Biopharmaceuticals Lecture 12 - Interferons PDF

Biopharmaceuti cal Science Biopharmaceuticals  Biopharmaceuticals Cytokines...

Biopharmaceuti cal Science Biopharmaceuticals  Biopharmaceuticals Cytokines Term coined in 1974 by scientist Stanley Cohen. Describes group of regulatory proteins secreted by hematopoietic (stems cells that develop into blood cells) and non-hematopoietic cells. Modulate the innate and adaptive immune systems. Multifunctional: immune system development, cell growth and differentiation, repair, the inflammatory cascade. Cytokines include: Interferons Interleukins Growth factors Kany, S., Tilmann Vollrath, J. and Relja, B. (no date) ‘Molecular Sciences Review Cytokines in Inflammatory Disease’. doi: 10.3390/ijms20236008. Chemokines Tumour necrosis factor alpha (TNF-alpha) and transforming growth factors alpha, beta and gamma (TGF-alpha, beta, gamma). 2 Biopharmaceuticals Interferons Family of glycosylated and non-glycosylated proteins. Part of the cytokine family. Molecular weight range 16.8 kDa to 22.1 kDa. Proteins produced by eukaryotic cells in response to: Viruses, parasites, bacteria and tumours Allows for communication between cells to trigger the protective defences of the immune system that eliminates pathogens or tumours. Are named after their ability to "interfere" with viral replication within host cells. 3 Biopharmaceut Induce variety of signal transduction pathways to illicit variety of responses icals including: Antiviral response. Inflammatory response. Inhibition or stimulation of cell growth. Initiation or inhibition of apoptosis. Modulation of immune response. 4 Biopharmaceuticals Interferons have other functions 1 2 3 4 Activate immune Increase recognition Increase the ability Regulate growth + cells, such as natural of infection or tumor of uninfected host differentiation of killer cells and cells by up- cells to resist new cells. macrophages regulating antigen infection by virus. Sustenance of presentation to T pregnancy in some lymphocytes animal species 5 Biopharmaceuticals Type I IFN 13 different IFN- α isoforms. Bind to common cell surface receptor: Type 1 IFN receptor (IFNR). However, act on different cell types, elicit different downstream responses. IFN-α 1 and IFN- α 13 are identical proteins. Also IFN-β, IFN-ε, IFN-κ and IFN- ω. Can all establish an anti-viral state in other cells. Involved in anti-microbial response. 6 Biopharmaceuticals Type II IFN IFN γ (immune interferon) - only one member of the family (IFN-γ 1). Essential for cell-mediated immune response. Produced by NK-cells, dendtritic cells, cytotoxic T-cells, progenitor Th0 cell and Th1 cells. Binds to IFN γ receptor (IFNGR) as a dimer. Type III IFN IFN lambda. 4 members of the family (IFN-lambda 1-4) Not well studied. IFN-α 2, IFN-β, IFN- γ most studied IFNs 7 Biopharmaceutica ls Interferon-α Produced - lymphocytes, monocytes, macrophages. Assumed to be the produce of one gene In humans at least 24 genes or pseudo genes exist that code for the production of 14 distinct mature IFN-α. Assigned to two families – type I and II Thirteen type I IFN-α and One type II IFN-α Mature type I IFN-α (166 amino acids) Mature type II IFN-α (172 amino acids) 8 Biopharmaceuticals All initially synthesised containing an additional 23 amino acid signal molecule. Isoelectric point is between 5 – 6.5. Individual IFN-α s exhibit in excess of 70% amino acid homology. Individual members each have identifying names. IFN-αA, IFN-αB etc. Some exceptions – may contain a number or a number + letter - IFN-α7, IFN-α2B. Several known by two different names. 9  Biopharmaceuticals 1 7 17 27 MALTFALLVA LLVLSCKSSC SVGCDLPQTH SLGSRRTLML LAQMRKISLF 37 47 57 67 77 SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD 87 97 107 117 127 ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT 137 147 157 165 LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE Signal sequence Human IFN-α2 O-Linked Glycosylation on T129 Mature peptide: 165 aa IFN alpha 2a K23 19.2 kDa IFN alpha 2b R23 pI 5.97 10 Biopharmaceuti Interferon- Produced in Fibroblasts. cals First to be purified. Humans synthesis one type of IFN-. Contains 166 amino acids. Exhibits 30% sequence homology to IFN-α. Mature molecule is a glycoprotein – carbohydrate chain is linked at amino acid 80 (asparagine) via a glycosidic bond. 11  Biopharmaceuticals 1 9 19 29 MTNKCLLQIA LLLCFSTTAL SMSYNLLGFL QRSSNFQCQK LLWQLNGRLE 39 49 59 69 79 YCLKDRMNFD IPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW 89 99 109 119 129 NETIVENLLA NVYHQINHLK TVLEEKLEKE DFTRGKLMSS LHLKRYYGRI 139 149 159 166 LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRN Signal sequence IFN-beta 1a C17; IFN-beta 1b S17 Human IFN-β Natural phosphorylation Y3 Mature peptide: 166 aa N-linked glycosylation site N80 20 kDa pI 8.93 12 Biopharmaceuti Interferon-γ Referred to as ‘immune’ interferon. cals Produced predominately by NK + T lymphocytes. Regulates all phases of immune and inflammatory responses, including the activation, growth and differentiation of T-cells, B-cells, macrophages, NK cells and other cell types such as endothelial cells and fibroblasts. It enhances MHC expression on antigen-presenting cells. A single IFN-γ gene exists – located on human chromosome 12. Mature IFN-γ - 143 amino acids. Glycosylated in three positions. 13 Biopharmaceuticals Interferon-γ protein Primarily a helical structure. Six helices in total. Comprise approximately 62% of the structure. No beta sheet. Dimeric in solution. 14  Biopharmaceuticals 1 7 17 27 MKYTSYILAF QLCIVLGSLG CYCQDPYVKE AENLKKYFNA GHSDVADNGT 37 47 57 67 77 LFLGILKNWK EESDRKIMQS QIVSFYFKLF KNFKDDQSIQ KSVETIKEDM 87 97 107 117 127 NVKFFNSNKK KRDDFEKLTN YSVTDLNVQR KAIHELIQVM AELSPAAKTG 137 143 KRKRSQMLFR GRRASQ Signal sequence and pro-peptide addition Human IFN-γ N-linked glycosylation site N25 and N97 Mature peptide: 138 aa Proteolytic processing sites G127, M134, G138 16.2 kDa pI 9.52 Dimer 15 Biopharmaceuticals Interferon Biotechnology Antiviral, anti proliferation, modulation of the immune system + anti -inflammatory responses thus potential medical applications. Approval for clinical use of several interferon's preparations. Expanding their uses by ongoing clinical trials. Medical Applications of interferon's goes back to the 1950s. However rendered impractical due to the low levels produced by the body. Large purification from blood non-viable. 16 Biopharmaceuticals ‘Interferon’ preparations consisted of a mixture of IFNs. Produced in the late 1970s by means of mammalian cell culture – especially cancer cell lines. Namalwa cell line - major industrial source (all IFNs present). Recombinant technology – production of IFNs in large quantities. Yielded a product to contain only one type of IFN. Produced in various expression systems E.Coli, fungi, yeast and some mammalian (CHO). Mostly produced in E.Coli. E.coli’s inability to carry out post translational modifications – glycosylation is irrelevant in the case of IFNα’s - as these are not glycosylated. However IFN-β and IFN-γ are normally glycosylated. 17  Interferon Alpha-2 Therapeutics Brand Name Interferon type Manufacturer Clinical Indication Besremi Ropeginterferon alpha 2b AOP Orphan Polycythaemia vera Pharmaceuticals IntronA Recombinant interferon MSD Chronic hepatitis B and C, hairy cell alpha 2b leukaemia, chronic myelogenous leukaemia, multiple myeloma, follicular lymphoma, carcinoid tumour ViraferonPeg Peginterferon alpha 2b Schering Plough Chronic hepatitis C PegIntron Peginterferon alpha 2b MSD Chronic hepatitis C Pegasys Peginterferon alpha 2a Roche Chronic hepatitis B and C 18 Biopharmaceuticals Besremi - Ropeginterferon-α2b Produced by AOP Orphan Pharmaceuticals. Orphan drug treatment for polycythaemia vera: Rare blood disease caused by mutation in JAK2 gene coding for JAK2 kinase enzyme. Over-production of red blood cells which causes blood thickening and can lead to blood clots. Can cause myelofibrosis which is scar tissue development in the bone marrow. Pegylated, recombinant interferon-α2b with additional proline residue at N-terminus. 166 aa; 19 kDa plus 40 kDa two-arm methoxypolyethylene glycol (mPEG) addition. Alpha-helical structure and 2 di-sulphide bonds as with other IFN-alpha proteins. mPEG 19 Biopharmaceuticals Ropeginterferon-α2b Mechanism of Action  Has inhibitory effect on the proliferation of hematopoietic and bone marrow fibroblast progenitor cells.  Acts as antagonist of growth factors and other cytokines involved in development of myelofibrosis.  Can decrease the mutated JAK2V617F allele burden in patients with polycythaemia vera i.e., reduce the proportion of mutated JAK2 alleles present in all patient blood cells. 20 Biopharmaceuticals ViraferonPeg - INF α Trade name ViraferonPeg. PEGylated form of Interferon α-2b. Approved for medical use in EU since 2000. Differs from human by the attachment of PEG. Indicated in the treatment of chronic Hep C. Administered in combination with the antiviral drug ribavirin. Produced in E.Coli cells carrying the Interferon α-2b gene. 21 Biopharmaceuti cals After fermentation – purified via crystallisation and multiple chromatography steps. Linked to chemically activated PEG – spontaneously forms a covalent link. Majority of IFNs are mono PEGylated. Product is lyophilised with sucrose, sodium phosphate and polysorbate as excipients. Administered once weekly s.c for a period of 6 months. Half life of 13-25 hours compared to unPEGylated molecule. 22 Biopharmaceuticals Normalizes liver function. Reduction in serum viral particle load. Combined with ribavirin it is more effective. Side effects include injection site reactions, weakness, weight loss, flu – like symptoms. Depression – being the most common reason for treatment discontinuation. Manufactured by Schering plough. 23  Interferon Beta-1 Therapeutics Brand Name Interferon type Manufacturer Clinical Indication Expression host Avonex IFN-beta1a Biogen Idec Ltd Multiple Sclerosis CHO (glycosylated) (MS) REBIF IFN-beta 1a Ares Serono Inc. Multiple Sclerosis CHO (glycosylated) (MS) Betaferon IFN-beta 1b Bayer AG Multiple Sclerosis E. coli (non- (Cys17Ser mutation) (MS) glycosylated) Extavia IFN-beta 1b Novartis Multiple Sclerosis E. coli (non- (Cys17Ser mutation) (MS) glycosylated) Plegridy Peg IFN-beta 1b Biogen Idec Ltd Multiple Sclerosis CHO (glycosylated (MS) and pegylated at N-terminus) 24 Biopharmaceuticals Rebif – INF β Trade name Rebif is an rIFN -1a approved in the EU (1998) and US (2002). Indicated in the treatment of relapsing MS - chronic disease of the nervous system. Delay the accumulation of physical disability. Produced by recombinant technology - CHO cells. Identically amino acid sequence as that of natural occurring IFN -1a – glycosylated. 25 Biopharmaceuti cals Purified after cell culture by a combination of chromatography steps. Formulated as a sterile product in prefilled syringes contain the excipients mannitol, HSA, sodium acetate, acetic acid and sodium hydroxide. Administered s.c – three times weekly, serum half life is 70 hours. Common side effects – injection site reactions and flu like symptoms + with severe potential side effects including depression. Manufactured by Ares Serono Inc. 26 Biopharmaceuticals Multiple Sclerosis (MS)  MS normally presents in young adults (more commonly women) aged 20-40 years.  Characterised by damage to the myelin sheath which surround neurons of the central nervous system.  Clinical presentations include shaky movement, muscle weakness, paralysis, defects in speech, vision and other mental functions.  Classified as an autoimmune disease – where elements of immunity cooperate in the destruction of the myelin sheath. 27  Interferon-Gamma Therapeutics Product Active substance Manufacturer Clinical Indication Expression host Actiimmune IFN-gamma1b Genentech Chronic E. coli (non- granulomatous glycosylated) disease and severe malignant osteoporosis 28 Biopharmaceuticals Actimmune – INF γ IFN γ-1b is a single-chain polypeptide containing 140 amino acids – non glycosylated Produced in genetically engineered E. Coli containing the human gene for IFN γ-1b Displays identical biological activity as the native form however its lacks the carbohydrate component. Sold in liquid form and manufactured by Genentech. Administered s.c three times weekly. 29 Biopharmaceuticals Used in the treatment of CGD – rare genetic condition with a population incidence of 1 in ¼ - 1 million. Phagocytic cells are poorly incapable / incapable of ingesting or destroying bacteria or protozoa. Results in patients suffering from repeated infections – many life threatening. Phagocytes from health individuals capable of producing substances such as hydrogen peroxide or hypochlorous acid – lethal to pathogens. 30 Biopharmaceuticals Traditional treatment include – prophylactic administration of antimicrobial agents to prevent occurrence of infection. Long term treatment with IFN γ-1b has proven effective in the treatment of this disease. Induces phagocytes to generate toxic products – promoting increased synthesis of these substances Also promotes increased expression of IgG Fc receptors on phagocytes - increase the phagocytes ability to destroy infectious agents. 31 Biopharmaceuticals PEGylation of IFNs Chemically activated PEG (methoxypolyethylene glycol, mPEG) Incubated with purified IFNs. Spontaneously forms a covalent linkage via selected protein amino acid groups. Function is to increase half-life of IFNs Increased half-life from 4 h to 13-25 h. mPEG 32 Biopharmaceuticals Interferon Toxicity Like most drugs, administration of Interferon's can elicit a number of unwanted side effects. Include the following: Range from flu like symptoms fever, headache, chills, hypersensitivity. Strong fatigue, insomnia, autoimmune reactions, cardiovascular complications, anorexia. Depression with rare instances promoting suicidal thoughts. 33