WO2019134866A1

WO2019134866A1 - Chimeric antigen receptors containing optimal spacer region

Info

Publication number: WO2019134866A1
Application number: PCT/EP2018/086665
Authority: WO
Inventors: Catia Traversari; Anna Stornaiuolo; Barbara Valentinis
Original assignee: Molmed Spa
Priority date: 2018-01-03
Filing date: 2018-12-21
Publication date: 2019-07-11

Abstract

The present invention relates to a Chimeric Antigen Receptor(CAR)containing a spacer having an optimal structure that generates an improvement of the antitumor effect of the molecule.Particularly, the CAR according to the present invention carries a spacer domain consisting of a fragment of the human low affinity nerve growth factor receptor (LNGFR) composed by a first sequence including the four TNFR-Cys domains, linked to a second sequence consisting of the first 11 amino acids of the serine/threonine rich stalk.

Description

Chimeric Antigen Receptors containing optimal spacer region

FIELD OF THE INVENTION

The present invention relates to the field of cancer immunotherapy, particularly, to a Chimeric Antigen Receptor containing a spacer having an optimal structure that generates an improvement of the antitumor effect of the molecule.

BACKGROUND TO THE INVENTION

Chimeric Antigen Receptors (CARs) are recombinant receptors that recognize a specific protein or antigen expressed on a target diseased cell. Once expressed in T lymphocytes or other cells of the immune system, CARs are able to redirect a specific immune response against all cells that express the antigen they bind to. The most largely explored clinical application of CARs is the cancer immunotherapy, which consists in the infusion of cells of the immune system, such as T cells or NK cells, carrying a CAR targeted to a tumor antigen. Such cells are able to generate a strong antitumor response against cells expressing the antigen targeted by the CAR (Sadelain et al., Cancer Discovery. 2013. 3(4):388-98).

Several CARs candidates are now in clinical development and different successful cases are emerging. The Food and Drug Administration (FDA) recently approved tisagenlecleucel (Kymriah™), the first CAR T-cell therapy indicated for the treatment of patients up to 25 years of age affected by B-cell precursor acute lymphoblastic leukemia (ALL) that is refractory or in second or later relapse.

CARs are recombinant chimeric proteins that consist of an ectodomain responsible of antigen recognition, commonly derived from a single chain variable fragment (scFv), a spacer region, a transmembrane domain, and an endodomain that transmits activation and costimulatory signals to the cells in which they are expressed. Depending on the number of signaling domains, CARs are classified into 1 st generation (one), 2nd generation (two), or 3rd generation (three) CARs (Dotti et al., Immunol Rev. 2014 Jan; 257(1 ): 10.11 1 1/imr.12131 ).

The“spacer” or“hinge” region, is the connecting sequence between the ectodomain and the transmembrane domain. The most common sequence used as spacer is the constant immunoglobulin lgG1 hinge-CH2-CH3 Fc domain. There are indications that the spacer region may affect CAR T-cell function by affecting the length and flexibility of the resulting CAR (Dotti et al., Immunol Rev. 2014 Jan; 257(1 ): 10.1 1 1 1/imr.12131 ). For example in Guest et al. J Immunother. 2005 May-Jun; 28(3):203-1 1 , CARs containing four different scFvs were used to target four different human tumor-associated antigens i.e. the carcinoembryonic antigen (CEA), the neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. T-cell populations expressing all CARs resulted to be active against their respective targets, but while the anti-5T4 and anti-NCAM CARs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region, the anti-CEA and anti-CD19 CARs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Moreover, Hudececk et al. Clin Cancer Res. 2013 Jun 15; 19(12):3153-64 compared the activity of CARs targeted to ROR-1 containing extracellular lgG4-Fc spacer domains of different lengths i.e.: CH2-CH3 hinge (229 aminoacids), CH3 hinge (1 19 aminoacids) and short hinge (12 aminoacids). The authors demonstrated that the CARs targeted to ROR1 having the short hinge have stronger antitumor effect. While these and other studies indicate that the spacer region has an impact on antitumor effect, the results appear to be conflicting. Therefore, depending on the target to be engaged, it is necessary to identify on a case by case basis the scFv/spacer domain combination required to determine the optimal CAR design (Dotti et al., Immunol Rev. 2014 Jan; 257(1 ): 10.1 1 1 1/imr.12131 ).

WO 2016/042461 discloses CARs comprising spacer regions deriving from the extracellular domain of the human low affinity nerve growth factor receptor (LNGFR). Among the large number of possible sequences falling within this genus, WO 2016/042461 discloses four specific species: (i) the entire extracellular domain of LNGFR (i.e.: including the four TNFR-Cys domains and the serine threonine rich stalk); (ii) a mutated version of the entire extracellular domain of LNGFR (such mutation consisting of the deletion of a fragment of the fourth TNFR-Cys domain substituted by three specific aminoacids); (iii) a fragment including only the four TNFR-Cys domains of the extracellular domain of LNGFR; (iv) a fragment including the first three TNFR-Cys domains of the extracellular domain of LNGFR and a mutated version of the fourth (such mutation consisting of the deletion of a fragment of the fourth TNFR-Cys domain substituted by three specific aminoacids).

WO 2016/042461 does not disclose other possible species of spacer neither how the structure or the length of the spacer can affect the antitumor effect of the CAR.

The present invention addresses the need to improve the antitumor activity of a CAR including a spacer derived from the extracellular domain of LNGFR by defining the optimal structure of this region. SUMMARY OF THE INVENTION

The present invention relates to the development of a CAR targeted to tumor antigens, containing a spacer with optimal structure to improve the antitumor effect of the molecule. Particularly, the spacer of the CAR of the present invention has a structure consisting of a fragment derived from the extracellular domain of the LNGFR, composed by a first sequence including the four TNFR-Cys domains, linked to a second sequence consisting of the first 1 1 amino acids of the serine/threonine rich stalk. As used herein“the first 1 1 amino acids” is the fragment starting from 5’ end of the serine/threonine rich stalk.

WO 2016/042461 discloses CARs comprising a spacer region derived from the extracellular domain of the LNGFR. The patent application discloses some preferential examples of LNGFR derived spacers. There is no indication in the application of a CAR containing a spacer according to the present invention. Moreover, it is not mentioned that the definition of the optimal structure of the LNGFR-derived spacer may have an impact on the in vitro and in vivo antitumor activity of the CAR nor the way in which an LNGFR derived spacer may be modified to achieve an improvement of the antitumor effect.

It was surprisingly found that a CAR targeted to a tumor antigen containing a spacer derived from LNGFR with a structure according to the present invention has an improved in vitro and in vivo antitumor activity, with respect to CARs targeted to the same tumor antigen and carrying different LNGFR derived fragments as spacer. Such improvement was observed in the treatment of haematological as well as solid tumors.

STATEMENTS OF THE INVENTION

According to a first aspect of the present invention there is provided a Chimeric Antigen Receptor (CAR) comprising:

(i) an antigen-specific targeting domain that targets a tumor antigen;

(ii) a spacer domain consisting of a fragment of the human low affinity nerve growth factor receptor (LNGFR) composed by a first sequence including the four TNFR-Cys domains, linked to a second sequence consisting of the first 11 amino acids of the serine/threonine rich stalk;

(iii) a transmembrane domain;

(iv) optionally at least one costimulatory domain; and

(v) an intracellular signaling domain. Preferably the antigen-specific targeting domain of the CAR comprises an antibody or fragment thereof, more preferably a single chain variable fragment.

Preferably the antigen-specific targeting domain targets a tumour antigen selected from CD44, CD19, CD20, CD22, CD23, CD123, CS-1 , ROR1 , mesothelin, c-Met, PSMA, Her2, GD-2, CEA, MAGE A3 TCR.

Preferably the tumour antigen is isoform 6 of CD44 (CD44v6).

More preferably the single chain variable fragment consist of Sequence ID NO:8.

In another preferred embodiment the single chain variable fragment consist of Sequence ID NO:6

In one preferred embodiment the CAR contains a spacer having a length of 173 amino acids and including the four TNFR-Cys domains and the first 1 1 amino acids of the serine /threonine-rich stalk

In a most preferred embodiment the CAR contains a spacer consisting of Sequence ID NO:1 .

Preferably the CAR of the invention comprises a transmembrane domain selected from any one or more of a transmembrane domain of a zeta chain of a T cell receptor complex, CD28, CD8a, CD4, CD244 (2B4), Dap10, DAP12 or combinations thereof.

Preferably the CAR of the invention further comprises one or more costimulatory domain selected from the intracellular domain of CD28, CD137 (4-1 BB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 , Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, CD244 (2B4), DAP10, DAP12 or combinations thereof.

Preferably the CAR of the invention contains an intracellular signaling domain selected from a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.

In a most preferred embodiment the CAR of the invention contains a targeting domain comprising a single chain variable fragment, a transmembrane domain derived from CD28, a costimulatory domain derived from CD28, an intracellular domain derived from human CD3 zeta chain.

More preferably the CAR of the invention consists of Sequence ID NO:3. In another aspect of the present invention there is provided a polynucleotide encoding a CAR of the invention and as defined herein.

More preferably the polynucleotide encodes for a CAR that consists of the Sequence ID NO:3.

In a further preferred embodiment there is provided a polynucleotide that consists of the Sequence ID NO: 4.

In another aspect of the invention there is provided a vector comprising the polynucleotide encoding the CAR of the invention. Preferably the vector is a viral vector, more preferably the viral vector is selected from a retroviral vector or a lentiviral vector.

In another embodiment there is provided a viral vector comprising a promoter operably linked to a polynucleotide encoding the CAR of the invention and a further promoter operably linked to a polynucleotide encoding a suicide gene.

In a preferred embodiment the suicide gene is the Herpes Simplex Virus Thymidine Kinase, more preferably the polynucleotide encoding the suicide gene consists of Sequence ID NO:19.

In another embodiment there is provided a cell comprising a CAR, a polynucleotide or a vector of the present invention. Preferably the cell is a T-cell, a Natural Killer (NK) cell or an NK-T cell

In another embodiment there is provided a pharmaceutical composition comprising a cell of the present invention.

In another embodiment there is provided a CAR, a polynucleotide, a vector or a cell of the present invention for use in treating tumours Preferably the tumours are selected from haematological or solid tumours. In a preferred embodiment there is provided a CAR, a polynucleotide, a vector or a cell of the present invention for use in treating tumours that express CD44v6. More preferably, the tumours that express CD44v6 are selected from haematological or solid tumours.

In another aspect of the present invention there is provided a method for treating tumours comprising administering a CAR, a polynucleotide, a vector or a cell of the invention to a subject in need of the same. Preferably the tumours are selected from haematological or solid tumors.

DESCRIPTION OF THE DRAWINGS Figure 1. Schematic structure of CD44v6 wild-type N2 (CD44v6-NWN2) The picture shows three CAR molecules all containing a CD44v6 binding domain, the transmembrane and co-stimulatory domain of CD28, the intracellular domain of CD3 z chain, but carrying different LNGFR derived spacer. CD44v6-NWL contains the LNGFR wild-type long spacer (including the four TNFR-Cys domains and the entire serine threonine rich stalk), CD44v6- NMS contains the LNGFR mutated short spacer (including the first three TNFR-Cys domains and a mutated version of the fourth consisting in the deletion of a fragment of the fourth TNFR-Cys domain substituted by three specific amino acids), CD44v6-NWN2, an example of CAR according to the present invention, contains the LNGFR optimized spacer according to the invention (including the four TNFR-Cys domains and the first 1 1 amino acids of the serine threonine rich stalk). Black: scFv. White: co-stimulatory domain CD28; Light grey TNFR-Cys domain: Grey: Oϋ3z chain.

Figure 2. CD44v6 wild-type N2 (CD44v6-NWN2) CAR cloning (A) schematic representation of the retroviral vector construct LTK-SCD44v6-NWL, derived from Moloney murine leukaemia virus (MoMLV), and containing the transcriptional promoter 5’ viral long terminal repeat (5’LTR), the viral sequence including the packaging signal and gag (Y+ gag), the polynucleotyde coding for the suicide gene HSV-TKMut2, the transcriptional promoter SV40 (Simian Virus 40), the CD44v6-NWL and the 3’ viral long terminal repeat (3’LTR). (B) schematic representation of the Pml l-Not I fragment, including the 3’ end of the NWN2 spacer, the CD28 and CD3 zeta-chain sequences (C) schematic representation of the retroviral vector construct LTK-SCD44v6-NWN2 obtained by cloning the Pml l-Not I fragment of (B) in the Pml I- Not I sites of the plasmid LTK- SCD44v6-NWL (A). (D) Polynucleotide sequence of Pml l-Not I fragment of (B).

Figure 3. CD44v6 wild-type N2 (CD44v6-NWN2) CAR sequences Bold: CD44v6- specific single-chain fragment. Italics and underlined: LNGFR (UNIPROT database (P08138, TNR16JHUMAN, position 29-250)). Bold and underlined: CD28 (UNIPROT database (P10747, CD28_HUMAN, position 153-220)). Italics: CD3 zeta-chain (UNIPROT database (20963, CD3Z_HUMAN, position 31 -143, without Q at position 80))

Figure 4. LNGFR wild-type N2 (NWN2) spacer sequences Underlined: TNFR cysteine- rich domain number 1 . Bold: TNFR cysteine-rich domain number 2. Bold and underlined: TNFR cysteine-rich domain number 3. Italics: TNFR cysteine-rich domain number 4. Italics and underlined: first 1 1 amino acids of Serine/Threonine rich stalk

Figure 5. CAR T memory differentiation phenotype Percentage of stem cell memory (SCM), central memory (CM), effector memory (EM) and terminally differentiated effector memory (TEMRA) T cells was defined by FACS analysis for the three different CD44v6- CAR T cells.

Figure 6. CD44v6-NWN2 CAR T cells antigen specific activity. Percentage of CD107a+, IFNy+ or TNF-a+ CAR T cells was analysed, by FACS analysis, in the two subpopulation of CD4 and CD8 T cells co-cultured with K562 clone#10 (CD44v6+) cells, K562 clone#19 (CD44v6-) cells, BV173 (CD44v6-) cells, or PMA+ionomycin (A). The same four CAR T cells were tested with three different CD44v6+ tumor cell lines: MR232, MSR3 and lgrov-1 (B). Graphs are representative of n=2-3 independent experiments.

Figure 7. In vitro, CD44v6-NWN2 CAR T cytotoxic activity. CD44v6-CAR T cells were incubated with GFP positive target K562 clone#10 (CD44v6+) and K562 clone #19 (CD44v6-), at the three different ratio E:T indicated. Percentage of GFP+ target cell dead was analysed by FACS analysis. Graphs are representative of n=3 independent experiments.

Figure 8. In vivo, CD44v6-NWN2 CAR T antitumor activity. CD44v6-NWN2 T cells mediate antileukemia effects in a well-established disease model. Liver appearance and weight in the different treatment groups at sacrifice (6 weeks) are shown. Results from unpaired T test are shown when statistically significant (^*P<0.05, ^**P<0.01 , ^***P<0.001 ).

Figure 9. In vivo, CD44v6-NWN2 CAR T antitumor activity against haematological tumor. Antileukemia effects of CD44v6-NWN2 T cells was confirmed, in the same disease model of Fig.8, with two additional experiments. Results from the three independent experiments are shown as liver weight in the different treatment groups at sacrifice. Results from unpaired T test are shown when statistically significant.

Figure 10. In vivo, CD44v6-NWN2 CAR T antitumor activity against solid tumor. CD44v6-NWN2 T cells mediate antitumor activity against a solid tumor. Tumor volumes (mm³) in the different treatment groups (5 mice/group) were measured at the indicated time points. Mean value with standard error are shown. Results from unpaired T test between the CD44vs-NWL or the CD44v6-NWN2 group and its corresponding CD19-NWL control group are shown when statistically significant (^*P<0.05, ^**P<0.01 ).

DETAILED DESCRIPTION OF THE INVENTION

Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M., and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M., and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

Chimeric Antigen Receptors

The present invention relates to compositions for treating tumors based on an immunotherapeutic approach consisting in the administration of cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combinations thereof or natural killer cells or NKT cells) genetically modified to express a chimeric antigen receptor (CAR). CARs are recombinant chimeric molecules that produce a specific immune response, by combining an antibody-based specificity for a target antigen of interest (e.g., tumor antigen) with a T cell receptor-activating intracellular domain. CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.

The CARs of the present invention comprise:

(i) an antigen-specific targeting domain that targets a tumor antigen;

(ii) a spacer domain consisting of a fragment of the human low affinity nerve growth factor receptor (LNGFR) composed by a first sequence including the four TNFR-Cys domains, linked to a second sequence consisting of the first 1 1 amino acids of the serine/threonine rich stalk;

(iii) a transmembrane domain;

(iv) optionally at least one costimulatory domain; and

(v) an intracellular signaling domain. Antigen-specific targeting domain

The extracellular domain of the CAR of the present invention comprises an antigen- specific targeting domain that has the function of binding to a tumor antigen.

The antigen-specific targeting domain may be any naturally occurring, synthetic, semi- synthetic, or recombinantly produced molecule, protein, peptide or oligo peptide that specifically binds to the tumor antigen.

Examples of possible antigen-specific targeting domains include antibodies or antibody fragments or derivatives, synthetic or naturally occurring ligands of the targeted receptor including molecules, binding or extracellular domains of receptors or binding proteins.

In a preferred embodiment, the antigen-specific targeting domain is, or is derived from, an antibody. An antibody is a protein, or a polypeptide sequence derived from an immunoglobulin able to bind with an antigen. Antibody as herein used includes polyclonal or monoclonal, multiple or single chain antibodies as well as immunoglobulins, whether deriving from natural or recombinant source. Methods to identify antibodies able to bind a selected protein are largely known in the art and include phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies.

An antibody-derived targeting domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen. Examples include a variable region (Fv), a complementarity determining region (CDR), a Fab, a single chain antibody (scFv), a heavy chain variable region (VH), a light chain variable region (VL) and a camelid antibody (VHH).

In a preferred embodiment, the binding domain is a single chain antibody (scFv). The scFv may be murine, human or humanized scFv.

"Complementarity determining region" or "CDR" of an antibody or antigen-binding fragment are highly variable domains in the heavy chain or in the light chain that determine specific antibody binding. There are three CDRs (CDR1 , CDR2 and CDR3), arranged non-consecutively, on the amino acid sequence of each of the heavy and light chain.

"Heavy chain variable region" or "VH" refers to the fragment of the heavy chain of an antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs. "Light chain variable region" or "VL" refers to the fragment of the light chain of an antibody that contains three CDRs interposed between framework regions.

"Fv" refers to the smallest fragment of an antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

"Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence.

As used herein the term tumor antigen includes antigens expressed on tumor cells including biomarkers or cell surface markers that are found on tumor cells and are not substantially found on normal tissues, or restricted in their expression in non-vital normal tissues. The term tumor antigen includes antigen expressed on solid tumors and/or hematological tumors. As used herein the term solid tumor means an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. As used herein the term hematological tumors includes malignancies, also called blood cancers that begins in blood-forming tissue such as the bone marrow, or in the cells of the immune system. Basically hematological malignancies originate from the proliferation and the survival of the two major blood cell lineages: myeloid and lymphoid cell lines. Examples of hematologic cancer are leukemia, lymphoma, and multiple myeloma.

Examples of tumor antigens that may be targeted by the CAR of the invention include but are not limited to any one or more of carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, ROR1 , mesothelin, c-Met, GD-2, and MAGE A3 TCR, 4-1 BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD152, CD200, CD22, CD19, CD22, CD123, CD221 , CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44, CD44 v6, CD51 , CD52, CD56, CD74, CD80, CS-1 , CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1 , GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatterfactor receptor kinase, IGF-1 receptor, IGF-I, IgGI, L1 -CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1 , integrin anb3, MORAb-009, MS4A1 , MUC1 , mucin CanAg, N-glycolylneuraminic acid, NPC-1 C, PDGF-Ra, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1 , SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-b, TRAIL-R1 , TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1 , VEGFR2 or vimentin.

In a preferred embodiment the antigen specific targeting domain targets the receptor CD44v6.

In a preferred embodiment the antigen specific targeting domain in the CAR of the invention is an anti-CD44v6 scFv.

The anti-CD44v6 scFv may be derived from the anti-CD44v6 antibodies disclosed in US 6’972’324.

An exemplary antigen-specific targeting domain is a CD44v6-specific single-chain fragment (scFv) such as described in Casucci M et al, Blood, 2013, Nov 14;122(20):3461 - 72.

In a preferred aspect of the invention the antigen-specific targeting domain is the anti- CD44v6 specific scFv having the following sequence:

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWLQQKPG QAPRILIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGG GTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFT FSSYDMSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSL RAEDTAVYYCARQGLDYWGRGTLVTVSS (SEQ ID NO:6 )

In one embodiment, the CD44v6-specific single-chain fragment comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO:6.

In a further preferred embodiment, the light chain variable region and the heavy chain variable region of the CD44v6-specific single chain fragments are connected to one another via a peptide linker having the following sequence GGGGSGGGGS (4GS2; SEQ ID NO:7). Such CD44v6-specific single chain fragment (CD44v6-4GS2) has the following sequence:

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWLQQKPG QAPRILIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGG GTKVEIKRGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVR QAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCA RQGLDYWGRGTLVTVSS (SEQ ID NO:8). Spacer domain

The CAR of the invention comprises an extracellular spacer domain that connects the antigen-specific targeting domain to the transmembrane domain. The spacer of the invention has an optimal structure that generates an improvement of the antitumor effect of cells genetically modified to express the CAR.

The spacer of the CAR of the invention is a fragment derived from the extracellular domain of human low affinity nerve growth factor (LNGFR). According to the present invention, the extracellular domain of LNGFR does not include the signal peptide and, therefore, it comprises amino acids 29-250 of LNGFR or a derivative thereof.

The sequence of the extracellular domain of LNGFR excluding the signal peptide is reported below:

Extracellular domain of the human LNGFR (UNIPROT # P08138, TNR16_HUMAN, position 29-250)

KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDWSATEPCKP CTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDK QNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITR STPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN (SEQ ID NO:9)

The extracellular domain of LNGFR comprises 4 TNFR-Cys domains (TNFR-Cys 1 , TNFR-Cys 2, TNFR-Cys 3 and TNFR-Cys 4) and a Serine/Threonine rich stalk. Sequences of the domains are exemplified below:

TNFR-Cvs 1 . (SEQ ID NO:1 Q):

ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC

TNFR-Cvs 2. (SEQ ID NO:1 1 Y.

PCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVC TNFR-Cvs 3. (SEQ ID NO:12):

RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVC TNFR-Cvs 4. (SEQ ID NO:13):

ECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC

Serine Threonine rich stalk (SEQ ID NO:14): IPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPVVTRGTTD

N.

The CARs of the present invention contain a spacer characterized by an optimal structure that causes an improvement of the antitumor effect of cells genetically modified to express these CAR molecules. Such optimal spacer has a structure consisting of a fragment derived from the extracellular domain of the LNGFR, composed by a first sequence including the four TNFR-Cys domains, linked to a second sequence consisting of the first 1 1 amino acids of the serine/threonine rich stalk.

The sequence of the first 1 1 amino acids of the serine/threonine rich stalk is here reported: IPGRWITRSTP (SEQ ID NO: 20).

Particularly, the spacer according to the present invention is a fragment derived from the extracellular domain of the LNGFR composed by, from 5’ to 3’ direction, a first sequence including the four TNFR-Cys domains and a second sequence consisting of the first 1 1 amino acids of the serine/threonine rich stalk, wherein the 3’ end of the first sequence is linked to the 5’ end of the second sequence to form the LNGFR derived fragment that constitutes the spacer.

In a preferred embodiment the spacer has a sequence composed of 173 aminoacids and includes the four TNFR-Cys domains and the first 1 1 amino acids of the serine /threonine- rich stalk. In a most preferred embodiment the spacer of the CAR of the present invention is the LNGFR wild-type N2 (NWN2) consisting of the polypeptide sequence disclosed in Figure 4 (Sequence ID NO:1 ).

In another preferred embodiment the spacer LNGFR wild-type N2 (NWN2) incorporated in the CAR of the present invention is encoded by the polynucleotide sequence disclosed in Figure 4 (Sequence ID NO:2).

WO 2016/042461 does not disclose CARs containing a spacer with the same optimal structure, length or sequence as those according to the present invention. Moreover, there is no indication in this application about how to modify the structure of the LNGFR derived spacer in order to achieve an improved antitumor effect of a CAR.

Transmembrane domain

The CAR of the invention comprises a transmembrane domain between the spacer domain and the signaling domain. The transmembrane domain may be derived eitherfrom a natural or from a synthetic source. The domain deriving from natural sources may comprise the transmembrane sequence from any membrane-bound or transmembrane protein including any of the type I, type II or type III transmembrane proteins. Transmembrane regions that may be used in the CAR of the present invention may be derived from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD244 (2B4), DAP10 or DAP12. The domain deriving from synthetic source will comprise predominantly hydrophobic sequence including residues such as leucine and valine.

Prior art discloses examples of transmembrane domain that can be used in a CAR such as : 1 ) the CD28 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Brentjens et al, CCR, 2007, Sep 15; 13(18 Pt 1 ):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.); 2) the 0X40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933- 41 ); 3) the 41 BB TM region (Brentjens et al, CCR, 2007, Sep 15; 13(18 Pt 1 ):5426-35); 4) the CD3 zeta TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Savoldo B, Blood, 2009, Jun 18; 113(25):6392-402.); 5) the CD8a TM region (Maher et al, Nat Biotechnol, 2002, Jan;20(1 ):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep 15; 13(18 Pt 1 ):5426-35; Milone et al, Mol Ther, 2009, Aug; 17(8): 1453-64).

Further examples of transmembrane domain may be applied by the skilled in the art to the CAR of the present invention.

In one embodiment the CAR of the invention comprises a transmembrane domain selected from any one or more of a transmembrane domain of a zeta chain of a T cell receptor complex, CD28, CD8a, CD4 or combinations thereof.

Preferably the transmembrane domain is derived from CD28.

More preferably the transmembrane domain of CD28 consists of sequence F W V LV W G G V LACY S L L VTVAF 11 F WV (SEQ ID NO: 15)

In one embodiment the transmembrane and intracellular signaling domain comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO:15.

Co-stimulatory domain

The CAR of the present invention may include, in the cytoplasmic tail, one or more co- stimulatory domains. Such domains may consist of the intracellular signaling domain of one or more co-stimulatory protein receptors (e.g., CD28, 41 BB, ICOS). The function of the co-stimulatory domain is to provide additional signals to the cells thus enhancing cell expansion, cell survival and development of memory cells. The CAR of the present invention may comprise one or more co-stimulatory domain selected from the group consisting of the intracellular domain of members of the TNFR super family, CD28, CD137 (4-1 BB), CD134 (0X40), DapIO, CD27, CD2, CD5, I CAM-1 , LFA-1 , Lck, TNFR-1 , TNFR-II, Fas, CD30, CD40, CD244 (2B4), DAP10, DAP 12 or combinations thereof. Co-stimulatory domains from other proteins may also be used with the CAR of the invention. Further examples of co-stimulatory domains may be employed by the skilled in the art in the CAR of the present invention.

In one embodiment the costimulatory domain is derived from the intracellular domain of CD28

In a preferred embodiment the transmembrane and costimulatory domains are both derived from CD28. In one embodiment the transmembrane and intracellular costimulatory domain comprise the sequence below:

Transmembrane and intracellular portion of the human CD28 (UNIPROT: P10747, CD28_HUMAN, position 153-220)

FWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRS (SEQ ID NO:16)

In one embodiment the transmembrane and costimulatory domains comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 16.

In one embodiment the intracellular costimulatory domain of the CAR is derived from the intracellular domain of CD28 and comprises the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:17).

Intracellular signaling domain

The CAR of the invention may also comprise an intracellular signaling domain. This domain may be cytoplasmic, transmits the activation signal and direct the cell to perform its specialized function. Examples of intracellular signaling domains include, but are not limited to, z chain of the T-cell receptor or any of its homologs (e.g., h chain, FceRl y and b chains, MB1 (Iga) chain, B29 (Ig3) chain, etc.), CD3 polypeptides (D, d and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell signal transduction, such as CD2, CD5 and CD28. The intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. In a preferred embodiment signaling domain comprises the intracellular signaling domain of human CD3 zeta chain.

In one embodiment the intracellular signaling domain of human CD3 zeta chain comprises the following sequence:

UNIPROT: P20963, CD3Z_HUMAN, position 31 -143

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO:18)

In one embodiment, the intracellular signaling domain comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO:18.

Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

In a preferred embodiment the spacer of the CAR of the present invention is the CAR CD44v6-NWN2 consisting of the polypeptide sequence disclosed in Figure 3 (Sequence ID NO:3)

In another preferred embodiment the CAR CD44v6-NWN2 is encoded by the polynucleotide sequence disclosed in Figure 3 (Sequence ID NO:4).

Key advantages of the invention

The present invention relates to an effective immunotherapy for the treatment of tumors consisting in the administration of cells of the immune system, such as T cells, NK cells or NK-T cells genetically modified to express a CAR containing a spacer with an optimal structure. The structure of the spacer causes an improvement to the antitumor effect of the CAR. It was surprisingly found that CARs targeted to a tumor antigen containing a spacer according to the present invention have stronger antitumor effect, both in vitro and in vivo, as compared to CARs targeted to the same tumor antigen and containing spacer derived from LNGFR, but having different structure and length. As shown in the examples, immune cells, genetically modified with a CAR containing a spacer according to the present invention, result to be more effective in the in vivo treatment of hematological tumors (figures 8 and 9 and example 6) an as well as of solid tumors (figure 10 and example 7).

Polynucleotides and vectors According to a further aspect of the invention, there is provided a polynucleotide encoding the chimeric antigen receptor described herein.

The term polynucleotide as used herein is defined as a polymer of nucleotides, which form a DNA or RNA fragment. One skilled in the art has the general knowledge that the 64 codons of the eukaryotic genetic code encode for only the 20 naturally-occurring amino acids and 3 stop codons, rendering the genetic code degenerate with respect to the encoding of amino acid residues. In view of the degeneracy of the genetic code, different polynucleotide sequences may encode the same polypeptide. By applying routine technique, it is possible to elaborate different polypeptide sequences that contain nucleotide substitutions and still encode for the polypeptide of the invention. Methods to modify polynucleotides are known in the art and may be applied by the skilled man in order to improve the polypeptide’s activity or stability, or to avoid splicing phenomenon.

Polynucleotides of the invention may be obtained by any means available in the art, including, without limitation, recombinant means, i.e. the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology, PCR™, and the like, and by synthetic means. The polynucleotides used in the present invention may be codon-optimised. Codon optimization is a technique known in the art (WO 1999/41397 and WO 2001/79518) aimed to increase or decrease the protein expression in a cell of interest. Multiple codons can often code for the same amino acid, but the preferential use of codons is different in each organism. Therefore, in each organism, t-RNAs corresponding to certain codons are more abundant than others. A polynucleotide may be synthetized or modified to increase protein expression in a host cell, by using codons matching with the most abundant degenerate tRNAs without affecting the amino acid sequence of the protein.

Vectors

In another aspect there is provided a vector comprising the polynucleotide of the invention.

A vector is a molecule used to deliver a polynucleotide into a cell. Numerous vectors are known in the art and may be employed in the present invention including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, mRNA molecules (e.g. in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses (i.e. viral vectors). Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, baculoviral vectors, herpes simplex viral vectors, retroviral vectors or lentiviral vectors. The vectors may include a promoter operably linked polynucleotide of the invention. The promoter modulates the expression of a physically adjacent polynucleotide. The expression operably linked refers to the functional linkage between a regulatory sequence (e.g. the promoter) and a polynucleotide sequence resulting in expression of the latter. Any kind of promoter may be employed in the present invention including but not limited to constitutive, inducible, tissue specific or synthetic promoters.

Vectors comprising polynucleotides of the invention may be introduced into cells using a variety of techniques known in the art, such as transformation, transfection and transduction. Several techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation.

Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell.

Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.

In a preferred aspect the vector comprising the polynucleotide of the invention is a viral vector, more preferably the viral vector is selected from a retroviral vector or a lentiviral vector.

Retroviral vectors

In one embodiment, polynucleotide of the invention are delivered to target cells using retroviral vectors. These vectors are commonly used and known to integrate a polynucleotide of interest into the genome of the target cell. Examples of retroviral vectors that may be employed include and are not limited to murine leukemia virus (MLV), human immunodeficiency virus (HIV-1 ), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A- MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV) and all other retroviridiae including lentiviruses. A detailed list of retroviruses may be found in Coffin et at., 1997, “retroviruses”, Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

Retroviral vectors derive from retroviruses. All retroviruses possess two copies of a single- stranded RNA and contain three major coding regions of the virion proteins gag, pol, and env. Simple retroviruses carry only this elementary information. On the contrary, the RNA genome of complex retroviruses contain coding sequence of additional regulatory proteins such as rev or RRE of HIV retroviruses.

The process of retroviral entry starts when the viral surface glycoproteins bind to a receptor expressed on the surface of the target cell. A series of molecular events follow that cause conformational changes in the viral glycoprotein, thus mediating the fusion between cell and viral membranes and allowing introduction of the genetic material of the virus into the host-cell cytoplasm. The process of reverse transcription starts after the entry of the RNA genome, and generates, in the cytoplasm, a double stranded DNA. Such DNA is co-linear with its RNA template, but it contains terminal duplications known as the long terminal repeats (LTRs). Viral DNA enter the nucleus and integrates into the genome of the host cells. Once integrated, the viral DNA is called proviral DNA or provirus. LTRs are involved in proviral integration and act as enhancer/promoter of viral genes.

Recombinant retroviral vector used for gene delivery are replication defective because their genome does not contain or contain non-functional variants of gag pol and env genes. The removed portions of the viral genome can be replaced by a polynucleotide of interest thus obtaining a virus that still integrates in the host cells, where it allows the expression of the polynucleotide of interest, but is not able to propagate itself due to the lack of structural proteins.

Manufacturing of retroviral vectors is performed using retroviral packaging cell lines in which viral Gag/Pol and Env proteins are encoded on separate helper expression plasmids, which lack all other retroviral components including the retroviral packaging signal or contain non-functional versions of them. Expression of viral proteins into the packaging cell line may be transient or stable. Examples of stable packaging cell lines are disclosed in the art such as GP+envAM12 (US 5,278,056) or PG13 (US 5,470,726) or the stable packaging cell line for lentiviral vectors disclosed in WO 2012/028681 . Production of retroviral vector is performed by delivering into the packaging cell line a recombinant vector carrying a packaging signal (y), the primer binding site (PBS) the long terminal repeats (LTR), and a polynucleotide of interest instead of genes encoding for structural and enzymatic retroviral proteins. The retroviral vector can be targeted to particular cells by modifying the retroviral Env protein. Examples of suitable env genes include, but are not limited to, VSV-G, a MLV amphotropic env such as the 4070A env, the RD1 14 feline leukaemia virus env or haemagglutinin (HA) from an influenza virus GALV env.

MLV

Preferably, the retroviral vector used in the present invention is a Murine Leukemia Virus (MLV) vector. Retroviral vectors derived from the amphotropic Moloney murine leukemia virus (MLV-A) are commonly used in clinical protocols worldwide. These viruses use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The genes are then maintained for the lifetime of the cell. Gene activity on MLV based constructs are easy to control and can be effective over a long time. Clinical trials conducted with these MLV -based systems have shown them to be well tolerated with no adverse side effects.

An example of an MLV vector for use in the present invention is a vector derived from SFCMM-3, which carries both the suicide gene HSV-TK and the marker gene ALNGFR (Verzeletti 98, Human Gene Therapy 9:2243). The original vector used in the preparation of SFCMM-3 is LXSN (Miller et al. Improved retroviral vectors for gene transfer and expression. BioTechniques 7:980-990, 1989) (Genebank accession #28248). LXSN vector was modified by the insertion of the HSV-TK gene into the unique Hpa I site (“blunt cut”), removal of the neo gene by digestion with Hind III and Nae I, and insertion of the cDNA encoding ALNGFR in this site.

In one embodiment of the invention there is provided a viral vector comprising a promoter operably linked to a polynucleotide encoding the CAR of the invention and a further promoter operably linked to a polynucleotide encoding a suicide gene. For example, the SFCMM-3 vector may be used since it contains two expression promoters i.e. the transcriptional promoter 5’ viral long terminal repeat (5’LTR) and transcriptional promoter SV40. Each of them may be used to independently to express the suicide gene and the CAR of the present invention.

In a preferred aspect of the invention the suicide gene is the Herpes Simplex Virus Thymidine Kinase (HSV-TK), more preferably a no splicing variant of the HSV-TK gene such as those disclosed in WO 2015/123912. In a further preferred embodiment the suicide gene is the HSV-TK Mut2, encoded by the following polynucleotide sequence: atggcttcgtacccctgccatcaacacgcgtctgcgttcgaccaggctgcgcgttctcgcggccatagcaaccgacgtacg gcgttgcgccctcgccggcagcaagaagccacggaagtccgcctggagcagaaaatgcccacgctactgcgggtttata tagacggtcctcacgggatggggaaaaccaccaccacgcaactgctggtggccctgggttcgcgcgacgatatcgtctac gtacccgagccgatgacttactggcaggtgctgggggcttccgagacaatcgcgaacatctacaccacacaacaccgcc tcgaccagggcgagatatcggccggggacgcggcggtggtaatgacaagcgcccagataacaatgggcatgccttatg ccgtgaccgacgccgttctggctcctcatgtcgggggggaggctgggagttcacatgccccgcccccggccctcaccctc atcttcgaccgccatcccatcgccgccctcctgtgctacccggccgcgcgataccttatgggcagcatgaccccccaggcc gtgctggcgttcgtggccctcatcccgccgaccttgcccggcacaaacatcgtgttgggggcccttccggaggacagaca catcgaccgcctggccaaacgccagcgccccggcgagcggcttgacctggctatgctggccgcgattcgccgcgtttacg ggctgcttgccaatacggtgcggtatctgcagggcggcgggtcgtggtgggaggattggggacagctttcggggacggcc gtgccgccccagggtgccgagccccagagcaacgcgggcccacgaccccatatcggggacacgttatttaccctgtttcg ggcccccgagttgctggcccccaacggcgacctgtataacgtgtttgcctgggccttggacgtcttggccaaacgcctccgt cccatgcacgtctttatcctggattacgaccaatcgcccgccggctgccgggacgccctgctgcaacttacctccgggatgg tccagacccacgtcaccaccccaggctccataccgacgatctgcgacctggcgcgcacgtttgcccgggagatggggga ggctaactga (Sequence ID NO: 19).

Lentiviral vector

In one embodiment, the vector of the present invention may be a lentiviral vector. A lentiviral vector as used herein refers to a genus within the family of retroviral vectors. Lentiviral vectors have a unique property among the retroviral vectors since they are able to infect non dividing cells. Lentiviral vectors offer the means to achieve significant levels of gene transfer in vivo.

A detailed list of lentiviruses may be found in Coffin et al (“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human acquired-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype “slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

An exemplary lentiviral vector for use in the present invention is the vector described in Amendola et al, Nat Biotechnol. 2005 Jan;23(1 ):108-16 that includes a bidirectional promoter for the expression of two coding sequences in opposite orientation, thus enabling efficient dual gene transfer. The bidirectional promoter is composed by minimal core promoter elements from the human cytomegalovirus (mCMV), joined upstream and in opposite orientation, to an efficient promoter derived from the human phosphoglycerate kinase (PGK) or polyubiquitin UBI-C gene. This lentiviral vector incorporating the bidirectional promoter may be used to express the CAR of the present invention and a suicide gene in one single construct.

Cells

The invention also provides genetically engineered cells, which comprise and stably express the CAR of the invention.

Genetically engineered cells, which may comprise and express the CARs of the invention include, but are not limited to, T-cells, naive T cells, stem cell memory T cells, central memory T cells, effector memory T cells, natural killer cells, NK-T cells, hematopoietic stem cells and/or cells capable of giving rise to therapeutically relevant progeny. In an embodiment, the genetically engineered cells are autologous cells. By way of example, individual T-cells of the invention may be CD4+/CD8-, CD4-/CD8+, CD4-/CD8- or CD4+/CD8+. The T-cells may be a mixed population of CD4+/CD8- and CD4-/CD8+ cells or a population of a single clone.

Genetically modified cells may be produced by stably transfecting cells with DNA encoding the CAR of the invention.

Various methods produce stable transfectants which express the CARs of the invention. In one embodiment, a method of stably transfecting and re-directing cells is by electroporation using naked DNA. By using naked DNA, the time required to produce redirected cells may be significantly reduced. Additional methods to genetically engineer cells using naked DNA encoding the CAR of the invention include but are not limited to chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle- based methods (e.g., impalefection, using a gene gun and/or magnetofection). The transfected cells demonstrating presence of a single integrated un-rearranged vector and expression of the CAR may be expanded ex vivo. In one embodiment, the cells selected for ex vivo expansion are CD8+ and demonstrate the capacity to specifically recognize and lyse antigen-specific target cells.

Viral transduction methods may also be used to generate redirected cells, which express the CAR of the invention.

Stimulation of the T-cells by an antigen under proper conditions results in proliferation (expansion) of the cells and/or production of IL-2. The cells comprising the CAR of the invention will expand in number in response to the binding of one or more antigens to the antigen-specific targeting regions of the CAR. The invention also provides a method of making and expanding cells expressing a CAR. The method may comprise transfecting or transducing the cells with the vector expressing the CAR after stimulating the cells with:

1 ) polyclonal stimuli such as cell-free scaffolds, preferably optimally-sized beads, containing at least an activating polipeptide, preferably an antibody, specific for CD3 alone or in combination with an activating polipeptide, preferably an antibody, specific for CD28;

2) tumor cells expressing the target antigen; 3) natural or artificial antigen presenting cells, and culturing them with cytokines including IL-2, IL-7, IL-15, IL-21 alone or in combination.

Therapeutic methods and pharmaceutical compositions

There are provided herein methods for treating a tumor associated with the antigen targeted by the CAR of the invention in a subject in need thereof. The method comprises administering an effective amount of the CAR, polynucleotide or vector encoding the CAR, or a cell expressing said CAR so as to treat the tumor associated with the antigen in the subject.

In a preferred embodiment there are provided herein methods for treating tumours that express CD44v6 using the CAR of the invention. The method comprises administering an effective amount of the CAR, polynucleotide or vector encoding the CAR, or a cell expressing said CAR so as to induce a specific immune response against tumor cells expressing CD44v6.

There is also provided a pharmaceutical composition comprising a CAR of the invention. The CAR of the invention in the composition may be any one or more of a polynucleotide encoding the CAR, a vector encoding the CAR, a protein comprising the CAR or genetically modified cells comprising the CAR.

A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent together with a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

EXAMPLES

Example 1 - Materials and Methods

LNGFR-spaced CD44v6 CAR constructs

The CAR constructs CD44v6-NWL and CD44v6-NMS are disclosed in WO 2016/042461 that includes detailed description of the sequences of such constructs. cDNA encoding the CARs CD44v6-NWL and CD44v6-NMS were purchased from the originators. The CAR construct CD44v6-NWN2 generated according to the present invention includes an optimized spacer structure.

A schematic version of the structures of the CAR constructs NWL, NMS and NWN2 is shown in figure 1 .

CD44v6-NWL consists of a CD44v6 binding domain, the LNGFR wild-type long spacer (including the four TNFR-Cys domains and the entire serine threonine rich stalk), the transmembrane and co-stimulatory domain of CD28 and the intracellular domain of CD3 ,c a\n.

CD44v6-NMS consists of a CD44v6 binding domain, the LNGFR mutated short spacer (a fragment including the first three TNFR-Cys domains of the extracellular domain of LNGFR and a mutated version of the fourth (such mutation consisting of the deletion of a fragment of the fourth TNFR-Cys domain substituted by three specific aminoacids) the transmembrane and co-stimulatory domain of CD28 and intracellular domain of CD3 z chain.

CD44v6-NWN2, an example of CAR according to the present invention, consists of a CD44v6 binding domain, the LNGFR optimized spacer LNGFR wild-type N2 (NWN2) (fragment of 173 amino acids in length including the four TNFR-Cys domains and the first 1 1 amino acids of the serine threonine rich stalk), the transmembrane and co-stimulatory domain of CD28 and intracellular domain of CD3 z chain.

The sequences of CD44v6-NWN2 are shown in figure 3 (Sequence ID NO: 4 nucleotide sequence, Sequence ID NO: 3 peptide sequence).

The sequences of optimal spacer LNGFR wild-type N2 (NWN2) are shown in figure 4 (Sequence ID NO: 2 nucleotide sequence, Sequence ID NO: 1 peptide sequence)

Cell lines

K562 myelogenous leukemia cells (ATCC CCL-243), BV173 lymphoblastoid cells (Pegoraro et al, J Natl Cancer Inst. 70:447, 1983), MR232 lung carcinoma, MSR3 melanoma (Lionello et al, Cancer Immunol Immunother. 56:1065, 2007), and lgrov-1 ovarian adenocarcinoma (Bernard J Cancer Res 45:4970, 1985), were cultured in RPMI 1640 (Lonza Biowhittaker) supplemented with 10%FBS (HyClone).

To generate CD44v6 positive cells, K562 cells were transduced with a retroviral vector expressing the v6 isoform of the CD44 gene. The transduced cells were immunoselected and cloned in limiting dilution conditions. The K562 clonel O (CD44v6+) and the K562 clone 19 (CD44v6-) were used for the study.

To generate green fluorescent protein (GFP) positive target cells, K562 clonel O (CD44v6+) and K562 clone 19 (CD44v6-) were transduced with a GFP-lentiviral vector at different MOI in order to obtain the transduction of the entire cell population. Green fluorescent protein (GFP) positive target cells were used in to perform the cytotoxic T cell killing as disclosed below.

Flow cytometry staining

We used mAbs for human CD3 (clone SK7), CD4 (clone SK3), CD8 Pacific Blue (clone RPA-T8), CD271 (clone H1100), CD45RA (clone L48), CD62L (clone SK1 1 ), IFN-g (clone B27), and TNF-a (clone Mab1 1 ) from BD Bioscience, anti-CD44v6 (clone #2F10) from R&D System, CD107a (clone H4A3) was from Miltenyi, and Fixable Viability stain 510 and 7-Amino-Actinomycin D (7-AAD) from BD Biosciences.

Transduction and culture conditions of T cells

T cells were activated with cell-sized CD3/CD28-beads (ClinExVivo, Invitrogen) plus IL- 7/IL 15 (5 ng/ml, Peprotech) and transduced with retroviral vectors, on RetroNectin pre- coated dish (Takara Bio Inc) at day 2 after stimulation. At day 3, beads were removed and T cells cultured in Xvivo-15 (Lonza Biowhittaker) supplemented with 3% human AB plasma (Kendrion) and IL-7 and IL-15. Surface expression of CD44v6-CAR constructs (NWL, NWN2 and NMS) was analysed at day6 using LNGFR-specific mAbs from BD Bioscience (Clone: C40-14579). At day6 after expression analysis, the cells were immunoselected with specific antibody.

CD44v6-CAR T cells can be immunoselected with three different methods:

1 ) LNGFR-specific mAb C40-14579 plus anti-PE paramagnetic beads (Miltenyi), that allows the isolation of CD44v6-NWL, CD44v6-NWN2 and CD44v6-NMS T cells.

2) LNGFR-specific mAb ME20.4 plus sheep anti-mouse dynabeads (ClinExVivo, Invitrogen), that allows the isolation of CD44v6-NWL and CD44v6-NWN2 T cells.

3) LNGFR-specific mAb ME20.4-coated paramagnetic beads (Miltenyi), that allows the isolation of CD44v6-NWL T cells.

The most used methods to prepare cells for in vitro and in vivo experiments were method #1 for CD44v6-NWN2 T cells and method #3 for CD44v6-NWL T cells.

CAR T memory phenotype analysis

Immunoselected T cells, expressing CD44v6-NWL, CD44v6-NWN2 and CD44v6-NMS, were cultured until day 10, then analysed for memory T cells differentiation phenotype. The percentage of stem cell memory (SCM; CD45RA+/CD62L+), central memory (CM; CD45RA-/CD62L+), effector memory (EM; CD45RA-/CD62L-) and terminally differentiated effector memory (TEMRA; CD45RA+/CD62L-) T cells was defined by FACS analysis.

Potency assay

The frequency of degranulating (CD107a+) or cytokine producing (TNF-a+ or IFNy+) CAR T cells was quantitated by a flow cytometry-based potency assay. CAR T cells (0.2x10⁶ cells/condition) were cultured in RPMI+10% FBS, alone or with the different target cells at E:T ratio of 1 :1 . Cells were stimulated with PMA (10 ng/ml; BD Biosciences) and ionomycin (1 pg/ml; Biolegend) as a positive control for T cells functionality. After 5 hours of incubation in the presence of CD107a Ab, monensin and brefeldin (Sigma), cells were stained with CD3, CD4, CD8 fluorescent Abs and Fixable Viability stain 510. Samples were than fixed and permeabilized according to manufacturer’s instruction (BD Cytofix/Cytoperm™; BD Biosciences) for intracellular IFN-g and TNF-a staining, and analysed by flow cytometry. The percentage of viable positive T cells co-cultured with the different targets or PMA+ionomycin (after subtraction of the percentage of positive T cells incubated alone) was analyzed in the two subpopulation of CD4 and CD8 T cells.

Cytotoxic T cell killing assay

Cytotoxic activity of CAR T cells was analysed by a flow cytometry measure of dead GFP+ target cells. CAR T lymphocytes were cultured in Xvivo-15, alone or with GFP-positive K562 clone#10 (CD44v6+) and K562 clone #19 (CD44v6) cells, at E:T ratio of 0.5:1 , 1 :1 and 2:1 (target cells 0.05 x10⁶ cells/condition). After 6 hours of incubation, surviving target cells were analysed by FACS after 7-Amino-Actinomycin D (7-AAD) live death staining. Cytotoxic activity was calculated as follows:

Cytotoxic activity= ((% dead target cells in the sample - % spontaneously dead target cells in the control)/(100 - % spontaneously dead target cells in the control)) X 100 (Allegra et al. Cytometry 2006).

In vivo xenograft models

To test the antitumor activity of CAR T in vivo, two different xenograft models were used. For haematological cancer, a well-established model of AML was used: NSG mice were infused intravenously with 1.5x10⁶ CD44v6+ THP-1 leukemia cells and, after two weeks, treated with the different CD44v6 CAR T cells (NWL, NWN2, NMS) or with T cells expressing a control CD19-NWL CAR. Two doses given 24 hrs apart for a total of 10- 13x10⁶ cells were infused intravenously into 3-6 mice per group. Liver appearance and weight in the different treatment groups were evaluated at sacrifice (6 weeks).

To evaluate CAR T killing activity against solid tumor, NSG mice were subcutaneously injected with 0.3x10⁶ CD44v6+ human ovarian cancer cells (IGROV-1 ) and, after 6 days, treated with the different CD44v6 CAR T cells (NWL, NWN2) or with T cells expressing a control CD19-NWL CAR. One dose of 4.5x10⁶ cells was infused intravenously into 5 mice per group. Tumors were measured by caliper and tumour volume was calculated using the equation l^2*L where I is the shortest diameter and L is the longest.

Example 2 - Generation of CD44v6-NWN2 construct.

The sequences of the LNGFR-based spacers were derived from the extracellular portion of the human low-affinity nerve growth factor receptor (LNGFR), excluding the signal peptide (P08138, TNR16_HUMAN). The wild-type long (NWL) design contains both the four TNFR cysteine-rich domains and the serine/threonine-rich stalk. The NWN2 spacer contains the four TNFR cysteine-rich domains and 1 1 aa (i.e. IPGRWITRSTP) of the serine/threonine-rich stalk. It was obtained from the CD44v6-NWL by deletion of aa 428 to aa 476. As a result of this modification, the spacer region still retains the binding ability to the antibody anti-LNGFR-ME20.4.

The polynucleotide encoding the CD44v6-NWL construct was cloned in the retroviral construct SFCMM-3, (Verzeletti 98, Human Gene Therapy 9:2243) together with the no- splicing variant of the Herpes simplex virus Thymidine Kinase HSV-TKMut2. The resulting retroviral construct LTK-SCD44v6-NWL (Figure 2A) comprises the transcriptional promoter 5’ viral long terminal repeat (5’LTR), the viral sequence including the packaging signal and gag (Y+ gag), a cDNA encoding for the suicide gene HSV-TKMut2, transcriptional promoter SV40 (Simian Virus 40), the polynucleotide encoding CD44v6- NWL and the 3’ viral long terminal repeat (3’LTR).

A 888bp Pml l-Not I fragment, including the 3’ end of the NWN2 spacer, the CD28 and CD3 zeta-chain sequences (schematic representation in figure 2B, polynucleotide sequence in figure 2D, Sequence ID N°), was entirely synthesized by Eurofins Genomics Sri, Italy, and cloned at the Pml I / Not I restriction site of the original LTK-SCD44v6-NWL retroviral construct replacing the corresponding sequences, thus generating the retroviral vector LTK-SCD44v6-NWN2 (Figure 2C) expressing the new chimeric CAR CD44v6- NWN2 protein.

The retroviral vector constructs LTK-SCD44v6-NWL, LTK-SCD44v6-NWN2 and LTK- SCD44v6-NMS (the last one having the same structure of the others but containing a polynucleotide encoding the CD44v6-NMS) were used to transiently transfect GP+E86 cells (ATCC # CRL-9642). The supernatants from such ecotropic producer cell lines containing the vectors (without plasmid backbone) were then harvested to stably transduce the amphotropic packaging cell line PG13 to obtain three stable producer cell lines able to produce retroviral vectors carrying polynucleotides encoding the three CARs CD44v6-NWL, CD44v6-NWN2 and CD44v6-NMS.

Example 3 - CAR T memory differentiation phenotype

Retroviral vectors carrying polynucleotides encoding the three CARs CD44v6-NWL, CD44v6-NWN2 and CD44v6-NMS were used to transduce T cells as detailed in Materials and Methods.

In order to verify that CD44v6-NWN2 T cells preserved their memory phenotype following transduction, purified CAR T cells were analyzed for CD62L and CD45RA expression by FACS analysis, at day10. CD44v6-NWN2 T cells show a memory phenotype intermediate between CD44v6-NWL and CD44v6-NMS T cells, with a percentage of SCM more similar to CD44v6-NMS CAR cells (Figure 5). This result indicates that modification of the spacer region in the CD44v6-CAR design may influence the preservation of the memory phenotype.

Example 4 - CD44v6-NWN2 CAR T cells antigen specific activity.

To evaluate the antigen-specific activity of CAR T cells expressing the three CD44v6-CAR (NWL, NWN2, and NMS), potency assays were performed using different stimulator cells expressing or not expressing the target antigen. CD44v6-CAR T cells were specifically activated by several target cells expressing the CD44v6 antigen, including leukemia, melanoma, ovary and lung carcinoma cell lines (Figure 6 A and B). In particular, the CD44v6-NWN2 T cells show a level of activity intermediate between CD44v6-NWL and CD44v6-NMS T cells, with all the CD44v6+ target cells tested. On the contrary, all the three CD44v6-CAR T cells show a comparable level of activation in response to the treatment with PMA plus ionomycin, an unspecific stimulation that bypass the TCR and CAR mediated activation signals (Figure 6A).

Example 5 - In vitro CD44v6-NWN2 CAR T cytotoxic activity.

To analyze their cytotoxic activity, CD44v6-CAR T cells were co-cultured with green fluorescent protein (GFP) positive K562 clone#10 (CD44v6+) and K562 clone #19 (CD44v6-) at different E:T ratio. After 6 hours of incubation, surviving target cells were analysed by FACS and cytotoxic activity was calculated. As shown in the graphs reported in figure 7, CAR-T cells expressing CD44v6-NWN2 have an antigen-specific cytotoxic activity always higher than that of CAR-T cells expressing CD44v6-NWL or CD44v6-NMS. Graphs are representative of n=3 independent experiments.

Example 6 - In vivo CD44v6-NWN2 CAR T antitumor activity against hematological tumor.

Antitumor activity of the different CAR T cells was evaluated in vivo in a well-established model of AML. NSG mice were infused with 1.5x10⁶ CD44v6+ THP-1 leukemia cells and, after 13 days, treated with the different CD44v6 CAR T cells (NWL, NWN2, NMS) or with T cells expressing a control CD19-NWL CAR. Two doses given 24 hrs apart of 3x10⁶ cells and 10x10⁶ cells were infused intravenously into 3-6 mice per group. As shown in the graphs reported in figure 8, CAR-T cells expressing CD44v6-NWN2 have antitumor effect higher than that of CAR-T cells expressing CD44v6-NWL or CD44v6-NMS. These results were confirmed with other two independent experiments. All three experiments are shown, together, in figure 9. Example 7 - In vivo CD44v6-NWN2 CAR T antitumor activity against solid tumor.

Antitumor activity of the different CAR T cells was evaluated in vivo, in a solid tumor model. NSG mice were infused, subcutaneously, with CD44v6+ human ovarian cancer cells (IGROV-1 ) and, after 6 days, treated with the different CD44v6 CAR T cells (NWL, NWN2) or with T cells expressing a control CD19-NWL CAR. CAR T cells expressing CD44v6- NWL or CD44v6-NWN2 were obtained, with their control CD19-NWL CAR, from two different donors. One dose of CAR T cells was infused intravenously into 5 mice per group. Tumor growth was monitored, and tumor dimension regularly measured. As shown in the graph reported in figure 10, CAR-T cells expressing CD44v6-NWN2 have a higher antitumor effect than that of CAR-T cells expressing CD44v6-NWL

Conclusions

The examples above compare the phenotype and the antitumor of activity of CD44v6- NWN2 T cells, (i.e. T cells containing a CAR molecule carrying a spacer region according to the present invention), with CD44v6-NWL T cells and CD44v6-NMS T cells (i.e.: T cells containing CAR molecules carrying LNGFR-derived spacers disclosed in prior art). Examples 3 and 4 shows that CD44v6-NWN2 T cells has memory phenotype and an activity profile in term of frequency of degranulation (CD107a+) or cytokine production (TNF-a+ or IFNy+) intermediate between CD44v6-NWL T cells and CD44v6-NMS T cells. These results shows that the structure of the spacer has an impact on the phenotype and on the activity profile of the cells. Considering that the activity profile is expected to correlate with antitumor effect of the cells, it may be expected that CD44v6-NWN2 T cells have an intermediate antitumor activity between CD44v6-NWL T cells and CD44v6-NMS T cells. Surprisingly, Example 5, 6 and 7 shows that CD44v6-NWN2 T cells has improved in vitro and in vivo antitumor activity in respect to CD44v6-NWL T cells and CD44v6-NMS against hematological and solid tumors. These results show that CARs targeted to a tumor antigen containing a spacer according to the present invention have stronger antitumor effect, both in vitro and in vivo, as compared to CARs targeted to the same tumor antigen and containing spacer derived from LNGFR, but having different structure and length.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described CARS, polynucleotides, vectors, cells and compositions of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.

Claims

1. A Chimeric Antigen Receptor (CAR) comprising:

(i) an antigen-specific targeting domain that targets a tumor antigen;

(ii) a spacer domain consisting of a fragment of the human low affinity nerve

growth factor receptor (LNGFR) composed by a first sequence including the four TNFR-Cys domains, linked to a second sequence consisting of the first 1 1 amino acids of the serine/threonine rich stalk;

(iii) a transmembrane domain;

(iv) optionally at least one costimulatory domain; and

(v) an intracellular signaling domain.

2. A CAR according to claims 1 wherein the tumor antigen is selected from CD44, CD19, CD20, CD22, CD23, CD123, CS-1 , ROR1 , mesothelin, c-Met, PSMA, Her2, GD-2, CEA, MAGE A3 or TCR.

3. A CAR according to claim 1 to 2 wherein the tumor antigen is isoform 6 of CD44 (CD44v6).

4. A CAR according to anyone of claims 1 to 3 wherein the antigen-specific targeting domain comprises an antibody or fragment thereof.

5. A CAR according to anyone of claims 1 to 4 wherein the antigen-specific targeting domain is a single chain variable fragment.

6. A CAR according to claim 5 wherein the single chain variable fragment consist of Sequence ID NO:8.

7. A CAR according to claim 5 wherein the single chain variable fragment consists of Sequence ID NO:6

8. A CAR according to anyone of claims 1 to 7 wherein the spacer has a length of 173 amino acids.

9. A CAR according to anyone of claims 1 to 8 wherein the spacer consists of

Sequence ID NO:1.

10. A CAR according to anyone of claims 1 to 9 wherein the transmembrane domain comprises one or more of a transmembrane domain of a zeta chain of a T cell receptor complex, CD28, CD8a, CD4 CD244 (2B4), DAP10, DAP12 or combinations thereof.

1 1. A CAR according to anyone of claims 1 to 10 wherein the costimulatory domain

comprises one or more of the intracellular domain of CD28, CD137 (4-1 BB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 , Lck, TNFR-I, TNFR-II, Fas,

CD30, CD40, CD244 (2B4), DAP10, DAP12 or combinations thereof.

12. A CAR according to anyone of claims 1 to 1 1 wherein the intracellular signaling domain is selected from a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.

13. A CAR according to anyone of claims 1 to 12 comprising a single chain variable fragment, the transmembrane domain of CD28, the costimulatory domain derived from intracellular domain of CD28 and the intracellular signaling domain of CD3 zeta chain.

14. A CAR consisting of Sequence ID NO:3.

15. A polynucleotide encoding a CAR of any one claims 1 to 14.

16. A polynucleotide encoding a CAR that consists of the Sequence ID NO:3.

17. A vector comprising the polynucleotide of claim 15 or 16.

18. A vector comprising a promoter operably linked to a polynucleotide of claim 15 or 16 and a further promoter operably linked to a polynucleotide encoding a suicide gene.

19. A vector according to claim 17 or 18 wherein the vector is a viral vector.

20. A vector according to claim 19 wherein the viral vector is a retroviral vector or a lentiviral vector.

21. A vector according to claim to anyone of claim 18 to 20 wherein the suicide gene is the Herpes Simplex Virus Thymidine Kinase.

22. A vector according to anyone of claim 18 to 21 wherein the suicide gene is the HSV- TK Mut2 that consists of Sequence ID NO: 19.

23. A cell comprising a CAR according to anyone of claims 1 to 14, a polynucleotide according to claim 15 or 16 or a vector according to anyone of claims 17 to 22.

24. A cell according to claim 23 wherein the cell is a T-cell, a Natural Killer cell or a NK-T cell.

25. A pharmaceutical composition comprising the cell of claim 23 or 24.

26. A CAR according to any one of claims 1 to 14, a polynucleotide according to claim

15 or 16, a vector according to anyone of claims 17 to 22, a cell of claim 23 or 24 or a pharmaceutical composition of claim 25 for use in treating tumours.

27. A CAR, a polynucleotide, a vector, a cell or a pharmaceutical composition for use according to claim 26 wherein the tumour is a haematological tumour.

28. A CAR a polynucleotide, a vector, a cell or a pharmaceutical composition for use according to claim 26 wherein the tumour is a solid tumour.