Patent Application
20220056536
The present application claims priority from Australian Provisional Application No. 20199900574 filed on 22 Feb. 2019 and Australian Provisional Application No. 2019900607 filed on 26 Feb. 2019, the entire contents of which are incorporated herein by way of reference.
The present disclosure relates to the field of immunotherapy and methods of modulating the immune response.
Immune balance determines the difference between the immune system recognizing self from non-self. It is recognised that loss, or a breakdown in the inhibitory signal can contribute to autoimmune disease, chronic inflammatory disease and transplant rejection. In cancer, these inhibitory signals are enhanced, thus suppressing the ability of the immune response to efficiently target and destroy tumours. In contrast, in autoimmune disease, these inhibitory signals are diminished, thus enhancing the ability of the immune system to attack when it should not otherwise.
In recent times the clinical development of novel immunotherapies has revolutionised the way many cancers are being treated. Immune checkpoint inhibitors including drugs that target anti-PD-1/PD-L1 and anti-CTLA-4, or immune activators like anti-CD40, have shown great improvements in patient outcomes for several types of cancers including melanoma of the skin, bladder cancer and Hodgkin's lymphoma. These results highlight the key principle that boosting the natural immune response either by removing immune suppressive pathways, or by directly enhancing the immune response, is a promising new approach to improve patient outcomes that could be applied to the treatment of cancer. Further, it highlights that the converse is true for the treatment of autoimmune disease.
A20 is encoded by the TNFAIP3 gene. It promotes microbial tolerance as a negative regulator of NF-κB signaling: an evolutionarily ancient and central pathway for activating innate and adaptive immune responses. It has a critical role in maintaining immune reactivity and function. However, little was known about the ability to modulate the immune response via controlling A20. The inventors have identified and functionally evaluated more than 20 SNVs within the A20 coding sequence that can differentially impact the immune system. The inventors have shown for the first time in both cell and animal models that expression of the identified A20 SNVs can be used to “tune” the immune system of a subject i.e., tune up or tune down the sensitivity and/or strength of the immune system of a subject in response e.g., to a treatment or pathogen. On the basis of these findings, the present inventors have developed new methods for modulating the immune system of a subject comprising modulating the A20 SNV expression profile in the subject, thereby “tuning” the immune system. The inventors have also developed reagents for use in such methods. The inventors envisage that the methods and reagents may have application in the treatment and/or management of diseases/conditions such as, for example, cancer, autoimmune disease, pathogenic infection, complement deficiency, and transplant rejection, where the strength of a subject's immune system plays an important role.
The present inventors have also shown that the A20 SNVs may be used as biomarkers to stratify subjects according to the strength of their immune system e.g., strong or poor immune response relative to a reference A20 genotype. The inventors therefore envisage that diagnostic methods which are designed to predict the strength of a subject immune response on the basis of A20 SNVs may be useful in formulating a treatment plan for a subject, as well as ongoing patient management.
Accordingly, the present disclosure provides a method of stratifying a subject according to the strength of their immune response, said method comprising:
In one example, the presence of a A20 single nucleotide variant (SNV) e.g., an A20 sequence comprising a non-synonymous SNV, in the subject relative to the reference A20 nucleotide sequence is indicative that the subject's immune response will be increased or decreased relative to if the subject had the reference A20 nucleotide sequence.
In one example, the presence of a A20 SNV corresponding to an A20 variant set forth in Table 1 in the subject is indicative that the subject's immune response will be increased or decreased relative to if the subject had a nucleotide sequence encoding the reference A20 protein.
In one example, the presence of an A20 SNV corresponding to an A20 variant set forth in Table 2 in the subject is indicative that the subject's immune response will be increased relative to if the subject had a nucleotide sequence encoding the reference A20 protein.
In one example, the presence of an A20 SNV corresponding to an A20 variant set forth in Table 3 in the subject is indicative that the subject's immune response will be decreased relative to if that subject had a nucleotide sequence encoding the reference A20 protein.
The present disclosure also provides a method of modifying the strength of an immune response in a subject, the method comprising administering to, or expressing in, the subject an A20 variant which will increase or decrease the strength of the subject's immune response relative to the subject's immune response in the absence of the A20 variant being administered or expressed.
In one example, the method comprises stratifying the subject according to the strength of their immune response based on the subject's A20 gene nucleotide sequence prior to administering to, or expressing in, the subject the A20 variant. For example, stratifying the subject according to the strength of their immune response may be performed according to the method described herein.
In one example, the A20 variant administered to, or expressed in, the subject is set forth in Table 1. For example, the A20 variant administered to, or expressed in, the subject may be a variant set forth in Table 2. For example, the A20 variant administered to, or expressed in, the subject may be a variant set forth in Table 3.
The A20 variant may be administered to, or expressed in, the subject in any number of ways. In one example, the method comprises administering to the subject an expression vector comprising a nucleic acid coding for the A20 variant. In one example, the method comprises administering to the subject the A20 variant protein. In one example, the method comprises administering to the subject a cell modified to express the A20 variant.
In accordance with an example in which a cell has been modified to express the A20 variant, the cell may be an autologous cell.
In accordance with another example in which a cell has been modified to express the A20 variant, the cell may be an allogeneic cell.
In one example, the cell has been modified to express the A20 variant using a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN) or a clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system.
In one example, the cell which has been modified to express the A20 variant is a T cell. For example, the T cell may be a chimeric antigen receptor (CAR) T cell.
In one example, the A20 variant is set forth in Table 2 and the strength of the subject's immune response is increased relative to if the subject expressed a wildtype A20 only or the subject expressed an A20 allele comprising the reference A20 nucleotide sequence only. In accordance with this example, the increase in the subject's immune response comprises an increase in NF-κB and/or c-Jun amino-terminal kinase (JNK) signalling relative to if the subject expressed a wildtype A20 allele only or the subject expressed an A20 allele comprising the reference A20 nucleotide sequence only.
In accordance with an example in which the strength of the subject's immune response is increased by performing the method, the subject may be suffering from cancer. For example, the subject may be suffering from cancer and is receiving treatment by immunotherapy or has been prescribed a treatment by immunotherapy. Accordingly, the present disclosure provides a method and reagents for treating cancer.
In accordance with another example in which the strength of the subject's immune response is increased by performing the method, the subject may be suffering from, or is at risk of, infection by a pathogen. Accordingly, the present disclosure provides a method and reagents for treating or preventing pathogenic infection. For example, the pathogen may be a disease-causing bacteria, virus, protist or fungus. In one example the pathogen is a bacteria. In one example the pathogen is a virus. In one example the pathogen is a protist. In one example the pathogen is a fungus.
In accordance with another example in which the strength of the subject's immune response is increased by performing the method, the subject may be suffering from complement deficiency. Accordingly, the present disclosure provides a method and reagents for treating complement deficiency.
In another example, the A20 variant is set forth in Table 3 and the subject's immune response is decreased relative to if the subject expressed a wildtype A20 allele only or the subject expressed an A20 allele comprising the reference A20 nucleotide sequence only. In accordance with this example, the decrease in the subject's immune response comprises a decrease in NF-κB and/or c-Jun amino-terminal kinase (JNK) signalling relative to if the subject expressed a wildtype A20 allele only or the subject expressed an A20 comprising the reference A20 nucleotide sequence only.
In accordance with an example in which the strength of the subject's immune response is decreased by performing the method, the subject may be suffering from an autoimmune disease.
In accordance with another example in which the strength of the subject's immune response is decreased by performing the method, the subject may have undergone or is about to undergo organ or tissue transplantation.
The present disclosure also provides a method of treating cancer in a subject in need thereof, comprising increasing a subject's immune response by performing the method of the disclosure and administering to the subject an immunotherapeutic agent.
In one example, the immunotherapeutic agent is selected from the group consisting of checkpoint inhibitors, monoclonal antibodies, chimeric antigen receptor (CAR) T cells, and small molecule immune agonists.
The present disclosure also provides a method of treating an autoimmune disease in a subject, comprising repressing a subject's immune response by performing the method of the disclosure.
In one example, the autoimmune disease is associated with activation of the innate immune system or auto-inflammatory response e.g., familial Mediterranean fever; Behcet disease. In another example, the autoimmune disease is associated with activation of the adaptive immune response or autoimmunity e.g., type-1 diabetes, lupus, irritable bowel syndrome (IBS), rheumatoid arthritis, multiple sclerosis, Hashimoto's disease, Celiac disease, Crohn's disease and asthma, for example.
The present disclosure also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an A20 variant set forth in Table 1. In one example, the isolated nucleic acid may comprise a nucleotide sequence encoding an A20 variant set forth in Table 2. In another example, the isolated nucleic acid may comprise a nucleotide sequence encoding an A20 variant set forth in Table 3.
The present disclosure also provides an expression vector comprising the isolated nucleic acid molecule of the disclosure.
The present disclosure also provides a delivery system e.g., a viral delivery system, comprising am isolated nucleic acid or expression vector described herein. For example, the delivery system may be an adeno-associated virus (AAV), a lentivirus, or an adenovirus.
The present disclosure also provides isolated protein comprising an A20 variant set forth in Table 1.
The present disclosure also provides isolated protein comprising an A20 variant set forth in Table 2.
The present disclosure also provides isolated protein comprising an A20 variant set forth in Table 3.
The present disclosure also provides T cell which is modified to express an A20 variant selected from Table 2. For example, the T-cell may be a chimeric antigen receptor (CAR) T cell.
The present disclosure also provides a pharmaceutical composition comprising (i) one or more of an isolated nucleic acid of the disclosure or an expression vector of the disclosure or an isolated protein of the disclosure or a T-cell of the disclosure, and (ii) a pharmaceutically acceptable carrier, diluent or excipient.
The present disclosure also provides a vaccine comprising an A20 protein selected from Table 2 or an isolated nucleic acid molecule comprising a nucleotide sequence encoding an A20 variant set forth in Table 2.
The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.
SEQ ID NO: 1 Amino acid sequence for reference human A20 protein
SEQ ID NO: 2 cDNA sequence encoding reference human A20 protein
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in immunology, molecular biology, oncology and biochemistry).
The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; J. F. Ramalho Ortigao, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York. 12. Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text.
The skilled artisan will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.
Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein, the term “stratify”, “stratifying” or similar in the context of a method of the disclosure shall be understood to mean classification or division of subjects tested into groups or categories indicative of the likely strength of that subject's immune response. For example, a subject on which the method of the disclosure is performed may be classified or identified as an individual which is likely to elicit a strong immune response or a weak immune response e.g., to an incoming treatment or antigen, relative to an immune response elicited by an individual having a reference A20 gene sequence.
As used herein, the term “subject” refers to any animal, for example, a mammalian aminal, including, but not limited to humans, non-human primates, livestock (e.g. sheep, horses, cattle, pigs, donkeys), companion animals (e.g. pets such as dogs and cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), performance animals (e.g. racehorses, camels, greyhounds) or captive wild animals. In a preferred example, the “subject” is a human. Typically, the terms “subject” and “patient” are used interchangeably, particularly in reference to a human subject.
As used herein, the term “immune response” will be understood to refer to an alteration of an organism's immune system upon exposure to, for example, an antigen, allergen, auto-antibody, pathogen, treatment agent or a graft. The term “immune response” encompasses, but is not limited to, an immune response involving antibody production (e.g., humoral immunity) and/or the induction of cell-mediated immunity (e.g., cellular immunity including helper T cell and/or cytotoxic T cell responses). The immune response may involve an activation of a subject's immune system or, alternatively, a suppression of a subject immune system.
As used herein, the term “single nucleotide variant” or “SNV” shall be understood to mean a nucleic acid sequence (DNA or RNA) which differs at a specific nucleotide position relative to a corresponding reference sequence. An “A20 SN V” as used herein shall be understood to mean a TNFAIP3 gene sequence encoding an A20 protein that comprises a non-synonymous SNV relative to a TAFAIP3 gene sequence encoding a wildtype reference A20 protein. As such, an A20 SNV or “A20 variant” of the present disclosure shall differ by at least one amino acid relative to the wildtype reference A20 protein sequence. An exemplary A20 reference amino acid sequence is set forth in SEQ ID NO: 1 and/or is encoded by the cDNA sequence set forth in SEQ ID NO: 2. Exemplary A20 SNVs are set forth in Tables 1-3 herein.
As used herein, the term “A20 allelic variant”, “A20 variant” or similar shall be understood to mean a variant of the A20 protein encoded by an A20 SNV sequence described herein. As described herein, an “A20 variant” of the present disclosure shall differ by at least one amino acid relative to the wildtype reference A20 protein sequence (i.e., as a result of the non-synonymous SNV). The “A20 variant” may be a nucleic acid comprising a SNV sequence which encodes an A20 variant protein, or may be provided as the A20 variant protein itself.
As used herein, the term “wild-type” refers to the most common polynucleotide sequence or allele for a certain gene in a population. Generally, the wild-type allele will be obtained from normal cells. Depending on the particular purpose or desire of the practitioner, the reference sequence is generally a wild-type sequence. Accordingly, a “wildtype A20 allele” shall be understood to mean a common, preferably the most common, allele of A20 in the population e.g., human population.
As used herein, an “autologous cell” shall be understood to related to a cell in which the donor and the recipient are the same. In contrast, an “allogeneic cell” shall be understood to mean a cell in which the donor and recipient are genetically non-identical individuals from the same species. Two or more cells are said to be allogeneic to one another when the genes at one or more loci are not identical. In some examples, allogeneic cells from individuals of the same species may be sufficiently unlike genetically to interact antigenically. In either context i.e., autologous or allogeneic, the cell may be any cell types including, but not limited to, a T-cell or a cell within transplant or graft tissue.
“T-cells” belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity and, to a lesser degree the adaptive immune response. Generally, T-cells are distinguished from other lymphocytes (e.g., B cells and natural killer cells) by the presence of T-cell receptors (TCRs). T-cells have diverse roles, which are accomplished by differentiation of distinct populations of T-cells, recognizable by discrete gene expression profiles.
The terms “CAR-T cell”, “CART cell” or similar shall be understood to mean a T-cell comprising a chimeric antigen receptor (CAR).
A “chimeric Antigen Receptor” or alternatively a “CAR”, refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as an intracellular signaling domain) comprising a functional signalling domain derived from a stimulatory molecule and/or costimulatory molecule. As used herein, the term “antigen-binding domain” shall be understood to mean a protein or region thereof that recognizes and binds to an antigen. An exemplary antigen binding domain is one which binds to a tumor antigen or a viral antigen expressed on a cell surface.
As used herein, the terms “treating”, “treat” or “treatment” and variations thereof, refer to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.
As used herein, the term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, blood cancers e.g., leukemia, lung cancer and the like. Further exemplary cancers are described herein. The term “cancer” includes all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The terms “tumor” and “cancer” are used interchangeably herein. Both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.
A “therapeutically effective amount” is at least the minimum concentration or amount required to effect a measurable improvement in the disease or condition to be treated or prevented e.g., cancer, infection by a pathogen, autoimmune disease or graft rejection. The skilled artisan will be aware that such an amount will vary depending on, for example, the disease to be treated (e.g., in the case of cancer, the specific type of cancer and/or stage thereof, or in the case of infection, the type of pathogen, or in the case of autoimmune disease, the type of autoimmune disease etc) and/or the particular subject and/or the type or severity of a condition being treated. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity or amount of A20 variant or cell population comprising same.
The present disclosure provides a method of modifying the strength of an immune response in a subject by administering to, or expressing in, the subject an A20 variant which will increase or decrease the strength of the subject's immune response relative to the subject's immune response in the absence of the A20 variant being administered or expressed.
A20 is encoded by the TNFAIP3 gene. It is acts a as a negative regulator of NF-κB signaling: an evolutionarily ancient and central pathway for activating innate and adaptive immune responses. A20 has multiple domains with inhibitory activity against NF-κB, primarily preventing activation of the central IκB kinase (IKK) by upstream proteins RIPK1, TRAF6 and NEMO. The A20 ovarian tumor (OTU) domain is a deubiquitin (DUB) protease that cleaves activating K63-linked ubiquitin chains from RIPKI, TRAF6 or NEMO. The A20 zinc finger 7 domain (ZnF7) binds linear polyubiquitin to suppress IKK activation, whereas ZnF4 promotes ligation of K48-linked ubiquitin chains to RIPK1, triggering RIPKI proteolysis. A20 feedback inhibition is induced at two levels: NF-κB proteins directly induce TNFAIP3 mRNA, and the inhibitory activities of A20 protein are enhanced by IκKβ-induced serine phosphorylation near the ZnF domains, notably S381.
Surprisingly, the inventors have shown that some non-synonymous SNV variants of the A20 protein decrease a subject's immune response relative to if the subject had a reference A20 protein sequence (e.g., a wild-type human A20 protein) and other non-synonymous SNV variants of the A20 protein increase a subject's immune response relative to if the subject had the reference A20 protein sequence (e.g., a wild-type human A20 protein). Accordingly, by controlling which A20 variant is expressed in or administered to a subject, the strength of the subject's immune response may be modified i.e., tuned up by expressing in or administering to the subject an A20 variant shown to increase the immune response relative to a wild-type or reference A20 protein, or tuned down by expressing in or administering to the subject an A20 variant shown to decrease the immune response relative to a wild-type or reference A20 protein.
In one example, the method of the disclosure comprises administering to, or expressing in, the subject a SNV variant of the A20 protein which increases or decreases NF-κB and/or c-Jun amino-terminal kinase (JNK) signalling in the subject relative to if the subject was administered or expressed the reference A20 protein sequence set forth in SEQ ID NO: 1. For example, the method may comprise administering to, or expressing in, the subject an A20 variant set forth in Table 1. Mutations in Table 1 are described relative to the reference A20 protein sequence set forth in SEQ ID NO: 1, which is the wildtype amino acid sequence for human A20 protein. Administering to, or expressing in, the subject an A20 variant set forth in Table 1 increases or decreases the subject's immune response relative to if the subject was administered or expressed the reference A20 protein sequence. In accordance with this example, an increase or decrease in the subject immune response comprises a corresponding increase or decrease in NF-κB and/or JNK signalling relative to if the subject was administered or expressed the reference A20 protein sequence.
In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject an A20 variant set forth in Table 2. Administering to, or expressing in, the subject an A20 variant set forth in Table 2 increases the subject's immune response relative to if the subject was administered or expressed the reference A20 protein. Similarly, administering to, or expressing in, the subject an A20 variant set forth in Table 2 increases the subject's immune response relative to if the subject was administered or expressed any one of the A20 variants set forth in Table 3. In accordance with this example, an increase in the subject's immune response comprises an increase in NF-κB and/or JNK signalling relative to if the subject expressed the reference A20 protein or any one of the A20 variants set forth in Table 3.
In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 1. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 2. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 3. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 4. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 5. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 6. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 7. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 8. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 9. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 10.
In another example, the method of the disclosure comprises decreasing the subject immune response by administering to, or expressing in, the subject an A20 variant set forth in Table 3. Administering to, or expressing in, the subject an A20 variant set forth in Table 3 decreases the subject's immune response relative to if the subject was administered or expressed the reference A20 protein. Similarly, administering to, or expressing in, the subject an A20 variant set forth in Table 3 decreases the subject's immune response relative to if the subject was administered or expressed any one of the A20 variants set forth in Table 2. In accordance with this example, a decrease in the subject's immune response comprises an decrease in NF-κB and/or JNK signalling relative to if the subject expressed the reference A20 protein or any one of the A20 variants set forth in Table 2.
In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 11. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 12. In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject A20 variant 13.
In one example, the A20 SNV variant for modifying the strength of an immune response in a subject as described herein is provided in the form of an A20 variant protein or a functional fragment or variant thereof which retains its NF-κB and/or JNK signalling function. In one example, the A20 variant protein is any variant protein which increases or decreases NF-κB and/or JNK signalling in the subject relative to if the subject was administered or expressed the reference A20 protein sequence set forth in SEQ ID NO: 1. Exemplary A20 variant proteins for modifying an immune response are set forth in Table 1. As described hereinabove, the variants presented in Table 1 are described relative to the reference A20 protein sequence set forth in SEQ ID NO: 1, which is the wildtype amino acid sequence for human A20 protein. In accordance with an example in which the A20 variant protein increases NF-κB and/or JNK signalling in the subject and thereby increases the immune response in a subject, the A20 variant may be an A20 variant protein set forth in Table 2 (i.e., a variant selected from A20 variants 1-10). In accordance with an example in which the A20 variant protein decreases NF-κB and/or JNK signalling in the subject and thereby decreases the immune response in a subject, the A20 variant may be an A20 variant protein set forth in Table 3 (i.e., a variant selected from A20 variants 11-13). Alternative splice variants of the A20 variant proteins described herein which retain A20 activity i.e., NF-κB and/or JNK signalling activity, are also contemplated for use in the methods of the disclosure.
In another example, an A20 variant protein of the disclosure comprises a sequence at least about 75% or 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant protein comprises the mutation of the corresponding variant set forth in Tables 1-3 and exhibits modified NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. For example, an A20 variant protein of the disclosure which increases an immune response in a subject may comprise a sequence at least about 75% or 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. For example, an A20 variant protein of the disclosure which decreases an immune response in a subject may comprise a sequence at least about 75% or 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1.
Functional fragments, analogs and/or derivatives of A20 variant protein set forth in Tables 1-3 are also contemplated for use in the methods of the present disclosure, provided that the fragments, analogs and/or derivatives retain the NF-κB and/or JNK signalling activity of the corresponding A20 variant protein set forth in Tables 1-3.
For example, the present disclosure contemplates the use of A20 variants set forth in Tables 1-3 comprising conservative substitutions or mutations in a small number of residues of the protein, provided that the variant retains the NF-κB and/or JNK signalling activity of the corresponding A20 variant set forth in Tables 1-3. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and/or hydropathicity and/or hydrophilicity.
Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indices are described, for example in Kyte and Doolittle J. Mol. Biol., 157: 105-132, 1982 and hydrophylic indices are described in, e.g., U.S. Pat. No. 4,554,101.
The present disclosure also contemplates the use of variants of the A20 proteins set forth in Tables 1-3 comprising non-conservative substitutions or mutations in a small number of residues of the protein, provided that the A20 variant proteins retain the NF-κB and/or JNK signalling activity of the corresponding A20 variant set forth in Tables 1-3. For example, of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or positively charged amino acids. In some examples, the A20 variant protein of the disclosure comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.
The present disclosure also contemplates the use of variants of the A20 protein set forth in Tables 1-3 comprising one or more non-naturally occurring amino acids or amino acid analogues. For example, an A20 variant protein may comprise one or more naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of an amino acid. For example, the A20 variant protein may comprise only D-amino acids. Exemplary non-coded amino acids include: hydroxyproline, 0-alanine, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylananine 3-benzothienyl alanine 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-tic isoquinoline-3-carboxylic acid β-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyric acid, ρ-aminophenylalanine, N-methylvaline, homocysteine, homoserine, 8-amino hexanoic acid, 6-amino valeric acid, 2,3-diaminobutyric acid.
The A20 variant protein or fragment, analog or derivative thereof may be provided as a conjugate. For example, the A20 variant protein, fragment, analog or derivative may be conjugated to a nonproteinaceous moiety known in the art for improving one or more pharmacokinetic properties of protein-based agents. Preferably, the moiety suitable for derivatization of the protein, fragment, analog or derivative is a physiologically acceptable polymer, preferably a water soluble polymer. Such polymers are useful for increasing stability and/or reducing clearance (e.g., by the target tissue or organ). Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or propropylene glycol (PPG).
Alternatively, or in addition, the A20 variant protein, fragment, analog or derivative described herein may be conjugated or linked to another protein, including another protein of the disclosure or an analog or derivative derived therefrom e.g., described herein. Other proteins are not excluded. Additional proteins will be apparent to the skilled artisan and include, for example, an immunomodulator or a half-life extending protein or a peptide or other protein that binds to serum albumin amongst others. When linked to another protein, the A20 variant protein, fragment, analog or derivative described herein may be a fusion protein or chimeric protein. Methods for producing fusion proteins and chimeric proteins are known in the art.
Conjugates of the A20 variant protein, fragment, analog or derivative and additional moieties can be made using a variety of bifunctional protein-coupling agents such as, but not limited to, 4-(4′acetylphenoxy)butanoic acid (AcBut), 3-acetylphenyl acidic acid (AcPac), 4-mercapto-4-methyl-pentanoic acid (Amide), N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene), and derivatives thereof.
The A20 variant protein, fragment, analog or derivative can be produced by recombinant means or synthetic means.
Recombinant means generally comprise operably linking a nucleic acid encoding the A20 variant protein to be expressed to a promoter to thereby form an expression construct, which can be an expression vector (e.g., a plasmid or phagemid). The present disclosure contemplates such an expression construct. The nucleic acid can be produced and/or isolated and cloned into an appropriate construct using methods known in the art and/or described in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) and/or (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
Suitable promoters and/or expression vectors will be apparent to the skilled person based on the cell/expression system to be used. For example, typical promoters suitable for expression in a mammalian cell include, for example a promoter selected from the group consisting of, retroviral LTR elements, the SV40 early promoter, the SV40 late promoter, the CMV IE (cytomegalovirus immediate early) promoter, the EF1α promoter (from human elongation factor 1α), the EM7 promoter, the UbC promoter (from human ubiquitin C). Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells); baby hamster kidney cells (BHK); Chinese hamster ovary cells (CHO); African green monkey kidney cells (VERO-76); or myeloma cells (e.g., NS/0 cells). For example, the cells are CHO cells.
Other elements of expression constructs/vectors are known in the art and include, for example, enhancers, transcriptional terminators, polyadenylation sequences, nucleic acids encoding selectable or detectable markers and origins of replication.
Of course, the present disclosure contemplates expression of the A20 variant proteins, fragments, analogs or derivatives thereof in any cell, including mammalian cells, bacterial cells, fungal cells, insect cells or plant cells.
Following production of a suitable expression construct, it is introduced into a suitable cell using any method known in the art. Exemplary methods include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
The present disclosure also encompasses recombinant cells expressing a A20 variant protein, fragment, analog or derivative. The cells are then cultured under conditions known in the art to produce the A20 variant protein, fragment, analog or derivative.
In one example, the A20 variant protein, fragment, analog or derivative is synthetic, and may therefore be produced using chemical methods known to a person skilled in the art.
For example, synthetic A20 variant proteins, fragments, analogs or derivatives may be prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and/or unnatural amino acids. Amino acids used for polypeptide synthesis may be standard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resin with the deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963, or the base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids described by Carpino and Han, J. Org. Chem., 37:3403-3409, 1972.
As the full length human A20 protein is 790 amino acids in length, convergent or stepwise synthesis techniques are also contemplated for synthesizing the A20 variant proteins, fragments, analogs or derivatives of the disclosure e.g., as described in Muir et al. (1993) Curr. Opin. Biotech., 4:420 and Miller et al., (1989) Science, 245:1149.
Following production/expression/synthesis, the A20 variant protein, fragment, analog or derivative may be purified using a method known in the art. Such purification provides the protein, fragment, analog or derivative substantially free of conspecific protein, nucleic acids, lipids, carbohydrates, and the like. For example, the protein, fragment, analog or derivative will be in a preparation wherein more than about 90% (e.g. 95%, 98% or 99%) of the protein in the preparation is the A20 variant protein, fragment, analog or derivative.
Standard methods of purification may be employed to obtain an isolated A20 variant protein or functional fragment, analog or derivative thereof, including but not limited to various high-pressure (or performance) liquid chromatography (HPLC) and non-HPLC peptide isolation protocols, such as size exclusion chromatography, ion exchange chromatography, phase separation methods, electrophoretic separations, precipitation methods, salting in/out methods, immunochromatography, and/or other methods.
Also provided herein is an isolated nucleic acid comprising a nucleotide sequence encoding an A20 variant protein or the functional fragment thereof, wherein the nucleotide sequence comprises an A20 SNV variant for modifying the strength of an immune response in a subject as described herein. Exemplary A20 SNV variants are set forth in Table 1-3 hereof. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 1 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 2 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 3 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 4 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 5 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 6 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 7 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 7 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 8 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 9 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 10 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 11 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 12 as described herein. In one example, an isolated nucleic acid of the discloses comprises a nucleotide sequence encoding A20 variant 13 as described herein.
Methods of producing isolated nucleic acid molecules are known in the art and described hereinabove, and shall be taken to apply mutatis mutandis to the present example of the disclosure.
In one example, the A20 SNV variant for modifying the strength of an immune response in a subject as described herein is provided in the form of an expression construct or vector comprising a nucleic acid encoding an A20 variant protein or functional fragment thereof (i.e., a functional fragment which retains the NF-κB and/or JNK signalling function of the full length variant), wherein the expression construct or vector is capable of expressing the A20 variant protein or the functional fragment thereof in a cell e.g., a mammalian cell. For example, the expression construct may comprise an isolated nucleic acid as described herein.
In one example, the expression construct or vector comprises a nucleic acid encoding an A20 variant protein which increases or decreases NF-κB and/or JNK signalling in a subject relative to if the subject was administered or expressed the reference A20 protein sequence set forth in SEQ ID NO: 1. Exemplary expression constructs or vectors comprise a nucleic acid encoding an A20 variant set forth in Table 1 (e.g., A20 variants 1-13). In accordance with an example in which the A20 variant increases NF-κB and/or JNK signalling in the subject and thereby increases the immune response in a subject, the expression construct or vector may comprise a nucleic acid encoding an A20 variant set forth in Table 2 (e.g., A20 variants 1-10). In accordance with an example in which the A20 variant decreases NF-κB and/or JNK signalling in the subject and thereby decreases the immune response in a subject, the expression construct or vector may comprise a nucleic acid encoding an A20 variant set forth in Table 3 (e.g., A20 variants 11-13).
In another example, the expression construct or vector comprises a nucleic acid encoding an A20 variant protein comprising a sequence at least about 75% or 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant protein comprises one of the mutations set forth in Tables 1-3 and exhibits modified NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1.
For example, an expression construct or vector of the disclosure may comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 75% or 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 75% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 80% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 85% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 90% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 97% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 98% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 2 and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising the sequence set forth in SEQ ID NO: 1 with the exception of one of the mutations set forth in Table 2, and exhibits increased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1.
For example, an expression construct or vector of the disclosure may comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 75% or 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 75% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 80% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 85% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 90% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 97% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 98% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising a sequence at least about 99% identical to the sequence set forth in SEQ ID NO: 1, provided that the A20 variant comprises one of the mutations set forth in Table 3 and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1. In one example, the expression construct or vector of the disclosure comprise a nucleic acid encoding an A20 variant protein comprising the sequence set forth in SEQ ID NO: 1 with the exception of one of the mutations set forth in Table 3, and exhibits decreased NF-κB and/or JNK signalling activity relative the sequence set forth in SEQ ID NO: 1.
Methods for producing expression constructs for expressing a protein of interest (such as an A20 variant protein of the disclosure) in a cell will be apparent to the skilled artisan. For example, the nucleic acid to be expressed in the cell i.e., a nucleic acid encoding the A20 variant protein described herein, will be operably-linked to a promoter for inducing expression of the A20 variant protein in the cell. For example, the nucleic acid will be linked to a promoter operable in a variety of cells of a subject, such as, for example, a viral promoter, e.g., a CMV promoter (e.g., a CMV-IE promoter) or a SV-40 promoter. Additional suitable promoters are known in the art and shall be taken to apply mutatis mutandis to the present example of the disclosure.
In one example, the nucleic acid will be prepared in the form of an expression construct. As used herein, the term “expression construct” refers to a nucleic acid that has the ability to confer expression on a nucleic acid to which it is operably connected, in a cell. Within the context of the present disclosure, it is to be understood that an expression construct may comprise or be a plasmid, minicircle, bacteriophage, phagemid, cosmid, virus sub-genomic or genomic fragment, or other nucleic acid capable of maintaining and/or replicating heterologous DNA in an expressible format. Accordingly, a plasmid, minicircle, bacteriophage, phagemid, cosmid, virus sub-genomic or genomic fragment vector may be used to deliver an A20 variant of the disclosure to a subject.
Methods for the construction of a suitable expression constructs of the disclosure will be apparent to the skilled artisan and are described, for example, in Ausubel et al., (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001). For example, each of the components of the expression construct is amplified from a suitable template nucleic acid using, for example, PCR and subsequently cloned into a suitable expression construct, such as for example, a plasmid or a phagemid.
Plasmid vectors suitable for use with such an expression construct are known in the art and/or described herein. For example, an expression vector suitable for the method of the present disclosure in a mammalian cell is, for example, a vector of the pcDNA vector suite supplied by Invitrogen, a vector of the pCI vector suite (Promega), a vector of the pCMV vector suite (Clontech), a pM vector (Clontech), a pSI vector (Promega), a VP 16 vector (Clontech) or a vector of the pcDNA vector suite (Invitrogen). The skilled artisan will be aware of additional plasmid vectors and sources of such vectors, such as, for example, Life Technologies Corporation, Clontech or Promega.
Means for introducing expression constructs and vectors into a cell for expression of a desired protein are also known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
In another example, the vector is a viral vector. Accordingly, a viral vector based on any appropriate virus may be used to deliver an A20 variant of the disclosure to a subject. In addition, hybrid viral systems may be of use. The choice of viral delivery system will depend on various parameters, such as the tissue targeted for delivery, transduction efficiency of the system, pathogenicity, immunological and toxicity concerns, and the like.
Commonly used classes of viral systems used in gene therapy can be categorized into two groups according to whether their genomes integrate into host cellular chromatin (oncoretroviruses and lentiviruses) or persist in the cell nucleus predominantly as extrachromosomal episomes (adeno-associated virus, adenoviruses and herpesviruses). In one example, a viral vector of the disclosure integrates into a host cell's chromatin. In another example, a viral vector of the disclosure persists in a host cell's nucleus as an extrachomosomal episome.
In one example, a viral vector is an adenoviral (AdV) vector. Adenoviruses are medium-sized double-stranded, non-enveloped DNA viruses with linear genomes that is between 26-48 Kbp. Adenoviruses gain entry to a target cell by receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells. Adenoviruses are heavily reliant on the host cell for survival and replication and are able to replicate in the nucleus of vertebrate cells using the host's replication machinery.
In one example, a viral vector is from the Parvoviridae family. The Parvoviridae is a family of small single-stranded, non-enveloped DNA viruses with genomes approximately 5000 nucleotides long. Included among the family members is adeno-associated virus (AAV). In one example, a viral vector used to deliver an A20 variant of the disclosure to a subject is an AAV. The AAV may be from any serotype known to infect humans, for example, serotype 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. AAV is a dependent parvovirus that generally requires co-infection with another virus (typically an adenovirus or herpesvirus) to initiate and sustain a productive infectious cycle. In the absence of such a helper virus, AAV is still competent to infect or transduce a target cell by receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells. Because progeny virus is not produced from AAV infection in the absence of helper virus, the extent of transduction is restricted only to the initial cells that are infected with the virus. It is this feature which makes AAV a desirable vector for the present disclosure. Furthermore, unlike retrovirus, adenovirus, and herpes simplex virus, AAV appears to lack human pathogenicity and toxicity (Kay, et al., Nature. 424: 251 (2003)). Since the genome normally encodes only two genes it is not surprising that, as a delivery vehicle, AAV is limited by a packaging capacity of 4.5 single stranded kilobases (kb).
Another viral delivery system useful for delivery of an A20 variant of the disclosure to a subject is a system based on viruses from the family Retroviridae. Retroviruses comprise single-stranded RNA animal viruses that are characterized by two unique features. First, the genome of a retrovirus is diploid, consisting of two copies of the RNA. Second, this RNA is transcribed by the virion-associated enzyme reverse transcriptase into double-stranded DNA. This double-stranded DNA or provirus can then integrate into the host genome and be passed from parent cell to progeny cells as a stably-integrated component of the host genome.
In some examples, the viral vector used to deliver an A20 variant of the disclosure to a subject is a lentivirus. Lentivirus vectors are often pseudotyped with vesicular somatitis virus glycoprotein (VSV-G), and have been derived from the human immunodeficiency virus (HIV); visan-maedi, which causes encephalitis (visna) or pneumonia in sheep; equine infectious anemia virus (EIAV), which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immunodeficiency virus (BIV) which causes lymphadenopathy and lymphocytosis in cattle; and simian immunodeficiency virus (SIV), which causes immune deficiency and encephalopathy in non-human primates. Vectors that are based on HIV generally retain <5% of the parental genome, and <25% of the genome is incorporated into packaging constructs, which minimizes the possibility of the generation of reverting replication-competent HIV. Biosafety has been further increased by the development of self-inactivating vectors that contain deletions of the regulatory elements in the downstream long-terminal-repeat sequence, eliminating transcription of the packaging signal that is required for vector mobilization. One of the main advantages to the use of lentiviral vectors is that gene transfer is persistent in most tissues or cell types, even following cell division of the transduced cell.
Other viral or non-viral systems known to those skilled in the art may be used to deliver an A20 variant of the disclosure to a subject, including but not limited to gene-deleted adenovirus-transposon vectors (see Yant, et al., Nature Biotech. 20:999-1004 (2002)); systems derived from Sindbis virus or Semliki forest virus (see Perri, et al, J. Virol. 74(20):9802-07 (2002)); systems derived from Newcastle disease virus or Sendai virus.
In one example, the present disclosure provides a cell e.g., a T-cell, which has been modified to express an A20 variant described herein. It is contemplated that such cell may be used to modulate a subject's immune response in a method of the disclosure. For example, a subject in need of an increased immune response may be provided a T-cell expressing an A20 variant protein of the disclosure e.g., an A20 variant set forth in Table 2, which will increase the strength of the subject's immune response relative to the subject's immune response in the absence of the A20 variant protein being expressed by the T-cell. The cell may be modified to express the A20 variant protein according to any suitable means known in the art. For example, the cell may be modified to express an A20 variant protein of the disclosure using a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN) or a clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system.
In one example, the cell is an autologous cell e.g., an autologous T-cell. In another example, the cell is an allogeneic cell e.g., an allogeneic T-cell lacking expression of a functional T-cell receptor (TCR).
In accordance with an example in which the modified cell is a T-cell, the T-cell may comprise or express a chimeric antigen receptor (CAR). Accordingly, a T-cell which is modified to express an A20 variant protein may be a CAR-T cell.
In one example, the CAR comprises an antigen binding domain in the extracellular region.
In one example, the antigen binding domain is a murine antibody or antibody fragment comprising an antigen binding domain. In one example, the antigen binding domain is a humanized antibody or antibody fragment comprising an antigen binding domain. In one example, the antigen binding domain is a human antibody or antibody fragment comprising an antigen binding domain.
The choice of an antigen binding domain can depend upon the type and number of ligands or receptors that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that may act as ligands or receptors include a cell surface marker associated with a particular disease state, e.g., cell surface makers for viral diseases, bacterial diseases parasitic infections, autoimmune diseases and disorders associated with unwanted cell proliferation, e.g., a cancer, e.g., a cancer described herein.
In certain examples, the antigen binding domain recognizes an antigen of a proliferative disorder e.g., cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer (e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer and the like. In some embodiments, the cancer is B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL), acute myelogenous leukemia (AML); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplasia syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia.
In one example, the antigen binding domain binds specifically to a tumor antigen which comprises one or more antigenic cancer epitopes immunologically recognized by tumor infiltrating lymphocytes (TIL) derived from a cancer tumor of a mammal.
Tumor antigens can be proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding domain of the disclosure will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA-IX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYP1B1, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2/neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WTi, prostate-carcinoma tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A 1, MAD-CT-1, MAD-CT-2, Melan A/MART 1, XAGEl, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephrinB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In one example, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof. In one example, the tumor antigen is CD19.
In one example, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target antigens include transformation-related molecules such as the oncogene HER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.
In some examples, the tumor antigen is a tumor antigen described in WO2015/120096, WO2015/142675, WO2016/019300, WO2016/011210, WO2016/109410 and WO2016/069283, the contents of which are incorporated by reference in their entirety.
Depending on the desired antigen to be targeted, the sequence encoding the CAR can be engineered to include the appropriate antigen binding domain that is specific to the desired antigen target.
The antigen binding domain of the CAR-T cell can be derived from an antibody molecule, e.g., one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-domain antibodies e.g., a heavy chain variable domain (VH), a light chain variable domain (VL) from e.g., human, and a variable domain (VHH). In some examples, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used in, e.g., for use in humans, it may be beneficial for the antigen binding domain of the CAR, described herein, to comprise a human or a humanized antigen binding domain.
In some examples, the antigen binding domain comprises a fragment of an antibody that is sufficient to confer recognition and specific binding to the target antigen. Examples of an antibody fragment include, but are not limited to, an Fab, Fab′, F(ab′)2, or Fv fragment, an scFv antibody fragment, a linear antibody, single domain antibody such as an sdAb (either VL or VH), a camelid VHH domain, and multi-specific antibodies formed from antibody fragments.
In other examples, the CAR-T cell comprises a bispecific CAR. Exemplary bi-specific CARs which are contemplated are described in WO2018/049471, the full contents of which is incorporated herein.
T-cells, including CAR-T cells, may be produced according to any methods known in the art. In one example, a T-cell e.g., a CAR-T cell, which expresses an A20 variant protein of the disclosure may be produced by introducing into the cell an expression construct or vector encoding an A20 variant protein as described herein. Methods of producing CAR-T cells which express genes of interest are described in, for example, n WO2018/049471, the full contents of which is incorporated herein.
In one example, the T-cell may be present in a subpopulation of T-cells which have been selected for particular properties e.g., based on HLA typing and/resistance to an immunosuppressant.
T-cells of the disclosure may be formulated for administration in adoptive T-cell therapy.
Formulation of the composition to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion) selected. An appropriate pharmaceutical composition comprising the composition of the present disclosure to be administered can be prepared in a physiologically acceptable carrier. A mixture of compositions can also be used. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.
The optimum concentration of cell populations in the chosen medium can be determined empirically, according to procedures well known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.
In one example, a plurality of T-cells described herein, or compositions comprising same, are provided in a bank. In one example, the T-cells in the bank have been modified to express A20 variant proteins of the disclosure. Different T-cells within the bank may be modified to express different A20 variant proteins as described herein, depending on the extent to which the immune system of a subject is to be increased. In addition, the T-cells in the bank may be CAR-T cells of the disclosure.
In accordance with this example, the T-cells of the disclosure may be “banked” for future use, at a cell bank or depository or storage facility, or any place where such as cells are kept cryopreserved, e.g., in liquid nitrogen, for safekeeping. Furthermore, appropriate computer systems can be used for data processing, to maintain records relating to donor information and to ensure rapid and efficient retrieval of cells from the storage repositories.
In one example, each of the storage containers (e.g., bags or tubes) can be tagged with positive identification based on a distinctive property associated with the donor, lines or cell type, prior to storing in a bank according to the disclosure. For example, DNA genetic fingerprint and HLA typing may be used with secured identification mechanism such as acceptable methods using microchips, magnetic strip, and/or bar code labels. This identification step may be included in the banking process.
In one example, at least one of the HLA alleles in the T-cells in each composition in the bank has been identified. In one example, the HLA is a HLA-DR allele.
At the time of use, only the required storage unit is retrieved, the number of units necessary to fulfil a desired dosage being selectable. Certain diseases may require cell therapy that includes a series of repeated treatments. The population of cells may be extracted from the bank and increased by cellular expansion before preparation of the pharmaceutical composition and administration to the subject.
T-cells of the disclosure which have been modified to express A20 variant proteins may be cryopreserved for adoptive T cell transfer.
The ability of an A20 variant to modify the strength of an immune response in a subject i.e., increase the strength of the immune response or decrease the strength of the immune response, may be determined using any suitable method or assay known in the art. For example, the effect of an A20 variant of the disclosure on the strength of the immune response may be determined by performing an NF-κB-luciferase reporter assay or by measuring JNK activation upon hTNFα stimulation as described in Examples 3 and 4. Alternatively, or in addition, the effect of an A20 variant of the disclosure on the strength of the immune response may be determined in vivo by performing experiments similar to those described in Example 6.
The A20 variant protein, expression construct or vector encoding same, or T cell as described herein may be provided in a pharmaceutical composition. The pharmaceutical composition (syn. active ingredient) may be administered parenterally, topically, orally, or locally or by aerosol administration, or transdermal administration, for prophylactic or for therapeutic treatment Formulation of the active ingredient to be administered will vary according to the agent, route of administration and formulation (e.g., solution, emulsion, capsule) selected. An appropriate pharmaceutical composition comprising the active ingredient to be administered can be prepared in a physiologically acceptable carrier. A mixture of active ingredients which increase A20 activity can also be used. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The active ingredient(s) of the disclosure can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.
The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.
The dosage ranges for the administration of the active ingredient are those large enough to produce the desired effect i.e., increase the strength of the immune response or decrease the strength of the immune response in a subject.
The dosage should not be so large as to cause adverse side effects, such as hyper viscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. Dosage can vary from about 0.1 mg/kg to about 300 mg/kg, such as from about 0.2 mg/kg to about 200 mg/kg, for example from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.
One or more active ingredients of the present disclosure can be administered to an individual by an appropriate route, either alone or in combination with (before, simultaneous with, or after) another drug or agent.
It will be appreciated by those skilled in the art that some active ingredients (e.g., A20 protein) of the present disclosure may be introduced into a subject by administering an expression construct of the disclosure or a cell expressing the active ingredient. A variety of methods can be used for introducing a nucleic acid encoding the active ingredient into a target cell in vivo. For example, the naked nucleic acid may be injected at the target site, may be encapsulated into liposomes, or may be introduced by way of a viral vector.
In one example, the pharmaceutical composition of the disclosure is a vaccine. In accordance with this example, the vaccine may comprise an A20 variant protein or an expression construct or vector encoding same which will increase the immune response in the subject to which it is administered. For example, the vaccine may comprise an A20 variant set forth in Table 2 or variant thereof as described herein. The vaccine will also comprise one or more antigens. In accordance with this example, the presence of the A20 variant in the vaccine will increase the immune response elicited against the antigen. Examples of suitable antigens include, but are not limited to those from ebola virus, influenza virus, pneumococcal, meningococcal, rotavirus, polio, hepatitis (e.g., HCV), chickenpox, small pox, measles, mumps, rubella, whooping cough, HPV, HIV, DENV, plasmodium sp., tetanus and diphtheria. However, it is contemplated that the A20 variant may be formulated with any antigen. In some examples, the vaccine may comprise an adjuvant. The adjuvant may be any adjuvant known in the art. Examples of suitable adjuvants include but are not limited to alum, pertussis toxin, lacto fucopentaose III, phosphopolymer, complete Freund's adjuvant, monophosphoryl lipid A, 3-de-O-acylated monophosphoryl lipid A (3D-MPL), aluminium salt, CpG-containing oligonucleotides, immunostimulatory DNA sequences, saponin, Montanide ISA 720, SAF, ISCOMS, MF-59, SBAS-3, SBAS-4, Detox, RC-529, aminoalkyl glucosaminide 4-phosphate, and LbeIF4A or combinations thereof.
In some examples, the pharmaceutical composition of the disclosure e.g., vaccine, comprises a surfactant. The surfactant may be any surfactant known in the art. Examples of suitable surfactants include, but are not limited to polysorbate 80 (Tween 80®), phospholipids, oleic acid (omega-9 fatty acid), Triton X-100 and Nonoxynol-9 In some examples, the pharmaceutical composition of the disclosure e.g., vaccine, comprises a stabiliser. The stabiliser may be any stabiliser known in the art. Examples of suitable stabilisers include, but are not limited to sugars such as lactose and sucrose, amino acids such as glycine and monosodiumglutamate (salts of amino acids), proteins such as recombinant human albumin (Recombumin®) derived from baker's yeast or fetal bovine (cow) serum and gelatin and partially hydrolysed collagen
The A20 variant may be formulated or administered in conjunction with one or more therapeutic, prophylactic or immunomodulatory agents.
In one example, an A20 variant of the disclosure e.g., as set forth in Table 2, may be formulated or administered in conjunction with a chemotherapeutic agent e.g., for cancer treatment. Suitable chemotherapeutic agents are known in the art. Examples of chemotherapeutic agents include, but are not limited to actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine and Vinorelbine.
In another example, an A20 variant of the disclosure e.g., as set forth in Table 3, may be formulated or administered in conjunction with an agent for treating or managing autoimmune disease. Suitable agents for treating or managing autoimmune disease are known in the art. Examples of such agents include, but are not limited to prednisone, prednisolone, hydrocortisone, dexamethasone, cyclosporine, budesonide, tacrolimus, sirolimus, everolimus, azathioprine, leflunomide, mycophenolate, abatacept, adalimumab, anakinra, certolizumab etanercept golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab and muromonab.
In another example, an A20 variant of the disclosure e.g., as set forth in Table 3, may be formulated or administered in conjunction with an agent for treating or managing transplantation tolerance and/or rejection. Suitable agents for treating or managing transplantation tolerance and/or rejection are known in the art. Examples of agents for treating or managing transplantation tolerance and/or rejection include, but are not limited to tacrolimus, cyclosporine, Mycophenolate Mofetil, mycophenolate sodium, azathioprine, sirolimus, prednisone, prednisolone, dexamethasone and leflunomide.
The inventors have shown e.g., in in vitro and in vivo models, that expression of A20 SNV variants described herein can be used to modulate NF-κB and/or JNK signalling. In doing so, the inventors have shown that some SNV variants of the A20 protein decrease a subject's immune response relative to if the subject expressed a reference A20 protein sequence (e.g., a wild-type A20 variant) and other variants of the A20 protein increase a subject's immune response relative to if the subject had the reference A20 protein sequence (e.g., a wild-type A20 variant). By controlling which A20 variant is expressed in or administered to a subject, the strength of the subject's immune response may be modified i.e., “tuned up” by expressing in or administering to the subject an A20 variant shown to increase the immune response relative to a wild-type or reference A20 protein, or “tuned down” by expressing in or administering to the subject an A20 variant shown to decrease the immune response relative to a wild-type or reference A20 protein. Exemplary A20 variants of the disclosure e.g., variants set forth in Tables 1-3 and functional fragments, analogs or derivatives thereof, have been described herein and shall be taken to apply mutatis mutandis to each and every examples describing a methods of treatment.
Accordingly, the present disclosure provides a method of modifying the strength of an immune response in a subject by administering to, or expressing in, the subject an A20 variant which will increase or decrease the strength of the subject's immune response relative to the subject's immune response in the absence of the A20 variant being administered or expressed.
In one example, the method of the disclosure comprises increasing the subject immune response by administering to, or expressing in, the subject an A20 variant set forth in Table 2 or a functional fragments, analogs or derivatives thereof as described herein.
In another example, the method of the disclosure comprises decreasing the subject immune response by administering to, or expressing in, the subject an A20 variant set forth in Table 3 or a functional fragments, analogs or derivatives thereof as described herein.
In one example, the A20 variant is provided in the form of a protein, as described herein. In one example, the A20 variant is provided in the form of an expression construct or vector encoding the A20 variant protein, as described herein. In one example, the method comprises administering to the subject a cell e.g., a T-cell, which has been modified to express the A20 variant, as described herein. The T-cell may be an autologous T-cell or an allogeneic T-cell. In one example, the T-cell is a CAR-T cell, as described herein.
In one example, the subject in whom the strength of the immune response is to be modified is suffering from cancer and the A20 variant administered to or expressed in the subject is an A20 variant set forth in Table 2 or a functional fragment, analog or derivative thereof as described herein. The cancer to be treated may be any cancer, including, but not limited to, lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasma cell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer.=
In accordance with an example in which the subject is suffering from cancer, the subject may be receiving treatment by immunotherapy or has been prescribed a treatment by immunotherapy. In this respect, it is contemplated that the A20 variant of the disclosure may be administered in conjunction with one or more other agents for treatment of cancer. Examples of anticancer agents include, but are not limited to, immune checkpoint inhibitor, a targeted antibody immunotherapy, a CAR-T cell therapy, an oncolytic virus, or a cytostatic drug. In one example, the methods of the present disclosure provide and A20 variant with an anti-cancer agent selected from the group consisting of. Yervoy (ipilimumab, BMS); Keytruda (pembrolizumab, Merck); Opdivo (nivolumab, BMS); MED14736 (AZ/MedImmune); MPDL3280A (Roche/Genentech); Tremelimumab (AZ/MedImmune); CT-011 (pidilizumab, CureTech); BMS-986015 (lirilumab, BMS); MEDI0680 (AZ/MedImmune); MSB-0010718C (Merck); PF-05082566 (Pfizer); MED16469 (AZ/MedImmune); BMS-986016 (BMS); BMS-663513 (urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110 (arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varlilumab; CellDex Therapeutics); TRX-518 (GITR Inc.); MK-4166 (Merck); JTX-2011 (Jounce Therapeutics); ARGX-115 (arGEN-X); NLG-9189 (indoximod, NewLink Genetics); INCB024360 (Incyte); IPH2201 (Innate Immotherapeutics/AZ); NLG-919 (NewLink Genetics); anti-VISTA (JnJ); Epacadostat (INCB24360, Incyte); F001287 (Flexus/BMS); CP 870893 (University of Pennsylvania); MGA271 (Macrogenix); Emactuzumab (Roche/Genentech); Galunisertib (Eli Lilly); Ulocuplumab (BMS); BKT140/BL8040 (Biokine Therapeutics); Bavituximab (Peregrine Pharmaceuticals); CC 90002 (Celgene); 852A (Pfizer); VTX-2337 (VentiRx Pharmaceuticals); IMO-2055 (Hybridon, Idera Pharmaceuticals); LY2157299 (Eli Lilly); EW-7197 (Ewha Women's University, Korea); Vemurafenib (Plexxikon); Dabrafenib (Genentech/GSK); BMS-777607 (BMS); BLZ945 (Memorial Sloan-Kettering Cancer Centre); Unituxin (dinutuximab, United Therapeutics Corporation); Blincyto (blinatumomab, Amgen); Cyramza (ramucirumab, Eli Lilly); Gazyva (obinutuzumab, Roche/Biogen); Kadcyla (ado-trastuzumab emtansine, Roche/Genentech); Perjeta (pertuzumab, Roche/Genentech); Adcetris (brentuximab vedotin, Takeda/Millennium); Arzerra (ofatumumab, GSK); Vectibix (panitumumab, Amgen); Avastin (bevacizumab, Roche/Genentech); Erbitux (cetuximab, BMS/Merck); Bexxar (tositumomab-1131, GSK); Zevalin (ibritumomab tiuxetan, Biogen); Campath (alemtuzumab, Bayer); Mylotarg (gemtuzumab ozogamicin, Pfizer); Herceptin (trastuzumab, Roche/Genentech); Rituxan (rituximab, Genentech/Biogen); volociximab (Abbvie); Enavatuzumab (Abbvie); ABT-414 (Abbvie); Elotuzumab (Abbvie/BMS); ALX-0141 (Ablynx); Ozaralizumab (Ablynx); Actimab-C (Actinium); Actimab-P (Actinium); Milatuzumab-dox (Actinium); Emab-SN-38 (Actinium); Naptumonmab estafenatox (Active Biotech); AFM13 (Affimed); AFM11 (Affimed); AGS-16C3F (Agensys); AGS-16M8F (Agensys); AGS-22ME (Agensys); AGS-15ME (Agensys); GS-67E (Agensys); ALXN6000 (samalizumab, Alexion); ALT-836 (Altor Bioscience); ALT-801 (Altor Bioscience); ALT-803 (Altor Bioscience); AMG780 (Amgen); AMG 228 (Amgen); AMG820 (Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110 (Amgen); AMG232 (adecatumumab, Amgen); AMG211 (Amgen/MedImmune); BAY20-10112 (Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen); AMP-514 (Amgen); MEDI575 (AZ/Medlmmune); MEDI3617 (AZ/Medlmmune); MEDI6383 (AZ/MedImmune); MEDI551 (AZ/MedImmune); Moxetumomab pasudotox (AZ/MedImmune); MEDI565 (AZ/Medlmmune); MEDI0639 (AZ/Medlmmune); MEDI0680 (AZ/MedImmune); MEDI562 (AZ/MedImmune); AV-380 (AVEO); AV203 (AVEO); AV299 (AVEO); BAY79-4620 (Bayer); Anetumab ravtansine (Bayer); vantictumab (Bayer); BAY94-9343 (Bayer); Sibrotuzumab (Boehringer Ingleheim); BI-836845 (Boehringer Ingleheim); B-701 (BioClin); BIIB015 (Biogen); Obinutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206 (Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest) BIL-010t (Biosceptre); MDX-1203 (BMS); MDX-1204 (BMS); Necitumumab (BMS); CAN-4 (Cantargia AB); CDX-011 (Celldex); CDX1401 (Celldex); CDX301 (Celldex); U3-1565 (Daiichi Sankyo); patritumab (Daiichi Sankyo); tigatuzumab (Daiichi Sankyo); nimotuzumab (Daiichi Sankyo); DS-8895 (Daiichi Sankyo); DS-8873 (Daiichi Sankyo); DS-5573 (Daiichi Sankyo); MORab-004 (Eisai); MORab-009 (Eisai); MORab-003 (Eisai); MORab-066 (Eisai); LY3012207 (Eli Lilly); LY2875358 (Eli Lilly); LY2812176 (Eli Lilly); LY3012217 (Eli Lilly); LY2495655 (Eli Lilly); LY3012212 (Eli Lilly); LY3012211 (Eli Lilly); LY3009806 (Eli Lilly); cixutumumab (Eli Lilly); Flanvotumab (Eli Lilly); IMC-TR1 (Eli Lilly); Ramucirumab (Eli Lilly); Tabalumab (Eli Lilly); Zanolimumab (Emergent Biosolution); FG-3019 (FibroGen); FPA008 (Five Prime Therapeutics); FP-1039 (Five Prime Therapeutics); FPA144 (Five Prime Therapeutics); catumaxomab (Fresenius Biotech); IMAB362 (Ganymed); IMAB027 (Ganymed); HuMax-CD74 (Genmab); HuMax-TFADC (Genmab); GS-5745 (Gilead); GS-6624 (Gilead); OMP-21M18 (demcizumab, GSK); mapatumumab (GSK); IMGN289 (ImmunoGen); IMGN901 (ImmunoGen); IMGN853 (ImmunoGen); IMGN529 (ImmunoGen); IMMU-130 (Immunomedics); milatuzumab-dox (Immunomedics); IMMU-115 (Immunomedics); IMMU-132 (Immunomedics); IMMU-106 (Immunomedics); IMMU-102 (Immunomedics); Epratuzumab (Immunomedics); Clivatuzumab (Immunomedics); IPH41 (Innate Immunotherapeutics); Daratumumab (Janssen/Genmab); CNTO-95 (Intetumumab, Janssen); CNTO-328 (siltuximab, Janssen); KB004 (KaloBios); mogamulizumab (Kyowa Hakko Kirrin); KW-2871 (ecromeximab, Life Science); Sonepcizumab (Lpath); Margetuximab (Macrogenics); Enoblituzumab (Macrogenics); MGD006 (Macrogenics); MGF007 (Macrogenics); MK-0646 (dalotuzumab, Merck); MK-3475 (Merck); Sym004 (Symphogen/Merck Serono); DI17E6 (Merck Serono); MOR208 (Morphosys); MOR202 (Morphosys); Xmab5574 (Morphosys); BPC-1C (ensituximab, Precision Biologics); TAS266 (Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031 (Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355 (Novartis); OMP-21M18 (Demcizumab, OncoMed); OMP52M51 (Oncomed/GSK); OMP-59R5 (Oncomed/GSK); vantictumab (Oncomed/Bayer); CMC-544 (inotuzumab ozogamicin, Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer); PSMA-ADC (Progenics); REGN1400 (Regeneron); REGN910 (nesvacumab, Regeneron/Sanofi); REGN421 (enoticumab, Regeneron/Sanofi); RG7221, RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636, RG7450, RG7593, RG7596, DCDS3410A, RG7414 (parsatuzumab), RG7160 (imgatuzumab), RG7159 (obintuzumab), RG7686, RG3638 (onartuzumab), RG7597 (Roche/Genentech); SAR307746 (Sanofi); SAR566658 (Sanofi); SAR650984 (Sanofi); SAR153192 (Sanofi); SAR3419 (Sanofi); SAR256212 (Sanofi), SGN-LIV1A (lintuzumab, Seattle Genetics); SGN-CD33A (Seattle Genetics); SGN-75 (vorsetuzumab mafodotin, Seattle Genetics); SGN-19A (Seattle Genetics) SGN-CD70A (Seattle Genetics); SEA-CD40 (Seattle Genetics); ibritumomab tiuxetan (Spectrum); MLN0264 (Takeda); ganitumab (Takeda/Amgen); CEP-37250 (Teva); TB-403 (Thrombogenic); VB4-845 (Viventia); Xmab2512 (Xencor); Xmab5574 (Xencor); nimotuzumab (YM Biosciences); Carlumab (Janssen); NY-ESO TCR (Adaptimmune); MAGE-A-10 TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (Juno Therapeutics); KTE-C19 CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (Bellicum Pharmaceuticals); BPX-601 (Bellicum Pharmaceuticals); ATTCK20 (Unum Therapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); HI10 (Shanghai Sunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences); Colo-Adl (PsiOxus Therapeutics); ProstAtak (Advantagene); Oncos-102 (Oncos Therapeutics); CG0070 (Cold Genesys); Pexa-vac (JX-594, Jennerex Biotherapeutics); GL-ONC1 (Genelux); T-VEC (Amgen); G207 (Medigene); HF10 (Takara Bio); SEPREHVIR (HSV1716, Virttu Biologics); OrienX010 (OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SVV-001 (Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eli Lilly), cisplatin, oxaliplatin, irinotecan, folinic acid, methotrexate, cyclophosphamide, 5-fluorouracil, Zykadia (Novartis), Tafinlar (GSK), Xalkori (Pfizer), Iressa (AZ), Gilotrif (Boehringer Ingelheim), Tarceva (Astellas Pharma), Halaven (Eisai Pharma), Veliparib (Abbvie), AZD9291 (AZ), Alectinib (Chugai), LDK378 (Novartis), Genetespib (Synta Pharma), Tergenpumatucel-L (NewLink Genetics), GV1001 (Kael-GemVax), Tivantinib (ArQule); Cytoxan (BMS); Oncovin (Eli Lilly); Adriamycin (Pfizer); Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene); Trelstar (Debiopharm); Taxotere (Sanofi); Nexavar (Bayer); IMMU-132 (Immunomedics); E7449 (Eisai); Thermodox (Celsion); Cometriq (Exellxis); Lonsurf (Taiho Pharmaceuticals); Camptosar (Pfizer); UFT (Taiho Pharmaceuticals); and TS-1 (Taiho Pharmaceuticals).
In one example, the subject in whom the strength of the immune response is to be modified is suffering from complement deficiency and the A20 variant administered to or expressed in the subject is an A20 variant set forth in Table 2 or a functional fragment, analog or derivative thereof as described herein.
In another example, the subject in whom the strength of the immune response is to be modified is suffering from an autoimmune disease and the A20 variant administered to or expressed in the subject is an A20 variant set forth in Table 3 or a functional fragment, analog or derivative thereof as described herein. Examples of autoimmune disease include, but are not limited to uveitis, multiple sclerosis; arthritis, such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis; neuromyelitis optica (Devic's disease); ankylosing spondylitis; spondyloarthritis; psoriasis; systemic lupus erythematosus; inflammatory bowel disease, such as Crohn's disease or ulcerative colitis; celiac disease; asthma, such as allergic asthma or neutrophilic asthma; chronic obstructive pulmonary disease (COPD); scleritis; vasculitis; Behcet's disease; atherosclerosis; atopic dermatitis; emphysema; periodontitis; allergic rhinitis; and allograft rejection.
In another example, the subject in whom the strength of the immune response is to be modified is suffering from a pathogenic infection or is in need or protection against pathogenic infection, and the A20 variant administered to or expressed in the subject is an A20 variant set forth in Table 2 or a functional fragment, analog or derivative thereof as described herein. The infection may be caused by any pathogen, including bacteria, virus, fungus. Examples of bacterial, viral and fungal infectious agents include but are not limited to Mycobacterium tuberculosis, Bordetella pertussis, Campylobacter jejuni, Chlamydia, Clostridium, Haemophilus, Neisseria, Pseudomonas, Salmonella, Shigella Staphylococcus, Streptococcus, Yersinia, varicella-roster virus, measles virus, rotavirus, norovirus, Hepatitis virus, Herpes, HIV, Rubella virus, Aspergillus, Candida, Coccidioides, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys
In another example, the subject in whom the strength of the immune response is to be modified is a transplant patient, such as for example, a subject who has undergone tissue or organ transplantation or is about to undergo tissue or organ transplantation. In accordance with this example, the A20 variant administered to or expressed in the subject is an A20 variant set forth in Table 3 or a functional fragment, analog or derivative thereof as described herein.
In accordance with any example in which an A20 variant, or functional fragment, analog or derivative thereof, as described herein is to be expressed in or administered to a subject as part of a combination therapy i.e., in conjunction with one or more other therapeutic agents or treatments as described herein, the A20 variant, or functional fragment, analog or derivative thereof, may be provided to the subject simultaneously or consecutive to the one or more other therapeutic agents or treatments.
Based on the finding that different A20 variants of the disclosure correlate with differing NF-κB and/or JNK signalling activity, the inventors also contemplate that the A20 SNV variants could be useful as biomarkers to stratify patients according to the likely strength of their immune response before undergoing treatment or during treatment. Such information may also be useful in selecting the type of and/or duration of treatment, and for ongoing patient management.
Accordingly, in one example, the present disclosure provides a method of stratifying a subject according to the strength of their immune response. In one example, the method of stratifying the subject comprises: (i) determining the nucleotide sequence of the A20 gene in the subject; (ii) comparing the nucleotide sequence of the A20 gene determined at (i) with a reference A20 gene nucleotide sequence; and (iii) predicting the strength of the subject's immune response on the basis of the subject's A20 gene nucleotide sequence being identical to the reference sequence at one or more nucleotide positions or on the basis of one or more differences in the nucleotide sequence of the A20 gene in the subject relative to the reference sequence. For example, the presence of an A20 SNV in the subject relative to the reference A20 nucleotide sequence e.g., an SNV encoding an A20 variant set forth in Table 1, will be indicative that the subject's immune response will be increased or decreased relative to if the subject had the reference A20 nucleotide sequence. In one example, the presence of an A20 SNV corresponding to an A20 variant set forth in Table 2 in the subject is indicative that the subject's immune response will be increased relative to if the subject had the reference A20 nucleotide sequence. Conversely, the presence of an A20 SNV corresponding to an A20 variant set forth in Table 3 in the subject is indicative that the subject's immune response will be decreased relative to if that subject had the reference A20 nucleotide sequence. As described herein, the reference A20 nucleotide sequence against which the subject's A20 nucleotide sequence is compared may be a sequence encoding a wild-type A20 protein e.g., a wild-type human A20 protein. The nucleotide sequence of the A20 gene, TNFAIP3, in the subject may be determined by any suitable sequencing methods known in the art. For example, the A20 nucleotide sequence may be determined by Sanger sequencing or high-throughput sequencing methods such as next-gen sequencing. The subject's A20 nucleotide sequence may be compared to the reference A20 nucleotide sequence by any suitable method known in the art. For example, the subject's A20 nucleotide sequence may be aligned (using any suitable sequence alignment or bioinformatics software) with the reference A20 gene sequence to identify differences between the subject's A20 nucleotide sequence and the reference sequence. It may also be readily determined whether any differences in nucleotide sequence translate into differences at the amino acid level i.e., whether SNVs are synonymous or non-synonymous. The method of stratifying a subject according to the strength of their immune response may be performed prior to administering to, or expressing in, the subject an A20 variant which modifies the immune response e.g., prior to performing a method of modifying the strength of an immune response in a subject as described herein, in order to determine whether the strength of the subject's immune response needs to be increased or decreased.
It will be appreciated by the skilled artisan that the methods of the present disclosure allows the stratification of a subject in order to be treated by the methods of treatment of the present disclosure.
Using available human genome sequence data from the ExAC database, the inventors utilised PolyPhen2 to determine the distribution of non-synonymous single nucleotide variations (nsSNVs) classified as benign or deleterious within the A20 locus. The results from this analyses are expressed in violin plot format (
The inventors applied the same kind of analysis on nsSNVs across the entire A20 locus, but now focusing on human A20 protein coding domains (functional and structurally connecting). Three main groupings emerged as is illustrated in
The A20 gene locus is characterized by many SNVs identified not only by GWAS or candidate-gene studies, but also by extensive sequencing studies contained within meta libraries like the ExAC database. The inventors combined variants from these various sources and analysed them by PolyPhen2, revealing a total of ˜262 rare missense protein coding SNPs in the OTU domain only.
The inventors also used bioinformatics resources to mine available online resources to make use of all the information on A20's genetic variability. The literature was also consulted for the most up to date variants in the A20 gene locus found through GWAS, NGS and WGS studies. Online databases like Ensembl.org were also extensively consulted for this purpose. For the identification of A20 gene variants identified through GWAS, databases like GWAS central and GWAS catalog were consulted. For a more extensive, non-GWAS centred, consultation of A20 variants, Ensembl online database was consulted. Ensembl is an aggregator of sources of genes information from literature, clinical observations and several other online databases (ExAC, gnomAD, dbSNP, SNPedia, 1000 genomes, HapMap). Furthermore, Ensembl provides, where possible, inferences on the deleteriousness of variants through the addition of prediction algorithms information regarding coding variants. In addition to these sources of gene variation, the inventors obtained exome sequencing data from 50 patients with type 1 diabetes (TID) sequenced at the Garvan Institute of Medical Research. From this analysis, 17,521 variants in TNFAIP3 gene locus were identified.
Given the difficulty and cost associated with functionally evaluating such a large number of SNVs, a series of filters was applied to reduce the working set of human A20 variants to functionally test (
Since the purpose of the functional testing was to assess the impact of A20 variation on protein function, non-coding variants of the A20 gene locus were discarded as a first filter. This ‘coding’ filter reduced the number of variants to 4580. A further filter based on the frequency of the variants in the population was then applied. In this case rare (<0.1% MAF) SNV were prioritised over common SNV. Rarity would allow the exploration of variants not detectable by GWAS, examining the hypothesis by which complex diseases might be explained by more rare variants in the population. This filter reduced the number of variants to 325.
Variants were then selected only if the point mutation caused a non-synonymous change in the protein sequence, filtering out the redundancy of variants coding for the same amino acid through the different nucleotide triplets. Using this filter, the number of variants was reduced to 205.
Lastly, PolyPhen2 was used to segregate variants that might have an effect on the protein function. PolyPhen2 served two purposes: the first was that it provided an unbiased method to select variants for functional analysis; and the second was that PolyPhen2 would intrinsically test the algorithm's success in correctly determining the functional consequence of a given SNV.
By applying this pipeline, 205 coding variants with a PolyPhen2 score were identified, collected and subsequently plotted against the A20 protein sequence. This offered a visual representation of the distribution of SNV across the A20 protein sequence, as well as a view of the range of functional consequences (benign 0-0.15, damaging >0.15, 0.85, deleterious ≥0.85) variants predicted by PolyPhen2 (
Together with the previously applied filters shown in
In addition to this set of 10 A20 SNVs, a further A20 variants were chosen for functional evaluation based on the following criteria:
These criteria a described in further detail below. This selection process brought the total number of variants tested in this study to 25. The 25 SNVs are presented in Table 4 below.
A group of six SNV that were coding, rare, non-synonymous, but predicted to be benign by PolyPhen2 (score <0.15) were chosen to compare with the set of rare-deleterious SNV. As well as providing important control data (as these were predicted to have no impact on function), these SNVs tested the PP2 algorithm for errors in predicting false negatives.
To draw the upper range of NF-κB activation across the different A20 SNV, a second group comprising two loss-of-function mutants was chosen, namely C103A and S381A. Given previous evidence that these sites may have a role on A20 activity and function, it was predicted that these mutants should define the maximal upper range of responses for any SNV that reduces A20's function in the NF-κB reporter assay.
A further group of variants includes the only two GWAS variants identified within A20's coding region, namely, A125V and F127C (Musone, Taylor et al. 2008, Lodolce, Kolodziej et al. 2010).
Also included in the panel of variants tested in functional assays were three variants from a cohort of 50 patients with TID whose exomes had been sequenced. These three variants, namely T108A, I207L and T647P, fulfil the criteria of rarity (MAF <0.1%) but were predicted to be benign by PolyPhen2 (scores 0.001, 0.132 and 0.000 respectively).
ENU Variant with an In Vivo Mouse Phenotype
The inventors also identified the I325N variant through an in vivo ENU-mutagenesis mouse screen for A20 LoF variants. This mutation changes a highly conserved isoleucine in the A20 OTU domain for an asparagine. The I325N mouse model showed a hyper-inflammatory phenotype including an increased frequency of activated T cells, spontaneous immune infiltrates in various organs, and when challenged with LPS these mice suffered premature death. For this study, the I325N variant was introduced into the A20 reference sequence by site-directed mutagenesis. Including this mutant in the panel for functional testing provided a benchmark to highlight whether the variant's effect on NF-κB inhibition could translate into an in vivo phenotype.
It was hypothesised that transfecting cells with human A20 variants would affect their ability to inhibit NF-κB upon hTNFα stimulation. Therefore, filtering variants for rarity and deleteriousness was undertaken in order to enrich for variants with an impaired NF-κB inhibitory activity. Variant choice would also serve another purpose, shedding some light on the current complex disease-variants hypothesis. If rare variants were found to confer an in vitro NF-κB phenotype, then there may be value in investigating the role of rare variants in complex diseases.
The next step was to test the A20 variants identified in Example 2 using a multi-model approach to elucidate the role of A20 in the context of human inflammatory diseases.
The impact of the 27 different sequence variants on A20's inhibitory function against NF-κB was tested in an NF-κB-luciferase reporter assay which is recognised as being a standard and robust approach for assessing the effect of A20 on NF-κB inhibition. Essentially, cells expressing a NFkB reporter are treated with TNF to activate the reporter. Reporter activation is then measured by analysing luciferase values. In some cases, cells also express A20, or one of the 27 A20 variants, and NFkB activation is measured after TNF stimulation. Thus, this assay requires transient transfection of mammalian cells with plasmids encoding:
The inventors conducted the luciferase reporter assay in HEK293T cells. This undifferentiated cell line was chosen because it is a human cell, originally derived from cultures of human embryonic kidney cells, and its widely reported propensity to transfection. The human derivation of this cell line makes it appropriate to test the function of human A20 variants. Also, the non-specialized nature of this cell line offered a ‘neutral’ environment for the testing of A20's variants.
Briefly, Lipofectamine 2000 was used to transiently transfect all cells (HEK293T) with NF-κB luciferase plasmid and j-gal reporter to correct for transfection efficiency. Selected wells were also transiently transfected with either an empty PCDNA3 plasmid (serving as non-template control) or human A20 plasmids coding for either the reference or the variants. Cells were stimulated with human-TNFα for 8 hours to stimulate the reporter, at the end of which cell lysates were tested for luciferase and β-galactosidase activity (luminescence) in technical duplicates and the luminescence values were recorded for each of the tested variants (±hTNFα stimulation) (
A representative read-out of the assay can be found in
Results from cells expressing different A20 variants are also presented (
To better facilitate a comparative analysis across the 27 variants, normalization of the data was performed by averaging the technical replicates from each experiment and then scoring each tested variant against each individual experiment's NTC (hTNFα stimulated), the TNF-stimulated NTC result was set to a value of 1, and the reference human-A20 (hTNFα stimulated) result was set to a value of 0. This method guaranteed self-contained consistency of results and allowed for comparability across experiments, increasing the robustness of the approach itself.
The variants reported in the representative bar graph (
To better facilitate a comparative analysis across the 27 variants, normalization of the data was performed by averaging the technical replicates from each experiment and then scoring each tested variant against each individual experiment's NTC (hTNFα stimulated), the TNF-stimulated NTC result was set to a value of 1, and the reference human-A20 (hTNFα stimulated) result was set to a value of 0. This method guaranteed self-contained consistency of results and allowed for comparability across experiments, increasing the robustness of the approach itself.
The relationship between normalized NF-κB activity of the A20 variants (left y-axis) and their predicted deleteriousness (right y-axis), reported as PolyPhen2 score (data from PolyPhen2 build September 2014), in HEK293T cells is illustrated in
The inventors then compared the functional data with the predictions of PolyPhen2 by plotting the PolyPhen2 scores for each variant against their respective NF-κB activity. Interestingly, hA20 variants with an impaired ability to inhibit NF-κB did not always report concordant deleterious (≥0.85) PolyPhen2 scores. Vice versa, hA20 variants with NF-κB inhibitory activity which is comparable or better to the reference human-A20 did not always report concordant benign (<0.15) PolyPhen2 scores. The relationship between normalized NF-κB activity of the A20 variants (left y-axis) and their predicted deleteriousness (right y-axis), reported as PolyPhen2 score (data from September 2014), in HEK293T cells is shown in
Variants were then re-organized from
As is apparent from
Interestingly, all transfected hA20 variants expressed in comparable levels to the ref-hA20 except for the N102S (
TNF is one of the cytokines that activates the NF-κB pathway, which in turn promotes the transcription of pro-inflammatory genes. There are also other molecular actors involved in the inflammatory response that are triggered by TNF. One of these is c-Jun N-terminal kinase (JNK), which belongs to the mitogen-activated protein kinases (MAPKs) family. While NF-κB's function is anti-apoptotic and cyto-protective, JNK's signalling pathway is involved in the mediation of apoptotic cell death in response to a variety of stress stimuli, such as cytokines, ultraviolet irradiation and changes in levels of reactive oxygen species.
When stimulated with TNF, cells can choose which fate to follow, either favouring a cell-protective fate (NF-κB pathway) or cell-death via the JNK pathway. These findings suggest that there are cell-intrinsic mechanisms that are able to discern which fate cells undergo. Studies have identified how the adaptor protein TRAF2 couples the membrane receptor to JNK, but the dynamics of its activation suggest a more complex network than the classic linear kinase cascade.
A20 fulfils both anti-inflammatory and anti-apoptotic roles and it is involved in both NF-κB and JNK pathways, although its inhibitory mechanisms in the latter pathway are unclear. Therefore, to complement the results obtained on NF-κB levels, the inventors used a cell-based assay to dynamically assess the impact of each hA20 variant on JNK activation upon hTNFα stimulation.
For this assay an mCherry Kinase Translocation Reporter HEK293T cell line was used. This cell line constitutively expresses the latter construct, which translocates from the nucleus to the cytoplasm when phosphorylated by JNK (
The inventors transiently infected HEK293T cells with human-A20 variants using JetPRIME reagent. Briefly, selected wells were transiently transfected with either an empty PCDNA3 plasmid (serving as non-template control) or human A20 plasmids coding for either the reference or the variants. A nuclear localization marker for JNK was given to the cells and then selected wells were stimulated with human-TNFα. Each well of transfected cells was assessed every 5 min for 1 hour. The ratio between cytoplasm JNK and nucleus JNK would be a measure of JNK activation upon hTNFα stimulation.
In
After the A20 variants were tested in this assay, values were normalized to be able to rank the variants based on their ability to suppress JNK activity at the 20 minute time point. The normalized activation of JNK in cells have been represented as a heatmap in
When considering the results obtained for the HEK293T cells in Example 3 for the dynamic inhibition of JNK although variants were ranked by their normalized JNK activity values at 20 minutes post-stimulation it is important to notice how some variants allow JNK hyper activation through time. The physiological kinetic of non-transfected, and NTC transfected, cells shown in
When considering a static snapshot of the variants ranked by their JNK activity values at 20 minutes it was possible to observe a polarization of the variants in respect to the ref-hA20. The previous NF-κB luciferase reporter assay measured the transcriptional responses accumulated after TNF stimulation (8 hours post-stimulation) while this assay reported real-time dynamic activation of JNK over a period of 60 minutes. Although there is a fundamental difference in the way each of the functional assay tests A20 effect on NF-κB or JNK, some of the most impaired variants (e.g. N102S, C243Y) reconfirmed their impairing nature in this assay, adding a dynamic aspect to the screening of human-A20 variants. Interestingly, the GoF variants from the NF-κB assay did not score less than the ref-hA20, one of which (V115L) was ranked 4th least protective. This might suggest a different role for these variants depending on their involvement according to the structural conformations the A20 protein needs to assume through time and across different pathways and molecular actors involved.
The inventors used the PolyPhen2 prediction algorithm as one of the filters applied to the final selection of the variants tested in this study. The in vitro functional consequences of amino acidic changes for each variant were then compared to PolyPhen2 predictions. This analysis was conducted on the HEK293T data due to the clarity of the subdivisions between variants' effects. Variants' normalized NF-κB luciferase values were ordered based on their ability to suppress NF-κB. This allowed for the identification of three groups of variants in the HEK293T cells: variants with GoF activity (highlighted in blue and ranging from −0.162 to −0.085), ref-hA20 comparable variants (highlighted in green and ranging from −0.018 to −0.0012) and variants with an impairing NF-κB inhibitory activity (highlighted in red and ranging from 0.005 to 1.105) (
Each variant was checked for conservation in the TNFAIP3 gene of 7 species (human, chimpanzee, cow, pig, dog, mouse, chicken and zebrafish) and an asterisk used (in
The GoF group included two common variants predicted to be benign (S184N and V 115L) and a rare variant predicted to be deleterious (R162Q), and their normalized NF-κB luciferase activity values. The ref-hA20 comparable group included most of the variants tested in this study (11 variants), yet no obvious correlation between conservation and effect emerged. The impairing variants (9 variants) were overall the most conserved among species and the most rarely observed in the general population (this group also included 1325N and S381A variants, which have never been observed in the general population, most likely due to their LoF effect).
One of the main challenges with the ever so increasing amount of variants discovered in the human genome is how to functionally annotate them. Being able to discern between what is disease causal and what is not will ultimately result in a better understanding of how the human genome works.
In this study, one of the filters used by the inventors to possibly enrich for the discovery of deleterious variants was using PolyPhen2 prediction algorithm. Aside from PolyPhen2, many other algorithms have been developed to help predict the functional effect of a protein non-synonymous missense mutation (Butkiewicz and Bush 2001, Boyko, Williamson et al. 2008, Di, Chan et al. 2009, Neale, Rivas et al. 2011, Adzhubei, Jordan et al. 2013, Itan and Casanova 2015, Miosge, Field et al. 2015), yet, their ability to correctly predict functional consequences is still at best ˜50% (Di, Chan et al. 2009, Miosge, Field et al. 2015). As new prediction algorithms are implemented it is interesting to see what is the concordance they display when compared to functional data and how these predictions change through time. In this section, the concordance between the functional results of the in vitro testing of the variants and PolyPhen2 predictions is explored.
The PolyPhen2 algorithm has been updated three times since the beginning of this study (September 2014, April 2017 and July 2017) but for consistency of results, variants were ‘locked’ with PolyPhen2 scores based on the September 2014 release. These PolyPhen2 predictions have been compared to predictions from other two extensively used online prediction tools, namely PROVEAN and SIFT, collected at the same time. These latter two prediction algorithms also assess the deleteriousness of an amino acidic substitution in a similar way to PolyPhen2, but have different degrees of strictness (Ng and Henikoff 2003, Choi and Chan 2015).
The A20 variants tested were then ordered according to their NF-κB inhibitory activity (
Although it was encouraging to see a positive increasing trend in the concordance of the PolyPhen2 scores when compared to the functional data, these studies were made on a relatively small set of variants. Therefore the observed concordance percentages might not reflect the reality when testing larger numbers of variants and the other filtering criteria used in the study are modified or removed. The concordance percentages would most likely also be strictly related to the type of assay conducted. Some of the variables that may have affected the accuracy of predictive algorithms with respect to functional analysis are listed and discussed in Table 5.
This analysis highlights another problem, the level of any given variants ‘deleteriousness’ can change over time, in the case of A125V, the call for this variant switched from benign to deleterious and back to benign with each new release of PolyPhen2. While the changes to PolyPhen2 have increased the overall level of concordance of the algorithm with the functional studies, at the same time the fluctuations in the specific calls hampers the scientific community from forming reliable assumptions about individual variants.
Improvements in the way these algorithms predict deleteriousness are constantly made and similar tools have emerged (Yachdav, Kloppmann et al. 2014). Another online prediction tool (PredictProtein) was used to visualise all the possible amino acidic substitution for the A20 protein (
A step further could be including insights on the spatial location of the mutation within the 3D architecture of the protein and information about the role each amino acid assumes in the protein context (catalytic sites, phosphorylation sites, site for docking of molecular partners, etc.).
Unfortunately not every protein has been fully biochemically characterized and/or has a solved protein structure. Even if a protein structure has been solved structure on Protein Data Bank (PDB) might have low definition protein structure (≥2.5 Å), which allows for a general visualization of the protein shape and secondary structures such as α-helices and β-sheets motifs spatial location. Higher resolutions (≤2.5 Å) can give information on side-chains and electron densities (useful for molecular dynamics studies).
In the case of A20, a partial structures of the protein (the OTU domain—amino acid 1 to 360) has been solved by other members within this laboratory team e.g. PDB ID: 4ZS5. In total three structures have been deposited on PDB, and they have resolutions of 2.5, 2.7 and 3.2 Å respectively. This information was used to localize the tested variants within the OTU domain and to correlate their effects on NF-κB inhibitory activity with their position within the protein. The position of each of the tested variants is annotated in
In respect to the position of the variants to their location on either α-helices or β-sheets motifs, GoF or ref-hA20 comparable variants were only found to be located on α-helices. Impaired variants were located either in the middle of a β-sheets motif (1325N) or at the junction between an α-helix and a β-sheet (I207L, C243Y, N102S). No clear pattern emerged in respect to the amino acid change, although the properties of each amino acid are shown in
To further investigate this aspect, the inventors performed molecular dynamics (MD) simulations for both the N102S and another deleterious candidate A20-C243Y. The first (N102) is of interest as it sits adjacent to the catalytic cysteine, C103. This is of further interest since the role of A20 variant C103A is of debate in the literature (Lu, Onizawa et al. 2013, De, Dainichi et al. 2014). The second (C243Y) might have a more obvious implication regarding local folding, this might be due to the substitution with a larger side chain.
A readout of the MD simulations is presented in
The root-mean-square-fluctuation (RMSF), a measure of the amplitude of atom motions, is presented in
Principal component analyses (PCA) of the simulation trajectories for the WT and the mutants are shown in
These behaviours were visualised by mapping the PCA onto an interaction network visualization of the chain structures (
The two mutants exhibited behaviours that are at the two opposite sides of the A20-WT mobility dynamics. N102S exhibits a reduced loop dynamics, which is likely to frustrate either binding to ubiquitin or disrupt the catalytic activity even if the active site geometry is still maintained, therefore, resulting in such a severe phenotype in vitro. C243Y also exhibited a reduced loop dynamics, which was even more than N102S, however, the regions surrounding the mutation (˜34, ˜280, ˜318) showed a slightly increased amount of non-native flexibility. The latter flexibility introduced near these loops could affect phosphorylation, hence disrupting protein function.
The A20 I325N allelic variant was identified in a genome-wide mouse ENU-mutagenesis screen segregating with increased frequencies of circulating CD44hi activated/memory T cells and regulatory T cells (
In wild-type mice transplanted with mixtures of mutant and wild-type A20 bone marrow, the I325N mutation acted cell autonomously to increase B cell activation and proliferation by LPS or antigen receptors, and to increase TCR and CD28-dependent formation of FOXP3+CD4+ regulatory T cells and their Helios+FOXP3− precursors within the thymus (
Consistent with heightened cellular markers of immunity, I325N mutant mice had greater resistance to Coxsackievirus B4 strain E2, a virus isolated from a human neonate with a disseminated fatal infection causing extensive pancreatic necrosis (35, 36). A virus dose that was lethal for 90% of wild-type C57BL/6 mice was not lethal for Tnfaip3I325N/I325N littermates, and caused less mortality in Tnfaip3I325N/+ mice (
The homogeneous genetic background of I325N mutant mice allowed testing if heightened immunity imposed a subclinical cost or altered the insulin anabolic axis. Tnfaip3I325N/+ mice were healthy, of normal weight, and fertile, producing homozygous mutant offspring at the expected ratio. Homozygotes also appeared healthy, although their body weight was 5-20% less than heterozygous or wild-type littermates (
In the rodent counterpart of smallpox, infection with the orthopoxvirus ectromelia virus was tolerated and controlled by wild-type mice, yet resulted in high mortality and higher viral titres in I325N homozygotes (
In bone marrow chimeric mice with a higher frequency of pancreatic islet-reactive CD4+ T cells, autoreactive T cells escaped deletion and precipitated diabetes when half the hematopoietic cells were homozygous for I325N (
Mice (C57BL/6) were engineered using CRISPR/Cas9 to express either the human I207L A20 variant or the C243Y A20 variant. Mice which were homozygous and heterozygous for each human A20 variants were produced for comparison to normal C57BL/6 mice.
Mice homozygous, heterozygous, or wild type for the three A20 variants I207L, I325N, or C243Y were challenged with a gram negative microbial stimuli of 2.5 mg/kg of LPS and serum IL-6 was measured as a marker of immune activation at time zero, 2 and 6 hours post treatment.
In an additional study, splenocytes were harvested from the I207L A20 variant mice at 6-8 weeks of age and CD4 and CD8 cells were analysed for CD44 expression. As will be apparent from
Thus, like the A20 I35N allelic variant mice described in Example 6, the A20 I207L allelic variant segregated with increased frequencies of circulating CD440 activated/memory T cells and regulatory T cells in otherwise healthy adult mice. CD44 is a marker of immune activation and indicates heightened immunity in these mice.
Using whole genome sequencing, the inventors identified in human subjects (parent and offspring trios) a pair of missense variants in cis, p.(T108A) and p.(I207L), which formed part of a larger haplotype consisting of multiple coding and non-coding variants (called ‘hap’; see
These human subjects were then used to assess whether TNFAIP3 variants can tune human immune responses. Peripheral blood mononuclear cells (PBMNC) were taken from subjects and exposed to TNF stimulation in cell culture experiments. At 0, 2 and 4 hours after TNF stimulation, the levels of inflammatory mRNAs were measured in the PBMNC by RT-PCR. The results of the RT-PCR analysis for TNFAIP3, TNF, ICAM1 and CXCL2 gene expression in PBMCs from Trios are shown in
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019900574 | Feb 2019 | AU | national |
| 2019900607 | Feb 2019 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2020/050155 | 2/21/2020 | WO | 00 |
