Lecture 10 - Biopharmaceuticals (Interlukins) 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: Interleukins (ILs) Interferons (IFs) Tumour necrosis factor alpha (TNF-) Kany, S., Tilmann Vollrath, J. and Relja, B. (no date) ‘Molecular Sciences Review Cytokines in Inflammatory Disease’. doi: 10.3390/ijms20236008. Growth factors (EGF, IGF) Chemokines Transforming growth factors alpha, beta and gamma (TGF- ,,) 2 Biopharmaceuticals Cytokine Signaling Pathway 1 Signal through binding to transmembrane receptors. Low affinity ligand-binding domain thus differentiating cytokines to ensure specificity. High-affinity effector domain thus activating target genes through intracellular signaling. 3 4 Biopharmaceuticals Cytokine Signaling Pathway 2 Cytokines can act in several different ways: Synergy - action together to induce a different response than either can induce alone. Antagonism - cytokines can counteract one another. Pleiotropy - action in a similar way on more than one “target” cell. Redundancy - more than one cytokine triggers identical or similar responses in each “target” cell. Pathway activation - triggered sequential induction or “cascade”. 5 Biopharmaceuticals Interleukins Interleukins represent a large group of cytokines with MWs from 15 to 30kDa. 33 different members (IL 1 to IL 33) which are mostly glycosylated except for IL-1. Produced by several different cells with a least 17 different cell types capable of producing IL -1. Soluble molecules that bind to specific receptors on the cell surface of target cells. Mostly exhibiting paracrine activity with some exhibiting autocrine activity. 6 Biopharmaceuticals  Primary function is to promote growth and differentiation of hematopoietic and immune cells.  Includes normal and malignant growth of cells.  Involved in all aspects of immune responses.  Regulation of inflammation.  Also regulates hematopoiesis, tissue remodeling and embryonic development.  As with Interferons, the advent of recombinant DNA technology facilitates production of these molecules in sufficient quantities to meet actual and potential needs. 7 Biopharmaceuticals IL – 1 Known as Lymphocyte activating factor (LAF) with two distinct form IL-1 α and IL-1 β. Both bind to the same receptor and induce similar biological activity however they only have 20% amino acid homology and are the product of different genes on human chromosome number 2. Both are secreted as a precursor molecules from which the mature polypeptide is released by proteolytic cleavage. Neither of these interleukins are glycosylated. 8 Biopharmaceuticals IL-1 α is secreted as a 271 amino acid precursor and when cleaved to its mature form it consists of 159 amino acids. IL -1 β is secreted as a 229 amino acid precursor and when cleaved to its mature form it consists of 153 amino acids. Both proteins are globular molecules, composed of six strands of antiparallel Beta sheets. A wide variety of cells product IL-1 such as endothelial cells, fibroblasts, monocytes, keratinocytes etc and it induces a wide variety of biological activities. It activates B-lymphocytes and may also play a role in the activation of T- lymphocytes. 9 Biopharmaceuticals IL -1 is a pro-inflammatory cytokine promoting the synthesis of a wide variety of enzymes such as proteases which are involved in generating inflammatory mediators, having paracrine activity at low levels of secretion. At high levels of secretion, it acts more in an endocrine manner, inducing fever and general body wasting which can be associated with some cancers. Many of these biological effects can also be associated with TNF. In addition to these interleukins, another third like IL-1 protein has been identified, termed the IL-1 antagonist which is capable of binding to the IL-1 receptor without triggering intracellular responses. 10 Biopharmaceuticals Human IL – 1 receptor Antagonist Member of the IL-1 family. Competitive inhibitor of IL-1 receptor. Competes for binding with IL-1alpha and IL- 1beta. Binds to the IL-1 receptor and prevents its dimerization with its co-receptor IL-1 receptor accessory proteins (alpha and beta) and prevents signaling. IL-1 alpha and beta are pro-inflammatory cytokine therefore IL-1 receptor antagonist suppresses this. 11 Biopharmaceuticals 5 15 25 MEICRGLRSH LITLLLFLFH SETICRPSGR KSSKMQAFRI WDVNQKTFYL 35 45 55 65 75 RNNQLVAGYL QGPNVNLEEK IDVVPIEPHA LFLGIHGGKM CLSCVKSGDE 85 95 105 115 125 TRLQLEAVNI TDLSENRKQD KRFAFIRSDS GPTTSFESAA CPGWFLCTAM 135 145 152 EADQPVSLTN MPDEGVMVTK FYFQEDE Human IL-1 – Receptor Antagonist Mature peptide 152 aa, 17.1 kDa, Signal sequence pI 5.42 C66-C116 Di-sulphide bond N-linked glycosylation N84 12 Biopharmaceuticals Because of IL-1 role in mediating acute chronic inflammation, downward modulation of IL-1 levels may prove effective in elevating the severity of these conditions such as rheumatoid arthritis. Several approaches may prove useful in this regards including the following: Administration of anti-IL-1 antibodies. Administration of soluble forms of the IL-1 receptor. Administration of the native form of the IL-1 receptor antagonist. 13 Biopharmaceuticals Kineret (anakinra) IL– 1 receptor Antagonist Manufactured by Swedish Orphan Biovitrum AB. Recombinant production in E. coli and is non-glycosylated in comparison to the native form which is glycosylated. Additional M residue at N-terminus (153 aa) which is a consequence of the prokaryotic expression system. Indicated for the treatment of moderate to severe rheumatoid arthritis. Slows progression and structural damage. Patients with rheumatoid arthritis have elevated IL-1 levels. 14 Biopharmaceuticals Purified product is presented as a solution containing sodium citrate, EDTA, sodium chloride and polysorbate as excipients. A daily injection of 100mg is recommended for patients with rheumatoid arthritis where this inflammatory condition is associated with high levels of IL-1 in synovial fluid of affected joints. 15 Biopharmaceuticals IL-2 IL-2, also known as T-cell growth factor, represents the most studied member of the interleukins family. First T-cell growth factor to be identified and it plays a central role in the immune response. Produced by T-cells in response to: Antigenic and mitogenic stimulation. Required for T-cell proliferation and other activities crucial to regulation of the immune response. Binds to several important immune cells and promote growth and differentiation in these cells: B-cells, monocytes, lymphokine-activated killer cells, natural killer cells, and glioma cells. 16 Biopharmaceuticals IL- 2 is a single chain polypeptide containing 133 amino acids. Glycoprotein with the carbohydrate component being attached via an O link glycosidic bond to threonine residue 3. Mature molecule has a molecular mass ranging from 15 – 20kDa –depending upon the extent of glycosylation. Carbohydrate chain is not required for biological activity. 17  Biopharmaceuticals 10 20 30 40 50 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN 60 70 80 90 100 YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL 110 120 130 140 150 RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT Human IL -2 Mature peptide 133aa AA 1 to 20: Signal sequence 15.4 kDa T23: O-linked glycosylation pI 7.05 C78-C125 di-sulphide bond 18 Biopharmaceuticals X-Ray diffraction analysis shows the protein to be a globular structure consisting of four alpha-helices stretches interrupted by bends and loops. It is devoid of beta conformation and contains a single di-sulphide linkage involving cysteine numbers 78 and 125. IL- 2 induces its characteristic biological activities by binding to a specific receptor on the surface of sensitive cells. The receptor consist of of three membrane spanning polypeptide chains - alpha, beta and gamma. 19  Biopharmaceuticals IL – 2 receptor Alpha and beta subunit bind IL-2 with low affinity. Alpha, beta, gamma trimer binds IL-2 with high affinity. 20  IL-2 receptor is associated with several cell types, mainly cells playing a central role in the immune response. Binding of IL-2 to the receptor induces growth and differentiation of these cells which include T-lymphocytes, NK cells, monocytes, macrophages etc. Biopharmaceutic als This cytokine behaves as a central molecular switch activating most aspects of the immune response. T-lymphocytes are stimulated largely by directly binding to an antigen fragment present on the surface of macrophages and this results in the stimulation of approx. 70 genes whose products are collectively important in immune stimulation 21 Biopharmaceuticals) Proleukin (Aldesleukin) As is the case with other Cytokines, medical appraisal/use of IL-2 was initially impractical due to the minute quantities in which it is normally produced. Recombinant production of IL-2 is now possible. Produced by Clinigen and approved for use in the US. Recombinant human IL-2 produced in E. coli. Non-glycosylated but this does not affect function. Lacks N-terminal alanine residue and has C125S substitution. Indicated for the treatment of adults with metastatic melanoma (mM) (since 1992 in US) and metastatic renal cell carcinoma (mRCC) (since 1992 in US). 22 Biopharmaceuti cals Displays typical IL-2 biological activities: Enhancement of lymphocyte mitogenesis and cytotoxicity. Induction of lymphocyte- activated (LAK)/ natural-killer (NK) cell activity. Induction of IFN-γ production. 23 Biopharmaceuti cals After extraction and chromatography purification, the product is formulated in phosphate buffer containing mannitol and low levels of the detergent SDS. Product displays IL-2 biological activities including the enhancement of lymphocyte mitogenesis and cytotoxicity. 24  The immunostimulatory activity of IL-2 has proven beneficial in the treatment of some cancer types. It is an effective anticancer agent and there exists direct evidence that the immune system mounts an immune response against most cancer types. Virtually all transformed cells (cancerous cells) express a novel surface Biopharmaceutic antigen not expressed by normal cells or expressed at greatly elevated levels and these antigens may have gone unnoticed by immune surveillance. als It has been demonstrated that both in-vitro and in-vivo, that immunostimulation can lead to enhanced tumour detection. It has been shown in early experiments that lymphocytes incubated with IL-2 could subsequently kill a range of cultured cancer cell lines, including myeloma and colon cancer cells. 25 Biopharmaceuticals IL – 11 IL-11 is an additional cytokine that has gained approval for general medical use. This cytokine is produced largely by IL-1 activated bone marrow cells and functions as a haemopoietic growth factor. Stimulates the production of platelets and growth and differentiation of bone marrow cells. It is a 23kDa polypeptide with 178 amino acids and it binds to a transmembrane receptor. 26  The rationale for assessing IL-11 as a potential therapeutic agent relates mainly to its ability to induce platelet synthesis. Platelet counts can be significantly low in certain conditions especially in patients undoing cancer chemotherapy. Biopharmaceutic Patients receiving IL-11 at 25ug per Kg of als body weight per day did see an increase in the production of platelets which clearly offset the chemotherapeutic effects. IL-11 promotes the proliferation of hepatocytes in response to liver damage. 27  Biopharmaceuticals 10 20 30 MNCVCRLVLV VLSLWPDTAV APGPPPGPPR VSPDPRAELD STVLLTRSLL 40 50 60 70 80 ADTRQLAAQL RDKFPADGDH NLDSLPTLAM SAGALGALQL PGVLTRLRAD 90 100 110 120 130 LLSYLRHVQW LRRAGGSSLK TLEPELGTLQ ARLDRLLRRL QLLMSRLALP 140 150 160 170 QPPPDPPAPP LAPPSSAWGG IRAAHAILGG LHLTLDWAVR GLLLLKTRL Human IL -11 Mature peptide 178 aa AA 1 to 21: Signal sequence 19.1 kDa L182 to R190 important for IL11RA pI 11.1 interaction 28  Neumega (Oprelevkin)  Produced by Pfizer.  Recombinant IL-11, which is produced in E. coli.  19 kDa and non-glycosylated. Biopharmaceutic  177 amino acid lacking N-terminal proline. als  Comparable activity to natural IL-11.  Indicated for prevention of severe thrombocytopenia and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in adult patients. 29  Biopharmaceutic als Presented in a freeze-dried format and excipients include phosphate buffer, salts and glycine. It is reconstituted with WFI to a concentration of 5mg/ml before it is administrated by s.c injection. 30