Patent Application
20200281977
The present invention refers to a stably or transiently IL-1R8 deficient isolated human cell, being a natural killer (NK) cell or T cell and to their medical use, preferably in the treatment of tumours and infections.
Interleukin-1 receptor 8 (IL-1R8, also known as single immunoglobulin IL-1R-related receptor, SIGIRR, or TIR8 [NCBI Gene ID: 59307; NM_001135053.1→NP_001128525.1; NM_001135054.1→NP_001128526.1; NM_021805.2→NP_068577.2, sequences shown below:
is a member of the IL-1 receptor (ILR) family with distinct structural and functional characteristics, acting as a negative regulator of ILR and Toll-like receptor (TLR) downstream signalling pathways and inflammation1.
The IL-1 system has a central role in both innate and adaptive immune responses and it is tightly controlled at different levels by antagonists, decoy receptors, scavengers, dominant negative molecules, miRNAs and other mechanisms, acting extracellularly or intracellularly. IL-1R8/TIR8/SIGIRR is an atypical receptor acting as a novel negative regulator of inflammatory and adaptive responses mediated by ligands of the IL-1 system. IL-1R8/TIR8/SIGIRR gene is localized on human chromosome 11 and on murine chromosome 7, and the protein (410 amino acids) is constituted by a single Ig extracellular domain with several N- and O-glycosylation sites, a transmembrane domain, an intracellular conserved TIR domain and a 95 amino acid-long tail at the C-terminal.
IL-1R8/TIR8/SIGIRR is widely expressed, in particular in epithelial tissues, such as the kidney, digestive tract, liver and lung, and in lymphoid organs by lymphoid cells.
IL-1R8/TIR8/SIGIRR has been reported to inhibit NF-kB, JNK and mTOR kinase activation following stimulation of IL-1 receptor or TLR family members. It negatively modulates the signal transduction activated by the IL-1 receptor family members IL-1R1, IL-18R, ST2, and several TLRs, such as TLR1/2, TLR3, TLR4, TLR7 and TLR9. The molecular mechanisms proposed include interference of the dimerization of IL-1R1 and IL-1RAcP through the extracellular Ig domain of IL-1R8/TIR8/SIGIRR, and binding of TIR-containing adaptor molecules through the TIR domain, which are no more available for signalling.
Natural killer (NK) cells are innate lymphoid cells which mediate resistance against pathogens and contribute to the activation and orientation of adaptive immune responses2-4. NK cells mediate resistance against haematopoietic neoplasms but are generally considered to play a minor role in solid tumour carcinogenesis5-7.
Several lines of evidence suggest that IL-1R8 interferes with the association of TIR module-containing adaptor molecules with signalling receptor complexes of the ILR or TLR family, tuning downstream signalling, thus negatively controlling inflammatory and immune responses and T helper cell polarization and functions1,8.
It has been previously shown that CD4+ T lymphocytes express IL-1R8 (Garlanda C et al, Trends Immunol (2009); Gulen et al Immunity (2010); Bulek et al J Immunol (2009); Bozza et al J Immunol (2008)). These studies reported that IL-1R8 is a negative regulator of CD4+T lymphocytes and their helper function was amplified when IL-1R8 was genetically silenced in mice. Helper activity can be exerted by different T subsets while among T lymphocyte subsets, cytotoxic activity is mostly exerted by CD8+T subsets. The molecular mechanisms regulating the cytotoxic potential of CD8+T lymphocytes differ from those involved in CD4+T lymphocytes and the functional activities of these two cell types are different, since CD4+ T cells have helper functions and CD8+ T cells cytotoxic activity. Therefore, the regulatory role of IL-1R8 in cytotoxic T cells has still to be investigated, in particular in CD8+T lymphocytes. Moreover, IL-1R8 is the co-receptor of IL-1R5/IL-18R for IL-37 and is required for the anti-inflammatory activity of this human cytokine9. Deregulated activation by ILR or TLR ligands in IL-1R8-deficient mice has been associated with exacerbated inflammation and immunopathology, including selected cancers, or autoimmune diseases10.
WO2005084696 refers to the use of an agent interacting with TIR8/SIGIRR for the preparation of a therapeutic composition for treating inflammation in the gastrointestinal tract and for stimulating mucosal or epithelial immunity.
WO2007034465 refers to the novel finding that IL-1 F5 (IL-1 delta) and polypeptides derived therefrom bind to the receptor SIGIRR, with this binding interaction serving to modulate the immune response by stimulating the production of the cytokine IL-4. This induces an anti-inflammatory immune response. It has been further shown that PPARgamma is a key mediator in downstream signalling from SIGIRR following activation by the IL-1 F5 ligand. Modulation of the immune response occurs following binding of SIGIRR by IL-1 F5 in neuronal tissue and according methods for the treatment of neurodegenerative diseases are described.
It is still felt the need of a method of treating tumours by using NK or T cells.
The present inventors found out that IL-1R8 serves as a checkpoint for NK cell maturation and effector function. Its genetic blockade unleashes NK-cell-mediated resistance to hepatic carcinogenesis, haematogenous liver and lung metastasis, and cytomegalovirus infection.
Inventors found that IL-1R8 acts as a checkpoint of NK cell anti-tumor and anti-viral activity. IL-1R8 genetic inactivation in NK cells has potential translational implications in NK cell-based cell therapies.
The inventors herein show that:
The inventors herein also show in NK cell-adoptive transfer experiments in preclinical models of liver and lung metastasis in mice that IL-1R8-deficient NK cells significantly and dramatically reduced the number and volume of metastasis (
Moreover, they found that IL-1R8 expression level inversely correlates with NK cell activation in humans (
IL-1R8 is also expressed in CD8+ T cells, indicating a wider role of IL-1R8 as a checkpoint molecule and potential implication of IL-1R8-inactivation in both NK and T cells (
It is therefore an object of the invention an isolated human cell, being a natural killer (NK) cell or T cell, wherein said cell is stably or transiently deficient in the expression and/or activity of IL-1R8. Said T cell is preferably a CD8+ T cell.
Said cell preferably produces greater amounts of effector molecules involved in anti-tumour immunity, preferably interferon-gamma (IFN-γ) and/or granzyme B and/or FasL and/or express higher levels of maturation markers, preferably CD44, than cells that do express IL-1R8.
The above cell is preferably further deficient in the expression and/or activity of at least one checkpoint for NK cell maturation and/or effector function. Said at least one checkpoint for NK cell maturation and/or effector function is preferably selected from the group consisting of: CIS, KIRs, PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT.
Further objects of the invention are a population of cells comprising the NK cells and/or T cells as above defined and a composition comprising the cells as above defined or the population of cells as above defined, preferably further comprising at least one physiologically acceptable carrier.
The cell, or the population, or the composition as above defined are preferably for use as a medicament, more preferably for use in the treatment and/or prevention of tumour and/or metastasis, or of microbial or viral infection.
The cell or the population or the composition as above defined are preferably used in Adoptive cell transfer (ACT), cell therapy treatment, mismatched bone marrow transplantation, mismatched NK cell infusion or cytokine-induced killer (CIK) cell infusion. Said NK cell or T cell is preferably previously isolated from the same treated subject or from a different subject.
Another object of the invention is a suppressor or inhibitor of IL-1R8 expression and/or activity for medical use, preferably for use in the treatment and/or prevention of tumour and/or metastasis, or of microbial or viral infection.
Said suppressor or inhibitor is preferably at least one molecule selected from the group consisting of:
a) an antibody or a fragment thereof;
b) a polypeptide;
c) a small molecule;
d) a polynucleotide coding for said antibody or polypeptide or a functional derivative thereof;
e) a polynucleotide, such as antisense construct, antisense oligonucleotide, RNA interference construct or siRNA,
e) a vector comprising or expressing the polynucleotide as defined in d) or e);
f) a CRISPR/Cas9 component, e.g. a sgRNA
g) a host cell genetically engineered expressing said polypeptide or antibody or comprising the polynucleotide as defined in d) or e) or the component of f).
Preferably said polynucleotide is an RNA inhibitor, preferably selected from the group consisting of: siRNA, miRNA, shRNA, stRNA, snRNA, and antisense nucleic acid, more preferably the polynucleotide is at least one siRNA selected from the group consisting of: AGU UUC GCG AGC CGA GAU CUU (SEQ ID NO:1); UAC CAG AGC AGC ACG UUG AUU (SEQ ID NO:2); UGA CCC AGG AGU ACU CGU GUU (SEQ ID NO:3); CUU CCC GUC GUU UAU CUC CUU (SEQ ID NO:4) (all 5′ to 3′), or a functional derivative thereof.
Said suppressor or inhibitor is preferably used in NK and/or T cell and/or in adoptive cell transfer (ACT), cell therapy treatment, mismatched bone marrow transplantation, mismatched NK cell infusion or cytokine-induced killer (CIK) cell infusion. Preferably, said suppressor or inhibitor is preferably used for the treatment of NK and/or T cells. Said host cell is preferably an NK or T cell.
A further object of the invention is a pharmaceutical composition comprising the suppressor or inhibitor as above defined and at least one pharmaceutically acceptable carrier, and optionally further comprising a therapeutic agent.
The above tumour is preferably a solid tumor or an hematological tumor, preferably selected from the group consisting of: Colon/Rectum Cancer, Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors In Adults, Brain/CNS Tumors In Children, Breast Cancer, Breast Cancer In Men, Cancer of Unknown Primary, Castleman Disease, Cervical Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Acute Lymphocytic (ALL), Acute Myeloid (AML, including myeloid sarcoma and leukemia cutis), Chronic Lymphocytic (CLL), Chronic Myeloid (CML) Leukemia, Chronic Myelomonocytic (CMML), Leukemia in Children, Liver Cancer, Lung Cancer, Lung Cancer with Non-Small Cell, Lung Cancer with Small Cell, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma In Children, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma—Adult Soft Tissue Cancer, Skin Cancer, Skin Cancer—Basal and Squamous Cell, Skin Cancer—Melanoma, Skin Cancer—Merkel Cell, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, uveal melanoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, Wilms Tumor, more preferably the tumour is a solid tumor, preferably colorectal cancer, and the metastasis are lung or liver metastasis.
The above infection is preferably caused by one of the following viruses or bacteria: herpesviruses, preferably cytomegalovirus, Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Hepatitis B Virus (HBV), West Nile virus (WNV), Salmonella, Shigella, Legionella, Mycobacterium.
Another object of the invention is a method to obtain the cell, or the population, or the composition as defined above, comprising the step of stably or transiently inhibiting or suppressing the expression and/or function of IL-1R8 in an NK or T cell or cell population comprising NK and/or T cells, and optionally further expanding in vitro the silenced population. Said T cell is preferably a CD8+ T cell. Said methods are preferably in vitro or ex vivo methods. Said NK or T cell or cell population is preferably previously purified from isolated peripheral blood mononuclear cell (PBMCs) and optionally expanded in vitro, preferably using Recombinant Human Interleukin-2 (rhIL-2).
The above method preferably further comprises the inhibition or suppression of the expression and/or function of at least one further checkpoint for NK cell maturation and/or effector function. Said at least one checkpoint for NK cell maturation and/or effector function is preferably selected from the group consisting of: CIS, KIRs, PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT.
In the above method the step of stably or transiently inhibiting or suppressing the expression and/or function of IL-1R8 in an NK or T cell or cell population is preferably carried out with at least one of the above defined suppressor or inhibitor.
In the context of the present invention a “CD8+ T cell” includes a cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell), a T lymphocyte (a type of white blood cell) that kills cancer cells, cells that are infected (particularly with viruses), or cells that are damaged in other ways. Most cytotoxic T cells express T-cell receptors (TCRs) that can recognize a specific antigen. Antigens inside a cell are bound to class I MHC molecules, which brings the antigen to the surface of the cell where they can be recognized by the T cell. In order for the TCR to bind to the class I MHC molecule, the former must be accompanied by a glycoprotein called CD8, which binds to the constant portion of the class I MHC molecule. Therefore, these T cells are defined as CD8+ T cells.
In the context of the present invention, a cell deficient in the expression and/or activity of IL-1R8 is a cell in which the levels of IL-1R8 (protein and/or mRNA) are reduced or completely inhibited permanently or transiently. A cell deficient in the expression and/or activity of IL-1R8 may be obtained e.g. by silencing using CRISPR/Cas9 system, siRNA, peptides or antibodies interfering with the interaction with other ILR/TLR receptors. Said deficient cell may be e.g. transformed using sgRNA, preferably said sgRNA being delivered into the cells with a CRISPR-Cas9 system.
In one embodiment, the NK and/or T cells deficient in the expression and/or activity of IL-1R8 express no detectable IL-1R8. In another embodiment, the NK and/or T cells deficient in the expression and/or activity of IL-1R8 express no immunologically detectable IL-1R8. In one embodiment, the NK and/or T cells deficient in the expression and/or activity of IL-1R8 express no detectable IL-1R8 mRNA. The NK and/or T cells deficient in the expression and/or activity of IL-1R8 (or lacking functional IL-1R8) can be prepared using any conventional method. In some embodiments, a cell deficient in the expression and/or activity of IL-1R8 is obtained by inhibiting or blocking IL-1R8 expression by, e.g., gene deletion, gene disruption, siRNA, shRNA or antisense approaches. In other embodiments, a cell deficient in the expression and/or activity of IL-1R8 is obtained by inhibiting or blocking IL-1R8 activity by, e.g., a IL-1R8 antagonist or antibody. In certain embodiments, a cell deficient in the expression and/or activity of IL-1R8 is obtained by blocking the expression of endogenous IL-1R8 by genetically modifying the immune cell. Although in some cases homologous recombination is used, in particular cases non-homologous end joining is used to edit the genome. Any suitable protocol to modify the genome of a particular immune cell is useful, although in specific embodiments gene modification is achieved using an engineered nuclease such as a zinc finger nuclease (ZFP), TALE-nuclease (TALEN), or CRISPR/Cas nuclease. Engineered nuclease technology is based on the engineering of naturally occurring DNA-binding proteins. For example, engineering of homing endonucleases with tailored DNA-binding specificities has been described, (see, Chames, et al. (2005) Nucleic Acids Res. 33(20):e178; Arnould, et al. (2006) J. Mol. Biol. 355:443-458). In addition, engineering of ZFPs has also been described. See, e.g., U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978; 6,979,539; 6,933,113; 7,163,824; and 7,013,219.
All the above definitions of “cell deficient in the expression and/or activity” apply, mutatis mutandis, also to the “cells deficient in the expression and/or activity of at least one checkpoint for NK cell maturation and/or effector function”.
The term “checkpoint for NK cell maturation and/or effector function” includes molecules which are fundamental for the regulation of immune-mediated responses e.g. the molecule known as CIS (cytokine-inducible SH2-containing protein), KIRs (killer cell immunoglobulin-like receptor), PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT (Hsu J et al, JCI (2018) https://doi.org/10.1172/JCI99317.; Guillerey C et al, Nat Immunol (2016) https://doi.org/10.1038/ni.3518; Delconte R B et al, Nat Immunol (2016) https://doi.org/10.1038/ni.3470). PD-1 blockade is known to favour an immune reactivation, being therefore protective and curative in tumor models and oncological patients; the other molecules (i.e. CTLA-4, PD-L1, KIRs, TIM-3, NKG2A, CD96, TIGIT, CIS) regulating different pathways and acting through different mechanisms, were previously described as inhibitory molecules in NK cells. Most of them are already in use in clinics, others are under development (e.g. CIS, CD96). PD-1 is the checkpoint molecule mostly used in the clinic and for which tools are available for preclinical studies in the mouse. The role of PD-1 as a checkpoint molecule of NK cells has recently been published (Hsu J et al, JCI (2018)). PD-1 is expressed in terminally differentiated and exhausted cytotoxic lymphocytes and it is induced upon chronic activation and in the tumor microenvironment as a mechanism of immunosuppression (Freeman G J et a. JEM (2000)). PD-1-dependent immune inhibitory activity depends on the interaction with the ligand (PD-L1) expressed on the target cell, in particular tumoral cells (Freeman G J et a. JEM (2000); Hsu J et al, JCI (2018)). Therefore, the inhibition of the PD-1/PD-L1 axis with checkpoint inhibitors (anti-PD-1 or anti-PD-L1 blocking antibodies) can be addressed only in presence of the cytotoxic cell type (e.g. NK cells, CD8+ T cells) and a target (e.g. tumoral cell).
In the context of the present invention an “effector molecule involved in anti-tumour immunity” is a molecule which mediates fundamental mechanisms of the immune response against tumor cells. Preferably it can be interferon-gamma (IFN-γ), granzyme B, FasL.
The population of cells according to the invention preferably comprises at least 50% of the NK cells and/or T cells as defined above.
In one embodiment, the composition or the cell population as defined above comprises more than 50% of NK and/or T cells deficient in the expression and/or activity of IL-1R8. In another embodiment, the composition or the cell population comprises more than 70% of NK and/or T cells deficient in the expression and/or activity of IL-1R8. In another embodiment, the composition or cell population comprises more than 80% of NK and/or T cells deficient in the expression and/or activity of IL-1R8.
The T cell of the invention is preferably a CD8+ T cell.
The above-mentioned cytokines are observed in vivo. Therefore, the expression “said cell produces” includes not only the direct production but also the indirect production of cytokines, relating to the final effect of the tumoral process, controlled differently between the two animal groups.
Every known method for obtaining/expanding mature NK or T cells may be used. Several strategies have indeed been developed to obtain/expand mature NK cells in vitro (see e.g. Fang F. et al. Semin Immunol 31 (2017) 37-54; Davis Z. B. et al. Semin Immunol 31 (2017) 64-75). As a way of example, NK cells may be purified from PBMCs and expanded in vitro using rhIL-2. IL-1R8 may be then silenced using any silencing method, e.g. CRISPR/Cas9 system or siRNA or neutralized with mAb. Pretreatment with cytokines may be preferably considered and NK or T cells may be infused in patients by any convenient administration route, e.g. through intravenous or intra-arterial injection. (see for instance Koehl U, et al. Front Oncol. 2013 May 17; 3:118. doi: 10.3389/fonc.2013.00118. eCollection 2013. Granzim N. et al. Front Immunol. 2017 Apr. 26; 8:458. doi: 10.3389/fimmu.2017.00458. eCollection 2017).
In the context of the present invention, “IL-1R8 activity” or “activity of IL-R8” comprises e.g. the interaction with other IL-1R family members and TLR family members, the negative regulation of TLR family members activation and signal transduction, inhibition of NF-kB, JNK and/or mTOR kinas activation, negative modulation of the signal transduction activated by the IL-1 receptor family member, e.g. IL-1R1, IL-18R, ST2, and TLRs, e.g. TLR1/2, TLR3, TLR4, TLR7 and/or TLR9.
IL-1R8 is a membrane receptor that interacts with other IL-1R family members and TLR family members, negatively regulating their activation and signal transduction. IL-1R8 activity has been e.g. inhibited by the present inventors through genetic deficiency in mice and genetic silencing using siRNA in humans using Dharmacon™Accell™ siRNA technology.
In addition, IL-1R8 activity may be inhibited by silencing using CRISPR/Cas9 system, other siRNA, by peptides or antibodies interfering with the interaction with other ILR/TLR receptors, as described for instance by Fang F. et al. Semin Immunol 31 (2017) 37-54. In the context of the present invention the term “activity” and “function” are interchangeable.
The NK cells of the invention include NK progenitors and mature and functional NK cells.
The NK progenitor cells can be differentiated into mature and functional NK cells recognizing a desired target by specific receptors on their surface known to the expert in the field (e.g. NKG2D, DNAM-1, NCRs, KIR-receptors). These mature and functional NK cells can be generated in vitro by extending the culture period 2-3 more weeks. However, as cellular therapeutic the injection of the primitive progenitors and maturation in vivo is preferred. These NK cells can be used in the treatment of tumors, cancer, in particular leukemias, ovarian, colon and skin cancers, Breast, Brain and Lung cancers, Cervical cancer and metastases of all kinds of cancer, particularly to the liver, as well as all viral diseases, in particular HIV, HCV, and other chronic viral diseases.
Doses for such pharmaceutical compositions are generally expressed in the number of viable cells present in such a composition. Said number should be between 1-9×106 NK-initiating cells or >1-10×108 mature NK-cells or 1-9×106 T cells per kg body weight of a subject to be treated. After pretreatment with cytokines, NK cells according to the invention may be infused in patients through intravenous or intra-arterial injection (see for instance Koehl U, et al. Front Oncol. 2013 May 17; 3:118. doi: 10.3389/fonc.2013.00118. eCollection 2013. Granzim N. et al. Front Immunol. 2017 Apr. 26; 8:458. doi: 10.3389/fimmu.2017.00458. eCollection 2017).
The polynucleotides as above described, as e.g. the siRNAs, may further comprise dTdT or UU 3′-overhangs, and/or nucleotide and/or polynucleotide backbone modifications as described elsewhere herein. In the context of the present invention, the term “polynucleotide” includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA, siRNA, shRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The polynucleotide may be single-stranded or double-stranded. The RNAi inhibitors as above defined are preferably capable of hybridizing to all or part of specific target sequence. Therefore, RNAi inhibitors may be fully or partly complementary to all of or part of the target sequence. The RNAi inhibitors may hybridize to the specified target sequence under conditions of medium to high stringency. An RNAi inhibitors may be defined with reference to a specific sequence identity to the reverse complement of the sequence to which it is intended to target. The antisense sequences will typically have at least about 75%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence identity with the reverse complements of their target sequences.
The term polynucleotide and polypeptide also includes derivatives and functional fragments thereof. The polynucleotide may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides).
In the context of the present invention, the genes as above defined (as IL-1R8) are preferably characterized by the sequences identified by their NCBI Gene ID and Gen Bank Accession numbers. However, they include also corresponding orthologous or homologous genes, isoforms, variants, allelic variants, functional derivatives, functional fragments thereof.
In the context of the present invention the term “gene” also includes corresponding orthologous or homologous genes, isoforms, variants, allelic variants, functional derivatives, functional fragments thereof. The expression “protein” is intended to include also the corresponding protein encoded from a corresponding orthologous or homologous genes, functional mutants, functional derivatives, functional fragments or analogues, isoforms thereof.
In the context of the present invention, the term “polypeptide” or “protein” includes:
i. the whole protein, allelic variants and orthologs thereof;
ii. any synthetic, recombinant or proteolytic functional fragment;
iii. any functional equivalent, such as, for example, synthetic or recombinant functional analogues.
The term “analogue” as used herein referring to a protein means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide.
A “derivative” may be a nucleic acid molecule, as a DNA molecule, coding the polynucleotide as above defined, or a nucleic acid molecule comprising the polynucleotide as above defined, or a polynucleotide of complementary sequence. In the context of the present invention the term “derivatives” also refers to longer or shorter polynucleotides and/or polypeptides having e.g. a percentage of identity of at least 41%, 50%, 60%, 65%, 70% or 75%, more preferably of at least 85%, as an example of at least 90%, and even more preferably of at least 95% or 100% with the sequences herein mentioned or with their complementary sequence or with their DNA or RNA corresponding sequence. The term “derivatives” and the term “polynucleotide” also include modified synthetic oligonucleotides. The modified synthetic oligonucleotide are preferably LNA (Locked Nucleic Acid), phosphoro-thiolated oligos or methylated oligos, morpholinos, 2′-O-methyl, 2′-O-methoxyethyl oligonucleotides and cholesterol-conjugated 2′-O-methyl modified oligonucleotides (antagomirs). The term “derivative” may also include nucleotide analogues, i.e. a naturally occurring ribonucleotide or deoxyribonucleotide substituted by a non-naturally occurring nucleotide. The term “derivatives” also includes nucleic acids or polypeptides that may be generated by mutating one or more nucleotide or amino acid in their sequences, equivalents or precursor sequences. The term “derivatives” also includes at least one functional fragment of the polynucleotide. In the context of the present invention “functional” is intended for example as “maintaining their activity”. The above defined antibodies comprise human and animal monoclonal antibodies or fragments thereof, single chain antibodies and fragments thereof and miniantibodies, bispecific antibodies, diabodies, triabodies, or di-, oligo- or multimers thereof. Also included are peptidomimetics or peptides derived from the antibodies according to the invention, e.g. they comprise one or several CDR regions, preferably the CDR3 region. Further included are human monoclonal antibodies and peptide sequences which, based on a structure activity connection, are produced through an artificial modeling process (Greer J. et al., J. Med. Chem., 1994, Vol. 37, pp. 1035-1054).
Preferably, the antibody is selected from the group consisting of an intact immunoglobulin (or antibody), a Fv, a scFv (single chain Fv fragment), a Fab, a F(ab′)2, an antibody-like domain, an antibody-mimetic domain, a single antibody domain, a multimeric antibody, a peptide or a proteolytic fragment containing the epitope binding region. The term “antibody” in the present invention is used in the most general sense, and encompasses various antibodies and antibody mimetic structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, deimmunized antibodies, chimeric antibodies, nanobodies, antibody derivatives, antibody fragments, anticalines, DARPins, affibody, affilins, affimers, affitines, alphabody, avimers, fynomers, minibodies and other binding domains, provided that they show desired binding activity for the antigen. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabody; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies consisting of antibody fragments. Fv of VH and VL are also called “nanobodies”. The term “mimetic antibody” refers to those organic compounds or binding domains that are not antibody derivatives but that can specifically bind to an antigen, in the same way of the antibodies. They include anticalines, DARPins, affibody, affilins, affimers, affitines, alphabody, avimers, fynomers, minibodies, and others. The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from one specific source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
The terms “full-length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain a Fc region as defined herein. A “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human being or a human cell or derived from a non-human source that uses repertoires of human antibodies or other sequences encoding human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. In humans, the antibody isotypes are IgA, IgD, IgE, IgG and IgM. An antibody “humanized” refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVR) and amino acid residues from the remaining human regions (FR: Framework Regions). In certain embodiments, a humanized antibody will comprise substantially at least an entire variable domain, and typically two, in which all or substantially all of the HVRs (for example, CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, for example, a non-human antibody, refers to an antibody subjected to humanization. An antibody “deimmunized” is an antibody with reduced immunogenicity based on the destruction of HLA binding, a basic requirement for the stimulation of T cells. A monoclonal antibodies to be used according to the present invention can be for example produced by a variety of techniques, including, but not limited to, the hybridoma method, methods based on recombinant DNA, phage display methods, and methods that use transgenic animals containing all or part of human immunoglobulin loci. In the context of the present invention, the antibody of the present invention includes modifications of the antibody according to the present invention able to maintain the specificity mentioned above. These changes include, for example, the conjugation to effector molecules such as chemotherapeutic or cytotoxic agents, and/or detectable reporter portions.
Bispecific antibodies are macromolecular, heterobifunctional cross-linkers having two different binding specificities within one single molecule. In this group belong, e.g., bispecific (bs) IgGs, bs IgM-IgAs, bs IgA-dimers, bs (Fab′)2, bs(scFv)2, diabodies, and bs bis Fab Fc (Cao Y. and Suresh M. R., Bioconjugate Chem., 1998, Vol. 9, pp. 635-644).
By peptidomimetics, protein components of low molecular weight are understood which imitate the structure of a natural peptide component, or of templates which induce a specific structure formation in an adjacent peptide sequence (Kemp D S, Trends Biotechnol., 1990, pp. 249-255). The peptidomimetics may, e.g., be derived from the CDR3 domains. Methodical mutational analysis of a given peptide sequence, i.e. by alanine or glutamic acid scanning mutational analysis, may be used. Another possibility to improve the activity of a certain peptide sequence is the use of peptide libraries combined with high throughput screening.
The term antibodies may also comprise agents which have been obtained by analysis of data relating to structure-activity relationships. These compounds may also be used as peptidomimetics (Grassy G. et al., Nature Biotechnol., 1998, Vol. 16, pp. 748-752; Greer J. et al., J. Med. Chem., 1994, Vol. 37, pp. 1035-1054).
The term antibody may also include proteins produced by expression of an altered, immunoglobulin-encoding region in a host cell, e.g. “technically modified antibodies” such as synthetic antibodies, chimeric or humanized antibodies, or mixtures thereof, or antibody fragments which partially or completely lack the constant region, e.g. Fv, Fab, Fab′ or F(ab)′2 etc. In these technically modified antibodies, e.g., a part or parts of the light and/or heavy chain may be substituted. Such molecules may, e.g., comprise antibodies consisting of a humanized heavy chain and an unmodified light chain (or chimeric light chain), or vice versa. The terms Fv, Fc, Fd, Fab, Fab′ or F(ab)2 are used as described in the prior art (Harlow E. and Lane D., in “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory, 1988).
The present invention also comprises the use of Fab fragments or F(ab)2 fragments which are derived from monoclonal antibodies (mAb), which are directed against IL-1R8 or other checkpoint for NK cell maturation and/or effector function. Preferably, the heterologous framework regions and constant regions are selected from the human immunoglobulin classes and isotypes, such as IgG (subtypes 1 to 4), IgM, IgA and IgE. In the course of the immune response, a class switch of the immunoglobulins may occur, e.g. a switch from IgM to IgG; therein, the constant regions are exchanged, e.g. μ from to γ. A class switch may also be caused in a directed manner by means of genetic engineering methods (“directed class switch recombination”), as is known from the prior art (Esser C. and Radbruch A., Annu. Rev. Immunol., 1990, Vol. 8, pp. 717-735). However, the antibodies according to the present invention need not comprise exclusively human sequences of the immunoglobulin proteins.
The antibodies of the present invention also include those for which binding characteristics have been improved by direct mutations, affinity maturation methods, phage display. The affinity or specificity can be modified or improved by mutations in any of the antibody CDRs of the present invention. The term “variable region” or “variable domain” refers to the domain of a heavy or light chain of antibody that is involved in the binding of the antibody to the antigen. The variable domains (or regions) of the heavy and light chain (VH and VL, respectively) of a native antibody generally have similar structures, each domain comprising four framework conserved regions (FR) and three hypervariable regions (HVR, see, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91, 2007). A single VH or VL domain can be sufficient to confer antigen binding specificity. Moreover, it is possible to isolate antibodies that bind to a specific antigen using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, for example, Portolano et al., J. Immunol. 150:880-887, 1993; Clarkson et al., Nature 352:624-628, 1991).
The antibody-like domain comprises binding proteins structurally related to antibodies, such as T cell receptors. The antibodies of the present invention also include functional equivalents that include polypeptides with amino acid sequences substantially identical to the amino acid sequence of the variable or hypervariable regions of the antibodies of the present invention. “The percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment in order to determine the percent of amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign software (DNASTAR). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. The antibody of the invention may e.g. have a dissociation constant (KD) of <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM or less, e.g. from 10−8 M to 10−−13 M, e.g., from 10−9 M to 10−13 M. Recombinant and/or biotechnological derivatives as well as fragments of the antibodies described above are included within the invention, provided that the binding activity of the antibodies and their functional specificity is maintained.
In the context of the present invention, the “cancer” or “tumour” includes primary and metastatic tumours, as well as refractory tumours, solid or non-solid tumours. A further aspect of the present invention is a nucleic acid encoding the antibody as defined above or hybridizing with the above nucleic acid, or consisting of a correspondent degenerated sequence.
It is within the scope of the invention an expression vector encoding the antibody as defined above, preferably comprising the nucleic acid as defined above. It is within the scope of the invention a host cell comprising the nucleic acid as defined above, or the vector as defined above.
The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. The host cells include “transformants” and “transformed cells,” which include the transformed primary cell and the progeny derived therefrom, without taking into account the number of steps. The progeny may be not completely identical in nucleic acid content to a parent cell, but may contain mutations. In the present invention mutant progenies are included, which have the same function or biological activity as that for which they have been screened or selected in the originally transformed cell. The nucleic acids of the invention can be used to transform a suitable mammalian host cell. Mammalian cells available as expression hosts are well known and include, for example, CHO and BHK cells. Prokaryotic hosts include, for example, E. coli, Pseudomonas, Bacillus, etc. Antibodies of the invention can be fused to additional amino acid residues, such as tags that facilitate their isolation. The term “vector”, as used in the present invention refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell in which it was introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. In the present such vectors are referred to as “expression vectors.” Any suitable expression vector can be used, for example prokaryotic cloning vectors such as plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC. Expression vectors suitable for expression in mammalian cells include derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences. The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the sequence or fragment of DNA that must be expressed. It is a further of the invention a pharmaceutical composition comprising at least the antibody or a synthetic or recombinant fragment thereof as defined above and pharmaceutical acceptable excipients, preferably said composition being for use by parenteral administration, in particular intravenously. The composition comprises an effective amount of the antibody and/or recombinant or synthetic antigen binding fragments thereof. The pharmaceutical compositions are conventional in this field and can be produced by the skilled in the art just based on the common general knowledge. The formulations useful in therapy as described herein may e.g. comprise the antibody as described above, in a concentration from about 0.1 mg/ml to about 100 mg/ml, preferably from 0.1 to 10 mg/ml, more preferably from 0.1 to 5 mg/ml. In other formulations, the antibody concentration may be lower, e.g. at least 100 pg/ml. The antibody of the invention is administered to the patient in one or more treatments. Depending on the type and severity of the disease, a dosage of e.g. about 1 mg/kg to 20 mg/kg of the antibody may be administered, for example in one or more administrations, or by continuous infusion. The antibodies of the present invention may be administered in combination with other therapeutic agents, in particular with antibodies able to neutralize other receptors involved in tumour growth or angiogenesis. Any method of administration may be used to administer the antibody of the present invention, in particular, for example, the administration may be oral, intravenous, intraperitoneal, subcutaneous, or intramuscular. The antibody according to the present invention may also be administered as a conjugate, which binds specifically to the receptor and releases toxic substances. In particular embodiments, the pharmaceutical composition of the present invention can be administered in the form of single dosage (for example, tablet, capsule, bolus, etc.). For pharmaceutical applications, the composition may be in the form of a solution, for example, of an injectable solution, emulsion, suspension, or the like. The vehicle can be any vehicle suitable from the pharmaceutical point of view. Preferably, the vehicle used is capable of increasing the entry effectiveness of the molecules into the target cell. In the pharmaceutical composition according to the invention, the inhibitor or suppressor may be associated with other therapeutic agents, such as antagonists of other growth factor receptors involved in tumorigenesis or angiogenesis, such as VEGFR-2, EGFR, PDGFR, receptor kinase inhibitors, BRAF inhibitors, MEK inhibitors, immunomodulatory antibodies, anticancer agents, such as: bevacizumab, ramucirumab, aflibercept, sunitinib, pazopanib, sorafenib, cabozantinib, axitinib, regorafenib, nintedanib, lenvatinib, vemurafenib, dabrafenib, trametinib, chemotherapeutic agents such as methylating agents (temozolomide, dacarbazine), platinum compounds (cisplatin, carboplatin, oxaliplatin), taxanes (paclitaxel, nab-paclitaxel, docetaxel), fluoropyrimidines (5-fluorouracil, capecitabine), topoisomerase I inhibitors (irinotecan, topotecan), poly(ADP-ribose) polymerase inhibitors (PARP) (e.g., olaparib), etc. The pharmaceutical composition is chosen according to the demands of treatment. These pharmaceutical compositions according to the invention may be administered in the form of tablets, capsules, oral preparations, powders, granules, pills, liquid solutions for injection or infusion, suspensions, suppositories, preparations for inhalation. A reference for the formulations is the book by Remington (“Remington: The Science and Practice of Pharmacy”, Lippincott Williams & Wilkins, 2000). The skilled in the art will choose the form of administration and the effective dosages, by selecting suitable diluents, adjuvants and/or excipients.
The term “pharmaceutical composition” refers to a preparation that is in such a form as to permit to the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation may be administered. It is a further aspect of the invention a method for producing the antibody or a synthetic or recombinant fragment thereof as defined above, comprising the steps of culturing the host cell and purifying the antibody or a synthetic or recombinant fragment thereof from the cell culture.
In the context of the present invention the term “comprising” also includes the terms “having essentially” or “consisting essentially”.
In the present invention, the herein mentioned “protein(s)” also comprises the protein encoded by the corresponding orthologous or homologous genes, functional mutants, functional derivatives, functional fragments or analogues, isoform, splice variants thereof.
In the present invention “functional” is intended for example as “maintaining their activity”.
As used herein “fragments” refers to polypeptides having preferably a length of at least 10 amino acids, more preferably at least 15, at least 17 amino acids or at least 20 amino acids, even more preferably at least 25 amino acids or at least 37 or 40 amino acids, and more preferably of at least 50, or 100, or 150 or 200 or 250 or 300 or 350 or 400 or 450 or 500 amino acids.
The present invention will be described by means of non-limiting examples, referring to the following figures:
293 ± 16.31
152 ± 20.14
Materials and Methods
Animals
All female and male mice used were on a C57BL/6J genetic background and were 8-12 weeks old, unless otherwise specified. Wild-type mice were obtained from Charles River Laboratories, Calco, Italy, or were littermates of Il1r8−/− mice. IL-1R8-deficient mice were generated as described31. Il1r1−/− mice were purchased from The Jackson Laboratory, Bar Harbour, Me., USA. All colonies were housed and bred in the SPF animal facility of Humanitas Clinical and Research Center in individually ventilated cages. Il1r1−/−/Il1r8−/− mice were generated by crossing Il1r1−/− and Il1r8−/− mice. Il1r8−/−/Il1r8−/− were generated by crossing Il1r8−/− and Il1r8−/− mice. Mice were randomized on the basis of sex, age and weight. Procedures involving animal handling and care conformed to protocols approved by the Humanitas Clinical and Research Center (Rozzano, Milan, Italy) in compliance with national (D.L. N.116, G.U., suppl. 40, 18 Feb. 1992 and N. 26, G.U. Mar. 4, 2014) and international law and policies (EEC Council Directive 2010/63/EU, OJ L 276/33, 22 Sep. 2010; National Institutes of Health Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). The study was approved by the Italian Ministry of Health (approval number 43/2012-B, issued on the 8 Feb. 2012, and number 828/2015-PR, issued on the 7 Aug. 2015). All efforts were made to minimize the number of animals used and their suffering. In most in vivo experiments, the investigators were unaware of the genotype of the experimental groups.
Human Primary Cells
Human peripheral mononuclear cells were isolated from peripheral blood of healthy donors, upon approval by the Humanitas Research Hospital Ethical Committee. Peripheral mononuclear cells were obtained through a Ficoll density gradient centrifugation (GE Healthcare Biosciences). NK cells were then purified by a negative selection, using a magnetic cell-sorting technique according to the protocols given by the manufacturer (EasySep Human NK Cell Enrichment Kit, Stem Cell Technology). Human monocytes were obtained from peripheral blood of healthy donors by two-step gradient centrifugation, first by Ficoll and then by Percoll (65% iso-osmotic; Pharmacia, Uppsala, Sweden). Residual T and B cells were removed from the monocyte fraction by plastic adherence. Monocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin and 100 ng ml−1 M-CSF (Peprotech) for 7 days to generate resting macrophages. T and B cells were obtained from peripheral blood of healthy donors using RosetteSep Human T Cell Enrichment Cocktail and RosetteSep Human B Cell Enrichment Cocktail (Stem Cell Technology), following the manufacturer's instructions. Neutrophils were enriched from Ficoll-isolated granulocytes, using an EasySep Human Neutrophil Enrichment Kit (StemCell Technologies), according to the manufacturer's instructions. To analyse pluripotent haematopoietic stem cells and NK cell precursors, human bone marrow mononuclear cells were collected from Humanitas Biobank, upon approval by the Humanitas Research Hospital Ethical Committee (authorization 1516, issued on 26 Feb. 2016). Frozen samples were thawed and vitality was assessed by trypan blue and Aqua LIVE/Dead-405 nm staining (Invitrogen), before flow cytometry analysis. Informed consent was obtained from all participants.
Fluorescence-Activated Cell Sorting Analysis
Single-cell suspensions of bone marrow, blood, spleen, lung and liver were obtained and stained. A representative NK cell gating strategy is reported in
Quantitative PCR
Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer's recommendations. RNA was further purified using an miRNeasy RNA isolation kit (Qiagen) or Direct-zol RNA MiniPrep Plus (Zymo Research). cDNA was synthesized by reverse transcription using a High Capacity cDNA Archive Kit (Applied Biosystems) and quantitative real-time PCR was performed using SybrGreen PCR Master Mix (Applied Biosystems) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). PCR reactions were performed with 10 ng of DNA. Data were analysed with the 2(−ΔCT) method. Data were normalized on the basis of GAPDH, β-actin or 18S expression, as indicated, determined in the same sample. Analysis of all samples was performed in duplicate. Primers were designed according to the published sequences and listed as follows: s18/S18: forward 5′-ACT TTC GAT GGT AGT CGC CGT-3′ (SEQ ID NO:5), reverse 5′-CCT TGG ATG TGG TAG CCG TTT-3′ (SEQ ID NO:6); Gapdh/GAPDH: forward 5′-GCA AAG TGG AGA TTG TTG CCA T-3′ (SEQ ID NO:7), reverse 5′-CCT TGA CTG TGC CGT TGA ATT T-3′ (SEQ ID NO:8); βactin/βACTIN: forward 5′-CCC AAG GCC AAC CGC GAG AAG AT-3′ (SEQ ID NO:9), reverse 5′-GTC CCG GCC AGC CAG GTC CAG-3′ (SEQ ID NO: 10); il1r8: forward 5′-AGA GGT CCC AGA AGA GCC AT-3′ (SEQ ID NO: 11), reverse 5′-AAG CAA CTT CTC TGC CAA GG-3′ (SEQ ID NO: 12); IL1R8: forward 5′-ATG TCA AGT GCC GTC TCA ACG-3′ (SEQ ID NO:13), reverse 5′-GCT GCG GCT TTA GGA TGA AGT-3′ (SEQ ID NO:14); il1r1: forward 5′-TGC TGT CGC TGG AGA TTG AC-3′ (SEQ ID NO: 15), reverse 5′-TGG AGT AAG AGG ACA CTT GCG AA-3′ (SEQ ID NO:16); il1r2: forward 5′-AGT GTG CCC TGA CCT GAA AGA-3′ (SEQ ID NO:17), reverse 5′-TCC AAG AGT ATG GCG CCC T-3′ (SEQ ID NO:18); il1r3: forward 5′-GGC TGG CCC GAT AAG GAT-3′ (SEQ ID NO:19), reverse 5′-GTC CCC AGT CAT CAC AGC G-3′ (SEQ ID NO:20); il1r4: forward 5′-GAA TGG GAC TTT GGG CTT TG-3′ (SEQ ID NO:21), reverse 5′-GAC CCC AGG ACG ATT TAC TGC-3′ (SEQ ID NO:22); il1r5: forward 5′-GCT CGC CCA GAG TCA CTT TT-3′ (SEQ ID NO:23), reverse 5′-GCG ACG ATC ATT TCC GAC TT-3′ (SEQ ID NO:24); il1r6: forward 5′-GCT TTT CGT GGC AGC AGA TAC-3′ (SEQ ID NO:25), reverse 5′-CAG ATT TAC TGC CCC GTT TGT T-3′ (SEQ ID NO:26); 16S: forward 5′-AGA GTT TGA TCC TGG CTC AG-3′ (SEQ ID NO:27), reverse 5′-GGC TGC TGG CAC GTA GTT AG-3′ (SEQ ID NO:28).
Purification of Mouse Leukocytes
Splenic NK cells and bone marrow neutrophils were enriched by MACS® according to the manufacturer's instructions (Miltenyi Biotec). Purity of NK cells was about 90% as determined by fluorescence-activated cell sorting. The purity of neutrophils was ≥97.5%. NK cells were stained (CD45-BV650, NK1.1-PE, CD3e-APC, CD11b-BV421, CD27-FITC) and sorted on a FACSAria cell sorter (BD Bioscience) to obtain high-purity NK cells and NK cell populations (CD11blowCD27low, CD11blowCD27high, CD11bhighCD27high and CD11bhighCD27low). Splenic B and T lymphocytes were stained (CD45-PerCP, CD3e-APC, CD4-FITC, CD8-PE, CD19-eFluor450) and sorted. The purity of each population was ≥98%. Resulting cells were processed for mRNA extraction or used for adoptive transfer or co-culture experiments. In vitro-derived macrophages were obtained from bone marrow total cells. Bone marrow cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin and 100 ng ml−1 M-CSF (Peprotech) for 7 days to generate resting macrophages. Bone marrow cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin and 20 ng ml−1 GM-CSF (Peprotech) for 7 days to generate dendritic cells.
Confocal Microscopy
Mouse splenic NK cells were enriched by magnetic cell sorting, left to adhere on poly-D-lysine (Sigma-Aldrich) coated coverslips, fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and incubated with blocking buffer (5% normal donkey serum (Sigma-Aldrich), 2% BSA, 0.05% Tween). Cells were then stained with biotin-conjugated goat polyclonal anti-SIGIRR antibody or biotin-conjugated normal goat IgG as control (both R&D Systems) (10 μg ml−1) followed by Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (Molecular Probes) and 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Coverslips were mounted with the antifade medium FluorPreserve Reagent (EMD Millipore) and analysed with an Olympus Fluoview FV1000 laser scanning confocal microscope with a 40× oil immersion lens (numerical aperture 1.3).
Stimulated Emission Depletion (STED) Microscopy
Human NK cells were enriched and left to adhere on poly-D-lysine (Sigma-Aldrich)-coated coverslips, stimulated with IL-18 (50 ng ml−1; 1 min, 5 min, 10 min), fixed with 4% PFA, incubated with 5% normal donkey serum (Sigma-Aldrich), 2% BSA, 0.05% Tween in PBS2+ (pH 7.4) (blocking buffer), and then with biotin-conjugated goat polyclonal anti-human IL-1R8 antibody or biotin-conjugated normal goat IgG (all from R&D Systems) and mouse monoclonal anti-IL-18Rα (Clone 70625; R&D Systems) or mouse IgG1 (Invitrogen), all diluted at 5 μg ml−1 in blocking buffer, followed by Alexa Fluor 488-conjugated donkey anti-goat IgG antibody and Alexa Fluor 555 donkey anti-mouse IgG antibody (both from Molecular Probes). Mowiol was used as mounting medium. STED xyz images were acquired in a unidirectional mode with a Leica SP8 STED3X confocal microscope system. Alexa Fluor 488 was excited with a 488 nm argon laser and emission collected from 505 to 550 nm applying a gating between 0.4 and 7 ns to avoid collection of reflection and autofluorescence. Alexa Fluor 555 was excited with a 555/547 nm-tuned white light laser and emission collected from 580 to 620 nm. Line sequential acquisition was applied to avoid fluorescence overlap. The 660 nm CW-depletion laser (80% of power) was used for both excitations. Images were acquired with Leica HC PL APO 100×/1.40 numerical aperture oil STED White objective at 572.3 milli absorption units (mAU). CW-STED and gated CW-STED were applied to Alexa Fluor 488 and Alexa Fluor 555, respectively. Collected images were de-convolved with Huygens Professional software.
3′-mRNA Sequencing and Analysis
Splenic NK cells (from six mice per genotype and pooled in pairs) were purified as described above and stimulated with IL-18 (MBL) (20 ng ml−1 for 4 h). RNA was prepared as described above. A QuantSeq 3′mRNA-seq Library Prep Kit for Illumina (Lexogen) was used to generate libraries, which were sequenced on the NextSeq (Illumina; 75 bp PE). The fastq sequence files were assessed using the fastqc program. The reads were first trimmed using bbduk in the bbmap suite of software32 to remove the first 12 bases and a contaminant kmer discovery length of 13 was used for contaminant removal. Regions of length 20 or above with average quality of less than 10 were trimmed from the end of the read. The reads were then trimmed to remove trailing polyG and polyA runs using cutadapt33 and the quality of the remaining reads reassessed with fastqc. The trimmed reads were aligned to the mm10 genomic reference and reads assigned to features in the mm10 annotation using the STAR program34. Differential expression analysis used the generalized linear model functions in the R/bioconductor35 edgeR package36 with TMM normalization. Gene set analysis used the romer37 function in the R/bioconductor package limma38. Metascape (http://metascape.org) was used to enrich genes for Gene Ontology biological processes, KEGG Pathway and Reactome Gene Sets.
Measurement of Cytokines
A BD Cytometric Bead Array (CBA) mouse inflammation kit (BD) or Duoset ELISA kits (R&D Systems) were used to measure cytokines.
In Vitro Functional Assays
Total mouse splenocytes or enriched mouse or human NK cells were cultured in RPMI-1640 medium supplemented with 10% FBS 1% L-glutamine, 1% penicillin/streptomycin and treated with IL-2, IL-12, IL-15 (Peprotech), IL-18 (MBL), IL-13 (Peprotech) and PMA-Ionomycin (Sigma-Aldrich), as specified. FasL expression was evaluated upon treatment for 45 min with IL-18 (50 ng ml−1), IL-15 (50 ng ml−1), IL-2 (20 ng ml−1) and IL-12 (10 ng ml−1). IFNγ production was analysed upon 16 h of treatment with IL-12 (20 ng ml−1) and IL-18 (20 ng ml−1) or IL-1β (20 ng ml−1), by intracellular staining using a BD Cytofix/Cytoperm Fixation/Permeabilization Kit, following the manufacturer's instructions, or by ELISA. Granzyme B and perforin intracellular staining was performed upon 18 h of stimulation with IL-12 (10 ng ml−1), IL-15 (10 ng ml−1) and IL-18 (50 ng ml−1−1), using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience). CD107a-Alexa Fluor 647 antibody was added during the 4 h culture and analysed by flow cytometry. BD GolgiPlug (containing Brefeldin) and BD GolgiStop (containing Monensin) were added 4 h before intracellular staining. PMA (50 ng ml−1) and ionomycin (1 μg ml−1) were added 4 h before intracellular staining, when specified.
NK-dendritic-cell co-culture experiments were performed as previously described39. Dendritic cells were treated with LPS from Escherichia coli 055:B5 (Sigma-Aldrich; 1 μg ml−1) or CpG ODN 1826 (Invivogen; 3 μg ml−1) and with anti-mIL-18 neutralizing antibody (BioXCell, Clone YIGIF74-1G7; 5 μg ml−1) or Rat Isotype Control (BioXCell, Clone 2A3).
IFNγ and CD107a expression upon viral infection was analysed by flow cytometry upon 4 h treatment with BD GolgiPlug, BD GolgiStop and IL-2 (500 U ml−1).
Phosphorylation of IRAK4, S6 and JNK was analysed upon 15-30 min stimulation with IL-18 (10 ng ml−1).
Human Primary NK Cell Transfection
Human NK cells were enriched from peripheral blood of healthy donors and transfected with Dharmacon Acell siRNA (GE Healthcare) using Accell delivery medium (GE Healthcare), following the manufacturer's instructions. SIGIRR-specific siRNA (1 μM) (On-Target Plus; Dharmacon, GE Healthcare) comprised 250 nM of the four following antisense sequences: I,
Generation of Bone Marrow Chimaeras
Il1r8−/− and Il1r8+/+ mice were lethally irradiated with a total dose of 900 cGy. Two hours later, mice were injected in the retro-orbital plexus with 4×106 nucleated bone marrow cells obtained by flushing of the cavity of freshly dissected femurs from wild-type or Il1r8−/− donors. Competitive bone marrow chimaeric mice were generated by reconstituting recipient mice with 50% CD45.1 Il1r8+/+ and 50% CD45.2 Il1r8−/− bone marrow cells. Recipient mice received gentamycin (0.8 mg ml−1 in drinking water) starting 10 days before irradiation and for 2 weeks after irradiation. NK cells of chimaeric mice were analysed 8 weeks after bone marrow transplantation.
Depletion and Blocking Experiments
Mice were treated intraperitoneally with 200 μg of specific mAbs (mouse anti-NK1.1, clone PK136; mouse isotype Control, clone C1.18.4; rat anti-mIL-18, clone YIGIF74-1G7; rat isotype Control, clone 2A3; rat anti-IFNγ, clone XMG1.2; rat IgG1 HRPN; mouse anti-IL-17A, clone 17F3; mouse isotype Control, clone MOPC-21; rat anti-CD4/CD8, clone GK1.5/YTS; rat isotype Control, clone LTF-2 (all from BioXCell)) and then with 100 μg once (anti-NK1.1) or three times (anti-IL-18, anti-IFNγ, anti-IL-17A, anti-CD4/CD8) a week for the entire duration of the experiment.
Microflora Depletion
Six-week-old mice were treated every day for 5 weeks by oral gavage with a cocktail of antibiotics (ampicillin (Pfizer) 10 mg ml−1, vancomycin (PharmaTech Italia) 10 mg ml−1, metronidazol (Societa Prodotti Antibiotici) 5 mg ml−1 and neomycin (Sigma-Aldrich) 10 mg ml−1). Control mice were treated with drinking water. A gavage volume of 10 ml/kg (body weight) was delivered with a stainless-steel tube without prior sedation of mice. DNA was isolated from bacterial faecal pellets with a PowerSoil DNA Isolation Kit (MO BIO Laboratories) and quantified by spectrophotometry at 260 nm. PCR was performed with 10 ng of DNA using SybrGreen PCR Master Mix (Applied Biosystems) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Data were analysed with the 2(−ΔCT) method (Applied Biosystems, Real-Time PCR Applications Guide).
Cancer Models
Mice were injected intraperitoneally with 25 mg/kg (body weight) of diethylnitrosamine (Sigma) at 15 days of age. They were euthanized 6, 8, 10 or 12 months later, to analyse liver cancer. Liver cancer score was based on the number and volume of lesions (0: no lesions; 1: lesion number <3, or lesion dimension <3 mm; 2: lesion number <5, or lesion dimension <5 mm; 3: lesion number <10, or lesion dimension <10 mm; 4: lesion number <15, or lesion dimension <10 mm; 5: lesion number >15, or lesion dimension >10 mm). Lung metastasis experiments were performed injecting intramuscularly the 3-MCA-derived mycoplasma-free sarcoma cell line MN/MCA1 (105 cells per mouse in 100 μl PBS)40. Primary tumour growth was monitored twice a week, and lung metastases were assessed by in vivo imaging and by macroscopic counting at the time of being euthanized 25 days after injection. Liver metastases were generated by injecting intrasplenically 1.5×105 mycoplasma-free colon carcinoma cells (MC38)21. Mice were euthanized 12 days after injection and liver metastases were counted macroscopically. MC38 cells were received from ATCC just before use. MN/MCA1 cells were authenticated morphologically by microscopy in vitro and by histology ex vivo. Tumour size limit at which mice were euthanized was based on major diameter (not more than 2 cm).
Viral Infections
Mice were injected intravenously with 5×105 plaque-forming units of the tissue-culture-grown virus in PBS. Bacterial artificial chromosome-derived MCMV strain MW97.01 has been previously shown to be biologically equivalent to MCMV strain Smith (VR-1399) and is hereafter referred to as wild-type MCMV41. Mice were euthanized 1.5 and 4.5 days after infection and viral titre was assessed by plaque assay, as previously described42,43. Newborn mice were infected intraperitoneally with 2,000 plaque-forming units of the MCMV strain MW97.01 and euthanized at day 7 after infection. Viral titre was assessed by plaque assay, as previously described42,43.
Adoptive Transfer
One million Il1r8+/+ or Il1r8−/− sorted NK cells were injected intravenously in wild-type adult mice 5 h before MN/MCA or MC38 injection, or intraperitoneally in newborn mice 48 h after MCMV injection. Adoptively transferred NK cell engraftment, proliferative capacity and functionality (IFNγ production and degranulation after ex vivo stimulation) were assessed 3 and 7 days after injection.
In Vivo Proliferation
In vivo proliferation was measured using a Click-iT Edu Flow Cytometry Assay Kit (Invitrogen). Edu was injected intraperitoneally (0.5 mg per mouse), mice were euthanized 24 h later and cells were stained following the manufacturer's instructions and analysed by flow cytometry.
Immunohistochemistry
Frozen liver tissues were cut at 8 mm and then fixed with 4% PFA. Endogenous peroxidases were blocked with 0.03% H2O2 for 5 min and unspecific binding sites were blocked with PBS+1% FBS for 1 h. Tissues were stained with polyclonal goat anti-mouse NKp46/NCR1 (R&D Systems) and a Goat-on-Rodent HRP polymer kit (GHP516, Biocare Medical) was used as secondary antibody. Reactions were developed with 3,3′-diaminobenzidine (Biocare Medical) and then slides were counterstained with haematoxylin. Slides were mounted with eukitt (Sigma-Aldrich). Images at 20× magnification were analysed with cell{circumflex over ( )}F software (Olympus).
In Vivo Imaging
After feeding with AIN-76A alfalfa-free diet (Mucedola, Italy) for 2 weeks to reduce fluorescence background, mice were intravenously injected with XenoLight RediJect 2-deoxyglucosone (PerkinElmer) and 24 h later 2-deoxyglucosone fluorescence was measured using a Fluorescence Molecular Tomography system (FMT 2000, Perkin Elmer). Acquired images were subsequently analysed with TrueQuant 3.1 analysis software (Perkin Elmer).
Statistical Analysis
For animal studies, sample size was defined on the basis of past experience on cancer and infection models, to detect differences of 20% or greater between the groups (10% significance level and 80% power). Values were expressed as mean±s.e.m. or median of biological replicates, as specified. One-way ANOVA or a Kruskal-Wallis test were used to compare multiple groups. A two-sided unpaired Student's t-test was used to compare unmatched groups with Gaussian distribution and Welch's correction was applied in cases of significantly different variance. A Mann-Whitney U-test was used in cases of non-Gaussian distribution. A ROUT test was applied to exclude outliers. P<0.05 was considered significant. Statistics were calculated with GraphPad Prism version 6, GraphPad Software.
Statistics and Reproducibility
Results
IL-1R8 is widely expressed10. However, inventors found strikingly high levels of IL-1R8 mRNA and protein in human NK cells, compared with other circulating leukocytes and monocyte-derived macrophages (
Mouse NK cells expressed significantly higher levels of Il1r8 mRNA compared with other leukocytes (
To assess the role of IL-1R8 in NK cells, inventors took advantage of IL-1R8-deficient mice. Among CD45+ cells, the NK cell frequency and absolute numbers were significantly higher in peripheral blood of Il1r8−/− compared with Il1r8+/+ mice, and slightly increased in liver and spleen. (
The enhanced NK cell maturation in Il1r8−/− mice occurred already at 2 and 3 weeks of age, whereas the frequency of NK precursors was similar in Il1r8−/− and Il1r8+/+ bone marrow, indicating that IL-1R8 regulated early events in NK cell differentiation, but did not affect the development of NK cell precursors12 (
Inventors next investigated whether IL-1R8 affected NK cell function. The expression of the activating receptors NKG2D, DNAM-1 and Ly49H was significantly upregulated in peripheral blood Il1r8−/− NK cells (
IL-18 is a member of the IL-1 family, which plays an important role in NK cell differentiation and function1,14. Enhanced NK cell maturation and effector function in Il1r8−/− mice was abolished by IL-18 blockade or genetic deficiency but unaffected by IL-1R1-deficiency (
The results reported above suggested that IL-1R8 regulated the IL-18 signalling pathway in NK cells and, indeed, an increased phospho-IRAK4/IRAK4 ratio was induced by IL-18 in Il1r8−/− NK cells compared with wild-type NK cells, indicating unleashed early signalling downstream of MyD88 and myddosome formation (
To obtain a deeper insight into the impact of IL-1R8 deficiency on NK cell function and on the response to IL-18, RNA sequencing (RNA-seq) analysis was conducted. IL-1R8 deficiency had a profound impact on the resting transcriptional profile of NK cells and on top on responsiveness to IL-18 (
To investigate the role of IL-1R8 in human NK cells (
To assess the actual relevance of IL-1R8-mediated regulation of NK cells, anticancer and antiviral resistance were examined. The liver is characterized by a high frequency of NK cells19 Therefore inventors focused on liver carcinogenesis. In a model of diethylnitrosamine-induced hepatocellular carcinoma, IL-1R8-deficient male and female mice20 were protected against the development of lesions, in terms of macroscopic number, size (
Evidence suggests that NK cells can inhibit haematogenous cancer metastasis5. In a model of sarcoma (MN/MCA1) spontaneous lung metastasis, Il1r8−/− mice showed a reduced number of haematogenous metastases, whereas primary tumour growth was unaffected (
Assessment of lung metastasis at the time of euthanasia and in vivo imaging analysis (
Liver metastasis is a major problem in the progression of colorectal cancer. Inventors therefore assessed the potential of Il1r8−/− NK cells to protect against liver metastasis using the MC38 colon carcinoma line21. As shown in
Finally, inventors investigated whether IL-1R8 affects NK cell antiviral activity, focusing on murine cytomegalovirus (MCMV) infection22. As shown in
NK cells belong to the complex, diverse realm of innate lymphoid cells (ILCs)23. Human and mouse non-NK ILCs express IL-1R8 mRNA and protein (ref. 24). Preliminary experiments were conducted to assess the role of IL-1R8 in ILC function. In the MCMV infection model, Il1r8−/− ILC1 showed increased IFNγ production, but represented a minor population compared with NK cells and one-thirtieth that of Il1r8−/− IFNγ-producing cells (
IL-1R8 deficiency was associated with exacerbated inflammatory and immune reactions under a variety of conditions1,10. NK cells engage in bidirectional interactions with macrophages, dendritic cells and other lymphocytes3,4,25,26. Therefore the role of NK cells in inflammatory and autoimmune conditions associated with IL-1R8 deficiency1,10 will need to be examined. IL-1R8-deficient mice show increased susceptibility to colitis and colitis-associated azoxymethane carcinogenesis27,28. The divergent impact on carcinogenesis of IL-1R8 deficiency in the intestine and liver is likely to reflect fundamental, tissue-dictated differences of immune mechanisms involved in carcinogenesis in these different anatomical sites. In particular, high numbers of NK cells are present in the liver19 and this physiological characteristic of this organ is likely to underlie this apparent divergence.
NK cells are generally not credited with playing a major role in the control of solid tumours6. Conversely there is evidence for a role of NK cells in the control of haematogenous lung metastasis5,29. The results presented here show that unleashing NK cells by genetic inactivation of IL-1R8 resulted in inhibition of liver carcinogenesis and protection against liver and lung metastasis. IL-1R8-deficient mice show exacerbated TLR and IL-1-driven inflammation10, and inflammation promotes liver carcinogenesis 30. Therefore, our results are probably an underestimate of the potential of removal of the NK cell checkpoint IL-1R8 against liver primary and metastatic tumours. Thus, NK cells have the potential to restrain solid cancer and metastasis, provided critical, validated checkpoints such as IL-1R8 are removed and the tissue immunological landscape is taken into account.
Materials and Methods
In Vitro NK Cell Functional Activation
Il1r8+/+ and Il1r8−/− splenic NK cells were enriched using a negative magnetic separation (NK cell isolation kit II, Miltenyi) (as described in example 1) and cultured for 8 days in RPMI 10% FBS with IL-2 (Peprotech, 20 ng/ml) plus IL-15 (Peprotech, 10 ng/ml) (Huang B Y et al, PloS ONE (2015). MC38 cells (as described in example 1) were pre-treated (24 hours) with IFNγ, in order to mimic the tumor microenvironment and induce the expression of PD-L1, as previously shown (Juneja V R et al, J. Exp. Med. (2017). NK cells were pre-incubated for 30 minutes (37° C.) with anti-PD1 blocking antibody or the relative isotype control (both BioxCell, 1 μg/ml). MC38 cells were washed and co-cultured with NK cells (1:2 ratio) for 3 hours. IFNγ and GranzymeB intracellular expression in NK cells was measured by flow cytometry.
Results
Effect In Vitro of the Combination of IL-1R8-Deficiency and PD-1 Blockade
Inventors herein show that the blockade of PD-1 drives an increased NK cell activation in IL-1R8-deficient NK cells compared to wild-type NK cells, when exposed to a tumoral target expressing the ligand (PD-L1), demonstrating that the combination of IL-1R8 and PD-1 blockade enforces NK cell effector functions (
Materials and Methods
IL-1R8 Expression in Human T Cells
Human peripheral mononuclear cells (PBMCs) were isolated from peripheral blood of healthy donors through a Ficoll density gradient centrifugation (GE Healthcare Biosciences), upon approval by Humanitas Research Hospital Ethical Committee. IL-1R8 expression was measured by flow cytometry in T cell subsets according to the expression of CD3, CD4, CD8, CCR7, CD45RO, CD127, CD25 (Gattinoni L. et al. Nature Medicine (2011).
Proliferation Assay
Il1r8+/+ and Il1r8−/− murine splenic T were enriched using a negative magnetic separation (Pan T cell isolation kit II, Miltenyi) and pre-incubated for 10 minutes (37° C.) with Vybrant® CFDA SE dye (Invitrogen, 1 μM). T cells were washed and cultured for 2 days in IMDM 10% FBS 0.1% BME (Gibco) with Dynabeads Mouse T-Activator CD3/CD28 (Gibco, 1 bead×cell) plus IL-2 (Proleukin, 20 ng/ml), IL-12 (Peprotech, 20 ng/ml), IL-18 (MBL, 20 ng/ml) alone or in combination (Hu B. et al. Cell Rep (2017); Freeman B. et al. PNAS (2012)). CFDA SE and CD44 expression in CD8 T cells was measured by flow cytometry.
T Cell Activation In Vitro
Il1r8+/+ and Il1r8−/− murine splenic CD8+ T cells were enriched using a negative magnetic separation (CD8a+ isolation kit, mouse, Miltenyi) and cultured for 2 days in IMDM 10% FBS 0.1% BME (Gibco) with Dynabeads Mouse T-Activator CD3/CD28 (Gibco, 1 bead×cell) plus IL-2 (Proleukin, 20 ng/ml), IL-12 (Peprotech, 20 ng/ml) alone or in combination. T cells were treated (overnight) with IL-18 (MBL, 20 ng/ml) and stimulated for 3 h with Cell Stimulation Cocktail (eBioscience) plus Golgi Plug (BD Biosciences) as specified (Hu B. et al. Cell Rep (2017); Freeman B. et al. PNAS (2012)). IFNγ and GranzymeB intracellular expression in CD8 T cells was measured by flow cytometry.
Results
Inventors hypothesized that CD8+T lymphocytes expressed IL-1R8 and that it played a negative regulatory activity in this cell type. Inventors first checked IL-1R8 expression in human T cells from healthy donors by flow cytometry. Here inventors show that human CD8+ T cells display a higher level of IL-1R8 compared to CD4+ T cells. Moreover, IL-1R8 expression is increased in effector/memory T cell subsets compared with naïve T cells, demonstrating that IL-1R8 expression is associated with the acquisition of the effector potential (
Science 331, 44-49 (2011).
| Number | Date | Country | Kind |
|---|---|---|---|
| 102017000120699 | Oct 2017 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/079188 | 10/24/2018 | WO | 00 |
