctaccggatgagcaatctggcc agcggcgtgcccgatagattttctggctctggcagcggcaccgccttcacactgagaatctctagagtggaagccgagga cgtgggcgtgtactactgtatgcagcacctggaataccctctgaccttcggagccggcaccaagctggaactgaaaacca aggccggaggcggaggctctggcggaggcggctctggcggaggcggctctggcggaggcggcagcgaaaaagtga agctgcagcagagcggccctgagctggttaagcctggcgcctctgtgaaggtgtcctgtaaagccagcggctacgccttt accagctacaacatgtactgggtcaagcagagccacggcaagtccctggaatggatcggctacatcgacccctacaac ggcgtgacctcctacaaccagaagttcaagggcaaagccacactgaccgtggacaagagcagctccaccgcctacat gcacctgaacagcctgaccagcgaggacagcgccgtgtactattgtgctagaggcggcggatccatctactacgccatg gattattggggccagggcaccaccgtgacagtgtctagcgatcccgccgagcccaaatctcctgacaaaactcacacat gcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagc gtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcc cccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggg atgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggga gagcaatgggcaaccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagc aagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc actacacgcagaagagcctctccctgtctccgggtaaaaaagatcccaaattttgggtgctggtggtggttggtggagtcct ggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgacta catgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagc ctatcgctccagggaccagaggctgccccccgatgcccacaagccccctgggggaggcagtttccggacccccatcca agaggagcaggccgacgcccactccaccctggccaagatcagagtgaagttcagcaggagcgcagacgcccccgc gtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagag acgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgca gaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgcctcctcgctaa

[0155] • Nucleotide sequence of the receptor CAR-TNC4 - SEQ ID NO: 3 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacgacatcgtgatgaca caagctgctcccagcgtgccagtgacacctggcgagtctgtgtccatctcttgccggagcagcaagagcctgctgcacag caacggcaatacctacctgtactggttcctgcagaggcccggacagtctcctcagctgctgatctaccggatgagcaatct ggccagcggcgtgcccgatagattttctggctctggcagcggcaccgccttcacactgagaatctctagagtggaagccg aggacgtgggcgtgtactactgtatgcagcacctggaataccctctgaccttcggagccggcaccaagctggaactgaa aggcggcggaggaagcggaggcggaggatctggtggtggtggatctgaaaaagtgaagctgcagcagagcggccct gagctggttaagcctggcgcctctgtgaaggtgtcctgtaaagccagcggctacgcctttaccagctacaacatgtactgg gtcaagcagagccacggcaagtccctggaatggatcggctacatcgacccctacaacggcgtgacctcctacaaccag aagttcaagggcaaagccacactgaccgtggacaagagcagctccaccgcctacatgcacctgaacagcctgaccag cgaggacagcgccgtgtactattgtgctagaggcggcggatccatctactacgccatggattattggggccagggcacca ccgtgacagtgtctagcaccacaacacccgctcctagacctccaacaccagctccaacaatcgccagccagcctctgtct ctgaggccagaagcttgtagacctgctgctggcggagccgtgcatacaagaggactggacttcgcctgcgacatctacat ctgggctcctctggctggcacatgcggagtgttgctgctgagcctggtcatcaccctgtactgcaagcggggcagaaaga aactgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagatggctgctcctgcagattc cccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatccgccgacgctcctgcctatcagcaggg acagaaccagctgtacaacgagctgaacctggggagaagagaagagtacgacgtgctggacaagcggagaggcag agatcctgagatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagctgcagaaagacaag atggccgaggcctacagcgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggactgtaccag ggcctgagcaccgccaccaaggatacctatgatgccctgcacatgcaggccctgcctccaagataa

[0156] • Nucleotide sequence of anti-EGFR scFv - SEQ ID NO: 4: gacattctgctgacccagagccccgtgatcctgagcgtttcaccaggtgagcgcgtctccttctcctgccgagcatcccagt ccataggcacaaacatccactggtaccagcagcgcaccaatggctccccgcgcctgctcatcaagtacgcgtccgaga gcatttccggcatcccctctcgcttcagcgggagtggatctggcaccgacttcactctgtctattaattctgtggagtctgaaga catcgcggactactattgtcagcagaacaacaactggcctaccacgtttggtgccggcactaagctcgagctgaaagga ggcggaggcagcggcgggggcggatctggcggcggtggttctcaagtgcagttgaagcagtccggacctggactggtg cagccgtcccagagtctttccatcacctgcaccgtgtccggcttctcgctgaccaactacggcgtccactgggtccgccagt cgcccggcaagggcctggagtggctcggcgtgatttggtctggaggcaacactgattacaacacgcccttcacctcccgc ttgagcattaacaaggacaactccaagagccaggtctttttcaagatgaattccttgcagagcaacgacaccgccatctac tattgtgcccgcgccctcacttactacgactacgagttcgcctattggggccagggaacattggtcaccgtgtccgct• Nucleotide sequence of anti-CD3 scFv - SEQ ID NO: 5 gacatcaaactgcagcagtcgggggcggagcttgctcgcccgggtgcttccgtgaaaatgtcctgtaagaccagcggct atacgttcacacgctacaccatgcattgggtgaagcagaggcctggacagggcctcgaatggatcggttacatcaaccc ctctcggggctacaccaactacaaccagaagtttaaagacaaggccacgctgacaaccgacaaaagctcgtccactgc ctacatgcagctgtcctcactgacctccgaagattctgccgtgtactactgcgctcgttactacgatgaccattactgcctgga ctattggggccagggcacaacactgaccgtctcctccgtggagggcggcagtggaggttctggtggatccggagggtca ggtggggtggatgacatccagcttacccagagtcccgctatcatgtctgcctccccaggggagaaggttactatgacctgt cgtgcttccagctccgtatcttacatgaactggtaccaacagaagagcggtacttctccgaagcgttggatctatgatacttc caaggtggcgtccggcgtcccctatcgttttagcgggagtggctccggcaccagctactcgctgaccatctcttctatggag gcggaggacgcagccacctactactgtcaacagtggtctagcaaccccctgaccttcggtgctggcaccaaactggagc tgaag

[0157] • Nucleotide sequence of the furin-T2A cleavage site of the vector comprising EGFRxCD3 / TNC5 - SEQ ID NO: 6 cgccgcaagcgcggcagcggtgagggccgtgggtctctgctaacctgcggggatgtagaggagaacccgggccct

[0158] • Nucleotide sequence of the furin-T2A cleavage site of the vector comprising TNC5 / EGFRxCD3 - SEQ ID NO: 7 cggcggaagcgtggttcgggcgagggccggggctcgctgctgacctgcggggacgtggaggagaatcccgggcca

[0159] • Nucleotide sequence of anti-TNC scFv in CAR-TNC5- SEQ ID NO: 8 gacatcgtgatgacacaagctgctcccagcgtgccagtgacacctggcgagtctgtgtccatctcttgccggagcagcaa gagcctgctgcacagcaacggcaatacctacctgtactggttcctgcagaggcccggacagtctcctcagctgctgatcta ccggatgagcaatctggccagcggcgtgcccgatagattttctggctctggcagcggcaccgccttcacactgagaatctc tagagtggaagccgaggacgtgggcgtgtactactgtatgcagcacctggaataccctctgaccttcggagccggcacc aagctggaactgaaaaccaaggccggaggcggaggctctggcggaggcggctctggcggaggcggctctggcgga ggcggcagcgaaaaagtgaagctgcagcagagcggccctgagctggttaagcctggcgcctctgtgaaggtgtcctgta aagccagcggctacgcctttaccagctacaacatgtactgggtcaagcagagccacggcaagtccctggaatggatcg gctacatcgacccctacaacggcgtgacctcctacaaccagaagttcaagggcaaagccacactgaccgtggacaag agcagctccaccgcctacatgcacctgaacagcctgaccagcgaggacagcgccgtgtactattgtgctagaggcggc ggatccatctactacgccatggattattggggccagggcaccaccgtgacagtgtctagc

[0160] • Nucleotide sequence of anti-TNC scFv in CAR-TNC4- SEQ ID No: 9gacatcgtgatgacacaagctgctcccagcgtgccagtgacacctggcgagtctgtgtccatctcttgccggagcagcaa gagcctgctgcacagcaacggcaatacctacctgtactggttcctgcagaggcccggacagtctcctcagctgctgatcta ccggatgagcaatctggccagcggcgtgcccgatagattttctggctctggcagcggcaccgccttcacactgagaatctc tagagtggaagccgaggacgtgggcgtgtactactgtatgcagcacctggaataccctctgaccttcggagccggcacc aagctggaactgaaaggcggcggaggaagcggaggcggaggatctggtggtggtggatctgaaaaagtgaagctgc agcagagcggccctgagctggttaagcctggcgcctctgtgaaggtgtcctgtaaagccagcggctacgcctttaccagct acaacatgtactgggtcaagcagagccacggcaagtccctggaatggatcggctacatcgacccctacaacggcgtga cctcctacaaccagaagttcaagggcaaagccacactgaccgtggacaagagcagctccaccgcctacatgcacctg aacagcctgaccagcgaggacagcgccgtgtactattgtgctagaggcggcggatccatctactacgccatggattattg gggccagggcaccaccgtgacagtgtctagc

[0161] • Nucleotide sequence of the signal peptide of human serum albumin (HSA) - SEQ ID NO: 10 atgaagtgggtcaccttcatctctctgctcttcctgttttcgagcgcctactcccgcggcgtgttccgccgt

[0162] • Nucleotide sequence of the “forward” primer for RT-qPCR for the TNC (5’-3’) transcript - SEQ ID NO: 11

[0163] actgtggacggaaccaagac

[0164] • Nucleotide sequence of the reverse primer for RT-qPCR for the TNC transcript (5 -3 ) - SEQ ID NO: 12

[0165] T gatgttggctgtcaccagg

[0166] • Amino acid sequence of the bispecific antibody complex (EGFRxCD3) - SEQ ID NO: 13

[0167] MKWVTFISLLFLFSSAYSRGVFRRDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWY QQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWP TTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSL TNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAGGGGSDIKLQQSGAELARPGASV KMSCKTSGYTFTRYTMHVWKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKS SSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSG GSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTS KVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKHH HHHH

[0168] • Amino acid sequence of the receptor CAR-TNC5 - SEQ ID NO: 14 METDTLLLVWLLLWVPGSTGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYL YWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQ HLEYPLTFGAGTKLELKTKAGGGGSGGGGSGGGGSGGGGSEKVKLQQSGPELVKPG AS VKVS C KAS G YAFTS YN M YWVKQS H G KS L E Wl G Yl D P YN G VTS YN Q KF KG KATLTV DKSSSTAYMHLNSLTSEDSAVYYCARGGGSIYYAMDYWGQGTTVTVSSDPAEPKSPD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK FWVLWVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0169] • Amino acid sequence of the receptor CAR-TNC4 - SEQ ID NO: 15

[0170] MALPVTALLLPLALLLHAARPDDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYL YWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQ HLEYPLTFGAGTKLELKGGGGSGGGGSGGGGSEKVKLQQSGPELVKPGASVKVSCK ASGYAFTSYNMYWVKQSHGKSLEWIGYIDPYNGVTSYNQKFKGKATLTVDKSSSTAY MHLNSLTSEDSAVYYCARGGGSIYYAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAmino acid sequence of the anti-EGFR scFv - SEQ ID NO: 16

[0171] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSR FSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSG GGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIW SGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWG QGTLVTVSA

[0172] • Amino acid sequence of the anti-CD3 scFv - SEQ ID NO: 17

[0173] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGY TNYNQKFKDKATLTTDDKSSSTAYMQLSSLTSESAVYYCARYYDDHYCLDYWGQGTT LTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMN WYQQKSGTSPKRWIYDTSKVASTGVPYRFSGSGSGTSYSLISSMEAEDAATYYCQQ WSSNPLTFGAGTKLELK

[0174] • *Amino acid sequence of the furin-T2A cleavage site in the vector comprising EGFRxCD3 / TNC5 - SEQ ID NO: 18

[0175] RRKRGSGEGRGSLLTCGDVEENPGP

[0176] • Amino acid sequence of the furin-T2A cleavage site in the vector comprising TNC5 / EGFRxCD3 - SEQ ID NO: 19

[0177] RRKRGSGEGRGSLLTCGDVEENPG P

[0178] • Amino acid sequence of the anti-TNC scFv in CAR-TNC5- SEQ ID NO: 20

[0179] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSN LASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELKTKAG GGGSGGGGSGGGGSGGGGSEKVKLQQSGPELVKPGASVKVSCKASGYAFTSYNMY VWKQSHGKSLEWIGYIDPYNGVTSYNQKFKGKATLTVDKSSSTAYMHLNSLTSEDSAVYYCARGGGSIYYAMDYWGQGTTVTVSS

[0180] • Amino acid sequence of the anti-TNC scFv in CAR-TNC4- SEQ ID NO: 21

[0181] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSN LASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELKGGG GSGGGGSGGGGSEKVKLQQSGPELVKPGASVKVSCKASGYAFTSYNMYVWKQSHG KSLEWIGYIDPYNGVTSYNQKFKGKATLTVDKSSSTAYMHLNSLTSEDSAVYYCARGG GS IYYAM DYWGQGTTVTVSS

[0182] • Amino acid sequence of the signal peptide of human serum albumin (HSA) - ID SEQ: 22

[0183] MKWVTFISLLFLFSSAYSRGVFRR

[0184] EXAMPLES

[0185] In the examples below, the bispecific antibody complex or fragment previously described are defined with the term BiTE.

[0186] Example 1 : Techniques for detecting the levels of tenascin-C (TNC)

[0187] In order to detect the extracellular or intracellular TNC protein by flow cytometry, live or fixed and permeabilized cells (Cytofix / Cytoperm kit, BD Bioscience) were stained with monoclonal mouse anti-human TNC antibody (BC-24) (Invitrogen), followed by staining with FITC-labelled goat anti-mouse IgG.

[0188] For the immunofluorescence in situ, the cells were seeded on sterile slides in 6-well plates in complete medium for 1-4 days. After incubation, the cells were fixed with 4% paraformaldehyde (Thermo Fisher), washed in PBS and then blocked with PBS+5% FBS. The cells were incubated with the monoclonal mouse anti-human TNC antibody (BC-24), followed by a Cy3-labelled secondary donkey anti-mouse antibody (Jackson ImmunoResearch, Ely) and, finally, with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI, Merck) for 10 minutes. The cells were washed three times in PBS after everystep. At the end, the slides were observed under an inverted fluorescence microscope (Axio Vert A1, Zeiss) and a confocal one (SP8 lightning, Leica).

[0189] In order to quantify the expression of the RNA of TNC, quantitative PCR (RT- qPCR) was performed on the RNA extracted from various cell lines with the RNeasy mini kit (QIAGEN, Hilden, Germany). The RNA was reverse transcribed using the Superscript IV VILO master mix (Thermo Fisher), following the manufacturer’s instructions. The amplifications were carried out in Power SYBR green PCR Master Mix (Applied Biosystems, Thermo Fisher) and the cycles included 10 initial minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute. The primers were designed to detect the RNA levels of TNC and two housekeeping genes, the [3-glucuronidase (GUS) gene and the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The primer sequences correspond to SEQ ID NO: 11-12. The respective expression levels of TNC among the various cell lines were analysed with the 2-AACt method.

[0190] Example 2: Measurement of TNC expression in tumour cell lines

[0191] In order to characterize the expression of the RNA and the TNC protein and select tumour cell lines for the functional assays, we examined 20 cell lines of haematological and solid tumours for TNC expression. The expression of the TNC protein was measured by immunofluorescence, both by flow cytometry of permeabilized cells in suspension, and by staining cells cultured on slides for several days and subsequently fixed. In some cases, the expression of TNC RNA was also measured by RT-qPCR. TNC positivity was observed more or less intensely in 4-5 lines of solid tumour cells: weakly in HT29, Hep-G2 and HT-1080 and strongly in the MDA-MB-231 breast cancer cell line (Table 1). The TNC expression levels observed by means of immunofluorescence were confirmed by qPCR (Table 1).

[0192] Among the blood cell lines analysed, all the cell lines of T-cell lymphoma and B-cell acute lymphoblastic leukaemia were negative when tested by surface flow cytometry (Table 1). In contrast, the more mature B cell lines, derived from chronic lymphocytic leukaemia (CLL), B-cell non-Hodgkin lymphoma (B-NHL) or multiple myeloma (MM) showed a variable surface TNC expression pattern, with different cell lines showing weak-to-medium expression levels. The maximum TNC positivity was observed in the Jekol mantle cell lymphoma (MCL) cell line and in the Burkitt lymphoma cell line BJAB.The surface TNC expression was confirmed with two different monoclonal anti-TNC antibodies that had different epitopes of the protein as their target. In contrast, RT-qPCR was negative for nearly all the haematopoietic cell lines, with the exception of a weak expression in the CLL and MCL cell lines (Table 1).

[0193] Table 1. TNC expression in tumour cell linesSlid t Tll li Bll liomors cenes cenesuu

[0194] Flow Immunofluoresce

[0195] Tumour cell

[0196] Tumour subtypes cytometry nee on slices PCR lines

[0197] (permeabilized) (fixed in formalin)

[0198] HT-29 CRC + + +

[0199] Hep-G2 Hepatocarcinoma ND + ND MDA-MB- BC + ++ ++ 231

[0200] MCF7 BC - ND ND

[0201] HT-1080 Fibrosarcoma + / - ND ND

[0202] HH CTCL - ND ND Hut-78 CTCL - ND ND Karpas 299 ALCL - ND - REH BCP-ALL - ND - TOM1 BCP-ALL - ND - 697 BCL-ALL - ND - MEC1 CLL + / - ND + / - Granta 519 MCL + / - ND - Jekol MCL +++ ND +

[0203] DOH H2 DLBCL ++ ND - SU-DHL4 DLBCL + / - ND - Albanes BL - ND - Namalwa BL ++ ND - BJAB BL +++ ND - AS283A AIDS BL + ND - KMS12 MM - ND - JJN3 MM + / - ND - RPMI 8226 MM - ND - OPM2 MM - ND - IM9 EBV-LCL - ND -

[0204]

[0205] Abbreviations. ALCL: Anaplastic large ce I lymphoma; BC: Breast carcinoma; BCD-ALL: B cell precursor acute lymphoblastic leukaemia; BL: Burkitt’s lymphoma; CLL: chronic lymphocytic leukaemia; CRC: colorectal carcinoma; CTCL: cutaneous T cell lymphoma; EBV-LCL: Epstein-Barr virus immortalized lymphoblastic cell line; DLBCL: diffuse large cell lymphoma; MCL:mantle cell lymphoma; MM: multiple myeloma.a:+++: >69%; ++: 30-59%; +: 20-29%; + / -: 5-19%; -: <5%

[0206] Example 3: Cell lines

[0207] HT-29 human colorectal adenocarcinoma cell lines and CEM lymphoblastic T cells were cultured in RPMI 1640 medium (Euroclone) with 10% fetal bovine serum (FBS) of the “heat-inactivated type (Euroclone), 2 mM of L-glutamine (Euroclone) and 100 pM of gentamycin (PHT Pharma). MDA-MB-231 human breast carcinoma and HEK-293 embryonic kidney cell lines were cultured in a-MEM medium (Gibco, Thermo Fisher Scientific) with 10% heat-inactivated FBS (Euroclone) and 100 pM gentamycin (PHT Pharma). The peripheral blood mononuclear cells (PBMCs) were purified from samples from healthy donors who gave informed consent and obtained from the buffy coat by Ficoll-Hypaque gradient centrifugation (Lympholyte-H; Cedarlane).

[0208] Example 4: Generation of cell lines stably expressing TNC

[0209] In order to generate target cells which stably express the TNC antigen for the functional analyses, an expression vector was synthesized (GeneArt, Thermo Fisher) which comprises the “EGFR-like” domain of TNC, fused with the transmembrane and intracellular region of heparin-binding epidermal growth factor (HB-EGF-TM). The intracellular region of HB-EGF was mutated to avoid the nuclear signalling pathway, as already reported in the literature.

[0024] The cDNA of this fusion protein was cloned downstream of the CMV promoter in the transposable pT4 vector, which leads the Sleeping Beauty transposase recognition sequences, in order to obtain the plasmid pT4-TNC-TM. The CEM and HEK-293 cells were then transfected with the plasmids pT4-TNC-TM and SB100X using the Nucleofector Kit V and the Amaxa lib nucleofection device (Lonza). After 7-10 days of expansion, the stably transfected NC-TM+T cells were purified by staining with a mouse anti-human TNC antibody and an FITC-labelled goat anti-mouse IgG, followed by immunoselection with anti-FITC magnetic micro beads (Miltenyi Biotec). After two cycles of immunoselection, the CEM-TNC-TM+ and HEK-293-TNC-TM+ cell lines were obtained; they stably express the fusion protein TNC-TM across >90% of the cells.

[0210] Example 5:

[0211] Generation ofanti-TNC CAR / EGFRxCD3 transposon plasmidsTwo plasmids encoding two different anti-TNC CAR constructs were generated. The two plasmids express the anti-TNC single-chain variable fragment (scFv) based on the VL-VH sequence of the monoclonal antibody ST2146 (tenanumomab), separated by a linker (G4Sx3 or G4Sx4), synthesized by GeneArt (Thermo Fisher).

[0212] The scFvs were fused to a combination of different domains. The CAR-TNC4 construct consists of a hinge domain and a transmembrane domain of CD8a, a 4-1 BB co-stimulatory domain and a CD3 T-cell activating domain, based on Novartis’ Tisagenlecleucel CAR-CD19, under the EF1a promoter (Fig. 1A).

[0213] The CAR-TNC5 construct consists of a human lgG1 hinge domain and the CH2-CH3 domain as a spacer element, a transmembrane domain and one of co-stimulation by CD28, a second OX-40 co-stimulatory domain and a CD3 T-cell activating domain, under the pTMNDU3 promoter, based on the CAR-CD19 described previously

[0017] , Both CAR sequences were subcloned in the pT4 vector (Fig. 1A).

[0214] In the double constructs, i.e. the ones comprising both the CAR and BiTE, the EGFRxCD3 BiTE consists of a sequence leader which codes for a peptide signal for the secretion thereof, followed by the VL-VH sequences of cetuximab linked by a linker (anti-EGFR scFv), followed by the VL-VH sequences of blinatumomab linked by a linker (anti-CD3 scFv) and, finally, by a TAG (His-tag) sequence solely for the in vitro BiTE detection function. This construct was synthesised by GeneArt and cloned in the pT4 vector. The BiTE sequence just described was also inserted upstream or downstream of CAR-TNC5, separated from the latter by a T2A sequence for cleavage of the two proteins produced (Figure 3A).

[0215] The transposase plasmids used are SB100X pCMV(CAT)T7-SB100 (Addgene #34879). The plasmids were verified by sequencing and purified with the Plasmid Maxiprep isolation kit (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s instructions.

[0216] Example 6: Generation of anti-TNC CARCIK cells

[0217] The CIK cells expressing CAR (CARCIK-TNC) were generated as previously described.

[0017] Briefly, PBMCs were co-transfected with the pT4-CAR-TNC4 (CARCIK-TNC4) or pT4-CAR-TNC5 (CARCIK-TNC5) and SB100X plasmids, using the Human T Cell Nucleofector kit (Lonza) and the Amaxa Nucleofector lib Device (Lonza). After nucleofection, the cells were expanded in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) with 20% FBS (Euroclone) and 2 mM of L-glutamine (Euroclone) and with theaddition of 1000 U / mL of IFN-y (Clinigen Healthcare Ltd) on day 0, and 50 ng / ml of anti-CD3 antibody (OKT-3, TakaraBio) and 300 ll / ml of recombinant human IL-2 (rhlL-2, Proleukin, Clinigen Healthcare Ltd) on day 1. Fresh medium with rhlL-2 was added twice a week until the 21stday for CIK expansion.

[0218] The CARCIK-TNC4 and CARCIK-TNC5 cells both showed an efficient expansion,

[0219] 6 6

[0220] reaching a mean of 221x10 and 233x10 total nucleated cells, respectively, after 21 days (Figure 1B). The CAR-TNC4 and CAR-TNC5 expression levels remained stable and after 21 days reached values of 34.5% and 43.8%, respectively (Figure 1 C), with a mean fluorescence intensity (MFI) of 24655 and 11657, respectively (Figure 1D). CAR-TNC4 is expressed in a slightly lower percentage, but with a fluorescence intensity that is significantly higher compared to CAR-TNC5 (p<0.05).

[0221] Considering the variability in CAR expression observed between experiments and between the two different CARs, the cells expressing the anti-TNC CAR were immunoselected for the purpose of performing functional assays with populations of CARCIK cells expressing comparable amounts of CAR. In these cases, 10 days after transfection, the cells expressing the anti-TNC CAR were treated with the human recombinant TNC protein labelled with a polyhistidine tag (R&D systems). Subsequently, the cells were labelled with an FITC-labelled anti-histidine antibody and then selected by means of anti-FITC magnetic micro beads (Miltenyi Biotec) and selected by means of a separation column (Miltenyi Biotec). The purification efficiencies of CARCIK-TNC4 and CARCIK-TNC5 showed to be comparable, as the result obtained was over 90% of cells expressing CAR-TNC (Figure 1E). The positive fraction was collected and maintained in culture as described above up to the 21stday. Unmodified CIK cells from the same donors were expanded in parallel as a control.

[0222] Example 7: Immunophenotypinci of CARCIK-TNC

[0223] The CARCIK-TNC cells were characterised with the following MAbs: anti-CD3-PerCP-Cy5.5 (clone SK7), anti-CD56-BV510 (clone NCAM16.2), anti-CD4-PE-Cy7 (clone SK3), anti-CD8-APC-H7 (clone SK1), and anti-CD45RA-FITC (clone L48), anti-CD62L-APC (clone SK11) (BD biosciences). CAR detection was carried out by first incubating the cells with the polyhistidine-tagged recombinant human TNC protein (R&D systems), followed by the FITC-, APC- or PE-conjugated anti-histidine antibody (Miltenyi Biotec, Inc).

[0224] The EGFRxCD3 BiTE secreted was detected in the supernatant of the cell culture byincubating the EGFR+cells for 15 minutes with 1 ml of supernatant of the cell culture, which was then stained with an FITC-, APC- or PE-conjugated anti-histidine antibody. An FACScanto II flow cytometer (BD Biosciences) with BD FACSDiva software was used to analyse the samples.

[0225] The percentages of CD4+, CD8+and CD3+CD56+subpopulations were comparable among the non-engineered CARCIK-TNC and CIK cells (Figure 1F). As regards the immunophenotype characteristic of the effector memory cells, the percentages of populations of the “naive” type and “EMRA” type were similar in the engineered CIK and control CIK cells. In contrast, the percentage of the central memory (CM) population was significantly higher for both the engineered CARCIK-TNC cells, with a mean of 63.5% for CARCIK-TNC4 and 61.2% for CARCIK-TNC5, compared to the nonengineered CIK cells (30.3%) (p<0.05). The percentage of effector memory (EM) cells was less represented in both CARCIK-TNC types versus the unmodified CIK cells (respectively 28.4% and 35.2% vs 67.6%, p<0.05, Figure 1G).

[0226] Overall, these data demonstrate that the method of culturing CARCIK-TNC cells is effective and that their phenotypes do not differ significantly from that of unmodified CIK cells, except for a few parameters.

[0227] Example 8: Cytotoxicity assay

[0228] The cytotoxicity of engineered CARCIK cells towards cells expressing TNC was assessed with a cytotoxicity assay. The TNC-expressing cells (TNC+) used were CEM-TNC-TM+ and HEK-293-TNC-TM+cell lines stably transfected with the TNC-TM construct and tumour cells naturally expressing TNC (HT-29 and MDA-MB-231). Cell lysis was assessed using the GFP-certified Apoptosis / Necrosis detection kit (Enzo Life Science). The target TNC-expressing cell lines were first stained with the fluorescent green dye 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma-Aldrich, Merck KGaA). The CEM-TNC-TM+and HEK-293-TNC-TM+cell lines were stained with 0.5 pM CFSE immediately before the test, whilst the HT-29 and MDA-MB-231 cell lines, which naturally express TNC, were stained with 1 pM of CFSE, plated and allowed to reach confluence for four days in culture. The engineered or nonengineered CIK cells were cultivated in a co-culture with the CFSE-labelled cells in a 5:1, 1:1, up to a 1:10 effector cell: target cell (E:T) ratio for 4-24 hours. The cells were then collected, washed and labelled with the apoptosis detection reagent (Annexin V-Enzo Gold) and the necrosis detection reagent for 10 minutes and analysed by flow cytometry. The percentage of dead cells was determined by calculating the overall percentage of Annexin V+and Necrosis Detection Reagent" in the CFSE+target cells co-cultured with the effector cells minus the spontaneous lysis of the target cells alone. The maximum lysis was measured by adding the apoptosis inducer (staurosporine) in the positive control wells.

[0229] Both types of engineered CARCIK-TNC cells showed to be highly cytotoxic against the CEM-TNC-TM4" line, to a significantly greater degree compared to non-engineered CIK cells (p<0.01, Figure 2A). The cytotoxic activity against HEK-293-TNC-TM " was significant only for CARCIK-TNC5 compared to the control CIK cells (p<0.05, Figure 2A). The CARCIK-TNC5 cells showed to be significantly more cytotoxic than the nonengineered CIK cells against the HT-29 and MDA-MB-231 cancer cell lines (respectively, p<0.01 and p<0.05, Figure 2B). In contrast, the CARCIK-TNC4 cells showed to be cytotoxic only against MDA-MB-231, which expresses higher levels of TNC (p<0.05, Figure 2B).

[0230] These data demonstrate that both CARCIK-TNC4 and CARCIK-TNC5 are more cytotoxic against cancer cell lines expressing TNC than non-engineered CIK cells, and that CARCIK-TNC5 is more effective overall than CARCIK-TNC4.

[0231] Example 9: Proliferation assays

[0232] The proliferation of CARCIK-TNC cells after recognition of the target cells was assessed using CFSE. Briefly, CIK and CARCIK-TNC cells were stained with 1 pM of CFSE for 10 minutes at 37°C. After washing they were plated at 0.4x10® cells / well in the presence or absence of target cells with E:T ratios of 10: 1 , 1:1 and 1:10. In order to test proliferation in co-culture with the HT-29 and MDA-MB-231 cell lines, the target cell lines were plated and left overnight before adding CARCIK-TNC to make them adhere to the plastic and express extracellular TNC. After 4 days of co-culture, the cells were collected and stained with CD3-PE, CD4- PE-Cy7 and CD8-APC-H7 antibodies (BD Bioscience) and the expression of CFSE was analysed by flow cytometry across different effector populations, using ModFit LT™ software to calculate the proliferation index.

[0233] The CARCIK-TNC5 cells showed a proliferation index that was often higher compared to non-engineered CIK cells or CARCIK-TNC4 (Figure 2C and D). In all cases,proliferation was higher with a low E:T ratio (1:10) as compared with the highest E:T ratio (10:1), indicating that CARCIK-TNC5 cells proliferate more significantly in the presence of a larger amount of target cells and thus of antigen. The CARCIK-TNC4 cells showed only a modest proliferative activity in all cases (Figures 2C and D).

[0234] Example 10: Induction of cytokines

[0235] The ability of CARCIK-TNC cells to produce cytokines in response to the target cells was assessed by intracellular staining and flow cytometry. HT-29 and MDA-MB-231 cells were plated four days prior to the test to enable them to reach confluence and express TNC in the extracellular microenvironment. The effector and target cells were co-cultured for six hours in an E:T ratio of 1:1 in the presence of BD GolgiStop solution (BD Bioscience). The cells were then collected, fixed and permeabilized using the BD Cytofix / Cytoperm kit (BD Bioscience), following the manufacturer’s instructions, and stained with antibodies: CD3-PerCP-Cy5.5, CD4-PE- Cy7, CD8-APC-H7 (BD Bioscience), IFN-y-FITC and IL-2-PE (Miltenyi Biotec). The samples were then analysed by flow cytometry.

[0236] The cytokines released by the CARCIK-TNC4 and CARCIK-TNC5 cells were measured in the presence of the CEM-TNC-TM+or MDA-MB-231 cell lines, i.e. in the presence of the target antigen.

[0237] After stimulation, both the CARCIK-TNC4 and -TNC5 CD8+cells produced more IFN-y compared to the non-engineered CIK cells (p<0.05, Figure 2E), with CARCIK-TNC5 demonstrating to be the most effective in secreting IFN-y.

[0238] The release of IL-2 was assessed in CD4+cells. CARCIK-TNC5 cells tend to induce IL-2 more than CIK or CARCIK-TNC4 cells, but statistical significance was not reached (Figure 2F). In short, the data show that CARCIK-TNC5 cells are more effective overall in vitro than CARCIK-TNC4 cells in terms of cytotoxicity, proliferation and cytokine release. Moreover, CARCIK-TNC5 cells showed to be more effective than nontransduced CIK cells in the majority of these functions.

[0239] Example 11 : Tumour models express! no TNC in vivo.

[0240] The in vivo experiments were authorized by the local ethics committee for animal experimentation and the Italian Ministry of Health. The animals were treated in accordance with European laws on animal experimentation. Briefly, NOD-SCID mice (6-week-old females, NOD. CB 17-Pr cfcsc / NCrHsd, Envigo) were inoculated subcutaneously with 5x10® MDA-MB-231 cells. When the tumours reached about 250 mg, the mice were treated with cyclophosphamide (250 mg / kg) and fludarabine (50 mg / kg) to complete lymphodepletion. After 48 hours, the mice were randomized into groups of 8 mice and treated intravenously with PBS, 10x10® non-engineered CIK cells or 10x10® CIK cells engineered with the specified expression vector. The treatment with the cells, generated from the same donor, was repeated on days 10 and 19. Tumour growth was measured twice a week, and the animals were sacrificed when the tumours reached >1000 mg.

[0241] The formalin-fixed paraffin-embedded (FFPE) tumour samples were analysed by immunohistochemistry to assess the expression of TNC and infiltration of human CD3+cells using automated devices.

[0242] Example 12: Effectiveness of CARCIK-TNC5 cells expressing the EGFRxCD3 BiTE (CARCIK-TNC5 / EGFRxCD3) in tumour models, in vitro and in vivo.

[0243] Given the apparent ineffectiveness of CARCIK-TNC5 cells in vivo, a new dual target strategy was developed.

[0244] The cDNA of a secretable form of a bispecific T cell-activating antibody (BiTE) -EGFRxCD3 - was placed upstream or downstream of the cDNA of CAR-TNC5, separated by a furin-T2A cleavable sequence, and inserted into the pT4 transposon plasmid (Figure 3A). The CIK cells transduced with these constructs thus express a CAR that recognizes TNC and a secretable BiTE that recognizes both the CD3 protein and the EGFR protein. As shown in Figure 3B, the CAR-TNC5 molecule was expressed better on the surface of CIK cells when the EGFRxCD3 BiTE sequence was positioned upstream rather than downstream of the CAR-TNC5 cells.

[0245] The cytotoxic activity of the double constructs was then tested in vitro against the MDA-MB-231 cell line as the target, positive for EGFR, with an effector celktarget cell (E:T) ratio ranging from 1:1 to a suboptimal 1:10. Figure 3C shows that both soluble EGFRxCD3 and CAR-TNC5 on their own induce about 55% cytotoxicity towards the MDA-MB-231 cells, with a 1 :2 ratio, as compared with about 30% for the nonengineered CIK cells. This percentage rises drastically to 80% when the CIK cells express both EGFRxCD3 and CAR-TNC5. Moreover, in a suboptimal E:T ratio of 1:10, the presenceof the two single molecules was not able to induce the killing of tumour cells, whereas the cytotoxicity showed to be significant (about 60%) when both CAR-TNC and BiTE were present. This clearly demonstrates the synergistic activity of EGFRxCD3 and CAR-TNC in inducing the cytotoxicity of tumour cells in vitro (Figure 3C).

[0246] The CIK cells were subsequently tested in vivo in a tumour model with the subcutaneous inoculation of MDA-MB-231 in lymphodepleted NOD-SCID mice.

[0247] The CARCIK-TNC5 cells on their own were demonstrated to reach and infiltrate the tumour (Figure 4A and B) in mouse models inoculated with the MDA-MB-231 cancer cell line, but they were unable to control tumour growth in the long term or improve the survival of treated mice (data not shown and Figure 4C). It is important to note that the cells were not toxic for the animals, as demonstrated by the measurement of body weight (Figure 4D) and survival (Figure 4C), despite the fact that the CAR-TNC expressed should also recognize mouse TNC, like its parent antibody tenatumomab. In particular, non-engineered CIK cells, cells expressing only BiTEs (EGFRxCD3), CARCIK-TNC5 cells and CARCIK cells expressing the double construct EGFRxCD3 / TNC5 were tested. The model is represented in Figure 5A, whereas the tumour growth curves are shown in Figure 5B. The data show that CIK cells transduced with the double construct EGFRxCD3 / CAR-TNC5 were very effective in controlling tumour growth, whereas the single constructs showed a partial activity. These data confirm the synergistic effectiveness of EGFRxCD3 secreted and CAR-TNC transduced in CIK cells as effectors for controlling TNC+tumours in vitro and in vivo.

[0248] Overall, these data suggest that CIK cells engineered so as to express both CAR-TNC and the EGFRxCD3 BiTE perform a synergistic action in vivo, thanks to the independent action of the two molecules. CAR-TNC enables localization in the tumour, cytotoxicity directed against tumour cells and the release of pro-inflammatory cytokines at the moment of recognition of the target. The link with the TNC antigen also induces the proliferation of CARCIK-TNC5. The secretable EGFRxCD3 BiTE, on the other hand, may induce a further cytotoxic effect on EGFR+cancer cells in situ, mediated both by the engineered CIK cells themselves and by non-transduced CIK cells, since the latter appear to be activated by the anti-CD3 part of the BiTE.

[0034] In a completely human context, the bispecific antibody or EGFRxCD3 BiTE secreted within the tumour appear also to allow endogenous cytotoxic T cells within the tumour to be activated and become cytotoxic against the tumour cells.Example 13: Statistical analyses

[0249] The results were compared using Student’s t-test. A p value <0.05 (*) was considered significant. In the case of in vivo assays, the growth curves were analysed using the t-test with the Satterthwaite method.

Claims

CLAIMS1. A vector comprising at least one polynucleotide coding for a bispecific antibody or a bispecific antibody fragment and at least one polynucleotide coding for a chimeric antigen receptor (CAR), wherein the CAR is able to recognize and bind at least one extracellular matrix protein, and wherein the bispecific antibody or antibody fragment is able to recognize and bind at least a portion of the human EGFR protein and at least a portion of the human CD3 protein.

2. The vector according to claim 1, comprising a signal sequence coding for a peptide suitable for allowing secretion of the antibody complex from a cell.

3. The vector according to claim 1 or 2, wherein the at least one extracellular matrix protein is human tenascin-C.

4. The vector according to any one of claims 1-3, wherein the at least one polynucleotide coding for the bispecific antibody or bispecific antibody fragment is complementary to a sequence having at least 90% identity with SEQ ID NO: 1, and / or wherein the at least one polynucleotide coding for a CAR is complementary to a sequence having at least 90% identity with SEQ ID NO: 2 or 3.

5. The vector according to any one of claims 1-4, wherein the at least one polynucleotide coding for the bispecific antibody or bispecific antibody fragment is complementary to a sequence which consists of SEQ ID NO: 1, and / or wherein the at least one polynucleotide coding for a CAR is complementary to a sequence which consists of SEQ ID NO: 2 or 3.

6. The vector according to any one of claims 1-5, wherein the at least one polynucleotide coding for a CAR further includes at least one sequence coding for a domain selected from the group consisting of:- an lgG1 CH2-CH3 hinge domain; and / or- a CD28 transmembrane domain; and / or- an QX-40 co-stimulator domain; and / or- a CD3 T-cell activating domain.

7. The vector according to any one of claims 1-5, wherein the at least one polynucleotide coding for a CAR further includes at least one sequence coding for a domain selected from the group consisting of:- a CD8a hinge domain; and / or- a CD8a transmembrane domain; and / or- a 4-1 BB co-stimulatory domain; and / or- a CD3 T-cell activating domain.

8. A host cell expressing a bispecific antibody or a bispecific antibody fragment and a chimeric antigen receptor ("CAR"), wherein the bispecific antibody or bispecific antibody fragment is able to recognize and bind at least a portion of the human EGFR protein and at least a portion of the human CD3 protein and wherein the CAR is able to recognize and bind human tenascin-C.

9. The host cell according to claim 8, wherein the bispecific antibody or bispecific antibody fragment is secretable.

10. The host cell according to one of claims 8 and 9, wherein the bispecific antibody or bispecific antibody fragment includes or consists of a sequence having at least 95% identity with SEQ ID NO: 13 and wherein the CAR includes or consists of a sequence having at least 95% identity with SEQ ID NO: 14 or 15.

11. The host cell according to any one of claims 8-10, wherein the host cell is an immune cell.

12. The host cell according to claim 11, wherein the host cell is selected from the group consisting of: peripheral blood mononuclear cell (PBMC), cytokine-induced killer (CIK) cell, T cell, and NK cell.

13. The host cell according to claim 12, wherein the immune cell is a CIK cell.

14. The host cell according to any one of claims 8-13 comprising the vector according to any one of claims 1 -10.

15. The vector according to any one of claims 1-7, or the host cell according to any one of claims 8-14, or a composition comprising them, for use as a medicament.

16. The vector according to any one of claims 1-7, or the immune cell according to any one of claims 8-15, or a composition comprising them, for use in the treatment or prevention or follow-up of tumours.

17. The vector or host cell or the composition comprising them, for use according to claim 16, wherein the tumour is a solid tumour selected from the group consisting of: a carcinoma, sarcoma, lipoma, myelofibroma, glioma, meningioma, tumour of the nervous system, and melanoma; or wherein the tumour is a haematological tumour selected from a T-cell lymphoma, B-cell lymphoma, or multiple myeloma.

18. A method for engineering a host cell comprising at least a step (I) of transferring into the host cell at least one vector according to any one of claims 1-7, preferably in combination with at least one vector coding for a transposase protein, and wherein the host cell is an immune cell preferably selected from the group consisting of: a PBMC, CIK cell, T cell, and NKcell.

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