BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 6 potx

essential role in the (in vitro or in vivo) biological activity of EPO. Interestingly, removal of the N-linked sugars, while having little effect on EPO’s in vitro activity, destroys its in vivo activity. The sugar components of EPO are likely to contribute to the molecule’s: . solubility; . cellular processing and secretion; . in vivo metabolism. Incomplete (N-linked) glycosylation prompts decreased in vivo activity due to more rapid hepatic clearance of the EPO molecule. Enzymatic removal of terminal sialic acid sugar residues from oligosaccharides exposes otherwise hidden galactose residues. These residues are then free to bind specific hepatic lectins, which promote EPO removal from the plasma. The reported plasma half-life (t 1/2 ) value for native EPO is 4–6 h. The t 1/2 for desialated EPO is 2 min. Comparison of native human EPO with its recombinant form produced in CHO cells reveal very similar glycosylation patterns. Circular dichroism studies show that up to 50% of EPO’s secondary structure is a-helical. The predicted tertiary structure is that of four anti-parallel helices formed by variable-sized loops, similar to many other haemopoietic growth factors. Development of bioassays and radioimmunoassays, along with the later development of specific mRNA probes, allowed determination of the sites of production of EPO in the body. It has now been established that EPO in the human adult is synthesized almost exclusively by specialized kidney cells (peritubular interstitial cells of the kidney cortex and upper medulla). Minor quantities are also synthesized in the liver, which represents the primary EPO-produci ng organ of the fetus. EPO is present in serum and (at very low concentrations) in urine, particularly of anaemic individuals. This cytokine/hormone was first purified in 1971 from the plasma of anaemic sheep, while small quantities of human EPO was later purified (in 1977) from over 2500 litres of urine collected from anaemic patients. Large-scale purification from native sources was thus impractical. The isolation (in 1985) of the human EPO gene from a genomic DNA library, facilitated its transfection into Chinese hamster ovary (CHO) cells. This now facilitates large- scale commercial production of the recombinant human product (rhEPO), which has found widespread medical application. EPO stimulates erythropoiesis by: . increasing the number of committed cells capable of differentiating into erythrocytes; . accelerating the rate of differentiation of such precursors; . increasing the rate of haemoglobin synthesis in developing cells. An overview of the be st-characterized stages in the process of erythropoiesis is given in Figure 6.5. The erythroid precursor cells, BFU-E (burst forming unit-erythroid), display EPO receptors on their surface. The growth and differentiation of these cells into CFU-Es (colony forming unit-erythroid), require the presence not only of EPO but also of IL-3 and/or GM-CSF. CFU-E cells display the greatest density of EPO cell surface receptors. These cells, not surprisingly, also display the greatest biological response to EPO. Progressively more mature erythrocyte precursors display progressively fewer EPO receptors on their cell surfaces. Erythrocytes themselves are devoid of EPO receptors. EPO binding to its receptor on CFU-E cells pro motes their differentiation into pro-erythroblasts and the rate at which this differentiation occurs HAEMOPOIETIC GROWTH FACTORS 265 appears to determine the rate of erythropoiesis. CFU-E cells are also responsive to insulin-like growth factor 1 (IGF-1). Although the major physiological role of EPO is certainly to promote red blood cell production, EPO mRNA has also been detected in bone marrow macrophages, as well as some multipotential haemopoietic stem cells. Although the physiological relevance is unclear, it is possible that EPO produced by such sources may play a localized paracrine (or autocrine) role in promoting erythroid differentiation. 266 BIOPHARMACEUTICALS Figure 6.5. Stages in the differentiation of haemopoietic stem cells, yielding mature erythrocytes. The EPO-sensitive cells are indicated. Each cell undergoes proliferation as well as differentiation, thus greater numbers of the more highly differentiated daughter cells are produced. The proliferation phase ends at the reticulocyte stage; each reticulocyte matures over a 2 day period, yielding a single mature erythrocyte The EPO receptor and signal transduction The availability of biologically active 125 I-labelled EPO facilitated the detection and study of cell surface receptors. In addition to erythroid precursors, various other cell lines were shown to express EPO receptors, at least when cultured in vitro. Many harboured two classes of receptors: a high-affinity and a low-affinity form. Most appeared to express between 1000 and 3000 recept ors/cell. Radiotracer experiments illustrated that the EPO receptor is rapidly internalized after ligand binding and the EPO–receptor complex is subsequently degraded within lysoso mes. The human EPO receptor is encoded by a single gene on chromosome 19. The gene houses eight exons, the first five of which appear to code for the 233 amino acid extracellular receptor portion. The sixth encodes a single 23 amino acid transmembrane domain, while the remaining two exons encode the 23 6 amino acid cytoplasmic domain. The mature receptor displays a molecular mass of 85–100 kDa. It is heavily glycosylated through multiple O-linked (and a single N-linked) glycosylation site. High- versus low-affinity receptor variants may be generated by self-association. The EPO receptor is a member of the haemopoietic cytokine receptor superfamily. Its intracellular domain displays no known catalytic activity but it appears to couple directly to the JAK 2 kinase (Chapter 4), which probably promotes the early events of EPO signal transduction. Other studies have implicated additional possible signalling mechanisms, including the involvement of G proteins, protein kinase C and Ca 2+ . The exact molecular events underlining EPO signal transduction remain to be elucidated in detail. Binding of EPO to its receptor stimulates the proliferation of BFU-E cells and triggers CFU-Es to undergo terminal differentiation. As well as such stimulatory roles, EPO may play a permissive role, in that it also appears to inhibit apoptosis (programmed cell death) of these cells. With this scenario, ‘normal’ serum EPO levels permit survival of a specific fraction of BFU-E and CFU-Es, which dictates the observed rate of haemopoiesis. Increased serum EPO concentrations permit survival of a greater fraction of these progenitor cells, thus increasing the number of red blood cells ultimately produced. The relative physiological importance of EPO stimulatory versus permissive activities has yet to be determined. Regulation of EPO production The level of EPO production in the kidneys (or liver) is primarily regulated by the oxygen demand of the producer cells, relative to their oxygen supply. Under normal conditions, when the producer cells are supplied with adequate oxygen via the blood, EPO (or EPO mRNA) levels are barely detectable. However, the onset of hypoxia (a deficiency of oxygen in the tissues) results in a very rapid increase of EPO mRNA in producer cells. This is followed within 2 h by an increase in serum EPO levels. This process is prevented by inhibitors of RNA and protein synthesis, indicating that EPO is not stored in producer cells, but synthesized de novo when required. Interestingly, hypoxia prompts increased renal and hepatic EPO synthesis in different ways. In the kidney, the quantity of EPO produced by an individual cell remains constant, while an increase in the number of EPO-producing cells is evident. In the liver, the quantity of EPO produced by individual cells appears to simply increase in response to the hypoxic stimulus. A range of conditions capable of inducing hypoxia stimulate enhanced production of EPO, thus stimulating erythropoiesis. These conditions include: HAEMOPOIETIC GROWTH FACTORS 267 . moving to a higher altitude; . blood loss; . increased renal sodium transport; . decreased renal blood flow; . increased haemoglobin oxygen affinity; . chronic pulmonary diseas e; . some forms of heart disease. On the other hand, hyperoxic conditions (excess tissue oxygen levels) promote a decrease in EPO production. The exact mechanism by which hypoxia stimulates EPO production remains to be elucidated. This process has been studied in vitro using an EPO-producing cancerous liver (hepat oma) cell line as a model system. These studies suggest the existence of a haem protein (probably membrane-bound), which effectively acts as an oxygen sensor (Figure 6.6). Adequate ambient oxygen concentration retains the haem group in an oxygenated state. Hypoxia, however, promotes a deoxy-configuration, which alters the haem conformation. The deoxy- form of haem is postulated to be capable of generating an active transcription factor which, upon migration to the nucleus, enhances transcription of the EPO gene. Evidence cited to support such a theory includes the fact that cobalt promotes erythropoiesis (cobalt can substitute for the iron atom in the haem porphyrin ring; Cobalt–haem, however, remains in the deoxy-conformation, even in the presence of a high oxygen tension). In addition to oxygen levels, a number of other regulatory factors can stimulate EPO synthesis, either on their own or in synergy with hypoxia (Table 6.6). Therapeutic applications of EPO A number of clini cal circumstances have been identified which are characterized by an often profoundly depressed rate of erythropoiesis (Table 6.7). Many, if not all, such conditions could be/are responsive to administration of exogenous EPO. The prevalence of anaemia, and the medical complications which ensue, prompts tremendous therapeutic interest in this haemopoietic growth factor. EPO has been approved for use to treat various forms of anaemia (Table 6.8). It was the first therapeutic protein produced by genetic engineering, whose annual sales value topped $1 billion. Its current annual sales value is now close to $2 billion. EPO used therapeutically is produced by recombinant means in CHO cells. Neorecormon is one such product. Produced in an engineered CHO cell line constitutively expressing the EPO gene, the product displays an amino acid sequence identical to the native human molecule. An overview of its manufacturing process is presented in Figure 6.7. The final freeze-dried product contains urea, sodium chloride, polysorbate, phosphate buffer and several amino acids as excipients. It displays a shelf-life of 3 years when stored at 2–8 8C. A pre-filled syrine form of the product (in solution) is also available, which is assigned a 2 year shelf-life at 2–8 8C. EPO can be administered intravenously or, more commonly, by subcutaneous (s.c.) injection. Peak serum concentrations are witnessed 8–24 h after s.c. administration. Although they are lower than the values achieved by i.v. administration, the effect is more prolonged, lasting for several hours. In healthy individuals, less than 10% of administered EPO is excreted intact in the urine. This suggests that the kidneys play, at best, a minor role in the excretion of this hormone. 268 BIOPHARMACEUTICALS HAEMOPOIETIC GROWTH FACTORS 269 Figure 6.6. Proposed mechanism by which hypoxic conditions stimulate enhanced EPO synthesis (see text for details) Table 6.6. Some additional regulatory factors that can promote increased EPO production. Other regulatory factors, including IL-3 and CSFs, which also influence the rate of erythropoiesis, are omitted as they have been discussed previously Growth hormone Thyroxine Adrenocorticotrophic hormone Adrenaline Angiotensin II Androgens and anabolic steroids More recently, an engineered form of EPO has gained marketing approval. Darbepoetin-a is its international non-proprietary name and it is marketed under the tradenames Aranesp (Amgen) and Nespo (Dompe ´ Biotec, Ital y). The 165 amino acid protein is altered in amino acid sequence when compared to the native human product. The alteration entails introducing two new N-glycosylation sites, so that the recombinant product, produced in an engineered CHO cell line, displays five glycosylation sites as opposed to the normal three. The presence of two additional carbohydrate chains confers a prolonged serum half-life on the molecule (up to 21 h as compared to 4–6 h for the native molecule). EPO was first used therapeutically in 1989 for the treatment of anaemia associated with chronic kidney failure. This anaemia is largely caused by insufficient endogenous EPO production by the diseased kidneys. Prior to EPO approval, this condition could only be treated by direct blood transfusion. It responds well, and in a dose-dependent manner, to the administration of recombinant human EPO (rhEPO). The administration of EPO is effective in the case of both patients receiving dialysis and those who have not yet received this treatment. Administration of doses of 50–150 IU EPO/kg three times weekly is normally sufficient to elevate the patient’s haematocrit values to a desired 32–35% (haematocrit refers to ‘packed cell volume’, i.e. the percentage of the total volume of whole blood that is composed of erythrocytes). Plasma EPO concentrations generally vary between 5 and 25 IU/l in healt hy individuals. One IU (international unit) of EPO activity is defined as the activity which promotes the same level of stimulation of erythropoiesis as 5 mmol cobalt. In addition to enhancing erythropoiesis, EPO treatment also improves tolerance to exercise, as well as patients’ sense of well-being. Furthermore, reducing/eliminating the necessity for blood transfusions also reduces/eliminates the associated risk of accidental transmission of blood-borne infectious agents, as well as the risk of precipitating adverse transfusion reactions 270 BIOPHARMACEUTICALS Table 6.8. EPO preparations that have gained regulatory approval or are undergoing clinical trials Product Status Company Epogen (rhEPO) Approved Amgen Procrit (rhEPO) Approved Ortho Biotech Neorecormon (rhEPO) Approved Boehringer–Mannheim Aranesp (rEPO analogue) Approved Amgen Nespo (rEPO analogue) Approved Dompe ´ Biotec rEPO In clinical trials Aventis Table 6.7. Diseases (and other medical conditions) for which anaemia is one frequently observed symptom Renal failure Rheumatoid arthritis Cancer AIDS Infections Bone marrow transplantation in recipients. An American study calculated the average cost of rhEPO therapy to be of the order of $6000/patient/annum, compared to approx. $4600 for transfusion therapy. However, due to the additional benefits described, the cost:benefit ratio appears to favour EPO therapy. The therapeutic spotlight upon EPO has now shifted to additional (non-renal) applications (Table 6.9). Chronic disease and cancer chemotherapy Anaemia often becomes a characteristic feature of several chronic diseases, such as rheumatoid arthritis. In most instances this can be linked to lower than normal endogenous serum EPO levels (although in some cases, a deficiency of iron or folic acid can also represent a contributory factor). Several small clinical trials have confirmed that admini stration of EPO increases haematocrit and serum haemoglobin levels in patients suffering from rheumatoid arthritis. A satisfactory response in some patients, however, required high-dose therapy, which could render this therapeutic approach unattractive from a cost:benefit perspective. HAEMOPOIETIC GROWTH FACTORS 271 Figure 6.7. Schematic overview of the production of the erythropoietin-based product Neorecormon. Refer to text for further details Severe, and in particular chronic, infection can also sometimes induce anaemia — which is often made worse by drugs used to combat the infection, e.g. anaemia is evident in 8% of patients with asymptomatic HIV infection. This incidence increases to 20% for those with AIDS-related complex and is greater than 60% for patients who have developed Kaposi’s sarcoma. Up to one-third of AIDS patients treat ed with zidovudine also develop anaemia. Again, several trials have confirmed that EPO treatment of AIDS sufferers (be they receiving zidovudine or not) can increase haematocrit values and decrease transfusion requirements. Various malign ancies can also induce an anaemic state. This is often associated with decreased serum EPO levels, although iron deficiency, blood loss or tumour infiltration of the bone marrow can be complicating factors. In addition, chemotherapeutic agents administered to this patient group often adversely affect stem cell populations, thus rendering the anaemia even more severe. Administration of EPO to patients suffering from various cancers/receiving various chemotherapeutic agents yielded encouraging results, with significant improvements in haematocrit levels being recorded in approximately 50% of cases. In one large US study (2000 patients, most receiving chemotherapy) s.c. administration of an average of 150 IU EPO/kg, three times weekly, for 4 months, reduced the number of patients requiring blood transfusions from 22% to 10%. Improvement in the sense of well-being and overall quality of life was also noted. The success rate of EPO in alleviating cancer-associated anaemia has varied in different trials, ranging from 32% to 85%. The EPO receptor is expressed not only by specific erythrocyte precursor cells but also by endothelial, neural and myeloma cells. Concern has been expressed that EPO, therefore, might actually stimulate growth of some tumour types, particular ly those derived from such cells. To date, no evidence (in vitro or in vivo) has been obtained to support this hypothesis. Additional non-renal applications Babies, especially babies born prematurely, often exhibit anaemia, which is characterized by a steadily decreasing serum haemoglobin level during the first 8 weeks of life. While multiple factors contribute to development of anaemia of prematurity, a lower than normal serum EPO level is a characteristic trait. In vitro studies indicate that BFU-E and CFU-E cells from such babies are responsive to EPO, and several pilot clinical trials have been initiated. Administration of 300–600 IU EPO/kg/week generally was found to enhanc e erythropoiesis and reduced the number of transfusions required by up to 30%. Patients who have received an allogeneic bone marrow transplant characteristically display depressed serum EPO levels for up to 6 months post-transplantation. Administration of EPO thus seems a logical approach to counteract this effect. Several clinical studies have validated 272 BIOPHARMACEUTICALS Table 6.9. Some non-renal applications of EPO (refer to text for details) Treatment of anaemia associated with chronic disease Treatment of anaemia associated with cancer/chemotherapy Treatment of anaemia associated with prematurity To facilitate autologous blood donations before surgery To reduce transfusion requirements after surgery To prevent anaemia after bone marrow transplantation this approach, observing accelerated erythropoiesis, resulting in attainment of satisfactory haematocrit levels within a shorter period post-transplant. Tolerability In general, rhEPO is well tolerated. The most pronounced adverse effects appear to be associated with its long-term administration to patients with end-stage renal failure. Particularly noteworthy is an increase in blood pressure levels in some patients and the increased risk of thromboembolic events (a thromboembolic event, i.e. a thromboembolism, describes the circumstance where a blood clot forms at one point in the circulation but detaches, only to become lodged at another point; Chapter 9). Most short-term applications of EPO are non-renal related, and generally display very few side-effects; i.v. administration can sometimes prompt a transient flu-like syndrome, while s.c. administration can render the site of injection painful. This latter effect appears, however, to be due to excipients present in the EOP preparations, most notably the citrate buffer. EPO administration can also cause bone pain, although this rarely limits its clinical use. Overall, therefore, rhEPO has proved both effective and safe in the treatment of a variety of clinical conditions and its range of therapeutic applications is likely to increase over the coming years. THROMBOPOIETIN Thrombopoietin (TPO) is the haemopoietic growth factor now shown to be the primary physiological regulator of platelet prod uction. Although its existence had been inferred for several decades, its purification from blood proved an almos t impossible task, due to its low production levels and the availability of only an extremely cumbersome TPO bioassay. Its existence was finally proved in the mid-1990s when thrombopoietin cDNA was cloned. This molecule is likely to represent an important future therapeutic agent in combating depressed plasma platelet levels, although this remains to be proved by clinical trials. Platelets (thrombocytes) carry out several functions in the body, all of which relate to the arrest of bleeding. They are disc-shaped structures 1–2 mm in diameter, and are present in the blood of healthy individuals at levels of approximately 250Â10 9 /l. They are formed by a lineage- specific stem cell differentiation process, as depicted in Figure 6.8. The terminal stages of this process entails the maturation of large progenitor cells termed ‘megakaryocytes’. Platelets represent small vesicles which bud off from the megakaryocyte cell surface and enter the circulation. A number of disorders have been identified that are primarily caused by the presence of abnormal platelet levels in the blood. Thrombocythaemia is a disease characterized by abnormal megakaryocyte proliferation, leading to elevated blood platelet levels. In many instances, this results in an elevated risk of spontaneous clot formation within blood vessels. In other instances, the platelets produced are defective, which can increase the risk of spontaneous or prolonged bleeding events. Thrombocytopenia, on the other hand, is a condition characterized by reduced blood platelet levels. Spontaneous bruising, bleeding into the skin (purpura) and prolonged bleeding after injury represent typical symptoms. Thrombocytopenia is induced by a number of clinical conditions, including: HAEMOPOIETIC GROWTH FACTORS 273 . bone marrow failure; . chemotherapy (or radiot herapy); . various viral infections. TPO should alleviate thrombocytopenia in most instances by encouraging platelet production. Currently, the standard therapy for the condition entails administration of 5 units of platelets to the sufferer (1 unit equals the quantity of platelets derived in one sitting from a single blood donor). TPO therapy is a particularly attractive potential alternative because: . it eliminates the possibility of accidental transmission of disease via transfusions; . platelets harvested from blood donations have a short shelf-life (5 days), and must be stored during that time at 228C on mechanical shakers; . platelets exhibit surface antigens, and can thus promote an tibody production. Repeat administrations may thus be less effective, due to the potential presence of neutralizing antibodies. The most likely initial TPO therapeutic target is thrombocytopenia induced by cancer chemo- or radiotherapy. This indication generally accounts for up to 80% of all platelet transfusions undertaken. In the USA alone, close to 2 million people receive platelet transfusions annually. Human TPO is a 332 amino acid, 60 kDa glycoprotein, containing six potential N-linked glycosylation sites. These are all localized towards the C-terminus of the molecule. The N- terminal half exhibits a high de gree of amino acid homology with EPO and represents the biologically active domain of the molecule. Sources of TPO include kidney and skeletal muscle cells but it is primarily produced by the liver, from where it is excreted constantly into the blood. This regulatory factor supports the proliferation, differentiation and maturation of megakaryocytes and their progenitors and 274 BIOPHARMACEUTICALS Figure 6.8. Simplified representation of the production of platelets from stem cells. CFU-megakaryocytes and in particular, mature megakaryocytes, are most sensitive to the stimulatory actions of TPO. These two cell types also display a limited response to IL-6, IL-11 and LIF [...]... slightly from one another The common form of IGF-1 is a 70 amino acid polypeptide displaying three intra-chain disulphide linkages and a molecular mass of 7 .6 kDa IGF-2 (67 amino acids; 7.5 kDa) also has three disulphide linkages IGF-1 and -2 display identical amino acid residues at 45 positions, and exhibit in excess of 60 % sequence homology Both display A and B domains, connected by a short C domain... expressed in the male and female reproductive tissue Thus, IGFs are believed to affect reproductive function by both (GH-stimulated) endocrine action and via paracrine- and autocrine-based activity In the human female, IGF-1 is expressed by follicular theca cells, while IGF-2 is synthesized by granulosa cells (Chapter 8) The IGF-1 and -2 receptors are widely expressed in ovarian tissue, and synthesis of... and being generally 294 BIOPHARMACEUTICALS Table 7.9 Molecules displaying neurotrophic activity in vivo and/ or with neurons in culture Nerve growth factor (NGF) Brain-derived neurotrophic factor (BDNF) Neurotrophin 3 (NT-3) Neurotrophin 4/5 (NT-4/5) Neurotrophin 6 (NT -6 ) Ciliary neurotrophic factor (CNTF) Glial cell-line-derived neurotrophic factor (GDNF) Fibroblast growth factors (FGFs) Platelet-derived... thrombopoietin cDNA and stimulation of platelet production in vivo Nature 369 , 565 – 568 2 76 BIOPHARMACEUTICALS Markham, A & Bryson, H (1995) Epoetin Alfa, a review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in non-renal applications Drugs 49(2), 232–254 Metcalf, D (1994) Thrombopoietin — at last Nature 369 , 519–520 Miyazaki, H & Kato, T (1999) Thrombopoietin: biology and clinical... functional receptor for NT-3 and NT-4/5 and is expressed widely throughout the central and peripheral nervous system Trk C (along with Trk A and B) represents the functional receptor for NT-3 This 145 kDa glycoprotein is expressed by various neuronal cell populations, both in the brain (particularly the hippocampus and cerebellum) and the peripheral nervous system The neurotrophin low-affinity receptor All... capable of blocking IGF-receptor binding IGF and fetal development IGFs 1 and 2, along with insulin, play an essential role in promoting fetal growth and development IGF-2, and its receptor is expressed by the growing embryo as early as the two-cell stage (i.e even before implantation in the womb wall) Later, the developing embryo also begins to synthesize IGF-1 and insulin These mitogens and their receptors... erythropoietin and a comparison with its receptor-bound conformation Nature Struct Biol 5, 861 Dranoff, G et al (1994) Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis Science 264 , 713–715 Duarte, R & Frank, D (2002) The synergy between stem cell factor (SCF) and granulocyte colony stimulating factor (G-CSF): molecular basis and clinical relevance Leukaemia Lymphoma 43 (6) ,... half-life Additional cytokines (IL-3, IL -6 , IL-11 and LIF) also promote a proliferative response in megakaryocytes Although these exhibit some ability to increase platelet levels, the physiological significance of this remains unclear TPO, on the other hand, induces a far swifter, greater and more specific response In one study, its administration to mice over a period of 6 days resulted in a four-fold... neurotrophic factors which all belong to the same gene family They include NGF, as well as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT-4/5) and neurotrophin -6 (NT -6 ) All are small, basic proteins sharing approximately 50% amino acid homology They exist mainly as homodimers and promote signal transduction by binding to a member of the Trk family of tyrosine kinase... survive and remain differentiated 2 96 BIOPHARMACEUTICALS Table 7.10 Biochemical characteristic of the neurotrophin family of neurotrophic factors Except for NT -6 , the molecular masses quoted are those of the homodimeric structure, which represents their biologically active forms See text for further details Neurotrophin Molecular mass (kDa) pI 26 27 27 28 15.9 10 10 9.5 10.8 10.8 NGF BDNF NT-3 NT-4/5 NT-6 . IGF-1 is a 70 amino acid polypeptide displaying three intra-chain disulphide linkages and a molecular mass of 7 .6 kDa. IGF-2 (67 amino acids; 7.5 kDa) also has three disulphide linkages. IGF-1 and. bind Ligand Receptor (R) IGF-I IGF-II Insulin IGF-I R A ˜ A ˜ A ˜ A ˜ A ˜ IGF-II R A ˜ A ˜ A ˜ – Insulin R A ˜ A ˜ A ˜ A ˜ for the insulin receptor, binding of ligand to the IGF-1 receptor a-subunit. paracrine- and autocrine-based activity. In the human female, IGF-1 is expressed by follicular theca cells, while IGF-2 is synthesized by granulosa cells (Chapter 8). The IGF-1 and -2 receptors