WO2019134866A1 - Récepteurs antigéniques chimériques contenant une région d'espaceur optimale - Google Patents

Récepteurs antigéniques chimériques contenant une région d'espaceur optimale Download PDF

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WO2019134866A1
WO2019134866A1 PCT/EP2018/086665 EP2018086665W WO2019134866A1 WO 2019134866 A1 WO2019134866 A1 WO 2019134866A1 EP 2018086665 W EP2018086665 W EP 2018086665W WO 2019134866 A1 WO2019134866 A1 WO 2019134866A1
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car
cells
domain
cd44v6
vector
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Catia Traversari
Anna Stornaiuolo
Barbara Valentinis
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Molmed Spa
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464428—CD44 not IgG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/892—Reproductive system [uterus, ovaries, cervix, testes]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • 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.
  • Chimeric Antigen Receptors 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).
  • 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.
  • scFv single chain variable fragment
  • 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.
  • 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 ).
  • CARs containing four different scFvs were used to target four different human tumor-associated antigens i.e.
  • CEA carcinoembryonic antigen
  • NCAM neural cell adhesion molecule
  • 5T4 oncofetal antigen 5T4
  • 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.
  • WO 2016/042461 discloses CARs comprising spacer regions deriving from the extracellular domain of the human low affinity nerve growth factor receptor (LNGFR).
  • LNGFR human low affinity nerve growth factor receptor
  • 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 species
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a Chimeric Antigen Receptor comprising:
  • 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;
  • LNGFR human low affinity nerve growth factor receptor
  • the antigen-specific targeting domain of the CAR comprises an antibody or fragment thereof, more preferably a single chain variable fragment.
  • 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.
  • 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.
  • tumour antigen is isoform 6 of CD44 (CD44v6).
  • single chain variable fragment consist of Sequence ID NO:6
  • 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
  • the CAR contains a spacer consisting of Sequence ID NO:1 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ITAM immunoreceptor tyrosine-based activation motif
  • 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.
  • the CAR of the invention consists of Sequence ID NO:3.
  • a polynucleotide encoding a CAR of the invention and as defined herein.
  • polynucleotide encodes for a CAR that consists of the Sequence ID NO:3.
  • polynucleotide that consists of the Sequence ID NO: 4.
  • a vector comprising the polynucleotide encoding the CAR of the invention.
  • the vector is a viral vector, more preferably the viral vector is selected from a retroviral vector or a lentiviral vector.
  • 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.
  • the suicide gene is the Herpes Simplex Virus Thymidine Kinase, more preferably the polynucleotide encoding the suicide gene consists of Sequence ID NO:19.
  • a cell comprising a CAR, a polynucleotide or a vector of the present invention.
  • the cell is a T-cell, a Natural Killer (NK) cell or an NK-T cell
  • composition comprising a cell of the present invention.
  • 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.
  • tumours comprising administering a CAR, a polynucleotide, a vector or a cell of the invention to a subject in need of the same.
  • the tumours are selected from haematological or solid tumors.
  • CD44v6-NWN2 CD44v6 wild-type N2
  • 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).
  • White co-stimulatory domain CD28;
  • Light grey TNFR-Cys domain Grey: O ⁇ 3z chain.
  • 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).
  • 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)).
  • NWN2 N2 spacer sequences Underlined: TNFR cysteine- rich domain number 1 .
  • 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.
  • 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).
  • FIG. 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 ).
  • FIG. 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.
  • FIG. 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 (mm 3 ) 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 ).
  • 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:
  • 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;
  • LNGFR human low affinity nerve growth factor receptor
  • 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.
  • 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.
  • 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).
  • the binding domain is a single chain antibody (scFv).
  • the scFv may be murine, human or humanized scFv.
  • CDR complementarity determining region
  • 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.
  • 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.
  • tumor antigen includes antigen expressed on solid tumors and/or hematological tumors.
  • 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.
  • 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.
  • tumor antigens examples 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,
  • CEA carcinoembryonic antigen
  • the antigen specific targeting domain targets the receptor CD44v6.
  • 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.
  • scFv CD44v6-specific single-chain fragment
  • the antigen-specific targeting domain is the anti- CD44v6 specific scFv having the following sequence:
  • the CD44v6-specific single-chain fragment comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO:6.
  • 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).
  • GGGGSGGGGS 4GS2; SEQ ID NO:7.
  • CD44v6-specific single chain fragment CD44v6-4GS2 has the following sequence:
  • 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).
  • LNGFR low affinity nerve growth factor
  • 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 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:
  • Serine Threonine rich stalk SEQ ID NO:14: IPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPVVTRGTTD
  • 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.
  • IPGRWITRSTP SEQ ID NO: 20.
  • 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.
  • 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.
  • 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 ).
  • 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.
  • 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.
  • 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.);
  • transmembrane domain may be applied by the skilled in the art to the CAR of the present invention.
  • 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.
  • the transmembrane domain is derived from CD28.
  • 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)
  • the transmembrane and intracellular signaling domain comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO:15.
  • 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).
  • 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.
  • the costimulatory domain is derived from the intracellular domain of CD28
  • transmembrane and costimulatory domains are both derived from CD28.
  • transmembrane and intracellular costimulatory domain comprise the sequence below:
  • the transmembrane and costimulatory domains comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 16.
  • the intracellular costimulatory domain of the CAR is derived from the intracellular domain of CD28 and comprises the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:17).
  • 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.
  • 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.
  • z chain of the T-cell receptor or any of its homologs e.g., h chain, FceRl y and b chains, MB1 (Ig
  • 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.
  • signaling domain comprises the intracellular signaling domain of human CD3 zeta chain.
  • intracellular signaling domain of human CD3 zeta chain comprises the following sequence:
  • the intracellular signaling domain comprises at least 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO:18.
  • 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)
  • the CAR CD44v6-NWN2 is encoded by the polynucleotide sequence disclosed in Figure 3 (Sequence ID NO:4).
  • 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.
  • 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).
  • polynucleotide encoding the chimeric antigen receptor described herein.
  • polynucleotide as used herein is defined as a polymer of nucleotides, which form a DNA or RNA fragment.
  • 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.
  • different polynucleotide sequences may encode the same polypeptide.
  • 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, PCRTM, 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • CFAs cationic facial amphiphiles
  • 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.
  • polynucleotide of the invention are delivered to target cells using retroviral vectors.
  • retroviral vectors are commonly used and known to integrate a polynucleotide of interest into the genome of the target cell.
  • retroviral vectors 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 lentiviruse
  • 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).
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HSV-TK Herpes Simplex Virus Thymidine Kinase
  • the suicide gene is the HSV-TK Mut2, encoded by the following polynucleotide sequence: atggcttcgtacccctgccatcaacacgcgtctgcgttcgaccaggctgcgcgttctcgcggccatagcaaccgacgtacg gcgttgcgccctcgcggcagcaagaagccacggaagtccgcctggagcagaaaatgcccacgctactgcgggtttata tagacggtcctcacgggatggggaaaccaccaccacgcaactgctggtgg
  • 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.
  • lentiviruses can be divided into primate and non-primate groups.
  • 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).
  • VMV visna/maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anaemia virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • 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.
  • 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.
  • the genetically engineered cells are autologous cells.
  • 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.
  • a method of stably transfecting and re-directing cells is by electroporation using naked DNA.
  • 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.
  • 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.
  • 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:
  • 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;
  • cytokines including IL-2, IL-7, IL-15, IL-21 alone or in combination.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1 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.
  • NWN2 LNGFR optimized spacer LNGFR wild-type N2
  • CD44v6-NWN2 The sequences of CD44v6-NWN2 are shown in figure 3 (Sequence ID NO: 4 nucleotide sequence, Sequence ID NO: 3 peptide sequence).
  • NWN2 optimal spacer LNGFR wild-type N2
  • 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).
  • 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.
  • Green fluorescent protein (GFP) positive target cells 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.
  • 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
  • TNF-a clone Mab1 1
  • anti-CD44v6 clone #2F10 from R&D System
  • CD107a clone H4A3
  • 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.
  • 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 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:
  • Immunoselected T cells expressing CD44v6-NWL, CD44v6-NWN2 and CD44v6-NMS, were cultured until day 10, then analysed for memory T cells differentiation phenotype.
  • CAR T cells 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 (0.2x10 6 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.
  • 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 x10 6 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).
  • mice were infused intravenously with 1.5x10 6 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.
  • NWL, NWN2, NMS CD44v6 CAR T cells
  • T cells expressing a control CD19-NWL CAR.
  • Two doses given 24 hrs apart for a total of 10- 13x10 6 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).
  • NSG mice were subcutaneously injected with 0.3x10 6 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.
  • IGROV-1 CD44v6+ human ovarian cancer cells
  • NWL, NWN2 CD44v6 CAR T cells
  • T cells expressing a control CD19-NWL CAR One dose of 4.5x10 6 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.
  • 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).
  • LNGFR human low-affinity nerve growth factor receptor
  • 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.
  • LTK-SCD44v6-NWL, LTK-SCD44v6-NWN2 and LTK- SCD44v6-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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • GFP green fluorescent protein
  • 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 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.
  • CAR-T cells expressing CD44v6-NWN2 have a higher antitumor effect than that of CAR-T cells expressing CD44v6-NWL
  • CD44v6-NWN2 T cells i.e. T cells containing a CAR molecule carrying a spacer region according to the present invention
  • 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.
  • Example 5 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.

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Abstract

La présente invention concerne un récepteur antigénique chimérique (CAR) contenant un espaceur ayant une structure optimale générant une amélioration de l'effet antitumoral de la molécule. En particulier, le CAR selon la présente invention porte un domaine espaceur constitué d'un fragment du récepteur du facteur de croissance nerveux humain à faible affinité (LNGFR) composé d'une première séquence comprenant les quatre domaines TNFR-Cys, liée à une seconde séquence constituée des 11 premiers acides aminés de la tige riche en sérine/thréonine.
PCT/EP2018/086665 2018-01-03 2018-12-21 Récepteurs antigéniques chimériques contenant une région d'espaceur optimale WO2019134866A1 (fr)

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CN114651068A (zh) * 2019-09-16 2022-06-21 麦克马斯特大学 嵌合共刺激受体及其方法和用途

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