SETDB1 histone methyltransferase inhibitors for combination cancer therapy
阅读说明：本技术 用于癌症联合治疗的setdb1组蛋白甲基转移酶抑制剂 (SETDB1 histone methyltransferase inhibitors for combination cancer therapy ) 是由 S·阿米格瑞纳 M·比尔巴格 D·洛奎藏 于 2019-03-06 设计创作，主要内容包括：本发明涉及H3K9组蛋白甲基转移酶SETDB1抑制剂与至少一种免疫检查点调节剂联合使用在治疗癌症中的用途。(The present invention relates to the use of an H3K9 histone methyltransferase SETDB1 inhibitor in combination with at least one immune checkpoint modulator in the treatment of cancer.)
Use of an H3K9 histone methyltransferase SETDB1 inhibitor in combination with at least one immune checkpoint molecule/protein modulator in the treatment of cancer.
2. The use of an inhibitor of H3K9 histone methyltransferase SETDB1 according to claim 1, wherein the inhibitor of H3K9 histone methyltransferase SETDB1 is selected from an organic small molecule, an aptamer, an intrabody, a polypeptide or an inhibitor of H3K9 histone SETDB1 gene expression.
3. The use of an inhibitor of H3K9 histone methyltransferase SETDB1 according to any one of claims 1 or 2, wherein the inhibitor of H3K9 histone methyltransferase SETDB1 is anthracycline.
4. The use of an H3K9 histone methyltransferase SETDB1 inhibitor according to claim 1, wherein the H3K9 histone methyltransferase SETDB1 gene expression is selected from an antisense oligonucleotide construct, an siRNA (microrna), an shRNA, and a ribozyme.
5. Use of an H3K9 histone methyltransferase SETDB1 inhibitor according to any one of claims 1 to 4 in combination with at least one immune checkpoint modulator, wherein the at least one immune checkpoint is an inhibitory immune checkpoint and/or a stimulatory immune checkpoint.
6. Use of an inhibitor of H3K9 histone methyltransferase SETDB1 in combination with at least one immune checkpoint modulator according to claim 5 wherein the inhibitory immune checkpoint is selected from PD-L1/PD1, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, LAG-3, TIM-3TIGIT, VISTA, CD96, CD112R, CD160, CD244 (or 2B4), DCIR (C-type lectin surface receptor), ILT3, ILT4 (immunoglobulin-like transcript), CD31(PECAM-1) (Ig-like R family), CD39, CD73, CD94/NKG2, GP49B (immunoglobulin superfamily), KLRG1, LAIR-1 (leukocyte-associated immunoglobulin-like receptor 1), CD305, PD-2, sirap-p 24 and sirap.
7. Use of an inhibitor of H3K9 histone methyltransferase SETDB1 in combination with at least one immune checkpoint modulator according to claim 5 or 6, wherein the stimulatory immune checkpoint is selected from the group consisting of CD27, CD40, OX40, GITR, ICOS, TNFRSF25, 41BB, HVEM, CD28, TMIGD2, CD226, 2B4(CD244) and the ligands CD48, B7-H6 Brandt (NK ligand), LIGHT (CD258, TNFSF14) and CD 28H.
8. The use of an inhibitor of H3K9 histone methyltransferase SETDB1 according to any one of claims 1 to 7, wherein said inhibitor is used in combination with at least one inhibitory immune checkpoint modulator and at least one stimulatory immune checkpoint agonist.
9. Use in combination with an H3K9 histone methyltransferase SETDB1 inhibitor according to any one of claims 1 to 8 and at least one immune checkpoint modulator, wherein the immune checkpoint modulator is an antibody or a fusion protein.
10. Use in combination with an H3K9 histone methyltransferase SETDB1 inhibitor according to any one of claims 1 to 9 and at least one immune checkpoint modulator, wherein the immune checkpoint modulator is an anti-PD-1 or anti-PD-L1 antibody.
11. A product containing the H3K9 histone methyltransferase inhibitor SETDB1 and at least one immune checkpoint modulator as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.
12. A method for classifying a cancer patient as responsive or hyporesponsive to immune checkpoint therapy, wherein the method comprises determining SETDB1 expression in a biological sample from the patient.
13. The use of an H3K9 histone methyltransferase SETDB1 inhibitor according to any one of claims 1-10, a product according to claim 11 or a method according to claim 12, wherein the cancer is selected from melanoma, glioblastoma, upper aerodigestive tract cancer, breast cancer, lung cancer, urothelial cancer, hodgkin's lymphoma, renal cancer, fibrosarcoma and gastric cancer.
Technical Field
The present invention relates to the treatment of cancer, in particular to the use of SETDB1 inhibitors in combination with immune checkpoint therapy.
Background
Immune checkpoints refer to a number of inhibitory and stimulatory pathways inherent in the immune system that are critical for maintaining self-tolerance and regulating the duration and magnitude of the physiological immune response in peripheral tissues, thereby minimizing collateral tissue damage. In fact, the balance between inhibitory and stimulatory signals determines lymphocyte activation and thus the modulation of the immune response (Pardoll DM, Nat Rev cancer.2012Mar 22; 12(4): 252-64).
It is now clear that tumors co-select (co-opt) certain immune checkpoint pathways as the primary mechanism of immune resistance, particularly against tumor antigen-specific T cells. Many immune checkpoints, since they are initiated by ligand-receptor interactions, can be easily blocked by antibodies or modulated by recombinant forms of the ligand or receptor. Therefore, agonists or antagonists of inhibitory signals of co-stimulatory receptors, both of which result in the expansion of antigen-specific T cell responses, are the primary agents in current clinical tests.
In this context, cancer immunotherapy is seen as a breakthrough in the field of cancer therapy, moving from tumor targeting to targeting of the immune system (Couzin-Frankel J., science.2013Dec20; 342(6165): 1432-3). Blockade of immune checkpoints with antibodies against CTLA-4, PD1 and PD-L1 has good clinical efficacy and manageable safety.
However, only a small fraction of patients respond to this treatment and there is therefore a need to improve cancer immunotherapy by new methods and/or by combining anti-checkpoint antibodies with other therapies (see Jenkins RW et al, BJC 2018118, 9-16 and Sharma P et al, Cell2017168(4): 707-723). Furthermore, anti-checkpoint antibodies can induce side effects, primarily autoimmunity, and thus there remains valuable medical benefit to administer combination therapies that help reduce the dose administered and the subsequent adverse events.
"epigenetic" is defined as a heritable change in gene expression caused by a chemical change in DNA or histone. Epigenetic events include DNA methylation, covalent histone modification, and non-covalent mechanisms such as histone variant integration, nucleosome localization, and remodeling.
Methylation of histone lysine and arginine residues is regulated by two classes of enzymes with opposite activities: histone methyltransferases and histone demethylases.
Histone Methyltransferases (HMTs) are histone modifying enzymes (e.g., histone-lysine N-methyltransferase and histone-arginine N-methyltransferase) that catalyze the transfer of one, two, or three methyl groups to lysine and arginine residues of histone. Attachment of methyl groups occurs primarily at specific lysine or arginine residues of histones H3 and H4. The class of lysine-specific histone methyltransferases is further subdivided into SET-containing and SET-free domains. Methylation of the N-terminal lysine residue of histone H3, particularly the formation of mono-, di-or trimethylated lysines at positions 4, 9, 27, 36 and 79, has been well documented. More than 30 histone methyltransferases have been described so far.
Epigenetic factors are associated with cancer, inflammatory and autoimmune diseases and have been considered as promising targets for drug development in the past few years. Several histone methyltransferases that methylate various lysine residues of histone H3 or H4 are cancer-related, e.g., MLL, SMYDD3, G9a, Suv39H1, STDB1, EZH2, NSD3, DNS1, DOT1L, SET8, Suv420H1, Suv420H 2. In contrast, various demethylases are also involved in cancer (Morera L et al, Targeting histomethylation mutants and demethylases in Clinical trials for cancer therapy, Clinical epidemics 2016; 8: 57). Histone methyltransferase inhibitor EZH2 has been proposed for the treatment of relapsed or refractory B-cell lymphoma (nature.2012dec 6; 492(7427): 108-12). Inhibitors of DNA methyltransferase (DNMT) or Histone Deacetylase (HDAC) are also currently approved clinically for the treatment of hematological malignancies. Two cytidine analogs, azacytidine (5-azacytidine or aza) and diazepam, nonspecifically inhibit DNA methyltransferase activity upon incorporation into DNA, resulting in loss of DNA methylation. Both of these drugs are approved for myelodysplastic syndrome (MDS) patients. Several studies in vivo and in vitro have shown that Aza treatment leads to a reduction in DNA methylation, although the degree of demethylation appears to be limited (Magnus Tobiason et al, Comprehensive mapping of the effects of azacitidine on DNA methylation, expressed/defective tissue marks and gene expression cells with MDS and MDS related disease on target,2017, Vol.8, (No.17), pp: 28812-.
It has also recently been proposed to use inhibitors of DNMT or HDAC in combination with other cancer therapies (e.g. immunotherapy) (WO2015035112, Chiapinelli KB et al, cell.2015Aug 27; 162(5): 974-86; Licht JD, cell.2015Aug 27; 162(5):938-9, but see Sharma P et al, Cell 2017 as previously described). In fact, DNA demethylating agents have been shown to elicit T cell mediated immune responses in solid tumors and therefore to act synergistically with anti-tumor immunotherapies such as checkpoint inhibitors (Roulois D, Yau HL, De CarvalhodD. pharmacological DNA methylation: immunology for cancer immunology. Onconomogy. Onconomogy.2016; 5(3): e 1090077). In addition, additional clinical findings indicate that non-small cell lung cancer patients pretreated with 5-azacytidine have a better clinical response to subsequent anti-PD 1 treatment (JuergensRA, Wrangle J, Vendetti FP, Murphy SC, ZHao M, Coleman B, Sebree R, Rodres K, HookerCM, Franco N, etc., Combination immunogenic thermal ha efficacy in tissues with reactive adsorbed non-small cell lung cancer. cancer Discov 2011; 1:598-, and the melanoma mouse model (B16) did respond better to the combination of 5-azacytidine plus anti-CTLA 4 than either 5-azacytidine alone or anti-CTLA 4 alone (see Chiapinelli KB et al, inhibition DNA Methylation Cancer Interferon Response in Cancer via dsRNA containing endogeneous research. cell 2015; 162: 974-86; and Roulois D et al, DNA-deletion assay target nucleic acid Cells by Inducing Viral nucleic acids transport. cell 2015; 162: 961. sub. 973; PMID: 26317465).
However, such epigenetic modulators areThe role in cancer immunity and immunotherapy is still poorly understood. In fact, the role of demethylating agents is diverse, and their identification of genes whose reactivation predicts or modulates the response remains elusive. Generally, the immunomodulatory effects of treatment with 5-azacytidine (a DNMT) are complex and dependent on the clinical setting and patient type (see for exampleTM and Hadrup SR, Mediators inflam.2015; 2015:871641).
Thus, there remains a need to implement combination therapies that can improve the efficacy of cancer immunotherapy with limited adverse side effects.
Summary of The Invention
The inventors demonstrate for the first time that the anti-tumor effect of immune checkpoint modulators is greatly enhanced in the absence of
Accordingly, the present invention relates to the use of an inhibitor of H3K9 histone methyltransferase SETDB1 in combination with at least one immune checkpoint protein modulator for the treatment of cancer in a patient.
Defining:
as used herein, "treatment" is defined as the application or administration of a therapeutic agent or combination of therapeutic agents (e.g., an inhibitor of SETDB1 and/or an immune checkpoint modulator) to a patient, or to an isolated tissue or cell line from a patient having cancer, with the aim of curing, healing, alleviating, altering, remediating, ameliorating, improving, or affecting the cancer or any symptom of the cancer. In particular, the term "treating" refers to reducing or alleviating at least one adverse clinical symptom associated with, for example, cancer, such as pain, swelling, low blood cell count, and the like.
In another embodiment, the term "treating" refers to slowing or reversing the progression of neoplastic uncontrolled cellular proliferation, i.e., shrinking an existing tumor and/or halting tumor growth.
The term "treating" also refers to inducing apoptosis in a cancer or tumor cell in a subject.
The term "treatment" is also used herein in the context of prophylactic administration of a therapeutic agent.
The term "effective amount" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.
As used herein, the term "therapeutically effective regimen" refers to a regimen of administration, timing, frequency and duration for administering one or more therapies (i.e., an inhibitor of SETDB1 and at least one immune checkpoint modulator) according to the present invention for the treatment and/or control of cancer or a symptom thereof. In particular embodiments, the protocol achieves one, two, three, or more of the following results: (1) stabilization, reduction, or elimination of a population of cancer cells; (2) stabilization or reduction of growth of a tumor or neoplasm; (3) a reduction in tumor formation; (4) eradication, removal or control of primary, regional and/or metastatic cancer; (5) a reduction in mortality; (6) disease-free, relapse-free, exacerbation-free, and/or an increase in overall survival, duration, or rate; (7) an increase in response rate, duration of response, or number of patients responding or remitting; (8) a reduction in hospitalization rate, (9) a reduction in hospitalization time, (10) the size of the tumor is maintained and does not increase or increases by less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 2%, and (11) an increase in the number of remitting patients.
As used herein, the term "combination" or "co-administration" in the context of the present invention refers to the administration of an inhibitor of SETDB1 and at least one immune checkpoint modulator to a patient to obtain a therapeutic benefit for cancer. In the context of administration, the term "combination" may also refer to the prophylactic use of a SETDB1 inhibitor when used with at least one immune checkpoint modulator. The use of the term "in combination" does not limit the order in which therapies (e.g., SETDB1 and at least one immune checkpoint modulator) are administered to a subject. One therapy can be administered prior to, concurrently with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) administration of a second therapy (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) to a patient already suffering from, or susceptible to cancer. The therapies are administered to the patient in a sequence and at time intervals so that the therapies can work together. In particular embodiments, the therapies are administered to a subject in a sequence and at time intervals such that they provide an increased benefit compared to other modes of administration. Any additional therapy may be administered in any order with other additional therapies.
These results of the invention establish the basis for a dual treatment of patients with an inhibitor of SETDB1 and at least one immune checkpoint modulator, such as an anti-PD-1 antibody. The two therapies need not be provided simultaneously, but may be provided sequentially, e.g., starting with a SETDB1 inhibitor, followed by immune checkpoint modulation. Thus, as used herein, the expression "use of an inhibitor of H3K9 histone methyltransferase SETDB1 in combination with at least one immune checkpoint modulator in the treatment of cancer" is used interchangeably with the expression "use of at least one immune checkpoint modulator in combination with an inhibitor of H3K9 histone methyltransferase SETDB1 in the treatment of cancer".
The terms "synergistic", "synergistic" or "synergistic effect" as used herein describe an effect that is of a magnitude greater than the sum of the effects. In some embodiments of the invention, the co-use of a SETDB1 inhibitor and an immune checkpoint modulator can provide a synergistic therapeutic effect on a neoplastic condition and/or cell growth in a patient. For example, if tumor growth is reduced by 10% using a SETDB1 inhibitor and 20% using only immune checkpoint modulators, the additive effect of reducing the growth of a neoplasm or tumor will be a reduction of 30%. Thus, in contrast, when a SETDB1 inhibitor and an immune checkpoint modulator are used together, the synergy will reduce the growth of the tumor or neoplasm to any degree greater than 30%.
As used herein, the term "antibody" refers to a protein that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody may comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, the antibody comprises two heavy (H) and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F (ab')2 fragments, Fd fragments, Fv fragments, and dAb fragments) as well as intact antibodies, such as full-length immunoglobulins of the intact and/or IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM types (and subtypes thereof). The light chains of immunoglobulins may be of the kappa or lambda type. In one embodiment, the antibody is glycosylated. The antibody may act on antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity, or on one or both of these activities.
Detailed Description
SETDB1 inhibitor
As used herein, the terms "SET domain bifurcate 1" or "
SETDB1 is a member of the SET domain-containing proteins involved in histone methylation, which are present in all eukaryotes. This family of proteins is characterized by a SET domain consisting of about 130 amino acids, named three Drosophila protein inhibitory factors, namely, variegation 3-9(Su (var)3-9), enhancer of zeste (E (z)), and the homeobox gene regulator trithorax (Trx). In this process, the SET domain uses the cofactor S-adenosyl-L-methionine (SAM) to methylate the-amino group of lysine residues.
The human SETDB1 gene (referenced ENSG00000143379 in the Ensembl database) is located on human chromosome 1q 21. The human SETDB1 gene consists of three isoforms.
The SETDB1 protein comprising 3 isoforms (produced by alternative splicing) is numbered Q15047 in UNIPROT. This protein (
According to the invention, the generic term SETDB1 also includes all homologs of the human SETDB1 protein.
According to the present invention, the SETDB1 inhibitor may be selected from any natural compound, or may not have the ability to inhibit SETDB1 activity or gene expression.
The inhibitory activity of a compound can be determined using various methods, such as Greiner d, et al Nat chembil.2005aug; l (3):143-5 or Eskeland, R. et al, biochemistry43,3740-3749 (2004). Typically, a SETDB1 inhibitor refers to a compound that inhibits SETDB1 activity in a subject (or in vitro cells) by at least 20%, 30%, 40%, 50%, 60%, preferably more than 70%, even more preferably more than 80%, more than 90%, more than 95%, more than 99% or even 100% (corresponding to undetected activity) compared to prior to or without administration of the compound.
The SETDB1 inhibitor can be selected from organic small molecule, aptamer, intracellular antibody, polypeptide or H3K9 histone methyltransferase SETDB1 gene expression inhibitor (Bennett RL, Licht JD. "" Targeting epitopes in cancer. Annu Rev Pharmacol Toxicol "Annu Rev Pharmacol Toxicol.2018Jan 6; 58: 187-207; Karanth AV et al," embedding role of SETDB1 as a therapeutic target "Expert Optic target. 201Mar7; 21(3): 319-331).
Generally, the H3K 9-histone methyltransferase SETDB1 inhibitor is a small organic molecule. The term "small organic molecule" refers to a molecule that is comparable in size to organic molecules commonly used in pharmaceuticals. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000Da, more preferably up to 2000Da, and most preferably up to about 1000 Da.
In a particular embodiment, the H3K9 histone methyltransferase SETDB1 inhibitor may be mithramycin (also known as plicamycin, MIT) (Ryu H et al, "ESET/SETDB 1gene expression and histone H3(K9) methylation in Huntington's disease"; Proc Natl Acad Sci USA.2006Dec 12; 103(50): 19176-81). In some embodiments, mithramycin may be conjugated to cystamine.
The identification of new small molecule inhibitors can be accomplished according to conventional techniques in the art. The current major method of identifying hit compounds is the use of High Throughput Screening (HTS). Small molecule reagents can be identified from small molecule libraries, which can be obtained from commercial sources, such as AMRI (Albany, n.y.), AsisChem Inc (Cambridge, Mass.), TimTec (Newark, Del.), or libraries known in the art.
In another embodiment, the SETDB1 inhibitor is an aptamer. Aptamers are a class of molecules that represent an alternative to antibodies in terms of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences that have the ability to recognize almost any kind of target molecule with high affinity and specificity. Such ligands can be isolated by systematic evolution of ligands by exponential enrichment (SELEX) of random sequence libraries, as described by Tuerk c. and Gold l., 1990. The random sequence library can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of unique sequence, which is optionally chemically modified. Possible modifications, uses and advantages of such molecules have been reviewed in Jayasena s.d., 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein such as E.coli thioredoxin A, which is selected from combinatorial libraries by two hybridization methods (Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. "Genetic selection of peptide aptamers that and inhibition-dependent kinase 2". Nature.1996Apr 11; 380(6574): 548-50).
The inhibition of SETDB1 in the cells according to the invention may also be achieved by intrabodies. An intrabody is an antibody that binds to its antigen within a cell after it has been produced in the same cell (see, e.g., review below, Marschall AL, Dubel Sand T“Specific in vivo knockdown of protein function byintrabodies”,MAbs.2015；7(6):1010-35.but see also Van Impe K,Bethuyne J,CoolS,Impens F,Ruano-Gallego D,De Wever O,Vanloo B,Van Troys M,Lambein K,Boucherie C,et al.“A nanobody targeting the F-actin capping protein CapGrestrains breast cancer metastasis”.Breast Cancer Res 2013；15:R116；Hyland S, Beerli RR, Barbas CF, Hynes NE, Wels W. "Generation and functional interactions of intracellular analytes with the kinase expression of human EGF receptor. oncogene 2003; 1557-67'; lobato MN, Rabbitts TH., "Intracellular antibodies and scales vibrating the use as therapeutics". Trends Mol Med 2003; 390-6, and Donnii M, Morea V, Desiderio A, PashkouloovD, Villani ME, Tramontano A, Benvenuto E. "Engineering stable cytoplasmatic intubations with designed specificity". J Mol biol.2003Jul 4; 330(2):323-32.).
Intrabodies can be generated by cloning the corresponding cDNA from existing hybridoma clones, or more conveniently, the novel scFvs/fabs can be selected from in vitro display techniques (such as phage display) which provide the necessary genes encoding the antibody from onset and allow for more detailed pre-design of the fine specificity of the antibody. In addition, bacterial-, yeast-, mammalian cell surface display and ribosome display can be employed. However, the most commonly used in vitro display system for selecting specific antibodies is phage display. Recombinant antibody phages are selected by incubating a pool of antibody phages with an antigen in a process called panning (affinity selection). This process is repeated several times to obtain an enriched antibody library containing specific antigen binders to virtually any possible target. To date, libraries of in vitro assembled recombinant human antibodies have produced thousands of novel recombinant antibody fragments. It is noted that the prerequisite for knock down of a specific protein by cytoplasmic intrabodies is the neutralization/inactivation of the antigen by antibody binding. Five different methods of producing suitable antibodies have emerged: 1) selection of functional intrabodies (antigen-dependent and independent) in eukaryotes (such as yeast) and prokaryotes (such as E.coli) in vivo; 2) producing antibody fusion proteins to improve cytoplasmic stability; 3) use of specific frameworks to improve cytoplasmic stability (e.g., by grafting CDRs or introducing synthetic CDRs into a stable antibody framework); 4) use of single domain antibodies to improve cytoplasmic stability; and 5) selecting stable intrabodies that do not contain disulfide bonds. Those methods are described in particular detail in Marschall, a.l et al, mAbs 2015, described above.
The most commonly used form of intrabody is the scFv, which consists of H chain and L chain variable antibody domains (VH and VL) held together by short and flexible linker sequences, usually (Gly4Ser)3, to avoid the need to express and assemble 2 antibody chains of a complete IgG or Fab molecule separately. Alternatively, a Fab format has been used which additionally comprises the C1 domain of the heavy chain and the constant region of the light chain. Recently, a new possible form of intrabody, scFab, has been described. The scFab format is expected to subclone the available Fab genes into intracellular expression vectors more easily, but it remains to be observed whether it offers any advantages over the well-established scFv format. In addition to scFv and Fab, bispecific formats have been used as intrabodies. Bispecific Tie-2x VEGFR-2 antibodies targeting the ER have an extended half-life compared to monospecific antibody counterparts. Bispecific transmembrane intrabodies were developed as a specific format to recognize both intracellular and extracellular epitopes of epidermal growth factor, binding to unique features of the relevant monospecific antibodies, namely inhibition of autophosphorylation and ligand binding.
Another form of intrabody particularly suitable for cytoplasmic expression is a single domain antibody (also referred to as nanobody) derived from a camel or consisting of a human VH domain or a human VL domain. These single domain antibodies generally have advantageous properties, such as high stability; good solubility; easy library cloning and selection; high expression yields in E.coli and yeast.
Intracellular antibody genes can be expressed in target cells following transfection with expression plasmids or viral transduction with recombinant viruses. Typically, the selection is intended to provide optimal levels of transfection and production of intrabodies. Successful transfection and subsequent intrabody production can be analyzed by immunoblot detection of the produced antibodies, however, to assess the correct intrabody/antigen interaction, co-immunoprecipitation of HEK 293 cell extracts transiently co-transfected with the corresponding antigen and the intrabody expression plasmid can be used.
As used herein, inhibition of SETDB1 gene expression includes any expression or reduction in protein activity or level of SETDB1 gene or the protein encoded by the SETDB1 gene, as compared to the situation where inhibition is not induced. The reduction may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% compared to SETDB1 gene expression or SETDB1 protein levels that are not targeted by inhibition. The inhibitor of H3K9 histone methyltransferase SETDB1 gene expression may also be selected from antisense oligonucleotide constructs, siRNA, shRNA, microrna (mirna), and ribozymes.
Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, will directly block translation of H3K 9-histone methyltransferase SETDB1, thereby preventing protein translation or increasing mRNA degradation, thereby reducing the level of H3K 9-histone methyltransferase SETDB1 and its activity in cells. For example, antisense oligonucleotides of at least about 15 bases and complementary to a unique region of an mRNA transcript sequence encoding H3K 9-histone methyltransferase SETDB1 can be synthesized, e.g., by conventional phosphodiester techniques, and administered, e.g., by intravenous injection or infusion. Methods for specifically inhibiting gene expression of genes of known sequence using antisense technology are well known in the art (see, e.g., U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
Small inhibitory rnas (sirnas) may also be used as expression inhibitors for use in the present invention. SETDB1 gene expression may be reduced by contacting the subject or cell with small double-stranded RNA (dsrna), or a vector or construct that causes the production of small double-stranded RNA, such that SETDB 1-histone methyltransferase gene expression is specifically inhibited (i.e., RNA interference or RNAi). Methods for selecting suitable dsRNA or dsRNA encoding vectors for genes of known sequence are well known in the art (see, e.g., Tuschl, T.et al (1999); Elbashir, S.M.et al (2001); Hannon, GJ. (2002); McManus, MT.et al (2002); Brummelkamp, TR.et al (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International patent publications WO 01/36646, WO 99/32619 and WO 01/68836). Advantageously, all or part of the phosphodiester bonds of the siRNA of the invention are protected. This protection is typically achieved by chemical means using methods known in the art. For example, the phosphodiester bond may be protected by a thiol or amine functional group or by a phenyl group. For example, advantageously, the siRNA of the present invention 5 '-and/or 3' -end also using the protection of phosphodiester bond technology to protect. The siRNA sequence advantageously comprises at least twelve consecutive dinucleotides or derivatives thereof.
As used herein, the term "siRNA derivative" with respect to a nucleic acid sequence of the invention refers to any nucleic acid having a percent identity of at least 90%, preferably at least 95%, e.g., at least 98%, more preferably at least 98% with erythropoietin or a fragment thereof.
As used herein, the expression "percent identity" between two nucleic acid sequences refers to the percentage of identical nucleic acids between the two sequences to be compared, obtained with optimal alignment of the sequences, which percentage is purely statistical, and the differences between the two sequences are randomly distributed over the nucleic acid sequences. As used herein, "optimal alignment" or "optimized alignment" refers to the alignment for which the percent identity (see below) determined is the highest. Sequence comparison between two nucleic acid sequences is typically achieved by comparing those sequences that have been aligned in advance according to an optimal alignment; the comparison is performed on the comparison segment to identify and compare similar local regions. In addition to manual operations, optimal sequence alignment can be achieved by using the global homology algorithm developed by SMITH and WATERMAN (Ad. App. Math., vol.2, P:482,1981), by using the local homology algorithm developed by NEDDLEMAN and WUNSCH (J.mol. biol, vol.48, P:443,1970), by using the similarity method developed by PEARSON and LIPMAN (Proc. Natl. Acd. Sci.USA, vol.85, P:2444,1988), by using Computer software employing these algorithms (Wisconsin Genetics software Package, Genetics Computer Group,575 SciencDr., Madison, GAP, STFIT, BLAST P, BLAST N, NuTA, TFASTA), by using the MUE multiple alignment algorithm (Edgar, Roeit Acerc, Research, Residh. 1792,2004. vol.32). To obtain the best local alignment, BLAST software is preferably used. The percent identity between two nucleic acid sequences is determined by comparing the two sequences in an optimized alignment, and to obtain an optimized alignment between the two sequences, the nucleic acid sequences can comprise additions or deletions relative to a reference sequence. Percent identity was calculated by: the number of identical positions between the two sequences is determined, then divided by the total number of positions compared, and the result is multiplied by 100 to obtain the percent identity between the two sequences.
shRNA (short hairpin RNA) can also be used as an expression inhibitor used in the present invention.
Micrornas (mirnas) are small (about 21-23 nucleotides) non-coding RNAs that regulate expression of a target gene post-transcriptionally through base pairing to partially complementary sites, thereby preventing protein accumulation by inhibiting translation or by inducing degradation of mRNA. These properties make it a possible tool for inhibiting protein translation. According to the invention, the miRNA may be selected from miR7 and miR9(Juanjuan Zhao et al, "MicroRNA-7: a optimizing new target in Cancer therapy", Cancer Cell International 2015; 15: 103; Zhang H et al, "MiR-7, inhibiting induced therapy by RNA HOTAIR,
Ribozymes may also be used as expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing RNA-specific cleavage. The mechanism of action of ribozymes involves sequence-specific hybridization of a ribozyme molecule to a complementary target RNA, followed by endonuclease cleavage. Thus, engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of the H3K 9-histone methyltransferase SETDB1 mRNA sequence are useful within the scope of the present invention. The specific ribozyme cleavage sites in any potential RNA target are initially identified by scanning the ribozyme cleavage sites of the target molecule, which sites typically include the following sequences: GUA, GUU and GUC. Once identified, predicted structural features, such as secondary structure, of short RNA sequences between about 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated, which would render the oligonucleotide sequence unsuitable.
Antisense oligonucleotides and ribozymes useful as expression inhibitors can be prepared by known methods. These include techniques for chemical synthesis, for example by solid phase phosphoramidite chemistry. Alternatively, antisense RNA molecules can be produced by in vitro or in vivo transcription of DNA sequences encoding the RNA molecules. Such DNA sequences may be incorporated into a variety of vectors which incorporate suitable RNA polymerase promoters (such as the T7 or SP6 polymerase promoters). Various modifications of the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' -0-methyl groups within the oligonucleotide backbone rather than phosphodiesterase linkages.
The antisense oligonucleotides, siRNA, shRNA and ribozymes of the present invention can be delivered in vivo alone or in combination with a vector. In the broadest sense, a "vector" is a vehicle capable of facilitating transfer of an antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to a cell, preferably to a cell expressing H3K 9-
Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by a gene of interest. Non-cellular viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by integration of the provirus into host cell DNA. Retroviruses have been approved for use in human gene therapy trials. Most useful are those replication-defective retroviruses (i.e., capable of directing the synthesis of the desired protein, but incapable of producing infectious particles). Such genetically altered retroviral expression vectors have general utility for the efficient transduction of genes in vivo. Kriegler,1990 and Murry,1991 provide standard protocols for the production of replication-defective retroviruses (including the incorporation of foreign genetic material into plasmids, transfection of plasmid-lined packaging cells, production of recombinant retroviruses from packaging cell lines, collection of viral particles from tissue culture media, and infection of target cells with viral particles).
For some applications, the preferred viruses are adenoviruses and adeno-associated viruses (AAV), which are double-stranded DNA viruses that have been approved for human gene therapy. Currently, 12 different AAV serotypes are known (AAV1-12), each with different tissue tropism (Wu, Z Mol Ther 2006; 14: 316-27). Recombinant AAV is derived from a dependent parvovirus AAV2(Choi, VW J Virol 2005; 79: 6801-07). Adeno-associated virus types 1-12 can be engineered to be replication-deficient and capable of infecting a variety of cell types and species (Wu, Z Mol Ther 2006; 14: 316-27). It also has the following advantages: such as thermal and lipid solvent stability; high transduction frequency of cells of various lineages including hematopoietic cells; and has no excessive infection inhibition effect, so that multiple series of transduction can be performed. It has been reported that adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability in inserted gene expression characterized by retroviral infection. In addition, wild-type adeno-associated virus infection was passaged more than 100 times in tissue culture without selective pressure, indicating that adeno-associated virus genomic integration is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.
Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, e.g., Sambrook et al, 1989. In recent years, plasmid vectors have been used as DNA vaccines for in vivo delivery of antigen-encoding genes to cells. They are particularly advantageous for this because they do not have the same safety issues as many viral vectors. However, these plasmids with promoters compatible with the host cell can express peptides from the operably encoded genes within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40 and pBluescript. Other plasmids are well known to those of ordinary skill in the art. In addition, restriction enzymes and ligation reactions can be used to custom design plasmids to remove and add specific DNA fragments. Plasmids can be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid may be injected intramuscularly, intradermally, subcutaneously, or otherwise. It can also be administered by intranasal spray or drops, rectal suppository and orally. It can also be applied to epidermal or mucosal surfaces using a gene gun. The plasmid can be provided in an aqueous solution, dried onto gold particles, or combined with another DNA delivery system, including but not limited to liposomes, dendrimers, cochlear delivery vehicles, and microencapsulation.
The antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences according to the invention are typically under the control of heterologous regulatory regions, such as heterologous promoters. The promoter may be specific for Muller glial cells, microglia, endothelial cells, pericytes, and astrocytes. For example, specific expression in Muller glial cells can be obtained by the promoter of the glutamine synthetase gene. For example, the promoter may also be a viral promoter, such as the CMV promoter or any synthetic promoter.
In the present invention, the inhibitor of the H3K9 histone methyltransferase SETDB1 according to the present invention is preferably selective for the H3K9 histone methyltransferase SETDB1 compared to other histone methyltransferases such as EZH2, G9A Suv39H1 or Suv39H 2. By "selective" is meant that the inhibitor has an affinity that is at least 10-fold, preferably 25-fold, more preferably 100-fold, even more preferably 500-fold higher than the affinity of other histone methyltransferases.
In general, the IC of the SETDB1 inhibitor of the invention50Less than 20M, preferably less than 10M, more preferably less than 5M, even more preferably less than 1M, in particular less than 0.5M or less than 0.1M. And in general, the IC of SETDB1 inhibitors for other methyltransferases (e.g. EZH2, G9A, Suv39H1 or Suv39H2), especially for H3K9 methyltransferases 50Greater than 5M, in particular greater than 10M, greater than 20M, or even greater than 50M. For example, the ID of the inhibitors of the invention for SETDB150May be less than 1M, in particular less than 0.5M, while for other methyltransferases (e.g. EZH2, G9A, Suv39H1 or Suv39H2), especially for H3K9 methyltransferases, their ID50May be greater than 10M, in particular greater than 20M.
Preferably, the inhibitor of SETDB1 according to the invention is not selected from triptolide, chaetocin and verticillium A.
Immune checkpoint modulators
As used herein, the term "immune checkpoint protein" (also referred to as immune checkpoint molecule) has its ordinary meaning in the art and refers to a molecule expressed by T cells and/or NK cells that either up-regulates a signal (stimulatory checkpoint molecule) or down-regulates a signal (inhibitory checkpoint molecule). Most preferably, according to the invention, the immune checkpoint molecule is expressed at least by T cells.
Immune checkpoint molecules are thought in the art to constitute an immune checkpoint pathway similar to CTLA-4 and PD-1 dependent pathways. The immune checkpoint molecules of the invention are described in particular in pardol, 2012.Nature Rev Cancer 12: 252-; mellman et al, 2011.Nature 480: 480-; chen L & Flies DB, nat. rev. immunol.2013april; 13(4): 227-. Examples of immune checkpoint molecules encompass mainly CD27, CD40, OX40, GITR, ICOS, TNFRSF25, 41BB, HVEM, CD28, TMIGD2, CD226, 2B4(CD244) and ligandCD4, B4-H4 Brandt (NK ligand), LIGHT (CD258, TNFSF 4), CD28 4, A2 4, B4-H4, BTLA, CTLA-4, CD277, IDO, KIRs, PD-1s, LAG-3, TIGIT-3, VISTA, CD4, CD112 4, CD160, CD4 (or 2B4) DCIR (C-type lectin surface receptor), ILT4 (immunoglobulin-like transcript), CD4 (PECAM-1) (family-like R), Ig-72, CD4, SIR 4, LARG-TIGIT 4, CD4, and CD4 related immunoglobulin receptor families.
Non-limiting examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIRs, PD-1, LAG-3, TIM-3TIGIT, VISTA, CD96, CD112R, CD160, DCIR (C-type lectin surface receptor), ILT3, ILT4 (immunoglobulin-like transcript), CD31(PECAM-1) (Ig-like R family, CD39, CD73, CD94/NKG2, GP49B (immunoglobulin superfamily), KLRG1, LAIR-1 (leukocyte-associated immunoglobulin-like receptor 1), CD305, PD-L1, and PD-L2.
The adenosine A2a receptor (A2aR) whose ligand is adenosine is considered an important checkpoint in cancer treatment, since adenosine in the immune microenvironment leading to activation of the A2a receptor is a negative immune feedback loop, and the tumor microenvironment has a relatively high concentration of adenosine. A2aR can be inhibited by antibodies that block adenosine binding or adenosine analogs, some of which have comparable specificity for A2 aR. These drugs have been used in clinical trials for parkinson's disease.
The B7 family is an important family of membrane-bound ligands that bind to costimulatory and inhibitory receptors. All members of the B7 family and their known ligands belong to the immunoglobulin superfamily. Many receptors have not been identified. B7-H3 (also referred to as CD276) was originally thought of as a costimulatory molecule, but is now thought of as a cosuppressive molecule. B7-H4 (also known as VTCN1) is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape.
CD160 is a Glycoprotein Phosphatidylinositol (GPI) -anchored member of the Ig superfamily protein with a restricted expression profile limited to CD56dim CD16+ NK cells, NKT cells, gamma T cells, cytotoxic CD8+ T cells lacking CD28 expression, a small fraction of CD4+ T cells, and all intraepithelial lymphocytes. Binding of CD160 to both classical MHC I and non-classical MHC I enhanced NK and CD8+ CTL function. However, binding of the herpes virus entry mediator (HVEM/TNFRSF14) to CD160 was shown to mediate inhibition of CD4+ T cell proliferation and TCR-mediated signaling.
The HVEM (herpes virus entry mediator) protein is a bimolecular switch that binds to the costimulatory LT-stimulatory entry mediator) protein and the co-inhibitory receptor BTLA/CD 160. Engagement of co-inhibitory receptors on T cells, BTLA and/or CD160, with HVEM expressed on DCs or Tregs transduces negative signals to T cells that are counteracted by co-stimulatory signals transmitted following direct engagement of HVEM on T cells with LIGHT expressed on DCs or more likely LIGHT expressed on other activated T cells (T-T cell cooperation). The interaction of HVEM with BTLA and CD160 predominates within the HVEM/LIGHT pathway and vice versa, probably due to differences in ligand/receptor affinity and differential expression patterns of these molecules on cell types at different stages of cell differentiation. LIGHT, BTLA, and CD160 have substantially different binding affinities and occupy spatially distinct sites upon interaction with HVEM receptors, which enables HVEM to function as a molecular switch. The net effect of the LIGHT/HVEM and HVEM/BTLA/CD160 interactions determines the outcome of the response when these different receptors and ligands are present together (see M.L.del Rio. fruits and ligands present together, resulting from the differential expression pattern of the molecules: adenosine 1) asics totumor immunothermal eye 480: 480-489; chen L & Flies DB, nat. rev. immunol.10; 87).
B and T Lymphocyte Attenuators (BTLA), also known as CD272, also have HVEM as their ligand. BTLA T cells are inhibited in the presence of their ligand, HVEM. During phenotypic differentiation of human CD8+ T cells from naive to effector cells, surface expression of BTLA was gradually down-regulated, but tumor-specific human CD8+ T cells expressed high levels of BTLA (kenneth m. murphy et al. balancing co-stimulation and inhibition with BTLA and hvem. nature Reviews Immunology 2006,6, 671-one 681).
CTLA-4 (cytotoxic T lymphocyte-associated protein 4, also known as CD152) is the first clinically targeted immune checkpoint. It is expressed only on T cells. It has been proposed that its expression on the surface of T cells attenuates T cell activation by competing for CD28 in binding to CD80 and CD86 and proactively delivering inhibitory signals to T cells. Expression of CTLA-4 on Treg cells is used to control T cell proliferation.
Ig-like transcripts-3 and-4 (ILT3 and ILT4) are inhibitory receptors expressed by monocytes, macrophages, and DCs. The corresponding ligand of ILT3 is not known, but it is likely to be expressed on T cells because ILT3 directly inhibits T lymphocyte function. In several cancers, ILT3 has been found to mediate immune escape mechanisms by impairing T cell responses. Furthermore, DCs expressing ILT4 block efficient CTL differentiation, a mechanism used by tumors that can upregulate ILT4 to escape the immune system (Vasaturo A et al, Front immunol.2013; 4: 417).
Platelet endothelial cell adhesion molecule-1 (PECAM-1), also known as CD31, is a type I transmembrane glycoprotein member of the immunoglobulin (Ig) gene superfamily, which contains six extracellular Ig domains and two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is limited to endothelial cells and hematopoietic cells (see Newman DK, Fu G, AdamsT, et al. the additive molecular PECAM-1 enzymes the TGF β -mediated inhibition of T cell function. science signaling.2016; 9(418): ra 27).
LAIR-1 is expressed at very high and relatively uniform levels in naive T cells, but at lower and more non-uniform levels in memory T cells. LAIR-1 consists of a 287 amino acid type I transmembrane glycoprotein with a single extracellular C2 type Ig-like domain and a cytoplasmic domain with two ITIM motifs. LAIR-1 can inhibit TCR-mediated signaling through recruitment of C-terminal Csk, one or more phosphatases SHIP, SHP-1 or SHP-2, and to some extent, inhibits signaling and ERK signaling by p38 MAP kinase (thoven iran T et al (2012) J Clin Cell Immunol S12: 004).
IDO1 (indoleamine 2, 3-dioxygenase 1) is a tryptophan catabolic enzyme. A related immunosuppressive enzyme. Another important molecule is TDO, tryptophan 2, 3-dioxygenase. IDO1 is known to suppress T and NK cells, generate and activate Treg and myeloid-derived suppressor cells, and promote tumor angiogenesis.
KIRs (killer cell immunoglobulin-like receptors) are the braided class of inhibitory receptors, which can be divided into two classes based on structure: killer cell immunoglobulin-like receptors (KIR) and C-type lectin receptors (type II transmembrane receptors). Although many receptors are expressed on T cells and APCs, these receptors were originally described as modulators of NK cell killing activity. Many KIRs are specific for a subset of mhc class i molecules and are allele-specific.
LAG3 (lymphocyte activation gene 3) has as its ligand an MHC class II molecule that is upregulated in some epithelial cancers, but is also expressed in tumor-infiltrating macrophages and dendritic cells. This immune checkpoint suppresses the immune response by acting on Treg cells as well as directly on CD8+ T cells.
PD-1 is a programmed death 1(PD-1) receptor with two ligands, PD-L1 and PD-L2. This checkpoint was the target for the FDA-approved Merck & co melanoma drug, Keytruda, obtained 9 months 2014. The advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.
TIM-3 is short for the T cell immunoglobulin domain and mucin domain 3 (also known as B7H5) and its ligand is galectin 9, which is expressed on activated human CD4+ T cells and modulates Th1 and Th17 cytokines. TIM-3 triggers cell death by interacting with its ligand galectin-9, thereby acting as a negative regulator of Th1/Tc1 function.
VISTA (short for V-domain Ig inhibitors of T cell activation), also known as c10orf54, PD-1H, DD1 α, Gi24, Dies1 and SISP1, is a member of the NCR B7 family and represents a new target for immunotherapy. Murine VISTA is a type I transmembrane protein with a single IgV domain that has sequence homology with its B7 relatives, with conserved segments being thought to be critical for IgV stability. VISTA is expressed on naive T cells, while PD-1 and CTLA-4 are not, which might indicate that VISTA functions to suppress T cell activity during an even earlier T cell priming phase. VISTA is expressed on both T cells and APCs, with high expression on myeloid cells. VISTA is restricted by the hematopoietic system and in various cancer models, VISTA is detected only on tumor-infiltrating leukocytes, but not on tumor cells. This unique surface expression pattern suggests that VISTA may act to limit T cell immunity at various stages. VISTA has been shown to exert ligand and receptor functions. First, VISTA can act as a ligand to down-regulate T cell activation. Secondly, VISTA has been shown to act as a receptor on T cells thereby down-regulating their activity. VISTA-/-CD4+ T cells had a more intense effect on polyclonal and antigen-specific stimulation than wild-type (WT) CD4+ T cells, resulting in increased proliferation and production of IFN-rich, TNF-rich and IL-17A. anti-VISTA monotherapy can be in a variety of preclinical models: reduction of tumor growth in B16OVA melanoma, B16-BL6 melanoma, MB49 bladder cancer and PTEN/BRAF-induced melanoma (see Deng J, Le Mercier I, Kuta A, Noelle RJ. "A New VISTA on Combination therapy for a new checkpoint
CD96, CD226(DNAM-1) and TIGIT belong to an emerging family of receptors that interact with Nectin and Nectin-like proteins. CD226 activates Natural Killer (NK) cell-mediated cytotoxicity, while TIGIT has been reported to counteract CD 226.
CD96 competes with CD226 for CD155 binding and limiting NK cell function by direct inhibition (Christopher JChan et al, "The receptors CD96 and CD226 oppose reach other in The regulation of natural killer cells functions", Nature Immunology 201415, 431-438).
TIGIT is also known as a T cell immunoreceptor with Ig and ITIM domains, or VSTM 3. TIGIT/VSTM3 is typically expressed by activated T cells, regulatory T (treg) cells, and Natural Killer (NK) cells. Poliovirus receptor (CD155/PVR) and Nectin-2(CD112) and CD 113 have been identified as related ligands. TIGIT/VSTM3 competes with molecules CD226 and CD96 for binding to CD155/PVR and CD112, respectively, but TIGIT/VSTM3 exhibits the strongest affinity for CD155/PVR among all the respective receptor-ligand combinations. TIGIT inhibited T cell activation in vivo (see Karsten Mahnke et al, TIGIT-CD155 Interactions in Melanoma: A Novel Co-inhibition Pathology with probability for Clinical interaction. journal of investigative Dermatology.2016; 136: 9-11).
CD112R (PVRIG) whose ligand is PVRL2 is a member of poliovirus receptor-like proteins that preferentially express on T cells and inhibit T cell receptor-mediated signaling.
Non-limiting examples of stimulatory checkpoint molecules include CD27, CD40L, OX40, GITR, ICOS, TNFRSF25, 41BB, HVEM, CD28, TMIGD2 and CD226, 2B4(CD244) and its ligands CD48, B7-H6 Brandt (NK ligand), CD28H and LIGHT (CD258, TNFSF 14).
CD27, CD40L, OX40, GITR, ICOS, HVEM, 2B4(CD244) and its ligands CD48, B7-H6 Brandt (NK ligand), LIGHT (CD258, TNFSF14), CD28H, and TNFSF25 are stimulatory checkpoint molecules that are members of the Tumor Necrosis Factor (TNF) receptor superfamily (TNFSF). TNFRSF proteins play an important role in B and T cell development, survival and anti-tumor immune responses. In addition, some TNFRSFs are associated with the inactivation of Treg cells. Thus, TNFRSF agonists can activate tumor immunity, and their combination with immune checkpoint therapy is promising. Several antibodies that act as agonists of TNFRSF have been evaluated in clinical trials (Shiro Kimbara and Shunsuke Kondo, "Immune chemistry architecture and therapy targets in genetic Carcinoma," World JGastrontenol. 2016Sep 7; 22(33): 7440. 7452, se expression for review Watts. TNF/TNFR family members in constraints of T cell responses. Annu RevImmunol. 2005; 23: 23-68.).
CD27 supports antigen-specific expansion of naive T cells and is crucial for the generation of T cell memory. CD27 is also a memory marker for B cells. The activity of CD27 is governed by the transient availability of its ligand, CD70, on lymphocytes and dendritic cells. Co-stimulation with CD27 is known to inhibit Th17 effector cell function.
CD40 the CD40L pathway is a costimulatory pathway affecting both humoral and cell-mediated immunity. CD40L (also known as CD154) is expressed primarily on T helper cells shortly after activation. Receptor 2B4(CD244) belongs to the Signaling Lymphocyte Activating Molecule (SLAM) subfamily within the immunoglobulin superfamily (IgSV). All members of this family contain two or more immunoreceptor tyrosine-based switch motifs (ITSM) in their cytoplasmic tails, including the receptors CD229, CS1, NTB-A and CD84[92 ]. Upon activation on CD8+ T cells, 2B4 is expressed by NK cells, gamma cells basophils and monocytes and binds with high affinity to CD48 on lymphoid and myeloid cells (Kemal Catakovic et al, cell communication and Signaling201715: 1).
TNFSF14/LIGHT/CD258 showed inducible expression and competed with Herpes Simplex Virus (HSV) glycoprotein D for the receptor herpes virus entry mediator expressed by T lymphocytes (HVEM/TNFRSF14), a recently identified member of the human and mouse TNF superfamily. TNFSF14/LIGHT/CD258 is a 29-kD type II transmembrane protein produced by activated T cells, monocytes and granulocytes as well as immature DCs. In vitro, the HVEM/LIGHT immune checkpoint pathway induces potent CD 28-independent costimulatory activity, leading to NF- κ B activation, production of IFN- γ and other cytokines, and T cell proliferation in response to allogeneic DCs. In vivo blocking studies showed that the HVEM/LIGHT immune checkpoint pathway is involved in promoting the response of cytolytic T cells to tumors and the development of GVHD, and that transgene overexpression of TNFSF14/LIGHT/CD258 in T cells leads to T cell expansion and causes a variety of severe autoimmune diseases (Qunrui Ye et al J Exp Med.2002Mar 18; 195(6): 795-.
CD28H was constitutively expressed on all naive T cells. B7 homolog 5(B7-H5) was identified as a specific ligand for CD 28H. B7-H5 is constitutively present in macrophages and can be induced on dendritic cells. The B7-H5/CD28H interaction selectively co-stimulates growth of human T cells and cytokine production by an AKT-dependent signaling cascade (Zhu Y et al, Nat Commun.2013; 4: 204).
OX40 (also known as CD134) has OX40L or CD252 as its ligand. Like CD27, OX40 promotes expansion of effector and memory T cells, however, OX40 also has the ability to inhibit T regulatory cell differentiation and activity, and may also regulate cytokine production. The value of OX40 as a drug target lies primarily in the fact that: i.e., transiently expressed after T cell receptor engagement, it is only upregulated by recently antigen-activated T cells within inflammatory lesions. An anti-OX 40 monoclonal antibody has been shown to have clinical utility in advanced cancers (Weinberg AD, Morris NP, Kovacscovics-Bank owski M, Urba WJ, CurtiBD (November 1,2011). "Science gene translation: the OX40 agonist store". Immunol Rev.244(1): 218-31).
GITR is short for glucocorticoid-induced TNFR family-related genes, which promotes T cell expansion, including Treg expansion. The ligand for GITR (GITRL) is predominantly expressed on antigen presenting cells. GITR antibodies have been shown to promote anti-tumor responses by losing Treg lineage stability (see Nocentini G, Ronchetti S, cuzzocorea S, ricccardi C (may1,2007). "GITR/GITRL: more than an effector T cell co-stimulation system". Eur J immunol.37(5): 1165-9).
ICOS is short for an inducible T cell costimulator, also known as CD278, which is expressed on activated T cells. Its ligand is ICOSL, expressed primarily on B cells and dendritic cells. This molecule appears to be important in T cell effector function (Burmeister Y, Lischke T, Dahler AC, Mages HW, Lam KP, Coyle AJ, Kroczek RA, HutloffA (January 15,2008). "ICOS controls the pore size of effector-memory and molecular T cells". J Immunol.180(2): 774-) 782).
Another stimulatory checkpoint molecule belonging to the B7-CD28 superfamily is in particular CD28 itself and TGMID 2.
CD28 is constitutively expressed in nearly all human CD4+ T cells and about half all CD 8T cells. Binding to its two ligands (CD 80 and CD86 expressed on dendritic cells) promotes T cell expansion.
TMIGD2 (also known as the CD28 homolog) modulates T cell function by interacting with its ligand HHLA 2; it is a newly identified B7 family member. TMIGD2 protein was constitutively expressed on all naive T cells and most Natural Killer (NK) cells, but not on T regulatory cells or B cells (see Yanping Xiao and Gordon J. Freeman, "Anew B7: CD28 family checkpoint target for cancer immunology: HHLA 2",
CD137ligand (CD 137L; also known as 4-1BBL and TNFSF9) is predominantly expressed on professional Antigen Presenting Cells (APCs), such as dendritic cells, monocytes/macrophages and B cells, and its expression is upregulated during activation of these cells. However, its expression has been demonstrated on a variety of hematopoietic and non-hematopoietic cells. Generally, 4-1BBL/CD137L is constitutively expressed on many cell types, but its expression level is low except for a few cell types. Interestingly, 4-1BBL/CD137L was co-expressed with CD137 (also known as 4-1BB and TNFRSF9) in various cell types, but expression of CD137/4-1BB strongly down-regulated expression of 4-1BBL/CD137L by cis-interaction between two molecules, resulting in endocytosis of 4-1BBL/CD137L (see Byungsuk Kwon et al. is CD137Ligand (CD137L) "signalling a Fine Tunerof Immune Responses?" Immune Net w.2015Jun; 15: 121-.
Finally, other immune checkpoint molecules according to the invention also include CD244 (or 2B4) and sirpa.
2B4/CD244 is a member of the Signaling Lymphocyte Activation Molecule (SLAM) -associated receptor family, also known as SLAMF4 and CD 244. All members of the SLAM family have similar structures, including the extracellular domain, the transmembrane region, and the tyrosine-rich cytoplasmic region. The 2B4 and CD48 immune checkpoint pathways can lead to signaling through two receptors. CD48/SLAMF2 signaling in B cells leads to homotypic adhesion, proliferation and/or differentiation, release of inflammatory effector molecules, and isotype class switching. Furthermore, all these processes are also initiated by CD48/SLAMF2 ligation in T cells, where their activation and/or cytotoxicity is promoted. 2B4 signaling requires Signaling Lymphocyte Activating Molecule (SLAM) associated protein (SAP) or EWS activated transcript 2 (EAT-2; also known as SH2D 1B). In CD 8T cells and NK cells, it has been reported that 2B4/CD244 exerts a positive or negative regulatory effect (see also Sebastian Stark. "2B 4(CD244), NTB-A and CRACC (CS1) catalytic oxidation button no promotion in human NK cells". int. Immunol.200618 (2): 241-247).
CD47 is a cell surface glycoprotein with a variety of functions, including regulation of phagocytosis by binding to macrophage and dendritic cell specific protein signal regulatory protein alpha (SIRP alpha). Binding of sirpa to CD47 (acting as a sirpa and CD47 immune checkpoint pathway) essentially sends a "do not eat me" message to macrophages by initiating signaling that inhibits phagocytosis. Increased expression of CD47 is thought to be a mechanism by which cancer cells evade immunodetection and phagocytosis. Targeting CD47 to cancer cells with anti-CD 47 blocking antibodies can promote phagocytosis of macrophages in vitro. Furthermore, in an in vivo xenograft model of non-hodgkin's lymphoma, treatment with anti-CD 47 blocking antibody in conjunction with rituximab treatment was used to promote phagocytosis in vitro and eliminate cancer cells. Further results indicate that CD47 expression is increased in a variety of human solid tumor types and that blockade of the SIRP α and CD47 immune checkpoint pathways with anti-CD 47 antibodies can promote phagocytosis of solid tumor cells in vitro and reduce growth of solid tumors in vivo (see Martina Seiffert et al, "Signal-regulated protein α (SIRP α) but not SIRP β is secreted in T-cell activation, binders to CD47with high affinity, and is expressed on animal CD34+ CD 38-hematology cells". 2001; Blood:97 (9)).
As used herein, the expression "modulator of an immune checkpoint protein" or "checkpoint modulator cancer immunotherapeutic" (both expressions are used interchangeably in the sense of the present invention) has the general meaning of the art and refers to any compound that inhibits the function of an immunosuppressive checkpoint protein (inhibitory immune checkpoint inhibitor or immune checkpoint inhibitor, as described previously) or stimulates the function of a stimulatory checkpoint protein (stimulatory immune checkpoint agonist or immune checkpoint agonist, used interchangeably). Inhibition includes reduced function and complete blockade.
Immune checkpoint modulators include peptides, antibodies, fusion proteins, nucleic acid molecules, and small molecules. For certain immune checkpoint proteins (i.e., immune pathway gene products), antagonists or agonists of such gene products, as well as small molecule modulators of such gene products, are also contemplated.
Preferred immune checkpoint inhibitors or agonists are antibodies or fusion proteins that specifically recognize an immune checkpoint protein or its ligand, as described previously.
According to the invention, various antibody mixtures directed against different epitopes of the same molecule or different targets on the same tumor cell; bispecific or multispecific Antibodies (Coralliza-Gorj Lou n I, Somovilla-Crespo B, Santamaria S, Garcia-Sanz JA, Kremer L.New Stratagenes Using antibody combinations to inhibitor Treatment efficacy. Frontiers in immunology.2017; 8: 1804; Liu H, Saxena A, Sidhu SS, Wu D.Fc Engineering for developing Therapeutic biological Antibodies and Novel polypeptides. ont Immunol.2017; 8:38.doi: 10.3389/mmu.2017.00038.E.2017.2017.Revim.) can be used.
Fusion proteins useful as immune checkpoint modulators can be prepared by fusing checkpoint molecules as described above to the crystallizable fragment (Fc) region of an immunoglobulin. Preferably, the antibody is a monoclonal antibody.
Many immune checkpoint inhibitors and agonists are known in the art and, like these known immune checkpoint protein modulators, alternative immune checkpoint modulators may be developed in the (near) future and used in combination with SETDB1 inhibitors according to the invention.
The immune checkpoint modulator according to the present invention results in the activation of the immune system, in particular in the expansion of antigen-specific T cell responses. In particular, the immune checkpoint modulator of the invention is administered to enhance the proliferation, migration, persistence and/or cytotoxic activity of CD8+ T cells in a subject, in particular to enhance tumor infiltration of CD8+ T cells in a subject. As used herein, "CD 8+ T cells" have their ordinary meaning in the art and refer to a subset of T cells that express CD8 on their surface. They are restricted by MHC class I and function as cytotoxic T cells. "CD 8+ T cells" are also known as Cytotoxic T Lymphocytes (CTL), T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is a cognate recognition element in major histocompatibility complex class I restriction interactions. The ability of immune checkpoint modulators to enhance T CD8 cell killing activity can be determined by any assay well known in the art. Typically, the assay is an in vitro assay in which CD8+ T cells are contacted with target cells (e.g., target cells recognized and/or lysed by CD8+ T cells).
For example, the ability of the immune checkpoint modulator of the invention may be selected to increase the specific lysis of CD8+ T cells by more than about 20%, preferably at least about 30%, at least about 40%, at least about 50%, or more specific lysis at the same effector to target cell ratio using CD8+ T cells or a CD8T cell line contacted with the immune checkpoint inhibitor of the invention. Examples of protocols for classical cytotoxicity assays are conventional.
The at least one immune checkpoint modulator according to the present invention may be a modulator of an inhibitory immune checkpoint molecule and/or a stimulatory immune checkpoint molecule.
For example, checkpoint modulators cancer immunotherapeutics may be agents that block (antagonists of) immunosuppressive receptors (i.e., inhibitory immune checkpoints) expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4(CTLA4) and programmed cell death 1(PDCD1, most commonly referred to as PD-1) or by NK cells, such as various members of the killer immunoglobulin-like receptor (KIR) family, or that block the primary ligands of these receptors, such as the PD-1 ligand CD274 (most commonly referred to as PD-L1 or B7-H1).
In some embodiments, the checkpoint blockade cancer immunotherapeutic agent is selected from the group consisting of: anti-CTLA 4 antibody,
Examples of anti-CTLA-4 antibodies are described in U.S. patent nos.: 5,811,097, respectively; 5,811,097, respectively; 5,855,887, respectively; 6,051,227, respectively; 6,207,157, respectively; 6,682,736; 6,984,720, respectively; and 7,605,238. One anti-CDLA-4 antibody is tiximumab (ticilimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also referred to as 10D1, MDX-D010), a fully human monoclonal IgG antibody that binds CTLA-4.
Examples of PD-1 and PD-L1 antibodies are described in U.S. patent nos.: 7,488,802, respectively; 7,943,743, respectively; 8,008,449; 8,168,757, respectively; 8,217,149, and PCT published patent application numbers: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO 2011161699. In some embodiments, the PD-1 blocking agent comprises an anti-PD-L1 antibody. In certain other embodiments, PD-1 blockers include anti-PD-1 antibodies and similar binding proteins, such as nivolumab (MDX 1106, BMS 936558, ONO4538), a fully human gG4 antibody that binds PD-1 and blocks PD-1 activation through its ligands PD-L1 and PD-L2; pembrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1; CT-011, a humanized antibody that binds PD-1; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559(MDX-1105-01) for PD-L1(B7-H1) blockade.
Other immune checkpoint inhibitors include lymphocyte activation gene 3(LAG-3) inhibitors such as IMP321 (a soluble Ig fusion protein) (Brignone et al, 2007, J.Immunol.179: 4202-4211).
Other immune checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors, in particular the anti-B7-H3 antibody MGA271(lo et al, 2012, clin.
Also included are TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al, 2010, j.exp.med.207:2175-86and Sakuishi et al, 2010, j.exp.med.207: 2187-94). As used herein, the term "TIM-3" has the ordinary meaning in the art and refers to molecule 3 which contains T cell immunoglobulin and mucin domains. Thus, the term "TIM-3 inhibitor" as used herein refers to a compound, substance or composition that inhibits the function of TIM-3. For example, the inhibitor may inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and/or block the binding of TIM-3 to its natural ligand galectin-9. Antibodies specific for TIM-3 are well known in the art and are typically described in WO2011155607, WO2013006490 and WO 2010117057.
In some embodiments, the immune checkpoint inhibitor is an indoleamine 2, 3-dioxygenase (IDO) inhibitor, preferably an IDO1 inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include, but are not limited to, 1-methyl-tryptophan (IMT), β - (3-benzofuranyl) -alanine, β - (3-benzo (b) thienyl) -alanine), 6-nitrotryptophan, 6-fluorotryptophan, 4-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, 5-methoxytryptophan, 5-hydroxytryptophan, indole 3-methanol, 3' -diindolylmethane, epicatechin gallate, 5-Br-4-Cl-
In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT (T cell immunoglobulin and ITIM domain) antibody.
In some embodiments, the immune checkpoint inhibitor is an anti-VISTA antibody, preferably a monoclonal antibody (LinesJL, Sempere LF, Wang L, et al. VISTA is an immune checkpoint polypeptide for human Tcells. cancer research. 2014; 74(7):1924-1932.doi:10.1158/0008-5472. CAN-13-1504).
In a preferred embodiment, the checkpoint modulator cancer immunotherapeutic is a CTLA4 blocking antibody, such as ipilimumab, a PD-1 blocking antibody (such as nigulumab or pembrolizumab), a PDL-1 blocking antibody, or a combination thereof. Typically, the checkpoint modulator cancer immunotherapeutic is a PD-1 blocking antibody (such as nigulumab or pembrolizumab), or a PDL-1 blocking antibody.
Checkpoint modulators cancer immunotherapeutics may also be agents that activate stimulatory immune checkpoint receptors expressed by activated T lymphocytes or NK cells, or agents that mimic the primary ligands of these receptors and also lead to expansion of antigen-specific T cell responses.
Thus, checkpoint modulators cancer immunotherapeutics are typically agonistic antibodies in general, especially monoclonal agonistic antibodies directed against stimulatory immune checkpoint molecules as described above, e.g. selected from the group consisting of: agonistic anti-4-1 BB, -OX40, -GITR, -CD27, -ICOS, -CD40L, -TMIGD2, -CD226, -TNFSF25, -2B4(CD244), -CD48, -B7-H6 Brandt (NK ligand), -CD28H, -LIGHT (CD258, TNFSF14), and-CD 28 antibodies.
Checkpoint agonists cancer immunotherapies may also be fusion proteins, such as 4-1BB-Fc fusion protein, Ox40-Fc fusion protein, GITR-Fc fusion protein, CD27-Fc fusion protein, ICOS-Fc fusion protein, CD40L-Fc fusion protein, TMIGD2-Fc fusion protein, CD226-Fc fusion protein, TNFSF25-Fc fusion protein, CD28-Fc fusion protein, 2B4(CD244) fusion protein, CD48 fusion protein, B7-H6 Brandt (NK ligand) fusion protein, CD28H fusion protein, and LIGHT (CD258, TNFSF14) fusion protein.
Several 4-1BB agonists have shown great potential for use in human cancers. For example, the fully humanized mAb BMS-666513 against 4-1BB has completed its phase I and II tests for its anti-cancer performance in patients with melanoma, renal cell carcinoma and ovarian cancer (Sznol M, Hodi FS, Margolin K, McDermott DF, Ernstoff MS, Kirkwood JM, et al. phase I study of BMS-663513, a full human anti-CD137 agglutin monoclonal antibody, in tissues (pts) with advanced cancer Caner (CA). J Clin Oncol 26:2008(May20 suppl; abs 3007).
Currently 7 OX40 agonists are being developed, 6 of which take the form of fully human monoclonal antibodies to address the problem of mouse antibodies. An OX40L-Fc fusion protein MEDI6383 is also undergoing clinical evaluation; this linked 2 molecules of OX40L to a portion of the fragment crystallizable (Fc) region of an immunoglobulin. In preclinical testing, the fusion protein appears to have a stronger effect than the OX40 antibody, probably because it can activate dendritic cells and vascular endothelial cells in addition to T cells. Examples of Ox40 agonists include MEDI6469, MEDI6383, MEDI0652, PF-04515600, MOXP0916, GSK3174998, INCAGNO 1949.
Agonistic antibodies to GITR have been developed, such as humanized anti-human GITR mAbs (TRX518.TolerxInc. inflammatory antibodies to human glucocorticoid-induced modulator receptors as potential modulators for the treatment of cancer and viral infection. Extra Optin therapeutics. 2007; 17: 567-575, seeellson Schaer DA, Murphy JT, Wolchok JD.Modulation of GITR for center modulation. curr Opin Immunol.2012Apr; 24(2): 217-24).
As an example of a CD27 agonistic antibody, another member of the TNF family includes fully human 1F5 mAb, which is currently undergoing phase I clinical trials for CD-B-1127 (varluumab) B cell malignancies, melanoma, and renal cell carcinoma (which analyze the properties of the anti-CD 27 monoclonal antibody (mAb) currently undergoing clinical trials) (Vitale LA, He L-Z, Thomas LJ et al.2012development of a human monoclonal antibody for potential therapy of CD 27-expressinglylymphoma and leukamia.Clin. cancer.18 (14), Res3812-3821).
Initial clinical trials of agonistic CD40 mAb showed very promising results in a single agent study without disabling toxicity. To date, four CD40 mabs have been studied in clinical trials: CP-870,893(Pfize and VLST), dacetuzumab (Seattle genetics), Chi Lob 7/4(University of south campton) and lucidum (novartis) (Vonderheide RH, FlaherKT, Khalil M, Stumacher MS, Bajordl, Hutnick NA, et al. clinical activity and immunity modification in canditatentis derived with CP-870,893, a novel CD40 acoustical monoclonal antibody. JIN Oncol.2007; 25: 876-83; Khuzhuchandani S, Czuzman MS, Hernanz-IlituriFJ. cetuzumab, a mannhagen NM 40, pharmaceutical excipient K577, antibiotic J.7, antibiotic J.S.A, antibiotic J.7. European molecular DNA 577, antibiotic J.7, antibiotic J.S, antibiotic J.7, antibiotic J., becker PS, et al, aphase 1 student of lucidumab, a full human anti-CD40 anti azoxyst monoclonal adsorbed induced to substrates with delayed or isolated microorganism Myeloma. br J haemal.2012; 159:58-66).
Checkpoint agonist Cancer immunotherapies may also be anti-ICOS agonist monoclonal antibodies (Kutlu Elpek, Christopher Harvey, Ellen Duong, Tyler Simpson, Jenny Shu, Lindsey Shallberg, MattWallace, Sriram Sathy, Robert Mabry, Jennifer Michaelson, and Michael Briskin, Abstract A059: impact of anti-ICOS agonist monoclonal antibodies in predictive therapeutic approaches, Abstract CRI-CITI-ELISA-International scientific Cancer assays for clinical diagnosis and analysis applications; CRI-CITI-EACR-I International Experimental Cancer assays for molecular diagnosis and Cancer metastasis assays; review by CD 20020. CD agonist antibody, see also for anti-ICOS agonist monoclonal antibodies in CD 20020. PD, Cancer Immunotherapy, see also for CD 20012.
According to the present invention, more than one modulator of an immune checkpoint protein may be used in combination with an inhibitor of SUV39H1 according to the present invention. For example, a modulator of at least one inhibitory immune checkpoint inhibitor (such as anti-PD-1 or anti-PD-L1) may be used in combination with at least one stimulatory immune checkpoint agonist as described above. Costimulatory and cosuppressive immune checkpoint molecules are described in particular in the review by ChenL & Flies B (Nat rev immuno, 2013, supra).
Patient's health
Typically, the patient according to the invention is a mammal, preferably a human.
Typically, the patient has, is in remission or is at risk of cancer. Patients in remission are typically patients whose cancer has been treated (e.g., removed by surgery) and is no longer present. Thus, in general, the combination therapy of the present invention may be administered to a patient who has undergone a therapeutic or primary procedure.
The cancer according to the invention is caused by uncontrolled division of abnormal cells in a part of the body.
The cancer may be a solid cancer or a cancer affecting the blood (i.e., leukemia). Leukemias include, for example, Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Acute Lymphocytic Leukemia (ALL), and chronic lymphocytic leukemia (including various lymphomas, such as mantle cell lymphoma, hodgkin's lymphoma, or non-hodgkin's lymphoma).
Solid cancers often involve malignant growth or tumors resulting from uncontrolled division of cells. Solid cancers primarily include cancers affecting one of the organs selected from: colon, retina (e.g., retinoblastoma), rectum, skin (e.g., melanoma, particularly advanced melanoma), endometrium, airway (including laryngeal carcinoma), gallbladder and biliary tract, lung (including non-small cell lung cancer), uterus, bone (e.g., osteogenic sarcoma, chondroma, ewing's sarcoma, fibrosarcoma, giant cell tumor, seminoma and chordoma), liver, kidney, esophagus, stomach, bladder (including urothelial and urinary tract cancers), pancreas, cervix, brain (e.g., meningioma, glioblastoma, low-grade astrocytoma, oligodendroglioma, pituitary tumor, schwannoma and metastatic brain cancer), ovary, breast (e.g., mucus cancer), cervical region, testis, prostate and thyroid. The term cancer also includes squamous cell carcinoma that may affect the skin, lung, thyroid, breast, esophagus or vagina, as well as fibrosarcoma. In some embodiments, the combination of the invention preferably targets melanoma, glioblastoma, respiratory cancer, breast cancer, lung cancer, urothelial cancer, hodgkin's lymphoma, renal cancer, fibrosarcoma and gastric cancer.
Dosage form
Preferably, the SETDB1 inhibitor and the immune checkpoint modulator are in effective doses.
Typically, the combination treatment regimens of the invention (i.e., the SETDB1 inhibitor and the at least one immune checkpoint modulator) are therapeutically effective. Currently available therapies and their dosages, routes of administration, and recommended use are known in the art and have been described in literature such as the physicians' Desk Reference (60 th edition, 2006). Routes of administration include parenteral, intravenous, subcutaneous, intracranial, intrahepatic, intranodal, intraureteral, sub-ureteral, subcutaneous, and intraperitoneal.
The dosage of one or more agents of the invention (e.g., SETDB1 inhibitors and immune checkpoint modulators) may be determined by one of skill in the art and may also be adjusted by the individual physician in the event of any complications.
Combination therapy
In a particular embodiment, cycling therapy involves administering a first cancer therapeutic for a period of time, followed by a second cancer therapeutic for a period of time, optionally followed by a third cancer therapeutic for a period of time, etc., and repeating the sequential administration, i.e., cycling, to reduce the development of resistance to one of the cancer therapeutics, to avoid or reduce side effects of one of the cancer therapeutics and/or to improve the efficacy of the cancer therapy.
When two combination therapies of the invention are typically administered to a patient simultaneously in a therapeutically effective regimen, the term "simultaneously" is not limited to administration of the cancer therapeutic agents at exactly the same time, but rather means that they are administered to the subject in a sequence and at intervals such that they can act together (e.g., can act synergistically to provide increased benefit as compared to administration in other ways). For example, two therapeutic agents may be administered simultaneously or sequentially at different time points, in any order; however, if not simultaneously, they should be administered in a sufficiently close time to provide the desired therapeutic effect, preferably in a synergistic manner. The combination cancer therapeutic agents may be administered separately in any suitable form and by any suitable route. When the components of the combination cancer therapeutic are not administered in the same pharmaceutical composition, it is understood that they may be administered in any order to a subject in need thereof. For example, a first therapeutically effective regimen can be administered prior to, concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) administration of a second cancer therapeutic agent according to the invention to a patient in need thereof (e.g., 5 minutes, 15 minutes, 30 minutes, 8 weeks, or 12 weeks), or (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks).
Preferably, administration of a SETDB1 inhibitor in combination with an immune checkpoint modulator according to the invention results in a synergistic anti-cancer effect.
Multi-component kit formulation
The present application also encompasses formulations comprising a SETDB1 inhibitor as described above and at least one immune checkpoint modulator as described above, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer. According to this formulation in the form of a "kit-of-parts", the individual active compounds, i.e. the SETDB1 inhibitor and the at least one immune checkpoint modulator, represent therapeutic agents and are physically separate, provided that the novel and unexpected combined therapeutic effects as described herein, which result from the simultaneous, separate or sequential use of these compounds, are not achieved by the compounds independently of each other. Indeed, as the results below show, the claimed combination of active ingredients does not merely represent a sum of known agents, but a new combination with the surprising valuable property that the antitumor effect of the combination is much more important than the simple addition of the antitumor effects observed when those active ingredients are used alone.
Thus, the two active ingredients may be formulated as separate compositions or as a single composition.
Therapeutic agents according to the present invention may be suitably formulated and introduced into a subject or cellular environment by any means recognized for such delivery.
Such compositions typically include the agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions.
The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
Method of treatment
The present invention also relates to a method of treating a cancer patient, wherein said method comprises administering a SETDB1 inhibitor in combination with at least one immune checkpoint modulator as described above. Typically, the co-administration is administered according to a therapeutically effective regimen.
Expression of SETDB1 was shown to be highly variable in patients, particularly in patients as defined in the present application (see Cuellar T L et al, JCB 2017, https:// doi.org/10.1083/jcb.201612160). The results of the present application now further demonstrate that the activity of immune checkpoint modulators (e.g. anti-PD 1 or anti-PDL 1) is greatly enhanced in the absence of
Thus, in one embodiment, the invention also relates to a method of classifying a patient as responsive or non-responsive to immune checkpoint therapy. Typically, the method comprises determining the expression level of SETDB1 in the patient. The expression level of SETDB1 may be compared to reference data. Typically, a patient may be classified as responding to an immune checkpoint treatment if the expression of SETDB1 is below the reference data. Alternatively, if the expression of SETDB1 is increased compared to the reference data, the patient may be classified as low-response to immune checkpoint therapy and may be treated with a combination of a SETDB1 inhibitor and at least one immune checkpoint modulator as defined herein.
Typically, the expression of SETDB1 in a patient may be determined from a biological sample of the patient. Biological samples refer to biological tissue, cell, or fluid samples (e.g., plasma or blood samples) generally known in the art.
Reference data may be obtained from SETDB1 expression measured in a reference sample. The reference sample may be obtained from a subject without cancer or the same patient at an earlier time point (e.g., prior to any cancer treatment or prior to the onset of cancer). Reference samples can also typically be obtained by pooling samples from multiple subjects to generate a standard within an average population, where the standard represents the average level of SETDB1 in an individual population. Thus, the levels of SETDB1 in the standards obtained in this way represent the average level of the marker in the general population or in the population with the disease (usually with cancer or a particular type of cancer).
Detection of SETDB1 may be achieved by any means of detecting expression of the polypeptide or a fragment thereof of the mRNA transcript of the polypeptide. Such detection methods are well known to those skilled in the art and involve conventional protein detection techniques such as immunohistochemistry, Western blot analysis, immunoblotting, enzyme-linked immunosorbent, immunoprecipitation, lateral flow immunodetection, radioimmunoassay, and transcript expression levels, e.g., measurement of messenger rna (mrna) expression by PCR procedures, RT-PCR, blot hybridization analysis, RNAese protection assay, and the like.
The present invention will be further explained based on the following experimental results.
Drawings
FIG. 1 shows the results obtained with WT, Suv39h1-/-Or SETDB1-/-WT mice were transplanted with B16OVA melanoma cells. When tumors were palpable (2 mm x 2 mm), animals were treated with
FIG. 2 shows the use of WT, Suv39h-/-Or SETDB1-/-WT C57BL6 mice were transplanted with B16OVA melanoma cells. When tumors were palpable (2 mm x 2 mm), animals were treated twice weekly with
FIG. 3 loss of Setdb1 in dendritic cells enhances the expression of Interferon Stimulated Gene (ISG) and promotes tumor rejection. (a) LPS treatment assignment After time, ISG, Ifi204 and Skivl2 were in SETDB1+/+(3 histograms from left) and SETDB1-/-(3 histograms from the right). (b) Mice conditionally excised with SETDB1 in dendritic cells using the Lox-cre system (CD11cre + SETDB 1)Flox/Flox(SETDB 1-/-) And CD11cre-SETDB1Flox/Flox(SETDB1+/+) Growth of medium MCA tumors.
FIG. 4 contains SETDB1-/-Dendritic cell mice are more responsive to PD-1 mediated tumor rejection. To SETDB1 in FIG. 2+/+And SETDB1-/-Mice were inoculated with MCA-OVA fibrosarcoma cells and tumor sizes were measured three times per week. PD-1 was administered when the tumor became palpable.
FIG. 5 is a schematic view of a circuit having Setdb1-/-Enhanced tumor rejection in dendritic cell mice requires CD8+T cells. In SETDB1+/+And SETDB1-/-MCA-OVA tumors were measured in mice three times a week. anti-CD 8 antibody was administered when the tumor became palpable.
Detailed Description
Mouse
A mouse strain (Setdb1tm1a (EUCOMM) Wtsi) carrying a loxP site flanking exon 4 of Setdb1, previously described (Collins 2015), was obtained from EUCOMM and was associated with CD11cre+Mice (B6.Cg-Tg (Itgax-cre)1-1 Reiz/J; Jackson laboratories) were crossed to generate mice with DC-specific deletions. Setdb1tm1a(EUCOMM)WtsiMice were also crossed Jax, B6 with mice expressing tamoxifen-inducible cre; 129-Gt (ROSA)26Sor tm1(cre/ERT)Nat/J) To provide a tissue donor for generating conditional Setdb1-/-BMDC。ERT-cre+Suv39h1WT/WTBone marrow served as a control. C57Bl/6N mice were originally from the Charles river laboratory.
Cell culture and stimulation
Bone marrow-derived dendritic cells were cultured in 20ng/ml GMCSF (Miltenyi) in IMDM (VWRI3390) (I-10 medium) supplemented with 10% fetal bovine serum (Eurobio), penicillin/streptomycin, 50. mu.M. beta. -mercaptoethanol, minimal non-essential amino acids, and 2mM glutamine (all from Life Technologies). Briefly, fresh bone marrow was collected by centrifugation from each two of the ilium, femur and tibia. 500 ten thousand bone marrow cells were seeded on an untreated 10cm plate (VWR) and cultured in 10ml of I-10 medium. An additional 10ml of I-10 medium was added on day 3, followed by collection and supplementation of 10ml on day 6. After 5 min incubation in PBS (REF) at 4 ℃, BMDC colonies were harvested on day 8, and then 2X10 per well in 2ml GMCSF-free I-10 medium in untreated 6-well plates (Sigma M9062-100EA)6Individual cells were stimulated. For setdb1-/-The production of BMDCs, Cre-mediated deletion induced by the addition of 20nb 4-OH-tamoxifen on day 3 of culture, supplemented on day 6 and maintained until day 8 harvest. Cell stimulation with LPS (100ng/ml) at indicated times; invivogen, tlrl-3 pelps).
MCA101 OVA-expressing tumor assay, immunotherapy and IFN γ ELISPOT
The tumor cell strain MCA101-sOVA verified previously is used1(fibrosarcoma secreting soluble OVA) was grown in a Roswell Park Memori Institute (Thermo Fisher, 10687010) supplemented with 10% FBS (eurobo), 100. mu.g/ml penicillin/streptomycin, beta-mercaptoethanol, 2mM L-glutamine and hygromycin. Cells were harvested by trypsinizing logarithmic growth phase cultures and cultured at 10%5Cells/100 μ Ι were resuspended in cold PBS for intradermal injection to the right side of recipient mice. Tumors were visible within 4-5 days, after which they were measured every two days until they reached 1000mm3(calculated as 0.5W L, W tumor width and L tumor length). 100 μ g of anti-PD-1 (Bio X Cell, RMP1-14) or anti-CD 8(Bio X Cell, 53-6.72) in PBS was delivered by intraperitoneal injection three times a week to the end of the experiment. Blood was collected from mice on day 13 post tumor inoculation and sterilized H2Rapid (5 sec) RBC lysis in O followed by quenching with 10-fold PBS to a final 1-fold concentration.
Production of lentiviral particles for CRISPR/Cas9 mutagenesis
HEK293-T cells were stored in Dulbecco's modified eagle's medium supplemented with 10% FBS (eurobo) and 100. mu.g/ml penicillin/streptomycin. Will be 8.105Each was inoculated in 6-well plates and transfected with 1G of psPax2, 0.4G of VSV-G packaging vector, and 1.6. mu.g of sgRNA cloned into the pCRISP-puro-v2 vector. Media was changed 14 hours after transfection. Viral supernatants were collected after 36 hours, filtered and immediately used for transduction of B16-OVA cells.
The sgRNA sequences used were:
generation of Suv39h1 and Setdb1 deficient B16OVA tumor cells
Melanoma cells expressing B16-F10 OVA were stored in a Roswell Park Molar Institute (RPMI) supplemented with 10% FBS, 100g/ml penicillin/streptomycin, and glutamine. 2.5.10 will be mixed5One was inoculated in 6-well plates. 24 hours after inoculation, the medium was replaced with 2ml of freshly prepared virus supernatant and the plates were spun in a centrifuge preheated to 30 ℃ for 30 minutes at 2500 rpm. The medium was changed 24 hours after transduction, and puromycin (2. mu.g/ml, invivogen) was added to the cells 48 hours after transduction.
Cells were screened with puromycin for two weeks, after which protein expression was checked by western blotting (Suv39h1 antibody, CellSignalling Technology, Setdb1 antibody from Abcam).
For tumor experiments, 2.5 × 105Tumor cells of the appropriate genotype were injected subcutaneously into C57BL6/J recipients (6-8 week females). When tumors were palpable (usually 5 days post-injection), animals were treated twice weekly with 200 μ G of anti-PDL 1(Bio X Cell, 10F9G2) or anti-PDL 1(PD-1(Bio X Cell, RMP1-14) 150G). Tumor was measured twice a week using an electronic caliper as tumorUp to 1000mm3Animals were sacrificed on volume (calculated as 0.5W L, W tumor width, L tumor length).
Reference to the literature
1Zeelenberg,I.S.et al.Targeting tumor antigens to secreted membranevesicles in vivo induces efficient antitumor immune responses.Cancer research68,1228-1235,doi:10.1158/0008-5472.CAN-07-3163(2008).
Results
-/- -/-SETDB1B16OVA cells were more sensitive to anti-PDL 1 treatment than WT or Suv39h1B16OVA cells
Suv39h1-/-Or Setdb1-/-B16OVA cells, an isogenic model of murine melanoma, grew similar to or slightly faster than WT B16OVA cells after adoptive transfer in B6 mice (see fig. 1A and 1C).
anti-PDL 1 treatment inhibited Suv39h1 compared to WT controls-/-Or SETDB1-/-B16OVA cells were very efficient in growth. Indeed, anti-PD-L1 treatment itself was inefficient in controlling WT B16OVA cell growth, with only a slight increase in survival.
anti-PD-L1 treatment resulted in Suv39h1-/-B16OVA cell growth decreased. In sharp contrast, anti-PD-L1 vs SETDB1-/-The effect of B16OVA cell growth was more intense as complete rejection was observed in more than 60% of mice.
The results show that inactivation of SETDB1 in tumor cells increases the efficacy of checkpoint blockade therapy with
-/-SETDB1B16OVA cells were highly sensitive to anti-PD 1 treatment compared to WT B16OVA cells
To further explore the response of Setdb 1-deficient tumors to checkpoint blockade, WT C57BL6 mice were injected with WT or Setdb1-KO B16OVA cells. When tumors were palpable, animals received
-/-Mice carrying the conditional mutation of Setdb1 in Dendritic Cells (DCs) had better control of tumor growth and and respond more strongly to anti-checkpoint treatment than littermate controls
Setdb1-/-Bone marrow-derived dendritic cells (BMDCs) produced more Interferon Stimulated Genes (ISGs) in response to LPS treatment, suggesting a stronger inflammatory phenotype. To test the potential physiological relevance of this phenotype in vivo, we correlated CD11c-cre expressing mice with SETDB1 Flox/FloxMice were combined to selectively delete SETDB1 in DCs and inoculated with MCA-OVA fibrosarcoma cells. CD11c-cre negative mice served as WT littermates. And Setdb1+/+Compared with mouse, Setdb1-/-Mice controlled tumor growth more effectively (figure 3). This shows enhanced SETDB1-/-inflammatory/Isg responses in myeloid cells promote better tumor rejection.
Using the same mouse strain conditionally deficient in DC for SETDB1, we performed a tumor experiment similar to fig. 3, but administered anti-PD-1 or PBS as a control (fig. 4). We observed that Setdb1-/-The response of mice to anti-PD-1 mediated tumor rejection was significantly stronger, indicating the potential benefit of combining anti-PD-1 treatment and inhibition of Setdb1 in DCs.
For testing at SETDB1-/-In mice enhanced tumor rejection to CD8+T cell demand, we administered anti-CD 8 weekly+Antibodies to deplete them. CD8+Depletion of T cells significantly increased tumor burden in WT and KO animals, which is required for CD8 to control MCA tumor rejection+T cells. In addition, these data will SETDB1-/-Phenotype and CD8+T cells were associated (fig. 5).
