Appunti Drugs Notes PDF

L19-IL2 (darleukin) and the F16-IL2 (teleukin): in this way, IL-2 is bound to the variable portion of an antibody that is able to recognize a tumoral antigen in a specific way, so that the delivery of IL-2 will be specific (in this cases, these Abs are vascular targeting Abs) and it will induce an i...

L19-IL2 (darleukin) and the F16-IL2 (teleukin): in this way, IL-2 is bound to the variable portion of an antibody that is able to recognize a tumoral antigen in a specific way, so that the delivery of IL-2 will be specific (in this cases, these Abs are vascular targeting Abs) and it will induce an immune modulation on immune effector cells and stromal cells only in the site of the tumor. This expedient is much better than administering simple IL-2 without a specific target, which can cause really dangerous side effects, as we saw. Darleukin has already been tested in clinical studies in melanoma and pancreatic cancer. More recently, combination of Darleukin with other immune-cytokines or with chemotherapeutic drugs has shown potent synergistic activity in pre-clinical studies, and therefore the current clinical trial strategy is based on this concept. On the other hand, Teleukin has previously been tested in clinical studies in various solid cancers. More recently, Teleukin has shown the potential to enhance the therapeutic performance of other agents, including taxanes. We saw that this is the only way through which we can use IL-2 has a therapeutic agent without it causing multiple serious side effects. But if we change our point of view we could think of using IL-2 as a target, not as a therapy. In fact, when too much IL-2 causes damage, prevention of IL-2 is necessary. A high amount of IL-2 can be present in autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus,...), because we have an over-activation of the immune system so also IL- 2 will be produced in higher quantities, but we could also have a high level of IL-2 in organ transplantation, and we can block its production to avoid host vs graft complications. IL-2 can be blocked by administering soluble forms of the IL-2 receptor (IL-2R), we can administer a mAb towards the IL-2 receptor, or against IL-2 itself, or we could administer IL-2 variants that fail to initiate the signal transduction, or we could deliver IL-2 coupled to bacterial toxins. The last method that we mentioned can be explained through the example of Ontak (denileukin diftitox): it is a really effective drug against IL-2, it is a fusion protein produced in E. coli formed by A and T fragments of the diphtheria toxin and by IL-2. In this way, the toxin is brought in close proximity to the cell and it will bind to the cell-surface IL-2 receptor through the IL-2 portion; in this way it will be internalized through endocytosis and it will cause the death of the cell. In fact, the acidic pH in the endocytic compartment causes a conformational change that enables the translocation of the A chain of DT to the cytosol. Once in this compartment, DT modifies elongation factor 2 (EF2) by adenosine diphosphate (ADP)-ribosylation, which leads to inhibition of protein synthesis and cell death. Another important cytokine is IL-1, a family of proteins characterized by an array of 12 beta strands and by the absence of a classical secretory N-terminus peptide sequence. The two main components of this family are the IL-1alpha and the IL-1beta: the first one is generally associated with the plasma membrane of the producing cell and so it acts locally, it has a widespread production (in endothelial cells and keratinocytes), it is important in priming T cells during contact hypersensitivity and for the induction of high levels of serum IgE and its pro-domain has a nuclear localization sequence (nuclear IL- 1a has transcriptional transactivating activity); the second one is generally secreted and it circulates systemically, it is mainly produced by monocytes and macrophages and since it can circulate to the brain, it is important for the induction of fever. They are both non-glycosylated, so they could be potentially really easy to produce directly in E. coli, however therapeutically they cannot be used because they give the same problems of IL-2, so they are mainly used as targets. The two proteins signal through the same receptor complex and have identical biological activities in solution. Because of their potency and extensive functions, their activity is tightly regulated (through mRNA induction, regulation of processing and secretion, expression of a receptor antagonist and of a decoy receptor). IL- 1 proteins are usually produced as longer peptides and then they undergo maturation through digestion with caspases (caspase 1, also called ICE, IL-1-converting enzyme). Stimulation of cells for the production of IL-1 leads to the production of inactive ICE which, when activated, cleaves inactive pro-IL- 1, which is released across the cell membrane in an activated mature form. In the case of IL-1alpha, calpain cleavage of active pro-IL-1 stimulates release of mature IL-1 across the cell membrane. Some clinical studies demonstrated that no significant anti-tumoral response was observed in the treatment of various cancer with IL-1, so they are actually more harmful than useful. So instead of studying them as biotechnological drugs they have been identified as targets: there are a lot of diseases where we have a major production of IL-1 and so by reducing its amount we can ameliorate the clinical severity of these disease, which are typically acute/chronic inflammatory diseases. Which are the functions of IL-1? It promotes the synthesis of eicosanoids and other inflammatory mediators, it activates B lymphocytes, and thus T cells, along with IL-6 it induces synthesis of acute- phase proteins in the liver, it induces the production of adhesion molecules in the endothelium and in fibroblasts, it acts as a co-stimulator of haematopoietic cell growth and differentiation and it stimulates the production of collagen from chondrocytes. Moreover, in the brain, it induces the raise of the body temperature. All of these activities correlate to an inflammatory state. To block these functions e could use the IL-1 receptor antagonist (IL-IRA), a protein that is able to bind to the same receptor as IL-1 but it doesn’t elicit any biological activity. A lot of studies have demonstrated that IL-1 is involved in a lot of pathologies, so we can consider it a sort of pathogenic molecule, because a lot of diseases with an inflammatory basis produce inflammatory mediators, of IL-1 is one of the main effectors. Some examples of pathologies in which IL- 1 is massively produced and so in which we can target it are sepsis syndrome, rheumatoid arthritis, inflammatory bowel disease, insulin-dependent diabetes mellitus, acute and chronic myelogenous leukemia and atherosclerosis. Other diseases include transplant rejection, graft vs host disease, psoriasis, asthma, osteoporosis, periodontal disease, autoimmune thyroiditis, alcoholic hepatitis, sleep disorder and premature labour secondary to uterine infection. Also neurological disorders can be included in the diseases in which IL-1 is implied: Alzheimer’s disease, Parkinson disease, epilepsy, stroke, … they all could present an increased expression of IL-1. In general, any pathology which presents an inflammatory state will present the production of IL-1. One inhibitor of IL-1 is called Anakinra: it is a human IL-1 receptor antagonist and is produced by recombinant DNA technology. It is non-glycosylated and is made up of 153 amino acids. With the exception of an additional methionine residue, it is similar to native human IL-1 receptor antagonist. This endogenous IL-1 receptor antagonist is a 17-kDa protein which competes with IL-1 for receptor binding and blocks the activity of IL-1. Anakinra (commercially called Kineret) is recommended for the treatment of severely active rheumatoid arthritis for patients 18 years of age or older. It is recommended for patients who have not responded well previously to the disease-modifying antirheumatic drugs. It reduces inflammation, decreases bone and cartilage damage and attacks active rheumatoid arthritis. The most serious side effects of Kineret are infections and neutropenia; other side effects may include headache, nausea, diarrhea, flu-like symptoms and abdominal pain. An increased risk of malignancies has also been observed. IL-1 in rheumatoid arthritis activates monocytes and macrophages causing the activation of the inflammatory process, it induces the fibroblast proliferation causing synovial pannus formation, it activates chondrocytes causing cartilage breakdown and it activates osteoclasts inducing bone resorption. Other IL-1 antagonist molecules for the treatment of rheumatoid arthritis are under development. IL-11 is the only interleukin which is produced with a therapeutic purpose, as it is useful in the bone marrow for the formation of platelets. It is not involved in inflammatory processes, so it does not cause the serious side effects that the other interleukins induce. It is usually produces by IL-1-actiavted bone marrow stromal cells and fibroblasts and it stimulates thrombopoiesis by inducing growth and differentiation of bone marrow cells. Commercially it is available under the name of Oprelvekin and it is mainly used in patients undergoing chemotherapy to stimulate platelet synthesis. Another drug used against interleukins is the Ustekinumab, used against IL-12/23. These cytokines are involved in the production of specific T cells that are implied in the inflammatory process of psoriasis. They usually bind to their specific receptor, activating T cells and inducing their differentiation into Th17 cells: these types of cells are a subtype of T cells that activate in some autoimmune diseases, like in psoriasis, inflammatory bowel diseases and colitis ulcers. The drug that we use is, as the name suggests, a monoclonal antibody and it is currently used in the treatment of psoriasis under the tradename of Stelara because of its ability to prevent the binding of the natural ligand to its receptor by attaching to the interleukins. Anther mAb that acts against an interleukin is tocilizumab, which isa humanized antibody able to bind to the IL-6 receptor and thus to prevent the binding of this interleukin to it. It is used in rheumatoid arthritis (especially in cases of RA resistant to other therapies), where IL-6 has a pathogenic role: it is involved in the control of the balance between autoimmunity and self-tolerance, favouring autoimmunity, in fact it usually stimulates the production of Th17 cells over Tregs (sentinel cells that prevent the hyperactivation of T cells, especially regulating Th17 cells, so these two populations of cells must always be balanced in number, if that doesn’t happen Tregs are not able to control Th17 cells anymore and so we have the development of autoimmune diseases). The side effects of this mAb therapy are infusion related reactions (flushing, headaches, fever, nausea and fatigue). The last cytokine against which we can act with biotechnological drugs is TNF (tumor necrosis factor), which is involved in a lot of autoimmune pathologies. We have two types of TNF, the alpha and the beta; it has been discovered in vitro as an anti-tumor molecule, that’s why it is called tumor necrosis factor, in fact it was also used for the treatment of some tumors. It is a pro-inflammatory cytokine involved in the activation of elements of both non-specific and specific immunity (macrophages, neutrophils and granulocytes), in the induction and regulation of inflammation (synthesis of IL-1, IL-6, IL-8; activation of neutrophils, expression of adhesion molecules, chemotaxis, synthesis of PGE and proteins of the acute phase), but it also presents a selective cytotoxicity against a range of tumoral cells and it mediates various pathological conditions like septic shock, cachexia and anorexia. TNF is known to mediate the symptoms of many diseases, like cancer, septic shock, rheumatoid arthritis and diabetes. However, the use of TNF as an anti-tumoral agent has failed, it can be used only in vitro because in vivo it causes also important side effect because its anti-tumoral action is not its only function. The only drug that is based on TNF that can be used in vivo is Beromun, a human TNF-alpha produced in E. coli identical to the native human protein that is used against soft tissue sarcoma to prevent necrosis and amputation of limbs. Side effects include nausea, liver toxicity, and locally (limb) oedema and infection. In every other case, TNF is used as a target, and we have come up with two strategies: first, we know that TNF has its own receptor (actually, we have one for TNF-alpha and one for TNF-beta), so we can produce its external portion as a recombinant soluble protein able to bind TNF and prevent its binding to the actual receptor, otherwise we can use mAbs. The mAbs against TNF are some of the oldest mAbs produced as biotechnological drugs. The soluble receptor drug is called Etanercept and it is a recombinant fusion protein made of a soluble version of TNFII bound to the Fc region of an IgG1 (actually it is made of two molecules of receptor because naturally the TNF receptor dimerizes to start the signal so in order to bind TNF it has to be dimerized), so the structure is actually pretty similar to an antibody but it is a recombinant protein. Infliximab (also called Remicade) is instead a mAb, a chimeric one (mouse-human) directed against TNF, it was the first chimeric antibody ever produced and it is still used in therapy; adalimumab (also called Humira) is a fully human mAb anti-TNF, it was the first totally human antibody ever produced; certolizumab pegol (also called Cimzia) is a PEGylated molecule constituted by Fab fragments, kept together by a linker peptide, of a fully human anti-TNF Ab; finally we have golimumab, a fully human mAb anti-TNF. How do we choose which drug to use? It depends on how the patient responds to the therapy, on their half-life (infliximab has an half-life of 7 to 9 days, adalimumab of 10 to 20 days, certolizumab pegol of 2 weeks) and also on how many times they need to be injected per month (for  example infliximab can be injected every 2 months while etanercept every month and adalimumab every week: this could be a problem for the patient, the lower is the frequency of injection per month the more compatible the therapy is for the life-style of the patients). Certolizumab pegol is a singular antibody: first of all it is PEGylated, to prolong its half-life (which, without PEGylation, would be much lower because it is a smaller antibody compared, for example, to adalimumab, so it would be digested in a faster way) and reduce its antigenicity, moreover recent studies have assessed that this mAb is not able to pass the placenta so it can also be used on pregnant women. In vitro, this antibody didn’t induce monocytes or lymphocytes apoptosis, complement activation or ADCC. In addition, along adalimumab, it is administered sub-cutaneously, which is a much easier administration route compared to the intravenous one of infliximab, because the patient can perform it even by himself without having to go to the hospital. Another particular aspect of mAb compared to the recombinant protein (Etanercept) is that antibodies (with the exception of certolizumab pegol) can form complexes because they have two Fab portions that can bind to two different molecules of TNF, meanwhile the recombinant protein only binds to a single molecule: the formation of immunocomplexes induces a higher immunogenicity, a higher clearance (through phagocytosis) and higher probability to have Fc-mediated effects. However, antibodies are able to bind to both the soluble and the membrane-bound TNF-alpha, while the recombinant proteins only binds to the soluble TNF. The ant-TNF drugs are used in inflammatory bowel diseases (like Chron’s disease), spondyloarthropathies (like psoriasis), juvenile rheumatoid arthritis and Adult Still’s disease (diseases against which they have a confirmed efficacy); we are now trying to use them also against vasculitis, scleroderma, graft vs host disease, inflammatory myositis, interstitial lung disease, Sjögren’s syndrome, inflammatory eye and ear disease, asthma, hepatitis C and sarcoidosis. Which are the disadvantages of these therapies? If TNF is removed from our body in pathogenic conditions, the pathology is reduced, but if we remove it in physiological conditions we could have some complications, like the reactivation of some infectious diseases (like Hepatitis B and tuberculosis), because TNF is produced in our body against infectious agents so if we have latent infections, we could make them reactivate if we deprive our body of TNF. Sometimes we could also have the reactivation not only of infectious diseases but also of tumors, like skin cancer. We could also have the development of autoimmune diseases (Lupus-like syndrome), demyelinating disorders, congestive heart failure and liver toxicity. Other side effects could be related to the immunogenicity of the drug that we use, especially with mAbs, and with the fact that if we interrupt the therapy, we could have the reoccurrence of the symptoms. Moreover, other disadvantages are related to the costs of the therapy (etanercept costs 15 000 euros per year per patient). [N.B. in this paragraph we talked about drugs used against autoimmune diseases but we have to remember that they cannot cure the disease, instead they are just able to ameliorate the symptoms, because autoimmune diseases are chronic: this is because they are due to genetic abnormalities which cannot be changed and so we cannot fully recover from an autoimmune disease] GROWTH FACTORS A lot of growth factors (GFs) are produced by our body (gamma-INF is also considered a growth factor because it promotes the clonal expansion of T cells), of these we have two specific haemopoietic growth factors which are erythropoietin and thrombopoietin, all the other growth factors have -GF as a suffix. We have a lot of growth factors that have been approved for medical use, of these the most important are G-CSF (granulocyte growth factors), GM-CSF (granulocyte-monocyte growth factor), platelet derived growth factor (PDGF); recently the epidermal growth factor (EGF) has been approved for the treatment of chronic wounds. Growth factors are really important for haemopoiesis, because they regulate the growth and the differentiation of the cell of the bone marrow. They are also used in vitro for the same process, so to obtain the growth of specific cell lineages. Which types of growth factors can we use for therapeutic purposes (obviously, excluding interleukins, which we already talked about)? We can use colony stimulating factors (CSF, like the G-CSF, M-CSF and GM-CSF), erythropoietin (for the production of red cells) and thrombopoietin (for the production of platelets). 1. CSF: they promote the differentiation and activation of haemopoietic progenitor cells. We have some conditions in which we have to use these molecules to stimulate the growing of the bone marrow cells: in the case of neutropenia we generally tend to use GM-CSF (or M-CSF or G-CSF), especially in cases of cancer patients treated with chemotherapy (all cells of the bone marrow can be attacked by chemotherapics so it is important to stimulate the production of granulocytes to avoid the death of the patient by bland infections); in the case of infectious diseases (especially the G-CSF), because granulocytes act against infective agents so we need to stimulate an increased production of these cells; in the case of bone marrow transplantation; in some forms of cancer (like acute leukemias and lymphomas). Neutropenia is, out of these conditions, the one were CSFs are generally used the most, and it could be caused by, as we said, treatments with chemotherapics, genetics, sever bacterial or viral infections, lukemias, lymphomas and autoimmune causes. The recombinant G-CSF is commercially called Filgrastim, it is a 175-amino-acid glycoprotein which differs from natural G-CSF due to lack of glycosylation and for the presence of an extra N-terminal methionine. Pegylated recombinant human G-CSF (peg-filgrastim) is also available. Since it doesn’t need glycosylation it can be produced in E. coli; it is administered either sub-cutaneously or intravenously (one infusion daily for several days) for the treatment of neutropenia caused by cancer chemotherapy. The only adverse effects that it presents are bone and muscle pain and local cutaneous reactions, so it is well tolerated and it is very helpful in reducing the morbidity and mortality rate associated with chemotherapy, possibly permitting higher doses and a greater antitumor response. Filgrastim is also used after autologous stem cell transplantation to treat neutropenia (it reduces the duration of neutropenia and lessens morbidity secondary to bacterial and fungal infections), for the treatment of severe congenital neutropenia, of neutropenia in patients with AIDS resulting from treatment with zidovudine and of patients donating peripheral blood stem cells for stem cell transplantation. The half-life of the normal Filgastrim is around 3.5 hours, while the PEGylated form can reach up to 80 hours of half-life (from 15 to 80 hours). For the same purposes we could also use the recombinant GM-CSF (Sagramostim), but Filgastrim is the most used (also because of its long half-life, since the one of the recombinant GM-CSF is of 60 minutes). In February 2019 a biosimilar for Pegfilgrastim has been approved (Pelgraz), so this means that it has been commercialized more than 20 years ago for the first time (one of the oldest biotech drug; the patent of an original drug expires after 20 years and then we can produce biosimilars). 2. Erythropoietin (EPO): it stimulates the growth of erythrocytes. It usually increases as a result of a negative feedback loop induced by low oxygen pressure in the blood, it is produced by kidneys (by peritubular cells) and by the liver (In the foetus only in the liver). Technically it is a cytokine but it’s role is just to induce the proliferation and the differentiation of red cells; an absence of EPO results in apoptosis of erythroid committed cells (so renal failure or any other kidney disease results in anaemia). Erythropoietin presents its own receptors (transmembrane proteins of the cytokine receptor superfamily, the biding of the ligand induces to the dimerization of the receptor and this leads to the activation of its tyrosine kinase activity) which are expressed by the cell in the stage of the BFU-E (bus forming unit); once these receptors have bound erythropoietin they induce the expression of a larger number of EPO receptor on the cells surface (stage of colony forming cells of the erythroid lineage) so EPO can bind to it and induce its proliferation and differentiation. The three stages of erythrocytes formation that are dependent on EPO are mature BFU, CFU and proerythroblasts, all the other cells do not express the EPO receptor. Erythropoietin also stimulates the formation of haemoglobin and releases reticulocytes in the circulation. During anaemia, the synthesis of erythropoietin reaches up to 100-fold the normal production because we have lower levels of oxygen and that is sensed by the receptors present on the surface of the peritubular cells of the kidneys. Before the synthesis of the recombinant EPO, erythropoietin was extracted by the urine of anaemic patients (because they have more EPO than normal people), however purifying a substance from urine is very complex (urine is toxic, not sterile and it can allow the collection of only a small amount of the substance). In 1985 the gene encoding for EPO was isolated and the cytokine was produced in CHO cells. The recombinant human EPO is called Epoetin, it is administered intravenously or sub- cutaneously and it usually remains in the plasma for 6 to 10 hours. It is used in anaemia conditions caused by kidney problems, rheumatoid arthritis, some cancers, AIDS, infections and bone marrow transplantation (so conditions in which anaemia is not the primary pathology but it is a consequence of the primary defect). It is also used in cases of heart failure, transplants, cancer patient undergoing chemotherapy (for the same reason as neutropenia) and elderly/chronic pathologies that cause anaemia. The treatment with recombinant EPO allows fewer blood transfusions, improved exercise capacity, improved cognitive functions and better quality of life. Another use of recombinant EPO could be the doping, however it is illegal: EPO (or blood transfusion) is used in these cases to increase the blood count, increasing the availability of oxygen to the exercising muscle and improving aerobic capacity and muscle endurance. Detection of recombinant EPO in athletes is really difficult because it is really similar to the endogenous molecule. It has been banned in the early 1990’s as a PED. Which are the side effects of recombinant EPO? It can cause uncontrolled hypertension, known hypersensitivity, thrombotic events, seizures, allergic reactions, red cell aplasia, bone pain and hyper- viscosity syndrome of the blood (especially if used for doping). The vascular obstruction is due to the production of too many erythrocytes, and these thrombotic events could case strokes, heart failure or even death. We can have different types of recombinant EPO (Epoetins): we have 5 different erythropoiesis- stimulating agents currently available, and they are the epoetin alpha, epoetin beta, epoetin omega, epoetin delta and darbepoetin alpha (erythropoietin analogue, carrying two additional glycosylation sites which produce a longer half-life and potency). These different epoetins have been produced by different pharma companies. - Epoetin alpha: it was the first epoetin produced and it was produced by the American genetic research corporation under the name of Epogen. It was first produced in E. coli and then attempts in CHO have been made too. It has three glycosylation sites, like the endogenous molecule, so they are immunologically and biologically indistinguishable. - Epoetin beta: produced in 1988 in a German pharmaceutical company (that’s why it is different from the alpha one) and marketed as NeoRecormon. It has a similar clinical efficiency than epoetin alpha. It is produced in CHO because it must be glycosylated. - Epoetin delta is one of the newest agents currently available, commercially called Dynepo and produced from human cells, it act like the other epoetins and it resembles human EPO so much that it cannot be detected by standard urine tests, so it is of great concern for the sports world. - Darbepoetin alpha: it presents 5 glycosylation sites, so its half-life is really long. Moreover, it also presents a rearranged aminoacidic sequence and a larger molecular weight, so it is structurally different from the endogenous erythropoietin but also from the other epoetins. It has a high content of syalic acids, a prolonged serum clearance and a half-life of 24 hours, which compared to the 8.5 hours of half-life of the epoetin alpha, is much longer. We also have to mention another molecule, which is the CERA (continuous erythropoietin receptor activator), which is a “special” erythropoietin that presents a half-life of 135 hours. It is a PEGylated form of epoetin beta (contain a single methoxy polyethylene glycol polymer of 30 kDa), so its prolonged survival in the circulation is due to its large molecular mass (60 kDa) and low EPO receptor binding affinity. It is chemically synthesized.  Biosimilars of epoetins have been produced, since it was one of the first marketed recombinant protein and so its patent has expired. However, not every biosimilar of epoetins work: there have been studies where samples from Brazil, Colombia, India, Indonesia, Iran, Jordan, Korea, Lebanon, Philippines, Thailand, Venezuela, Vietnam, and Yemen have been tested against the European quality specifications for epoetin alpha, and they have pointed out how several biosimilar tested were found to be inconsistent in quality and potency and 26 out of 31 didn’t conform to all European specification for epoetin alpha. Some of them contained additional forms (basic isoforms), which reduce the clinical efficacy of the final product, moreover 2 samples contained endotoxins (safety concern) and 17 of them contained more than 2% of aggregates (concerns for immunogenicity). So, even if biosimilar versions of epoetin have been available in developing countries for many years and they are widely used for economic reasons, we have to be sure that the biosimilar that we produce is actually conform to all the safety rules of the European biosimilars, which are finely regulated. 3. Thrombopoietin (TPO): it is produced in the liver, it stimulates the production of platelets by megakaryocytes. There are some medical conditions in which platelets are not sufficiently produced and in general these conditions are all grouped under the term of thrombocytopenia (low platelet quantity); but we may need to produce platelets also for the transfusion (transfusion through blood is expensive and could lead to complications). TPO is able to induce the growth and the differentiation of the megakaryocyte progenitors and it is needed until we have the final production of platelets and their release in blood circulation from the megakaryocyte. We have two forms of recombinant TPO: one is the full molecule, it is a highly glycosylated protein, which for this reason is produced in mammalian cells, fully identical to the endogenous one; the other is a shorter PEGylated molecule made out of only the portion of the growth factor that binds to the receptor (PEGylated recombinant human megakaryocyte growth and development factor), which is produced in E. coli. The advantage of the second molecule is that it has a longer half-life because of the PEGylation. Another type of TPO for therapeutic use is the Romiplostin: it is a “peptibody”, which is made of 4 identical portions of 14 amino acids of the TPO protein bound to the Fc portion of an IgG4 molecule, kept together by disulphide bonds. It is able to bind easily to the receptor because it has a higher affinity to the receptor than the normal protein (the Fc portion is only used to create the molecule, it doesn’t have any therapeutical activity). It is used for treatment of thrombocytopenia in patients with chronic immune thrombocytopenia/idiopathic thrombocytopenic purpura (ITP) who have had an insufficient response to corticosteroids or immunoglobulins. Finally, we can say that thrombopoiesis can also be elicited by a chemical molecules, which is highly used, called Eltrombopag. Trials of a modified recombinant form, megakaryocyte growth and differentiation factor (MGDF), were stopped when healthy volunteers developed autoantibodies to endogenous TPO and then developed thrombocytopenia themselves. Romiplostim and Eltrombopag, structurally different compounds which stimulate the same pathway, are used instead. The potential clinical users of these biotech drugs are people that are affected by cancer and are undergoing chemotherapy (solid tumors or leukemia), have underwent a bone marrow transplantation, radiation therapy, anaplastic anemia or bone marrow failure states, immune thrombocytopenic purpura (ITP and thrombocytopenia of HIV, harvesting of peripheral blood progenitor cells and platelet apheresis. Side effects of thrombopoietin therapy imply thrombocytosis, thrombosis, marrow fibrosis, veno- occlusive disease, interaction with other growth factors. 4. Wound healing growth factors: a lot of growth factors generally cooperate to heal a wound alongside various types of cells (keratinocytes, fibroblast,...), but there are some pathological conditions in which this is not possible, like for example in diabetic patients, in patients affected by varicose ulcers, ulcerous cancers like the rodent ulcer, peptic ulcers and decubitus ulcers, so wounds due to continuous pressure on a particular area of the skin. The phases of wound healing involve coagulation, inflammation, migration and proliferation of the cellular component, angiogenesis, epithelization, contraction, fibroplasia and remodelling. The most important growth factors involved in this process are the FGF (fibroblast growth factor), TGF (transforming growth factor), PDGF (platelet-derived growth factor), IGF (insulin-like growth factor),  EGF (epidermal growth factor). They all stimulate the cell division and proliferation and they promote cell survival, the ones that are released by activated cells can stimulate the production of a number of different cells; they can promote cell migration, differentiation and tissue remodeling and be involved in different steps of wound healing. From clinical trials we were able to demonstrate that only one growth factors cannot be used for the treatment of wounds, if we don’t use a cocktail of growth factors, we will never obtain the healing of the wound. The only approved biotech drug for diabetic ulcers is Regranex, which is a recombinant platelet-derived growth factor, but we also have Kepivance, a drug made of a recombinant keratinocyte growth factor produced in E. coli, which decreases the incidence and duration of severe oral mucositis in patients with hematologic malignancies. Endogenous KGF is a member of the “fibroblast growth factor family” that triggers proliferation, differentiation and migration of epithelial cells; the recombinant molecule differs from native KGF because the first 23 N-terminal amino acids have been deleted, and this improves the stability of the product. Kepivance is even used in chemotherapy and radiotherapy treatment since it can reduce the damage to epithelial cells by thickening the epithelial layer and speeding up the healing process. The therapy with this growth factor implied an intravenous administration daily. RECOMBINANT BLOOD PRODUCTS 1. Coagulation factors: they are 13 factor and, with the exception of factor IV which is made of calcium atoms, they are all proteins. There are some diseases in which we have a lack of expression or an altered aminoacidic sequence of one of the clotting factors, and they can have serious clinical consequences, like prolonged haemorrhages. Up to 90% of these diseases are related to the deficiency of factor VIII and much of the remaining is due to deficiency of factor IX. Before the production of the recombinant missing factor, the therapy for these conditions was based on the administration of blood derived products extracted from donors, either of whole blood or of blood concentrates. The problem of this therapy is that we could possibly develop serious infectious diseases because we are dealing with blood of donors. Factor VIII is usually produced from the liver, it is able to bind to von Willebrand factor and get activated (so in healthy conditions both factor VIII and von Willebrand factor are needed for haemostasis). Conditions in which we have an alteration of this process are: haemophilia A (lack of factor VIII) and von Willebrand disease (lack of von Willebrand factor). Patients expressing 5% or above of the normal complex levels experience less severe clinical symptoms of these conditions and their coagulation cascade is almost normal. Lower percentages of these factor induce a really critical symptomatology. Haemophilia A affects 1 over 10 000 males in USA, and before the recombinant DNA technology, factor VIII for therapy was obtained from blood, however it was really difficult to use because 8000 pints (pint= half a litre) per year were needed for patient and the risk of transmission of infectious diseases, like HIV, was really high, and, in a period were the wide-spread screening for HIV was not available yet, a lot of haemophilic people unfortunately developed AIDS. Right now we don’t just have a recombinant factor VIII but we also dispose of a PEGylated form of the protein, which also misses of one domain (deletion of the B-domain), approved in January of 2018 by EMA. This form has a prolonged half-life because its degradation is delayed: normally factor VIII is degraded in the hepatocytes after the binding of a specific receptor present on the surface of these cells; the PEGylation avoids this binding, hence the degradation is slowed down. The cDNA coding for the human factor VIII is expressed in a variety of eukaryotic cells (CHO, BHK, …) because human FVIII must be glycosylated (it contain 25 potential glycosylation sites, so glycosylation is important for the function of the protein). The recombinant factor VIII is also called facto VIIIC (it means activated but not bound to von Willebrand factor), it has the same affinity as the endogenous one for von Willebrand factor, however some patient may develop antibodies against factor VIII. This is because these haemophilic patients need to be treated with this factor for their whole life, so they can easily develop an immune response against it even if it is human. If this happens, the factor will be bound to antibodies so it will not be able to act anymore and the therapy will stop being effective. In these cases we prefer to administer factor Xa or factor VIIa. Factor VIII is very sensitive to temperature, in fat room temperature could degrade the protein, hence, it needs to be produced and stored at 4°. A lot of studies are currently being carried out to try and improve factor VIII, for example with the deletion of B-domain, which can increase the half-life of the mRNA during the production phase of the protein, or with modifications that could increase the secretion of the product (this modification is, again, useful for the production process, because factor VIII is a really big molecule and really unstable so new approaches must be attempted when producing it to facilitate the procedure), its potency, resistance to inactivation, its half-life and reduce its immunogenicity. It has been proved that porcine factor VIII has a reduced immunogenicity compared to the human one (which is a paradox because it is of another species). The factor VIII deprived of the B-domain is constituted by two subunities linked together by a peptide linker of 14 amino acids, it is commercially called Refacto. We have a biotech drug also for the von Willebrand disease, called Vonvendi: it is a recombinant von Willebrand factor and it is used as an on-demand treatment no safety concerns were identified in the trials (the most common adverse reaction was generalized itching). Another type of haemophilia is the B haemophilia, caused by the lack of factor IX. Recombinant factor IX is called Benefix, it is produced in CHO cells because it needs glycosylation, hence the final product displays an identical aminoacidic sequence and similar post-translational modification profile to the one of the native protein. It is administered intravenously (like any other recombinant factor of coagulation), because it needs to go straight into the blood. It could be also used for haemophilia A, as we saw before, if the patient develops an immune reaction against factor VIII, so that the common pathway of the coagulation cascade is still activated even in the absence of factor VIII. There are some studies ongoing to improve also factor IX, for example to allows its secretion by the cells that produce it (increasing the mRNA levels or finding alternative expression systems), to increase its potency or to increase the half-life (through PEGylation or with the generation of fusion proteins of factor IX with the Fc fragment or with albumin; this last expedient is used to slow down the release of the drug in the blood and its diffusion in the tissues, moreover the fusion with the Fc portion of an antibody can allow a delay of the degradation because of its binding with the Fc-Rn neonate receptor; the drug composed of the fusion protein between factor IX and albumin is called Idelvion, while the one with factor IX fused to the Fc portion is called Alprolix). Factor IX can also be produced in the mammary glands of a pig (in the milk of pigs): 16 pigs in one year can supply the entire amount of recombinant factor IX needed in the United States, so it could be really advantageous, but the thus obtained factor IX has not been marketed yet. The last factor that we use for therapeutic reasons is factor VII: we use it in haemophilia A patients when they develop antibodies against factor VIII because in this way we are still able to activate the common pathway of the coagulation cascade in an alternative way (even without factor VIII). The recombinant factor VII is produced in BHK cells and it differs only slightly from the native molecule. Another approach that we could apply to haemophilia is the use of an antibody instead of the recombinant factor: in physiologic conditions factor VIII binds to factor IX and factor X and induces their activation (really high affinity of the factors); if instead of using recombinant factor VIII we use a bispecific antibody that activates factor IX and factor X in substitution of factor VIII, we can have an  effective alternative to the recombinant factor because an antibody has a much longer half-life than the normal protein, so we can administer it once a month (or even more) and ameliorate the life-style of the patient. There are some differences between the action of the antibody and the recombinant factor, for example the recombinant factor is specifically able to bind only to the non-activated forms of factor IX and X, while the antibody is not able to distinguish between the zymogen and the enzyme. Moreover, factor VIII activated has an on-off mechanism (once it is activated it become inactive and does not perform its action until further required), instead the antibody doesn’t have an on-off mechanism. There is also a different in affinity, because factor VIII has a higher affinity to factor IX and X compared to the antibody. However, the antibody is still really used because the advantages are more than the disadvantages: if we had to choose between a protein and an antibody in terms of faster absorption, a protein should be preferred, but since we are talking about products that need to reach the blood stream and act there, we should favour a molecule with a longer half-life, and so in this case the antibody is the best option. 2. Anticoagulants: we mainly use two proteins, heparin and hirudin, however heparin is not an engineered anticoagulant but it is an extracted protein (from the pancreas of pigs or lungs of beef). Heparin is one of the most used anticoagulant protein: it is able to bind to antithrombin and enhance its activity by attaching to thrombin (factor IIa) or factor Xa, inhibiting their actions, thus preventing the coagulation cascade and stopping it (it can also inhibit factor XII, XI, IX, VIII and V). We have also a low molecular weight heparin available for the same purposes, except that it is only able to increase the action of antithrombin on factor X and not on thrombin. Heparin, although efficacious (especially in patients that underwent a surgery, to prevent the formation of thrombi), presents clinical disadvantages, including the need for a cofactor (antithrombin-AT III) and poorly predictable dose- response effects (it is unpredictable because its effect are not linked to the dose). That’s why recombinant anticoagulants have been developed: hirudin is the protein naturally produced by the leech as an anticoagulant, which is able to directly inhibit thrombin without binding to antithrombin first (it doesn’t require co-factors). Its effects depend on the dose (differently from heparin) and it is less likely to indue unintentional haemorrhages compared to the other anticoagulants that we have. Recombinant hirudin is slightly different from the natural one because the first two amino acids have been substituted (Val, Val -> Leu, Thr) and it is devoid of the sulphate groups normally present in tyrosine. It is safe and effective, it is administered intravenously and it is produced in Saccharomyces cerevisiae. It is available with two different names: in USA it is called lepirudin, while in Europe it is called desirudin. It has a short half-life but its action is more controllable than the one of heparin, so we know exactly which is the therapeutic window for each patient and we know that if we give too much hirudin we can cause bleeding (the dose is monitored and adjusted to give an aPTT ratio of 1.5-2.5 as above this range there is an increased risk of bleeding). Its clearance is mostly renal. Lately a new recombinant hirudin has been developed: it is called bivalirudin, it is a really short protein (20 amino acids), because it just contains the portion of the molecule that is able to bind to thrombin. It has the same effect as the whole molecule: its binding to thrombin (both fibrin-bound and circulating thrombin) prevents the formation of fibrin fibres so that the coagulation cascade is prevented; then the thrombin digests a piece of the recombinant protein and so, once the hirudin is removed, it can work again to form the clots and so the coagulation cascade becomes normal again (in this way, the use of the molecule is restricted for, for example, a period following a n intervention and it doesn’t last for too much time increasing the bleeding risk). Hirudin is used in cases of heparin induced thrombocytopenia (whether complicated by thrombosis or not, also in patients with cardiopulmonary bypass and vascular surgery), thrombosis prophylaxis after major orthopaedic surgery and in case of acute coronary syndrome and percutaneous coronary intervention. We could also produce recombinant antithrombin, which is a natural inhibitor of coagulation because it is physiologically able to bind to thrombin and to inhibit it, as well as to factors IXa and Xa. It is the only marketed recombinant protein produced in the milk of a goat. Its oligosaccharide composition usually varies. It is used for the treatment of hereditary and acquired anti-thrombin deficiency. Another factor that could lead to the formation of clots if absent is protein C: Xigiris is a recombinant human activated protein C produced in an engineered mammalian cell line and characterized by the presence of several gamma-carboxyglutamate and beta-hydroxylated residues. It is indicated for the  treatment of severe sepsis, in order to prevent multiple organ failure that can be triggered by sepsis- associated blood clot formation. 3. Thrombolytic agents: they are proteins that are able to cut the fibrin meshes, so the thrombus structure. In general, thrombolytic drugs are used in cases in patients that have suffered acute myocardial infarction, in cases of ischaemic stroke, peripheral arterial thrombosis, acute massive pulmonary embolism and of occluded haemodialysis shunts. The most used in the therapeutic field is the tissue plasminogen activator (tPA), a serine protease found physiologically on endothelial cells, and it was the first recombinant protein produced in the milk of a goat, but never marketed as one. It is instead produced in other production cells. It is able to convert plasminogen in plasmin, which then digests fibrin clots in small dimers. tPA is a glycosylated protein, it present 4 potential glycosylation sites, 3 of which are normally glycosylated, and they mediate the hepatic uptake of the molecule and hence its clearance from plasma. Because of these post- translational modifications, it was firstly produced in CHO in 1987, with the name of Alterplase. It was one of the first proteins engineered together with insulin and growth hormone. Its half-life is really short because it is just of 3 minutes, however this short period is enough to activate plasminogen and dissolve the clots. Some modified forms of this molecule are available: they have been produced because the tPA is 527 amino acids long, so it is a huge molecule. Even if its administration is directly intravenous (they are delivered directly in the site where they need to work), so the size is not so important for the administration, the protein needs to be shortened to improve its half-life: for this reason we have an alternative version of alterplase called reteplase, which is 335 amino acids long (it just contains the tPA domains responsible for the fibrin selectivity and for the catalytic activity) and has an half-life of 20 minutes, which facilitates the administration as a single bolus injection, instead of having to perform a continuous infusion with alterplase. Reteplase is a non-glycosylated protein so it can be produced in E. coli, where it accumulates in the form of inclusion bodies. Finally, we have another version of tPA called Tenecteplase, it is produces in CHO cells, it differs in sequence by 6 amino acids so it has a prolonged half-life of 15-19 minutes. It is glycosylated, and this gives to the molecule an increased resistance to PAI-1 (plasminogen activation inhibitor 1). However, since the production of t-PA in cell culture is a difficult and expensive process, t-PA is an expensive drug. t-PA’s main competition in the thrombolytic (clot busting) market is streptokinase. This protein costs 1/10th the price of t-PA, because it is an extracted protein, and it seems to do an equivalent job. Streptokinase is derived from the bacterium Streptococcus haemolyticus, which is called by this name because it dissolves clots thanks to this protein. It binds specifically and tightly to plasminogen, thereby activating and converting plasminogen into plasmin. In some condition it may elicit immune responses, but since its purification from bacteria is really simple and cheap it is preferred to tPA. The same can be said for urokinase, a serine protease that does the same job as tPA, it is produced by the kidneys, and it is generally purified directly from human urine. However, as we said a lot of times, dealing with urine is very tricky, so also urokinase can be produced through the recombinant DNA technology. But we have another protein that has the same action as tPA and can be extracted from bacteria, and it is the Staphylokinase. This protein is produced by a number of strains of S. aureus, but also in E. coli by recombinant DNA technology (but the costs are obviously higher). Covalent attachment of PEG reduces the rate of serum clearance of this protein, whereas we can have a lot of domain-delated variants which display significantly reduced immunogenicity levels. All of these enzymes work properly as thrombolytic agents, so when we compare them is just in terms of costs and process of production. OTHER RECOMBINANT PROTEINS Asparaginase is an enzyme which induces apoptosis in transformed cells because it catalyses the hydrolysis of L-asparagine in aspartic acid and ammonia, and cells without asparagine die by apoptosis. It has been approved for the treatment of refractory childhood acute lymphoblastic leukemia. PEG-coupled enzymes are often preferred. DNase is an enzyme used for the treatment of cystic fibrosis (disease where we have an abnormal response of granulocytes against infections in the lungs; the lung become full of dead granulocytes that release their DNA, which is viscous and obstructs the airways). It induces the hydrolysis of long DNA chains into small fragments, it is produced in CHO cells and it is administered as an aerosol. Glucocerebrosidase is a protein approved for the treatment of Gaucher disease (lysosomal storage disease affecting lipid metabolism, specifically the degradation of glucocerebrosides), which is characterized by the lack of this enzyme. The recombinant form of glucocerebrosidase is produced in CHO cells, and an engineered form with mannose is used to facilitate the uptake by macrophages. It is the first approved biotech drug that was produced in carrots. THERAPEUTIC ANTIBODIES Antibodies are very useful as therapeutic agents, even if they have some disadvantages related to their big molecular mass, because they have multiple advantages. Antibodies were first produced in mice, by the immunization of the animal, the isolation of the antibody- forming cells from its spleen and, after the formation of hybridomas with tumoral cells and the expansion of these hybridomas in vitro, the collection of the antibodies from the cell culture. However, we already saw that mouse antibodies cannot be used because they are immunogenic. How can we produce fully human antibodies? One way is through the phage display technology. We start from the creation of a library, and it is a non-immune library: it is produced by using B lymphocytes obtained from non-immunized donors as a source of genes of antibodies. The difference with an immune library is that the immune library is obtained by cloning antibodies or antibody fragments-coding sequences derived from B lymphocytes of donors (from their spleen), previously immunized with the target antigen. We use non-immune libraries because since we have to produce fully human antibodies, we cannot immunize people to obtain our library, that’s too dangerous. So, we exploit antibodies that people already have produced in different occasions. Therefore, from the blood of donors, we isolate the B lymphocytes, we extract the RNA from these cells, and we isolate the RNA encoding for the light and heavy variable chains of the antibodies that could be produced by the lymphocyte. This isolated RNA is cloned into specific phage vectors in the form of cDNA, in frame with a surface protein. In this way, after the expression of the antibody, it will be expressed on the surface of the phage. Hence from this process we obtain a lot of different phage particles that display a different variable region of a different antibody on their surface. So eventually our library will me made out of all the variable portion of every antibody already produced by the B lymphocyte of our donors. Thanks to a technique called panning, I can isolate the phage particle carrying our variable fragment of interest simply by exposing our phage library to the antigen of interest (binding by affinity), immobilized on a surface. Then we elute the specific phage that we were able to isolate thanks to the panning technique by removing the phages that did not bind and the ones who bind un-specifically and we amplify it. Finally, we make these infect a E. coli cell line to produce a high quantity of this antibody for further analyses. The library that we produce at the beginning of this process usually comes from the antibodies of a lot of different donors, so it is very large, moreover we can add also mutations and rearrangements in vitro to increase the complexity of the library. In fact, usually, during cloning, different light (L) and heavy (H) chains are cloned and then the DNA of one H and one L chain are cloned into the same vector. I this way, many different combinations of H and L chains are cloned together in the same vector. Lambda is not useful for producing large amounts of proteins. The L and H chains are excised from Lambda and cloned into an E. coli plasmid and the recombinant plasmid transformed into E. coli. The production of mAbs as therapeutic agents has developed during the years, especially since fully human antibodies have been developed, and it is still growing. To avoid the problem of immunogenicity, some strategies have been developed. Inside an antibody, we have different epitopes, because immunogenicity can be triggered by the antibody in many different region of the molecule (we can have neutralizing antibodies produced against the epitopes in the Fab portion, or antibodies produced against the epitopes of the Fc portion, …). It has been proved that the Fc portion induce the activation of Tregs (regulatory cells), instead effector T cells are triggered and activated by epitopes on the Fab portion of the therapeutic antibody, and once they get activated they produce neutralizing antibodies against these epitopes. So how can we engineer the mAbs to avoid the stimulation of the effector T cells but to also stimulate, at the same time, the activation of the Tregs in order to control the immune reaction induced by the Fc portion of the antibody? We could add some epitopes (stretches of sequences of amino acids) to the Fc portion to induce the stimulation of Tregs (phenomenon called tolerization, the epitopes that we add are called Tregitopes), and then we can remove some epitopes of the Fab portion to reduce its immunogenicity (especially the ones that we know being immunogenic and triggering for effector T cells; phenomenon called de-immunization). So we could potentially engineer the mAb molecule to obtain a better immunogenicity profile and a better regulation from the Tregs. An example of this process is Aletuzumab, a mAb which was engineered through the addition of specific amino acids which lower the triggering of effector T cells and of other amino acids which, instead, induced the activation of Tregs, with an overall minimal change in the 3D structure of the molecule. However, it was a failure: even though it was modified, this mAb for the treatment of chronic B cell lymphocytic leukemia was still neutralized by multiple antibodies in 75% of the patients. How can we use monoclonal antibodies? They can have multiple functions, like the induction of passive immunity, diagnostic imaging purposes and therapeutic ones. 1. Diagnostic purposes: we can use them for imaging of vascular pathologies (for example cells of an heart that underwent a myocardial infarction expose myosin on their surface, so we can use a specific monoclonal antibody against myosin bound to a specific molecule for the detection of the signal, to localize which is the portion of the heart that has undergone an heart attack), for the visualization of site of focused bacterial infections (if we produce antibodies against bacterial surface cells and we bind them to molecules for the detection of the signal; this method is available for hepatitis, influenza, herpes, streptococcus and chlamydia). One example is Nofetumumab, an antibody bound to a radioisotope, directed against the antigens of small cell lung cancer. The same principal is used for rapid tests for the diagnosis of infections (for example covid tests): we have a membrane of nitrocellulose where antibodies directed against another antibody that bound the antigen of our interest are bound. 2. Therapeutic use: the main application is against tumor. We can possibly modify the antibody to bind any molecule that could be helpful for the specific application that we want to obtain. From the point