WO2012127464A2 - Lymphocytes t constitutivement activés pour l'utilisation dans une thérapie cellulaire adoptive - Google Patents

Lymphocytes t constitutivement activés pour l'utilisation dans une thérapie cellulaire adoptive Download PDF

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WO2012127464A2
WO2012127464A2 PCT/IL2012/000125 IL2012000125W WO2012127464A2 WO 2012127464 A2 WO2012127464 A2 WO 2012127464A2 IL 2012000125 W IL2012000125 W IL 2012000125W WO 2012127464 A2 WO2012127464 A2 WO 2012127464A2
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cells
linked
peptide
transmembrane
seq
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WO2012127464A3 (fr
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Gideon Gross
Alon Margalit
Aviad PATO
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Gavish-Galilee Bio Applications Ltd
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    • 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
    • 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
    • A61K39/464—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
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A61K2239/57—Skin; melanoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • the present invention relates in general to central nervous system (CNS) injuries and, in particular, to sub-populations of human monocytes useful in the treatment of CNS injuries, methods for isolation of these sub-populations and treatment of patients suffering from spinal cord injury.
  • CNS central nervous system
  • the present invention relates to the field of adoptive cell therapy and the use of activated T cells expressing constitutively active (ca) CD40 molecules, caTLR4 molecules, membrane-bound cytokines and combinations thereof.
  • adoptive cell therapy involves the administration of large numbers of highly selective cells with high avidity for tumor antigens.
  • T cells can be programmed and activated ex vivo to exhibit antitumor effector functions.
  • T cell infusion may be preceded by 'conditioning' of the patient with lymphodepleting chemotherapy or total body irradiation, which enables the diminution of immunosuppressive cell types/factors followed by the infusion of tumor-specific T cells.
  • TILs tumor-infiltrating lymphocytes
  • APCs artificial antigen-presenting cells
  • beads coated with T cell ligands and activating antibodies or cells isolated by virtue of capturing target cell membrane
  • allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR)
  • non-tumor-specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules displaying antibody-like tumor recognition capacity known as "T-bodies”.
  • ACT is currently the most effective treatment for patients with metastatic melanoma, and is extensively explored for the treatment of other human cancers.
  • major obstacles which limit the clinical benefit and broader application of this approach.
  • some of the intrinsic difficulties are attributable to the particular method employed for isolation, propagation or generation of the effector lymphocytes, others, such as the exhaustion of the proliferative and survival potential of fully differentiated T cells, seem to be a more general phenomena related to the effector phenotype.
  • Thl- promoting cytokines such as interleukin (lL)-2, IL-12 and IL-15
  • cytokines such as interleukin (lL)-2, IL-12 and IL-15
  • CTLA-4 or PD-1 contact- dependent T cell suppression by CTLA-4 or PD-1
  • depletion or attenuation of Tregs blockade of TGF- ⁇
  • costimulatory signaling pathways such as CD28, 4- IBB and OX40
  • introduction of anti-apoptotic genes e.g. Bcl-2, Bcl-xL
  • TLR agonists e.g. Bcl-2, Bcl-xL
  • the present invention relates, in one aspect, to polynucleotides comprising a sequence encoding a chimeric protein comprising a cytoplasmic domain of a T cell costimulatory receptor of the tumor necrosis factor receptor (TNFR) family that is linked to a heterologous polypeptide comprising at least one self-assembly domain, said chimeric peptide being linked to an integral membrane or a transmembrane oligopeptide that allows the anchorage of the chimeric polypeptide to the cell membrane.
  • TNFR tumor necrosis factor receptor
  • the invention relates to polynucleotides comprising a sequence encoding a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to the cell membrane, and said transmembrane oligopeptide, if existent, is linked, optionally via a bridge peptide, to a peptide tag or oligopeptide tag.
  • the invention relates to polynucleotides comprising a sequence encoding a chimeric protein comprising a cytokine linked through its carboxyl terminus via a flexible bridge peptide to an integral membrane or transmembrane oligopeptide that allows the anchorage of the cytokine molecule to a cell membrane, with the proviso that said chimeric protein does not comprise IL-2 linked to a glycoinositol phospholipid, IL-12 linked to the costimulatory molecule CD80 (B7.1) or IL-15 linked to IL-15 RA.
  • the polynucleotide encoding a chimeric protein comprising a cytoplasmic domain of a T cell costimulatory receptor of the TNFR family or the chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 is each linked to an integral membrane or a transmembrane oligopeptide that allows the anchorage of the chimeric polypeptide to the cell membrane, wherein said transmembrane oligopeptide is linked via a flexible bridge peptide to the carboxyl terminus of a cytokine.
  • the present invention relates to expressions vectors and compositions comprising the above-identified polynucleotides or any combination thereof, and to activated T cells expressing at least one chimeric protein encoded by these polynucleotides.
  • the present invention relates to methods for treating cancer, comprising administering to a cancer patient a therapeutically effective amount of activated tumor specific T cells expressing the chimeric proteins of the present invention; and to methods for preparing activated T cells comprising transfecting said T cells with a polynucleotides or expression vectors as defined herein, whereby the polynucleotide is expressed within said T cells, thus obtaining the activated T cells.
  • the present invention provides a method for propagating T cells, comprising transfecting said T cells with a polynucleotide comprising a sequence encoding a chimeric protein comprising IL-2 linked through its carboxyl terminus via a flexible bridge peptide to an integral membrane or transmembrane oligopeptide that allows the anchorage of the IL-2 molecule to a cell membrane.
  • Fig. 1 depicts flow cytometry analysis showing expression of GFP in human CD8 T cells following mRNA transfection. Fluorescence was analyzed 24 hours after transfection against the same cells undergoing an identical treatment but receiving no RNA (filled black histogram). Ml, Marker 1 : the range used to calculate specific, relative to total, fluorescence and MFI (mean fluorescence intensity).
  • Figs. 2A-B depict schemes of the basic genetic construct (A) and the resulting MHC-I molecules at the cell surface (B).
  • FIG. 3 shows a bar graph demonstrating that human peripheral blood lymphocyte (PBL)-derived CD4 and CDS T cells produce interferon-7 (IFN- ⁇ ) following transfection with 5 /ig caTLR4- (but not caTLR2- or EGFP-) encoding mRNA.
  • PBL peripheral blood lymphocyte
  • CDS T cells produce interferon-7 (IFN- ⁇ ) following transfection with 5 /ig caTLR4- (but not caTLR2- or EGFP-) encoding mRNA.
  • NT non- treated
  • LPS lipopolysaccharide
  • Pam3, Pam3CysS 4 is a bar graph demonstrating that human peripheral blood lymphocyte (PBL)-derived CD4 and CDS T cells produce interferon-7 (IFN- ⁇ ) following transfection with 5 /ig caTLR4- (but not caTLR2- or EGFP-) encoding mRNA.
  • NT non- treated
  • LPS lipopolysaccharide
  • FIGs. 4A-B demonstrates that as little as 1 ⁇ g caTLR4 mRNA activates human CD4 (A) and CD8 (B) T cells derived from PBLs of healthy HLA-A2+ (A2+) and HLA- A2- (A2-) donors.
  • Transfection of either peptide-p2m-TLR4 (construct 500) or 2m-TLR4 only (construct 931) were performed with 1 or 5 /xg in-vitro-transcribed mRNA.
  • Non- treated cells or cells similarly transfected with EGFP-encoding mRNA (GFP) served as a negative control, and cells transfected with EGFP mRNA and treated with LPS (1 ⁇ g ml) served as a reference.
  • GFP EGFP-encoding mRNA
  • Figs. 5A-B show a flow cytometry analysis for the expression of CD25 by the tumor specific CTL clone, 1C9, (A) and the expression of CD69 by the primary TIL 431-4 (B), following transfection of 5 ⁇ g mRNA encoding either 2m-HLA-A2 (construct 541), caTLR4 (construct 931) or GFP (construct 540; irrelevant RNA). GFP served as an indicator for transfection efficiency (upper panel).
  • N G2D an activating receptor that is expressed by T cells, as well as by natural killer ( ) cells and macrophages; CD 137, a lymphocyte activation marker also known as 4- IBB.
  • Figs. 6A-C depict the design of oligomeric derivatives of hCD40.
  • A The basic scheme of the genetic construct.
  • B The expected organization of the GCN4 coiled coil motifs.
  • C The expected oligomerization of the encoded polypeptides.
  • Fig. 7 depicts a scheme of the chimeric gene encoding for single chain IL-12. Pr, Promoter; Spe I, Xma I, Xho I and Not I, restriction sites.
  • Fig. 8 shows CFSE dilution assay for cell proliferation.
  • CD8 T cells were prepared from PBLs of a healthy donor, grown for 4 days in OKT3 and IL-2 and separated to CD8 and CD4 T cells. Following 24 h rest were transfected or treated as indicated. Results are presented as % of live cells with decreased CFSE staining relative to the initial histogram.
  • Fig. 9 shows that here is functional synergy between genetic adjuvants.
  • Melanoma TILs grown the presence of 2,000 U/ml IL-2 for 4 days, were electroporated with 10 ⁇ g each mRNA. 24 hours post-transfection growth medium was collected for IFN- ⁇ ELISA. No, non-treated cells; N.R., irrelevant mRNA. GFP mRNA served as an additional negative control and as a reference for transfection yield.
  • Figs. 10A-B show restoration of TIL-425 functional activity by the genetic adjuvants.
  • Anti-melanoma CD8 TILs were transfected with mRNA encoding caCD40, memIL3 ⁇ 4 caTLR4, their indicated combinations and an irrelevant mRNA as a negative control. 3 days post transfection the cells were incubated with relevant M427 melanoma (A) or irrelevant M171 melanoma (B). Flow cytometry analysis for IFN- ⁇ (intracellular staining, upper panels) and cytolysis-associated degranulation (CD107, lower panels).
  • adoptive cell therapy is one of the most promising approaches being developed for treating cancer.
  • ACT adoptive cell therapy
  • a network of suppressive mechanisms operating at the tumor site, T cell exhaustion, down regulated effector mechanisms and low persistence of the transferred cells are critical factors which limit clinical efficacy and broader use of ACT.
  • the present invention is based on a novel genetic approach designed to overcome these complications and maximize the curative potential of tumor-reactive T cells in ACT.
  • the central components of this approach are based on the concept of severing T cell activation from the ligand-dependent activation of T cell activating factors by expressing in the T cells constitutively active factors; and on the concept of confining the activity of T cell promoting cytokines, which are naturally secreted from the cells, preventing their undesired consumption by competing cells (mainly Tregs) and coupling their activity to that of other T cell activating factors, by expressing these cytokines in T cells as integral membranal proteins.
  • T cells T cells
  • TNFR tumor necrosis factor receptor
  • the present invention is thus directed to a polynucleotide comprising a sequence encoding a chimeric protein comprising a cytoplasmic domain of a T cell costimulatory receptor of the tumor necrosis factor receptor (TNFR) family that is linked to a heterologous polypeptide comprising at least one self-assembly domain, said chimeric peptide being linked to an integral membrane or a transmembrane oligopeptide that allows the anchorage of the chimeric polypeptide to the cell membrane.
  • TNFR tumor necrosis factor receptor
  • polypeptide encoded by the polynucleotides defined above is termed herein "self- aggregating TNFR polypeptide", for example as in “self-aggregating CD40", “constitutively active T cell costimulatory receptor of the TNFR family” or "caTNFR”.
  • the candidates in this family that are most relevant to the present invention are CD40, CD27, 4-1BB (CD137), OX40 (CD134), herpesvirus entry mediator (HVEM; also known as TNFRSF14), CD30 and glucocorticoid-induced TNFR-related protein (GITR).
  • HVEM herpesvirus entry mediator
  • TNFRSF14 glucocorticoid-induced TNFR-related protein
  • GITR glucocorticoid-induced TNFR-related protein
  • 4-1BB is expressed on activated human T cells. Its ligation by cognate ligand expressed on the surface of antigen-presenting cells (APCs) or by antibodies or soluble ligand promotes T cell survival by upregulating anti-apoptotic genes, induces cell division, augments Thl cytokine production, protects T cells from antigen-induced cell death, induces memory formation and confers resistance to Treg suppression. Results from different experimental systems have described the beneficial anti-tumor effect of 4-1 BB stimulation in- vivo.
  • APCs antigen-presenting cells
  • soluble ligand promotes T cell survival by upregulating anti-apoptotic genes, induces cell division, augments Thl cytokine production, protects T cells from antigen-induced cell death, induces memory formation and confers resistance to Treg suppression.
  • OX40 is a late costimulatory receptor expressed on activated CD4 and CD8 T cells. OX40 ligation promotes T cells survival, proliferation, migration to sites of inflammation, memory formation and abrogation of suppression. Similarly to 4- IBB, a large body of evidence maintains that OX40 stimulation augments anti-tumor T cell reactivity.
  • CD40 is also expressed on both CD8 and CD4 T cells, playing an intrinsic role in T cell costimulation, memory formation, rescue from exhaustion and contra-Treg activity.
  • the T cell costimulatory receptor of the T FR family is selected from CD40, CD27, 4- IBB (CD137), OX40 (CD 134), HVEM, CD30 or GITR, preferably the human homolog of these receptors.
  • TNFR-associated factor (TRAF) proteins via the NF-/3B, p38 MAPK or JNK/SAPK pathways.
  • Signaling entails receptor homo-oligomerization, which is induced by engagement with their respective homo- oligomeric (typically homotrimeric) ligands.
  • a skilled artisan may easily assess if a cell expresses a molecule that has ligand-independent characteristic TNFR activity as measured for example by the activation of downstream signal pathways as recited above.
  • the self-assembly domain that mediates the aggregation of the chimeric protein comprising the full or partial intracellular domain of a TNFR molecule may be any such self-assembly domain known in the art, such as a coiled-coil domain.
  • coiled coils comprise two or more a-helices that supercoil around each other to form rope-like structures.
  • these motifs usually have a repeating pattern of seven residues called the heptad repeat, and often designated abcdefg. Residues at a and d positions are predominantly hydrophobic, which results in a hydrophobic stripe along each participating helix.
  • a coiled-coil is the leucine zipper motifs present in many DNA-binding proteins transcriptional regulators, some of which are listed in Miller (2009), incorporated herein by reference.
  • leucine zipper is that of the C/EBP a basic region: leucine zipper (bZIP) protein of the amino acid sequence of SEQ ID NO: 1 ; each seven amino acid residues of which forms a heptad.
  • Non-limiting examples of human bZIP protein families mentioned in the Miller paper are C/EBP a, AP-1 (including the Jun subfamily, Fos subfamily and ATF2&4 subfamily), C EB/ATF, ATF6, PAR, MAF and CNC families.
  • C/EBP a C/EBP a
  • AP-1 including the Jun subfamily, Fos subfamily and ATF2&4 subfamily
  • C EB/ATF C EB/ATF
  • ATF6, PAR MAF and CNC families.
  • the self-assembly domain of each of these proteins, but not limited to them, is contemplated by the present invention for use in the chimeric polypeptide of the present invention.
  • association domains tetramerization domains of the vanilla receptor as taught by Garci'a-Sanz et al. (2004).
  • the coiled-coil domain may be a yeast GCN4 leucine zipper DNA-binding motif, which depending on its particular amino acid sequence, is capable of forming homodimers, homotrimers, or homotetramers.
  • a CD40 genetic construct as a template, and incorporated the GCN4 binding motifs in 3 configurations encoding dimers, trimers and tetramers, based on Harbury et al, (1993; 1995).
  • the self-assembly domain is a yeast GCN4 leucine zipper DNA-binding motif, in particular the yeast GCN4 leucine zipper DNA- binding motif of SEQ ID NO: 2 forming homodimers, SEQ ID NO: 3 forming homotrimers or SEQ ID NO: 4 forming homotetramers.
  • the self-aggregating TNFR polypeptide could conceivably be anchored by any transmembrane oligopeptide known in the art (see below).
  • the cytoplasmic constitutively active domain of the TNFR molecule of the chimeric protein of the present invention is linked to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TNFR molecule to a cell membrane.
  • the TNFR moiety may be linked to an oligopeptide which is buried in the lipid bilayer and does not protrude to the other side of the membrane.
  • the transmembrane oligopeptide of the TNFR chimeric protein is the transmembrane oligopeptide of CD40.
  • the term “contralateral” is used herein to describe the side of the membrane which is opposite to the side of the constitutively active TNFR signal conducting moiety.
  • the contralateral side is the extracellular side.
  • the term “contralateral protein” is used herein to describe a protein or peptide attached to the transmembrane stretch on the contralateral side of the membrane.
  • the contralateral protein is a peptide tag or oligopeptide tag.
  • the contralateral protein to which the transmembrane stretch may be attached via the bridge peptide is not essential for the proper activation of the T cells by the TNFR molecule and could thus be chosen according to its desired function such as, but not limited to, detection or purification of the expressed protein (e.g. a His-tag (e.g. His 6 ), a (His-Asn) tag, a Flag tag, a Ha tag, a fluorescent protein, such as enhanced green fluorescent protein (EGFP), or any other tag that may facilitate the purification and identification of the chimeric protein), or an enzymatic function.
  • peptide, oligopeptide and peptide-tag as used herein are defined as a peptide or oligopeptide that has no functional activity and that can be detected with a specific detecting agent, such as a specific antibody.
  • a specific detecting agent such as a specific antibody.
  • the tag may be His 6 .
  • the transmembrane oligopeptide of the TNFR chimeric protein oligopeptide is linked, optionally via a bridge peptide comprises an amino acid stretch of about 5 amino acids to about 15 amino acids, preferably 13 amino acids of the membrane-proximal amino acids of human HLA-A2, to a peptide tag.
  • the peptide tag may be selected from, but is not limited to, Ha tag, a His tag or a Myc tag.
  • CD40 constitutively active, ligand-independent CD40, which confers a constitutively activated state on transfected T cells, by linking CD40 to the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions, and thus induces self-oligomerizing.
  • TNFR members mentioned here bear structural similarities and they all signal through adaptor TNFR-associated factor (TRAF) proteins via the NF-0B, p38 MAP or JNK/SAPK pathways, one may assume that also the other TNFR members, when linked to a self-assembly domain, will become constitutively active, that are capable of conferring a constitutively activated state on transfected T cells.
  • TNF TNFR-associated factor
  • self- ggregating TNFR polypeptide or “constitutively active T cell costimulatory receptor of the TNFR family” as used herein thus refer to any polypeptide with minor variations in its amino acid sequence as compared with the self- aggregating TNFR polypeptides defined herein, that have ligand-independent characteristic TNFR activity as measured for example by the activation of downstream signal pathway elements such - NF-jSB, p38 MAPK or JNK/SAPK.
  • the polynucleotide encoding the constitutively active T cell costimulatory receptor of the TNFR family comprises the following nucleotide sequences: (a) the sequence of SEQ ID NO: 5 encoding for the leader peptide of the human /32m gene; (b) a sequence encoding for a peptide tag for detection selected from: (i) Ha tag of SEQ ID NO: 6; (ii) Myc tag of SEQ ID NO: 7; or (iii) His tag of SEQ ID NO: 8; (c) the sequence of SEQ ID NO: 9 encoding a bridge peptide; (d) the sequence of SEQ ID NO: 10 encoding for the transmembrane domain of human CD40; (e) the sequence of SEQ ID NO: 11 encoding for a GCN4-derived homo-oligomerizing domain forming homotrimers; and (f) a sequence encoding for the cytosolic signaling domain of a T cell cost
  • nucleic acid sequences i.e. polynucleotides
  • polypeptide encoded by the first nucleic acid has activity that is identical to, or characteristic of, the polypeptide encoded by the second nucleic acid.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes , “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5xSSC, and 1% SDS, incubating at 42° C, or, 5xSSC, 1% SDS, incubating at 65° C, with wash in 0.2xSSC, and 0.1% SDS at 65° C.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in l SSC at 45° C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., Current Protocols in Molecular Biology , ed. Ausubel, et al.
  • the nucleic acid sequences are at least about 80%, 90%, 95%, or 97-98%, or 100% identical to the sequences of SEQ ID NOs: 5-20 and 23- 29, respectively, as assessed using a computer program such as BLAST (available at the National Center for Biotechnology Information, USA) that finds regions of similarity between biological sequences.
  • BLAST available at the National Center for Biotechnology Information, USA
  • the polynucleotide encoding the constitutively active T cell costimulatory receptor of the TNFR family comprises the sequence of (iv) encoding for the cytosolic signaling domain of CD40.
  • T cells refers to T-lymphocytes taken directly from a living mammal; they do not include in their scope immortalized T cell lines, such as Jurkat cells.
  • TLR2 engagement on these cells triggers interferon (IFN)-Y production, lowers the activation threshold, enhances cell proliferation and survival, maintains T cell memory, attenuates regulatory T cell (Treg)-mediated suppression and renders effector T cells resistant to this suppression.
  • IFN interferon
  • DCs Dendritic cells
  • APCs antigen presenting cells
  • TLRs TLRs
  • the particular TLRs engaged by these cells in the periphery polarize the ensuing response towards the Thl, Th2, Thl7 or the Treg course, and the elucidation in recent years of the functional consequences of TLR engagement on particular cell subsets is harnessed in vaccine development.
  • Cumulative findings ascribe a critical role for Tregs in the suppression of tumor-reactive T cells. Treg activity can be modulated by agonists of TLR2, TLR5 and TLRS, and these can be exploited in cancer immunotherapy.
  • TLR2 which appears to be a key TLR family member that can directly stimulate T cell survival and proliferation and boost effector functions.
  • TLR2 which appears to be a key TLR family member that can directly stimulate T cell survival and proliferation and boost effector functions.
  • BLP bacterial lipopeptide
  • CD4+ CD45RO+ memory T cells constitutively expressed TLR2 produced IFN- ⁇ and manifested elevated proliferative capacity in response to BLP.
  • Cottalorda et al. (2006) showed that BLP stimulation of mouse CD 8 T cells led to increased cell proliferation and survival, and these were associated with a sustained expression of IL-2 receptor ct-chain (IL-2Ro: or CD25) and enhanced expression of the anti-apoptotic protein Bcl-xL.
  • IL-2Ro IL-2 receptor ct-chain
  • BLP upregulated IFN- ⁇ and granzyme B secretion augmented the overall cytotoxic activity of antigen- activated T cells and lowered the threshold of their initial activation by APCs.
  • TLR2 triggered by 2 different ligands
  • IFN-7 IFN-7 production
  • cell proliferation and cell survival in mouse Thl cells
  • TLR2 ligands induced IFN- ⁇ also by CD8 T cells pre-stimulated with anti-TCR and CD28 Abs.
  • Asprodites et al. (2008) showed that BLP augmented the anti-melanoma activity of TLR2-proficient, but not deficient, CD8 T cells from the TCR transgenic OT-1 mice.
  • TLR3 was shown to be expressed by human effector CD8 T cells (Tabiasco et al, 2006).
  • the bacterial TLR3 agonist poly(I:C) increased IFN- ⁇ secretion induced by TCR-dependent and -independent stimulation of these cells, but without affecting proliferation or specific cytolytic activity.
  • TLRs Removal of the ligand-binding domain of most, if not all, TLRs results in constitutive signaling triggered by the truncated derivatives, referred to as constitutively active (ca), or dominant positive (DP) TLRs.
  • TLR ligands of microbial origin as immunological adjuvants to directly augment T cell effector functions either in- vivo or ex-vivo is intriguing; however, this may produce undesirable side effects and is not favored from a regulatory point of view.
  • the inventors have also utilized the genetic platform for targeting antigenic peptides to MHC-I presentation in antigen presenting cells.
  • the selected antigenic peptides are fused to the N-terminus of /32m which is anchored to the membrane as described above.
  • WO 01/091698 teaches antigenic peptides related to an autoimmune disease and WO 03/106616, of the same applicants, teaches antigenic peptides not related to an autoimmune disease, i.e. the antigenic peptide is derived from a tumor associated, bacterial, viral, fungal or parasite antigen.
  • US 2008/0286312 and WO 08/041231 teaches a construct encoding /32m anchored to the membrane by the means of the intracellular and transmembrane regions of TLR4, which confers activation of antigen presenting cells expressing the construct.
  • WO01091698, WO 03/106616, US 2008/0286312 and WO 08/041231 are all hereby incorporated by reference in their entirety as if fully disclosed.
  • TLR4 is known in the art to be expressed in T cells as described above.
  • expression of constitutively active (transmembranal and cytosolic domains) (caTLR4) caTLR4 mRNA transfected T cells causes extraordinary activation of both CD4 and CD8 T cells.
  • TLR4 is not believed to be functionally expressed in T cells as evidenced by the absence of a response of T celjs exposed to TLR4 ligands such as LPS as described above.
  • TLR4 is capable of activating Jurkat cells (Medzhitov et al, 1997), has always been considered in the art to be a specific feature of this immortalized cell line, and not as a feature shared by T cells due to the latter' s refractoriness to LPS, and has not prompted researchers to attempt expressing TLR4 in T cells.
  • TLR2 and TLR4 signaling pathways differ, T cells in general would not be expected to possess all the downstream components required for TLR4 signaling.
  • TLR2- mediated signaling is solely transduced through the recruitment of the MyD88 and TIRAP adaptor proteins, which leads to the activation of the transcription factors NF- ⁇ and AP- 1.
  • TLR4 also harnesses the TRIF-TRAM set of adaptors, a pathway which culminates in the activation of interferon regulatory factors.
  • the T cells activated by these two constitutively active molecules may thus be transfected with a single polynucleotide or vector encoding for both molecules, or it may be transfected with two separate molecules, e.g. RNA molecules, each one encoding for one of the two molecules.
  • the present invention provides a polynucleotide comprising a sequence encoding a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to the cell membrane, and said transmembrane oligopeptide, if existent, is linked, optionally via a bridge peptide, to a peptide tag or oligopeptide tag.
  • the polynucleotide encoding the constitutively active T cell costimulatory receptor of the TNFR family comprises a further sequence encoding a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino tenninus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to the cell membrane, and said transmembrane oligopeptide, if existent, is linked, optionally via a bridge peptide, to a peptide tag or oligopeptide tag.
  • caTLR4 refers to any polypeptide with minor variations in its amino acid sequence as compared with transmembranal and cytosolic domains of human TLR4 (based on the sequence as set forth at GBA NM138554), that have ligand-independent characteristic TLR4 activity as measured for example by the activation of downstream signal pathway elements such myD88, TRIF-TRAM or NFKB.
  • the cytoplasmic constitutively active domain of the TLR4 molecule could conceivably be anchored by any transmembrane oligopeptide known in the art (see below).
  • the cytoplasmic constitutively active domain of the TLR4 molecule of the chimeric protein of the present invention is linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to a cell membrane.
  • the TLR4 moiety may be linked to an oligopeptide which is buried in the lipid bilayer and does not protrude to the other side of the membrane.
  • the TLR4 moiety is preferably anchored to a cell membrane by its ow native transmembrane oligopeptide.
  • the TLR4 is a human TLR4.
  • the bridge peptide (which is linked to the TLR4 transmembrane oligopeptide) comprises an amino acid stretch of about 5 amino acids to about 15 amino acids, preferably 13 amino acids of the membrane-proximal amino acids of human HLA-A2.
  • the peptide tag is selected from, but is not limited to, Ha tag, a His tag or a Myc tag.
  • the polynucleotide encoding for the caTLR4 may comprise the following nucleotide sequences: (a) the sequence SEQ ID NO: 17 encoding for the 5' non- translated region of ⁇ 2 ⁇ ; (b) the sequence SEQ ID NO: 18 encoding for the human ⁇ 2 ⁇ leader peptide; (c) a sequence encoding for a peptide tag for detection selected from: (i) Ha tag of SEQ ID NO: 6; (ii) Myc tag of SEQ ID NO: 7; or (iii) His tag of SEQ ID NO: 8; (d) the sequence of SEQ ID NO: 9 encoding a bridge peptide; (e) the sequence SEQ ID NO: 19 encoding for human transmembrane and cytosolic domains of TLR4; and (f) the sequence
  • Thl immunostimulatory cytokines are IL-la, IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, TNF- ⁇ 3 ⁇ 4 IFN-o IFN-0 and IFN- ⁇ .
  • IL-2 is an essential factor for the growth, differentiation and survival of antigen-selected T cells. It is a 15.5 kDa protein comprising a single polypeptide chain of 133 amino acids. IL-2 is naturally produced by activated T cells and functions in an autocrine or paracrine manner. It binds to a heterotrimeric high affinity receptor comprising IL-2Ra (CD25), IL-2/15R/3 and the common cytokine-receptor ⁇ -chain (7c). IL-2 is evidently the most widely explored cytokine in cancer immunotherapy and particularly in ACT.
  • IL-2 substantially augments the TLR-2-mediated enhancement of the functional and proliferative status of T cells.
  • IL-12 is a Thl polarizing cytokine, driving the differentiation of CD4 Thl and CD8 T cells and natural killer (NK) cells and is also known. for its anti-angiogenic activity. It stimulates IFN- ⁇ and TNF-a production from T and NK cells.
  • IL-12 is a heterodimeric cytokine, consisting of two gene products: p35 and p40. It is produced mainly by activated DCs and binds to its heterodimeric receptor comprising IL-12R-/31 and IL-12R-
  • the anti-rumor activities of IL-12 have been explored in cancer immunotherapy, gene therapy and ACT .
  • IL-12 was capable of amplifying the observed influence of TLR2 ligands on T cell effector functions.
  • the production of different forms of recombinant IL-12 or its gene delivery were facilitated by the development of single-chain derivatives of this cytokine, which retain the functional properties of the native protein (Chakrabarti et al (2004), Lode et al (1998), Lee et al, 1998).
  • IL-15 is a T cell (and NK cell) stimulating cytokine. It induces the generation of primary and memory antigen-specific CD8. T cells, mediates CD4 T cell help to promote longevity of CD8 cells and prevent their TRAIL-induced apoptosis. In particular, IL-15 induces telomerase activity in CD8 T cells and prolongs their proliferation. IL-15 synergizes with TLR2 in promoting proliferation of, and IFN-7 production by, human CD4 T cells. IL-15 is a key cytokine used in ACT in ex- vivo generation, and propagation of tumor-reactive effector T cells.
  • T cells engineered to express IL-15 exhibited improved anti-rumor activity and prolonged survival in- vivo (Quintarelli et al (2007), Hsu et al (2005), Klebanoff et al., 2004). It has been shown that in addition to IL-2 consumption, Tregs can bind and eliminate IL-15 (and IL-4 and IL-7).
  • IL-2 As an integral membranal constituent of transfected cells, the inventors have genetically engineered IL-2 as an integral membranal constituent of transfected cells.
  • Membranal (mem) IL-2 was designed with a flexible spacer so as to engage its cell surface receptor in-cis (that is, on the same cell). As long as both the gene product and its endogenous IL-2 receptor are expressed at the cell surface this closed system is expected to signal constitutively, precluding IL-2 intake by tumor-resident Tregs and other cells and avoiding systemic dissemination and subsequent side effects.
  • the T cells activated by these two molecules may thus be transfected with a single polynucleotide or vector encoding for both molecules, or it may be transfected with two separate molecules, e.g. RNA molecules, each one encoding for one of the two molecules.
  • the T cells may be transfected with a single polynucleotide encoding a cytokine, grafted via a bridge peptide and a transmembrane oligopeptide, to the cytoplasmic domain of the self-aggregating TNFR polypeptide of the present invention, or the cytoplasmic domain of the caTLR4 of the present invention.
  • the polynucleotide of the present invention encodes for the constitutively active T cell costimulatory receptor of the TNFR family, or the constitutively active TLR4, wherein the constitutively active T cell costimulatory receptor of the TNFR family, or the constitutively active TLR4, is linked through a transmembrane oligopeptide via a flexible bridge peptide to the carboxyl terminus of a cytokine.
  • the polynucleotide of the present invention may also encode for a chimeric protein comprising a cytoplasmic domain of a T cell costimulatory receptor of the tumor necrosis factor receptor (TNFR) family that is linked to a heterologous polypeptide comprising at least one self-assembly domain, said chimeric peptide being linked to an integral membrane or a transmembrane oligopeptide that allows the anchorage of the chimeric polypeptide to the cell membrane, and a chimeric protein comprising a cytokine linked through its carboxyl terminus via a flexible bridge peptide to a cytoplasmic constitutively active domain of a TLR4 molecule.
  • TNFR tumor necrosis factor receptor
  • the polynucleotide of the present invention may encode for a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to the cell membrane, and said transmembrane oligopeptide, if existent, is linked, optionally via a bridge peptide, to a peptide tag or oligopeptide tag, and a chimeric protein comprising a cytokine linked through its carboxyl terminus via a flexible bridge peptide to a self-aggregating cytoplasmic domain of a T cell costimulatory receptor of the tumor necrosis factor receptor (TNFR) family.
  • TNFR tumor necrosis factor receptor
  • the polynucleotide of the present invention encoding for the constitutively active T cell costimulatory receptor of the TNFR family, or the constitutively active TLR4 comprises a further sequence encoding a chimeric protein comprising a cytokine linked through its carboxyl terminus via a flexible bridge peptide to an integral membrane or transmembrane oligopeptide that allows the anchorage of the cytokine molecule to a cell membrane, with the proviso that said chimeric protein does not comprise IL-2 linked to a glycoinositol phospholipid, IL-12 linked to the costimulatory molecule CD80 (B7.1) or IL-15 linked to IL-15 RA.
  • a polynucleotide encoding a certain self-aggregating TNFR polypeptide may also encode for additional different self-aggregating members of the TNFR family, e.g. but not limited to, self-aggregating CD40 and self- aggregating 4- 1BB.
  • a polynucleotide encoding a membrane bound cytokine may also encode for additional membrane bound cytokines, e.g., but not limited to, membrane bound IL-2 and membrane bound IL-12.
  • the present invention is directed to a polynucleotide encoding a chimeric protein comprising a cytokine linked through its carboxyl terminus via a flexible bridge peptide to an integral membrane or transmembrane oligopeptide that allows the anchorage of the cytokine molecule to a cell membrane, with the proviso that said chimeric protein does not comprise IL-2 linked to a glycoinositol phospholipid, IL-12 linked to the costimulatory molecule CD80 (B7.1) or IL-15 linked to IL-15 RA.
  • said cytokine is selected from IL-2, IL-12 or IL-15, and in other embodiments the cytokine is a human cytokine.
  • the flexible bridge peptide may comprise an amino acid stretch of about 5 amino acids to about 15 amino acids, preferably 8 amino acids of the membrane-proximal amino acids of human HLA-A2, and in certain embodiments it further comprises the 13 amino acid flexible linker Gly 4 Ser(Gly 3 Ser) 2 (SEQ ID NO: 21).
  • the cytokine molecule of the chimeric protein of the present invention is linked to an integral membrane or transmembrane oligopeptide that allows the anchorage of the cytokine to a cell membrane.
  • the cytokine may be linked to an oligopeptide which is buried in the lipid bilayer and does not protrude to the other side of the membrane.
  • the TNFR, caTLR molecule or cytokine of the present invention could conceivably be anchored by any transmembrane oligopeptide known in the art.
  • it may be linked through its amino terminus via a glycosylphosphatidylinositol (GPI)-anchor sequence, such as the GPI-anchor peptide of SEQ ID NO: 22, of the sequence FTLTGLLGTLVTMGLLT (from the protein DAF - complement decay- accelerating factor precursor or CD55 antigen; SWISSProt ID P08174, positions 365-381).
  • GPI glycosylphosphatidylinositol
  • the transmembrane oligopeptide may be predicted by algorithms.
  • transmembrane stretch of amino acids could be any such stretch predicted by the above mentioned algorithm or a different algorithm with similar prediction and assignment ratings.
  • the cytokine is linked to a transmembrane oligopeptide
  • the transmembrane oligopeptide of HLA-A2 it is, in certain embodiments, the transmembrane oligopeptide of HLA-A2 and it may be linked to the cytoplasmic domain of HLA-A2.
  • the cytokine may be linked to, and therefore anchored to the cell membrane by > the transmembrane and cytoplasmic portion of HLA-A2.
  • the polynucleotide encodes for a membrane bound interleukin, wherein the polynucleotide comprises the following nucleotide sequences: (a) the sequence of SEQ ID NO: 23 encoding for the transmembrane and cytoplasmic portion of HLA-A2; (b) the sequence of SEQ ID NO: 24 encoding for a bridge peptide of a 21 amino acid spacer to the cell membrane, comprising the 13 amino acid flexible linker Gly 4 Ser(Gly 3 Ser) 2 (SEQ ID NO: 21), followed by the 8 membrane-proximal amino acids of the HLA-A2 connecting peptide with an Xhol restriction site; (c) a sequence encoding for the extracellular domain of a cytokine selected from IL-2, IL-12 or IL-15, wherein: (i) the full human IL-2 and its leader peptide sequence is encoded by the sequence of SEQ ID NO: 25; (ii) the chimeric single
  • the polynucleotide encoding the chimeric protein of the present invention is an in vitro transcribed RNA molecule, and it may be polyadenylated, capped or both.
  • RNA molecule of the present invention could be synthesized by any method known in the art, such as, but not limited to, transcription of an expression vector comprising the relevant DNA sequence either in a eukaryotic cell or, preferably, in vitro using purified RNA polymerase.
  • the present invention provides an expression vector comprising a polynucleotide as defined herein above.
  • the expression vector comprises a nucleic acid sequence encoding an RNA molecule in a manner which allows transcription of said nucleic acid sequence in vitro.
  • the present invention provides a composition comprising at least one polynucleotide or the expression vector as defined herein above.
  • the composition comprises at least one polynucleotide, i.e. a DNA or RNA molecule, comprising a sequence encoding: (a) a chimeric protein comprising a cytoplasmic domain of T cell costimulatory receptor of the TNFR family that is linked to a heterologous polypeptide comprising at least one self-assembly domain, said chimeric peptide being linked to an integral membrane or a transmembrane oligopeptide that allows the anchorage of the chimeric polypeptide to the cell membrane; (b) a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to the cell membrane, and said transmembrane oligopeptide, if existent, is linked, optionally via a bridge peptide, to a peptide tag or
  • the composition comprises polynucleotides encoding for a T cell costimulatory receptor of the TNFR family selected from CD40, CD27, 4-1 BB (CD137), OX40 (CD134), herpesvirus entry mediator (HVEM, TNFRSF14), CD30 or glucocorticoid-induced TNFR-related protein (GITR), or combinations thereof.
  • a composition comprising a polynucleotide encoding for a self- aggregating TNFR polypeptide as defined in (a) to (1) may also include combinations of polynucleotides encoding for different self-aggregating members of the TNFR family, e.g.
  • compositions comprising a polynucleotide encoding for a membrane bound cytokine as defined in (a) to (1) may also include combinations of polynucleotides encoding for different cytokines, e.g., but not limited to, membrane bound IL-2 and membrane bound IL-12.
  • the cytokine encoded by the polynucleotide in the composition is selected from IL-2, IL-12 or IL-15, or combinations thereof.
  • the present invention relates to activated T cells expressing at least one chimeric protein encoded by the polynucleotide or the expression vector as defined herein above.
  • the activated T cells express (a) a chimeric protein comprising a cytoplasmic domain of T cell costimulatory receptor of the TNFR family that is linked to a heterologous polypeptide comprising at least one self-assembly domain, said chimeric peptide being linked to an integral membrane or a transmembrane oligopeptide that allows the anchorage of the chimeric polypeptide to the cell membrane; (b) a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to the cell membrane, and said transmembrane oligopeptide, if existent, is linked, optionally via a bridge peptide, to a peptide tag or oligopeptide tag; (c) a chimeric protein comprising an extracellular domain of a cytokine linked through its
  • a TLR4 molecule y active domain of a TLR4 molecule;
  • a chimeric protein comprising a cytokine linked through its carboxyl terminus via a flexible bridge peptide to a cytoplasmic domain of a self- aggregating T cell costimulatory receptor of the tumor necrosis factor receptor (TNFR) family;
  • TNFR tumor necrosis factor receptor
  • a combination of (a) and (b) a combination of (a) and (c);
  • h a combination of (b) and (c);
  • the cells express polynucleotides encoding for a T cell costimulatory receptor of the TNFR family selected from CD40, CD27, 4-1BB (CD137), OX40 (CD134), herpesvirus entry mediator (HVEM, TNFRSF14), CD30 or glucocorticoid-induced TNFR-related protein (GITR), or combinations thereof.
  • a cell expressing a self-aggregating TNFR polypeptide as defined in (a) to (1) may also express combinations of polynucleotides encoding for different members of the TNFR family, e.g. but not limited to, self-aggregating CD40 and self- aggregating 4- IBB.
  • a cell expressing a membrane bound cytokine as defined in (a) to (1) may also express combinations of polynucleotides encoding for different cytokines, e.g., but not limited to, membrane bound IL-2 and membrane b ound IL-12.
  • the cell express a cytokine selected from IL-2, IL-12 or IL-15, or combinations thereof.
  • the activated T cells express a chimeric protein comprising a cytoplasmic and transmembrane polypeptide of CD40 that is linked to a yeast GCN4 leucine zipper DNA-binding motif self-assembly domain and/or a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through a TLR4 transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to the cell membrane.
  • the cytokine is linked to the transmembrane and cytoplasmic portion of HLA-A2.
  • the T cells used in the present invention may be autologous T cells that are naturally reactive against a tumor (tumor infiltrating lymphocytes or TILs), tumor-specific T cells isolated from peripheral blood lymphocytes (PBLs) or non-tumor-specific autologous or allogeneic cells genetically reprogrammed to express tumor-reactive TCR or chimeric TCR molecules displaying antibody-like tumor recognition capacity.
  • the polypeptide may be a chimeric T cell receptor, a T-body (T-bodies are genetically engineered T cells armed with chimeric receptors whose extracellular recognition unit is comprised of an antibody-derived recognition domain and whose intracellular region is derived from lymphocyte stimulating moiety(ies)), a natural or genetically-modified TCR (Morgan et al., 2006), or a TCR-like antibody optionally fused to a toxin, that is specific to a TAA.
  • T-bodies are genetically engineered T cells armed with chimeric receptors whose extracellular recognition unit is comprised of an antibody-derived recognition domain and whose intracellular region is derived from lymphocyte stimulating moiety(ies)
  • TCR-like antibody optionally fused to a toxin, that is specific to a TAA.
  • Tumor infiltrating CTLs are a mixed population of effector and effector memory cells. These cells, which are highly selective to tumor antigens, are suppressed or at anergic state and do not elicit cytotoxic activity. TIL potentiation is currently a major goal of the immune research.
  • the activated T cells are tumor-specific T cells.
  • the present invention provides a method for treating cancer, comprising administering to a cancer patient, a therapeutically effective amount of tumor specific activated T cells as defined herein above.
  • the T cells may be transfected ex vivo, i.e. the T cells are removed from the patient, transfected with the polynucleotide in a laboratory using standard techniques and then returned to the patient; or in situ, using recent techniques that allow for the transfection of the T cells in the patient's body.
  • treating refers to the alleviation, reduction of progression or complete cure of the disease, or to the reduction of symptoms related to or caused by the disease.
  • the basic TIL-ACT protocol for the treatment of melanoma includes selected tumor-reactive TILs, employing IFN- ⁇ secretion in response to tumor antigens as readout for specificity.
  • TILs are expanded by vigorous TCR stimulation followed by lengthy culturing periods with IL-2, resulting in reduced survivabilit. Difficulties in generating autologous melanoma cultures, failure of cultured TILs to produce IFN- ⁇ and exacerbation of disease during selection contribute to considerable patients' dropout and limit the widespread use of sTIL-ACT.
  • the TILs are autologous T cells, in particular tumor infiltrating lymphocytes such as young tumor infiltrating lymphocytes.
  • the T cells are directed to tumor cells selected from melanoma, breast cancer; chronic myeloid leukemia; acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, chronic lymphocytic leukemia, mature B cell neoplasms, mature T cell and natural killer cell neoplasms; liver, stomach, pancreas, intestine and kidney tumors; or bladder, glandular epithelia, lung squamous cell, gut, prostate, testis, thymus, bone marrow, and lymph nodes carcinomas.
  • the present invention further provides methods for preparing activated T cells comprising transfecting the T cells with the polynucleotide, the expression vector or the composition as defined herein above, whereby the polynucleotide is expressed within said T cells, thus obtaining the activated T cells.
  • the present invention is directed to a method for propagating T cells, comprising transfecting said T cells with a polynucleotide comprising a sequence encoding a chimeric protein comprising IL-2 linked through its carboxyl terminus via a flexible bridge peptide to an integral membrane or transmembrane oligopeptide that allows the anchorage of the IL-2 molecule to a cell membrane.
  • This method which obviates the need for adding soluble cytokines to the T cells in order for the cells to propagate and survive, solves the two-sided problem of systemic IL-2 toxicity and insufficient IL-2 support for cells transferred during ACT.
  • the method does not involve contacting the cells with a soluble cytokine, i.e. the cells are grown in the absence of soluble cytokines.
  • the present invention also contemplates TLR4-activated T cells, wherein said T-cells are obtained by transfecting T cells with an RNA molecule including a ribonucleotide sequence encoding a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to a cell membrane, whereby the RNA molecule is expressed within said T cells, thus obtaining the TLR4-activated T cells.
  • an RNA molecule including a ribonucleotide sequence encoding a chimeric protein comprising a cytoplasmic constitutively active domain of a TLR4 molecule linked through its amino terminus to an integral membrane or transmembrane oligopeptide that allows the anchorage of the TLR4 molecule to a cell membrane, whereby the RNA molecule is expressed within said T cells, thus obtaining the TLR4
  • the present invention further considers nucleic acid molecules that comprise DNA sequences encoding for the caTLR4, self- aggregating TNFRs and membrane bound cytokines, and combinations thereof, operably linked to a promoter.
  • Any suitable mammalian expression vector can be used such as, but not limited to, the pCI mammalian expression vectors (Promega, Madison, WI, USA), pCDNA3 expression vectors (Invitrogen, San Diego, CA) and pBJl-Neo.
  • the expression vector may also be a plasmid DNA in which the polynucleotide sequence is controlled by a virus, e.g. cytomegalovirus, promoter, or, most preferably, the expression vector is a recombinant viral vector such as, but not limited to, pox virus or adenovirus or adeno-associated viral vector.
  • transfection any of the techniques which are available in the art may be used to introduce the recombinant nucleic acid encoding the polypeptide into the antigen presenting cell. These techniques are collectively referred to as transfection herein and include, but are not limited to, transfection with naked or encapsulated nucleic acids, cellular fusion, protoplast fusion, viral infection, cellular efidocytosis of calcium-nucleic acid microprecipitates, fusion with liposomes containing nucleic acids, and electroporation. Choice of suitable vectors for expression is well within the skill of the art. Antigen expression may be determined by any of a variety of methods known in the art, such as immunocytochemistry, ELISA, Western blotting, radioimmunoassay, or protein fingerprinting.
  • T cells The activation of T cells is manifested in naive cytotoxic T-lymphocytes (CTLs) by differentiation into effector cells that can kill antigen-bearing target cells using granzymes, perforin and FAS ligand, and can rapidly produce inflammatory cytokines, such as IFNy and TNFa upon TCR ligation and activation.
  • CTLs cytotoxic T-lymphocytes
  • Type II interferon IFN- ⁇ plays a key role in adaptive immunity to viral and bacterial infections and in the antitumor response.
  • IFN- ⁇ functions include the enhancement of antigen presentation by both MHC-I and MHC-II products, suppression of the Thl- antagonistic activities of Th2 cells and promotion of effector CD4 Thl and CD8 T cell differentiation.
  • IFN- ⁇ is also known for its anti-proliferative, apoptotic and angiogenic effects, all potentially contributing to its well documented antitumor activity.
  • An additional cytokine which is involved in T cell activation is IL-2. This cytokine is released from T cells and bind to a specific T cell IL-2 receptor to further promote the activation and proliferation of T cells in an autocrine fashion.
  • effector CTLs can adopt multiple cell fates, such as short-lived effectors or memory precursors.
  • Short-lived cells effectors can either become senescent effectors and die by apoptosis in several days, or persist into early memory as short-lived effector memory cells.
  • Memory precursors by contrast, are long-lived and can be differentiated into effector memory cells or central memory cells. Although the exhausted cell is depicted as sharing a common effector cell precursor with other cell fates, it is currently unclear at what point during effector cell differentiation this fate branches.
  • the TLR-activated CD4 T cells are capable of conferring immune memory to activated CD8 T cells, i.e. the constitutive activation of both CD4 and CD8 T cells by the means of the method of the present invention not only produces armed killer T cells but may also produce memory T cells that are capable of sustaining a prolonged attack on the target cells.
  • the marker used herein to measure T cell activation is mainly the measurement of the level of secreted IFN- ⁇ ; however, the level of other markers such as, but not limited to, IL-2, TNFa, granzyme B, perforin and FAS ligand secretion and transcription factor NF- B activity, or combination of markers, for T cell maturation could also be assessed. It should be clear to a person skilled in the art that a CD8 T cell that secretes high levels of IFN- ⁇ is an activated, fully mature CD8 T cell, i.e.
  • a cytotoxic T cell also known as TC, CTL, T-Killer cell, cytolytic T cell, or killer T cell
  • TC cytotoxic T cell
  • CTL CTL
  • T-Killer cell cytolytic T cell
  • killer T cell a CD4 T cell that secretes high levels of IFN- ⁇
  • Thl -cell also known as effector Thl cell or Thl cell
  • the phrase "preparing TLR-activated T cells" is used herein to describe the production of T cells exhibiting the hallmarks of a mature phenotype, for example secreting high levels of IFN- ⁇ .
  • the activated T cells of the present invention are preferably prepared by transfecting T cells with an RNA molecule encoding constitutively active factors and/or membrane cytokines.
  • ACT procedures which require the genetic reprogramming of human T cells utilize either retroviral or lentiviral vectors.
  • retroviral or lentiviral vectors the clinical implementation of such vectors entails complicated and lengthy procedures, while the number and size of genes that can be introduced is restricted.
  • the use of these vectors in the clinic raises serious safety concerns which further limit their wide application.
  • mRNA should only gain entry into the cell cytoplasm where it is ready for translation, thus requiring milder transfection conditions; the introduced RNA is similar in composition to the endogenous mRNA and does not carry any inherent danger signals; pre-defined mixtures of mRNA can be easily prepared and introduced simultaneously, rendering this approach highly versatile; andj mRNA offers maximal safety.
  • mRNA may not be the ideal vehicle for conferring durable tumor specificity through the transfer of anti-tumor TCR or T-body genes. Nonetheless, this consideration is irrelevant to other ACT approaches which utilize T cells that are naturally reactive against the tumor (TILs, membrane-capturing, allogeneic and T cells activated by tumor or tumor antigens ex-vivo). In contrast, modification of the functional status of T cells (and other immune cells) is usually accomplished in the course of a natural immune response following a transient, rather than a constitutive, stimulus.
  • peptide as used herein refers to a short amino acid stretch of between 2 and about 10 amino acids and the term oligopeptide as herein refers to a short amino acid stretch of between 2 and about 20 amino acids.
  • oligopeptide refers to a short amino acid stretch of between 2 and about 20 amino acids.
  • the term "about” as used herein refers to a maximum deviation of ⁇ 10% from the value preceded by this term.
  • RNA preparation and transfection of DCs and APC clones Genes of interest were cloned into the multiple cloning site of a special vector designed for in-vitro production of mRNA ⁇ pEGM4Z/GFP/A64 ⁇ , following removal of the GFP gene.
  • This vector described in detail in Boczkowski et al., 2000. Cancer Res 60:1028-1034, is based on the vector pGEM4Z (Promega, Madison, WI).
  • Messenger RNA was prepared from cloned templates using the AmpliCap-Max T7 High Yield Massange Marker Kit (Epicenter) following plasmid linearization with Spel.
  • Immature and mature DCs were harvested and washed two times in cold PBS, then resuspended in cold Opti-MEM at 2-3 xl06 cells/100 ⁇ and were mixed in 2 mm gap cuvette (Bio-Rad) along with 5-10 Dg of in-vitro transcribed mRNA . The mixture was then subjected to appropriate conditions in a square wave electroporator (BTX-ECM 830,San Diego, CA).
  • Example 2 Expression via mRNA transfection of caTLR4, but not caTLR2, potently activates human CD4 and CD8 T cells to secrete IFN- ⁇ .
  • Cells were separated by CD4 magnetic beads (BD IMag anti-human CD4 particles) positive selection, grown for additional 24 hours in the presence of 0.5 ⁇ g/ml each anti-CD3 and anti-CD28 mAbs and subjected to electroporation with 5 /ig mRNA using a square- wave pulse at 400 V, 0.95 msec in 0.1 ml in 2 mm cuvettes.
  • Control cells were either non-treated (NT), incubated in the presence of LPS or Pam3 (1 ig ml each) or transfected with EGFP-encoding mRNA as a negative control. Secretion of IFN- ⁇ the growth medium was monitored by ELISA (R&D systems Human IFN-gamma Duo Set Economy Pack) 24 h post-transfection.
  • the caTLR4 constructs we used in the last experiment encode an HLA-A2- binding tumor- associated peptide linked to ⁇ 2 ⁇ , while the donor was HLA-A2+.
  • caTLR4 is active in CD4 and CDS T cells of both donors.
  • a Melan-A27- 35/HLA-A2-specific CTL clone, 1C9 isolated from a human melanoma patient by tetramer sorting, were thawed and incubated in culture medium and 6000 u/ml IL-2 was added every 2 days (for the method of tetramer sorting, see for example Ogg and McMichael (1998) Current Opinion in Immunology, 10: 393-396). Electroporation was preformed 5 days post thawing. TIL 122, isolated from a human melanoma tumor, were thawed and incubated for 2 days in culture medium containing 6000 u ml IL-2.
  • CD4 positive magnetic beads separation was preformed (BD IMag anti-human CD4 particles). After separation the cells were incubated in 6 ml culture medium and 6000 u/ml IL-2 was added every 2 days. Electroporation was preformed 7 days post thawing. After the electroporation the cells were incubated in 12 well-plates in 3 ml culture medium in the absence of IL-2. 24 h later the supernatant was collected and IFN-7 was measured using R&D systems' Human IFN-gamma DuoSet Economy Pack.
  • IL-2Ra CD25
  • CD69 elevation is linked to the effector phenotype of CTLs and was reported as an activation marker following CTL incubation with the TLR2 ligand, Pam3.
  • Example 3 Generation of constitutively active oligomeric CD40 (caCD40) based on the yeast leucine zipper motif GCN4,
  • DCs In order to gain full CD8 T cell priming capacity, DCs must complete their maturation and secrete IL-12. Acquisition of this capacity requires initial triggering of the DC via TLRs (or other innate pathogen-recognition receptors), followed by licensing signals mediated by the engagement of trimeric CD40 ligand (CD40L) at the surface of activated Thl cell with the DC CD40 receptor.
  • TLRs or other innate pathogen-recognition receptors
  • CD40L trimeric CD40 ligand
  • primers for PCR-mediated cloning having the following sequences: For the dimers, primers 1 133 and 1134 of SEQ ID NO: 30 and 31, respectively; for the trimers, primers 1135 and 1136 of SEQ ID NO: 32 and 33, respectively; and for the tetramers, primers 1137 and 1138 of SEQ ID NOs: 34 and 35, respectively.
  • primers 1 128 and 1 129 having the sequences of SEQ ID NOs: 36 and 37, respectively, were used.
  • Example 4 Generation of membrane bound cytokines and characterization of their activity in transtransfected T cells alone or in combination with caTLR4 or caCD40.
  • These chimeric genes comprises the sequence of SEQ ID NO: 23 encoding for the transmembrane and cytoplasmic portion of HLA-A2 and the sequence of SEQ ID NO: 24 encoding for a bridge peptide;
  • IL-2 the full human IL-2 and its leader peptide sequence is encoded by the sequence of SEQ ID NO: 25;
  • the chimeric single chain derivative of human IL-12 is encoded by the sequence of SEQ ID NO: 26 encoding for the full human IL-12 p40, including the leader peptide, the sequence of SEQ ID NO: 27 encoding for the linker peptide, and the sequence of SEQ ID NO: 28 encoding for the human IL-12 p35 without the leader peptide (see Fig. 7); and
  • chimeric human IL-15 is encoded by SEQ ID NO: 5 encoding for the leader peptide of human j8 2 m, and SEQ ID NO: 29 encoding for the mature peptide of human IL-15.
  • SEQ ID NO: 5 encoding for the leader peptide of human j8 2 m
  • SEQ ID NO: 29 encoding for the mature peptide of human IL-15.
  • Membranal (mem) IL-2, IL-12 and IL-15 are designed with a flexible spacer so as to engage its cell surface receptor in-cis (that is, on the same cell).
  • CD 8 T cells from PBLs of a healthy donor, were grown for 4 days in O T3 and IL-2 and separated to CD8 and CD4 T cells (O T3 (Janssen-Cilag, or collected from a hybndoma available in our lab) is a mouse monoclonal that binds to and blocks function of T lymphocytes responsible for antigen recognition) Thereafter, the cells were electroporated with memIL-2 and irrelevant mRNA(s) or treated as indicated (Fig. 8).
  • CFSE carboxyfluorescein succinimidyl ester
  • melanoma TILs were thawed and grown in complete medium in the presence of 2,000 U/ml IL-2.
  • Four days post-thawing cells were electroporated with 10 /zg of each mRNA.
  • 24 hours post-transfection growth medium was collected for IFN- ⁇ ELISA.
  • Fig 9 shows that memIL-2, memIL-12 and caCD40 work synergistically with caTLR4, as can be seen by the tremendous increase in secreted IFN- ⁇ .
  • caCD40 and to a lesser extent, caTLR4 are capable of activating the TILs alone.
  • M427 is a primary melanoma cells that are recognized by its autologous TIL- 425. Although these cells recognize its target, they are considered exhausted and exhibit very low killing activity.
  • TIL-425 were thawed and cultured with 3000 u/ml IL-2 for 4 days. The cells were grown for an additional 3 days to wash out the direct mRNA effect (measured by IFN- ⁇ production). Thereafter 1X10 5 cells were incubated at 1/1 ratio with its targets melanoma cells for 5 hrs. The Ml 71 melanoma cells served as a negative control.
  • Example 5 ACT using tumor-specific T cells expressing membranal cytokines, caTNFR or caTLR4, or their combinations.
  • mice are used for comparative evaluation of T cell tumoricidal activity in-vivo, using a transplantable human melanoma.
  • human melanoma cell line 624mel For this purpose we utilize the human melanoma cell line 624mel. Briefly, male CDlnu/nu mice are injected s.c. in the upper back with lxlO 6 624mel mlanoma cells admixed 1 :1 with Matrigel. Five days later mice are divided into groups of ten and each mouse is injected in the tumor area or IV with lxlO 6 624mel- reactive CD8 T cells cloned from anti-melanoma TILs.
  • T cells from each group Prior to injection, all T cells from each group (>10 l0 6 ) will be electroporated in a single cuvette with the respective in- vitro transcribed mRNA under investigation, or irrelevant mRNA as control and then divided for cell transfer. Additional control groups receive none-modified T cells or no T cells. Mice are injected with 1,000 units IL-2 into the tumor site twice daily for 5 days and survival is monitored daily.
  • the in-vitro transcribed mRNA tested in this experiment encode for caTLR4, caCD40, caCD27, ca4-lBB (CD137), caOX40 (CD134), caHVEM, caCD30 and caGITR as well as mem IL-2, memIL-12 and memIL-15. Also T cells transfected with combinations thereof will be used. First, caTLR4, caCD40 and mem-IL2 expressing T cells will be examined, and later other combinations.
  • Besser M. J., R. Shapira-Frommer, A. J. Treves, D. Zippel, O. Itzhaki, E. Schallmach, A. Kubi, B. Shalmon, I. Hardan, R. Catane, E. Segal, G. Markel, S. Apter, A.

Abstract

L'invention concerne des polynucléotides codant pour des protéines chimériques comprenant le TLR4 constitutivement actif, un élément constitutivement actif du récepteur de costimulation des lymphocytes T de la famille du récepteur du facteur de nécrose tumorale (TNFR) ou d'une cytokine liée à la membrane, ou des combinaisons de ceux-ci. Les polynucléotides sont utiles pour la transfection des lymphocytes T et ainsi les propagent sans avoir besoin de cytokines solubles pour activer constitutivement les lymphocytes T et pour le transfert cellulaire des lymphocytes T constitutivement activés dans le traitement du cancer.
PCT/IL2012/000125 2011-03-23 2012-03-22 Lymphocytes t constitutivement activés pour l'utilisation dans une thérapie cellulaire adoptive WO2012127464A2 (fr)

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