Appunti drugs-66-79.pdf

of view of the specificity they are much better than any possible chemical drug (lower off-target side effects). In an antibody, the Fc portion is not just the portion of the molecule that keeps together the two Fab portion arms, but it’s also the portion that can be recognized by the Fc-Rg...

of view of the specificity they are much better than any possible chemical drug (lower off-target side effects). In an antibody, the Fc portion is not just the portion of the molecule that keeps together the two Fab portion arms, but it’s also the portion that can be recognized by the Fc-Rgamma, receptors expressed on a variety of different cells of our immune system (T cells, NK cells, dendritic cells, macrophages, …). This plays an important role in the therapeutic function of mAb, especially in the oncologic field: the immune system plays an important role in the development of a cancer, in fact a tumor grows only when the immune system is not able to control and suppress is growth anymore (immune escape of the tumor). So an immune cell can react against a cancer cell, but only if it is triggered to do so: if we send a mAb against a tumor we can use its Fc portion to induce the activation of effector cells against tumoral cells (ADCC and CDC). So, the Fc portion of a lot of mAb used in therapy as anti-tumoral antibodies is the most important part of the molecule in this approach. This type of therapy is called immune therapy, and it is used to induce cancer cell apoptosis, inhibit its growth or interfering with its function. To implement the efficacy of this therapy we can add molecules to the antibody: radionucleotides, toxins, cytokines (which stimulates the activation of the immune system even more), prodrugs (that with the addition of an enzyme, become drugs directed against the cell that uptakes it), … in general, the rationale behind this application is to bring molecules potentially toxic or able to activate the immune system in the close proximity of the target, so the cancer, cell. Finally, one last approach is to design bispecific antibodies that can bind with their Fab regions both to the target antigen (so to the target cell) and to an effector cell antigen, activating the latter against the tumor. Antibodies can kill the cell by inducing their apoptosis in multiple ways: they can bind directly to one of their receptor (which could be growth factor receptors for example, so we first antagonize its growth), they can target specific surface antigens and directly elicit the apoptotic signalling inside the cell, or they could induce the activation of the immune system against them. We have a lot of antibodies that have been developed for a therapeutic use: the one of pharmaceutical antibodies is the fastest growing segment of the biopharmaceutical market, and it is estimated that more than 500 antibody-based therapies are currently under development. They are directed not only against cancer but also against other pathologies, especially chronic autoimmune diseases. - ANTI-TNF: we already talked about them (check the paragraph). - ANTI-CD20: the main example is rituximab (commercially called Rituxan). It is an anti-CD20 antibody so, since CD20 is a B cell antigen, it is an anti-B cell antibody. CD20 usually induces the progression of the cell in the cell cycle, the binding of the antibody causes the induction of apoptosis. It was first approved in 1997 to treat B-cell lymphoma, and it is administered in combination with Methotrexate (the response rates of the mAb alone are of 50-70%, when used in combination they raise up to 90-100%). It is directed for patients who do not respond to anti-TNF treatments. It is also used in rheumatoid arthritis because this disease has a B cell component to its pathology. It is a chimeric antibody (-ximab), so it contain the human IgG1 and murine variable region (in chimeric antibodies, 1/3 of the molecule is murine). Now we have a fully human anti-CD20 antibody that does the same job as rituximab, and it is called Ofatumumab: it is a second generation anti-CD20 antibody which has been demonstrated to be safe and effective in patients with NHL, CLL, RA and MS. It targets CD20 but on a newly described epitope and it shows lower off-rates and improved CDC effect than rituximab. In fact, rituximab binds to the larger of two extracellular loops within the CD20 molecule; this loop includes the alanine-N-proline (ANP) residues at positions 170 to 172. Instead, Ofatumumab binds to two sites on the CD20 molecule: the smaller extracellular loop and positions 159 to 166 on the larger loop. This unique binding pattern of ofatumumab, which results in increased proximity of the anti-body to the cell membrane, may account for the greater potency of the drug in inducing complement-mediated lysis. Three mechanisms of action of anti-CD20 antibodies have been proposed: in complement- dependent cytotoxicity, the first component of complement (C1) binds to the Fc portion of the anti-CD20 molecule, resulting in the activation of the complement cascade and cell lysis  through the formation of membrane attack complexes (MAC). In antibody-dependent cell- mediated cytotoxicity (ADCC), effector cells, such as natural killer cells or macrophages, bind to the Fc portion of the anti-CD20 molecule through Fcγ receptors; the effector cells then release effector molecules such as perforin, which cause cell lysis. In direct cytotoxicity, the anti-CD20 antibody induces internal signaling within the tumor cell, causing antiproliferative effects or cell death, which may involve apoptosis or other cell-death pathways. - VASCULAR ENDOTHELIAL GORWTH FACTOR (VEGF): Bevacizumab is a mAb that is able to prevent the mechanism of angiogenesis. This process is used by tumoral cells to generate new vessels that can be able to bring oxygen and nutrients to the tumoral mass (tumoral mass composed of necrotic cells in the centre, because they don’t receive enough oxygen and nutrients, and vascularized cells on the outside, but this has to be maintained during the growth of the tumor). Angiogenesis is regulated by VEGF: if we use an antibody against it we block the formation of new vessels and the ones that are being generated can be destroyed. In this way the tumor becomes weaker (no sufficient supply of nutrients and oxygen), it grows with a lower rate and so in this way the immune system can easily act against it. Agents targeting VEGF are: bevacizumab (directly acts on VEGF), soluble VEGF receptor (recombinant protein, called VEGF-TRAP, not really used in therapy), anti-VEGF receptor antibodies (called IMC-1121B, still not approved yet) and small molecules inhibitors of the VEGF receptors (they are not proteins, they are chemical molecules; one example is vatalanib, it can be taken orally). The inhibition with any of these molecules prevents the dimerization of the VEGF receptors and thus they block the pathway that induces cell growth on endothelial cells. Bevacizumab is a humanized monoclonal antibody (commercially called Avastin), it is 935 human and 7% murine, it can recognize all isoforms of VEGF and has a terminal half-life of 17- 21 days. It was approved for the treatment of patients suffering from advanced colorectal cancer, advanced ovarian cancer and also glioblastoma (even if it is harder to use in this case because glioblastoma is a CNS pathology). Even if the “preferential” cancers are the ones that we mentioned for this molecule, it can also be used for any type of cancer that presents angiogenesis. - HER2: the main example is trastuzumab (also called Herceptin). It is mainly used in HER2+ breast cancer, it a humanized IgG that can target the HER2 receptor (ErbB2). HER2 receptor is typically overexpressed on the surface of tumoral cells in some types of breast cancer (25-30% of all breast cancer cases). HER2 usually amplifies signals generated by ErbB family members (growth factor receptors) by forming heterodimers with them. Trastuzumab down-regulates HER2 and disrupts its signalling (of cell growth), and it induces an Ab-dependent cytotoxicity (in particular, ADCC; this means that it both block the activity of the receptor and activates the immune system) and inhibits angiogenesis (in vitro); moreover, following resection, it enhances the efficacy of chemotherapy and reduces the rate of recurrence by 50%. In addition, it prevents the shedding of the receptor (because HER2 can be cut from the cancer cell so that its soluble form, constituted by the extracellular domain, can be released; trastuzumab prevents it). ErbB family members are human epidermal growth factor receptors, in which we have HER1, HER2, HER3 and HER4. HER2 is also known as neu (really important denomination for laboratory practice because we have some neu+ mouse which spontaneously develop breast cancer so we can use them to study the disease). They are tyrosine kinase receptors and they can have multiple different ligands (except for HER2, whose main role is actually to act a dimer partner for the full activation of the other epidermal growth factor receptors, so if we block HER we also block all the other HER receptors). Trastuzumab is not the only mAb that we have against HER2, in fact we can also use Pertuzumab, which targets the same receptor but on different domains. This means that they can also be sued together (because they target different epitopes of the same receptor), to double their efficacy. It is especially used for patients with HER2-positive metastatic breast cancer and it is intended for patients that have not received prior treatments for metastatic breast cancer with another anti HER2 therapy or with chemotherapy.  However, even if it is really effective against breast cancer, Trastuzumab could have some side effects: first of all, the binding of trastuzumab with the HER2 receptor induces a reduced expression of eNOS (nitric oxidase synthetases), which produced NO (nitric oxide), a molecule able to relax the smooth muscles of the vessels. Moreover, the binding of trastuzumab to HER2 induces a concomitant increase in angiotensin II (vasoconstrictor) levels and in ROS production. All of these factors lead to endothelial dysfunctions, with a reduced vasodilatatory capacity and an enhanced vasoconstriction tone, which could cause endothelial injury and dysfunction, vascular resistance, coronary microvascular blood flow and consequent myocardial workload, leading to congestive heart failure. All of this is due to the fact that when trastuzumab inhibits HER2, the dimerization of neuregulin and HER4 complex is prevented, and eNOS is not expressed anymore. This is why all women under trastuzumab therapy need to undergo frequently heart and vascular check-ups. However, this is the only severe side effects that trastuzumab could cause, the advantages/disadvantages ratio is all in favour of advantages, in fact we know that between the 80s and 90s, more than 100 mAb were generated against this receptor, but only trastuzumab pass the clinical trials. - DUAL SPECIFIC ANTIBODIES AGAINST EPIDERMAL GROWTH FACTOR RECEPTORS: dual specific antibodies are antibodies with two identical arms that recognize two different antigens (so they are not like bispecific antibodies, where one arm recognizes one antigen and the other a different one). In this way we can bind two different antigens with the same antibody (both in the case in which only one of these antigens is present or in the case in which both are present). Both mono-specific and Two-in-One antibodies bind antigens at low density with the affinity intrinsic to a Fab arm. At sufficiently high antigen densities, a Two-in-One antibody will benefit from an avidity effect of bivalent binding regardless of the probable variations in each receptor’s distribution. The avidity effect, which increases the apparent affinity, is a combination of a slower apparent off rate (koff) of the bivalently bound antibody and a faster apparent on rate (kon) of the second Fab arm binding to adjacent receptor on the cell membrane (not so important to know). We mention them because one example of these antibodies is the one directed against both HER3 and EGFR. - EGFR: epidermal growth factor receptors (EGFR) are a part of the family of HER, they are very important in the growth of many tumor cells so we can target it to weaken the cancer. They are again tyrosine kinase receptors, so once they bind the ligand they dimerize and activate (they can both homodimerize and heterodimerize). The activation of these receptors leads to an increase in the transcription of genes related to cell growth, inducing the loss of regulatory mechanisms for receptor signalling; so they promote cell survival (anti-apoptosis), angiogenesis, metastasis, proliferation and chemotherapy resistance. EGFR can be targeted by EGFR tyrosine kinase inhibitors, anti-EGFR mABs, anti-EGFR ligands mAbs (so anti-growth factor antibodies, in particular anti-EGF and anti-TGF-alpha), bispecific antibodies (which bring effector cells to the close proximity of tumor cells so they can be activated). In this way we prevent the dimerization and thus the activation of these receptors. Different types of tumor cells have different levels of expression of EGFR: they are highly expressed in a lot of tumors (even if in different ways), and they generally correlated to poor- prognosis solid tumors (NSCLC, colorectal cancer, glioblastoma, breast cancer, prostate cancer, …). For this reason we have several antibodies directed against these receptors: some examples are cetuximab (chimeric IgG1), matuzumab (humanized IgG1, in trial for recurrent ovarian cancer, NSCLC and colorectal cancer), panitumumab (fully human IgG2, in trial for colorectal cancer, NSCLC). Cetuximab was the first antibody anti-EGFR developed, it exerts also CDC and ADCC functions (other than blocking the dimerization of the receptor and its pathway of signal  transduction and other than stimulating the receptor internalization), inducing apoptosis, block of cell growth and decrease of EGF production. It has been demonstrated to be effective in combination with chemotherapy in patients with metastatic colorectal cancer, squamous cell carcinoma of the head and neck and NSCLC. The most common side effect of cetuximab is an acne-like rash, and some allergic and anaphylactoid reactions have been reported with cetuximab administration. The development of human anti-chimeric antibodies (HACAs) has been reported in 4 of 120 patients (4%) in the trial; 3 of the 4 patients had neutralizing antibodies. - CHECKPOINT INHIBITORS: the first example is the Lirilumab, which targets the KIR receptor, expressed on NK cells. NK cells are a part of the innate immune system, so their recognition of an antigen is not specific, they just activate against non-self-antigens through the summation of multiple different stimuli that could come from activating receptors or inhibiting receptors, like KIR. So they could get activated against tumors and their antigens, however tumors can escape this actions through the expression of some particular receptors that inhibit the triggering of NK cells by stimulating their inhibitory receptors. In fact, if antigens present on the tumor cell bind to KIR (for example MHC I molecules), the NK cells are suppressed. So if we use antibodies against KIR we prevent this interaction and so the NK cells can still be activated. Several studies in hematologic and solid cancers are ongoing, but of particular interest are trials in which lirilumab is being combined with anti-PD-1 (nivolumab) or with anti-CTLA-4 (ipilimumab). PD-1 is the programmed cell death receptor 1, it is a typical negative co-stimulatory receptor of activated T cells that is useful to regulate the activity of these cells, to avoid an autoimmune reaction. The binding of PD-1 to its ligands (PD-L1 and PD-L2) inhibits the effector T cell function. Unfortunately, some tumors could express PD-L1 on their surface, in this way they suppress immune surveillance and allow the neoplastic growth. Nivolumab is a mAb directed against PD-1, which will not bind PD-L1 on the surface of the tumoral cells anymore. In this way the T cells will only receive positive (activating) signals and so they will activate against the cancer. It has been approved for the treatment of Non-Small Cell Lung Carcinoma (NSCLC), metastatic renal cell carcinoma and advanced melanoma. Another antibody directed against PD-1 is pembrolizumab (humanized antibody), which is a gain really used in the treatment of NSCLC, because this type of tumor usually highly expresses PD-L1 on the surface of its cells. It has been approved as a monotherapy but it is frequently associated to chemotherapy so that we can have better results with lower doses of chemotherapeutic drugs (which could be toxic). We could also target the ligand, so PD-L1, instead of PD-1, and we obtain the same results. One example is Atezolizumab, which has PD-L1 as a target. Since a lot of cancer express PD-L1 and so a lot fo cancer are able to suppress the activity of effector T cells, we have a lot of mAbs directed against this checkpoint inhibitor pathway: other than the ones we just saw we also have dovalumab and avelumab (both PD-L1 inhibitors). Not all of them work in the same way but they can all be used in combination with chemotherapy. Then we could have antibodies against another checkpoint inhibitor, which is the CTLA-4: it is an intracellular protein of T cells which, only in the case of the activation of T cells, it gets exposed on the surface of the cell. Once it is exposed, the B7 ligand of antigen presenting cells, which participates also in the activation of T cell by binding to CD28 (co-stimulatory receptor), will ligate to CTLA-4 and induce the inhibition of T cell (regulatory mechanism to avoid autoimmune reactions). Tumors can exploit this mechanism to repress the activation of T cells against them, so if we send antibodies directed against the CTLA-4 receptor we can avoid its binding with B7 and thus we can prevent the tumor-induced repression of T cells. One antibody that is produced with this purpose is ipilimumab, a fully human monoclonal antibody which blocks the CTLA-4 receptor, potentiating the T cell activation, and is administered intravenously for the treatment of advanced melanoma. Ipilimumab improves overall survival to this disease compared to controls (treated with gp100, a vaccine peptide derived from a melanoma antigen  that is used to trigger a response against the tumor). However, at the same time, ipilimumab induced more serious adverse events, so this means that it is toxic. Another antibody directed against CTLA-4 is tremalimumab, which can act with different strategies: the main aim is to reactivate the T cells and make them proliferate, so that we a lot of CD4+ and CD8+ T lymphocytes that can react against tumor cells. In this sense, tremalimumab could either reactivate T effector cells by binding to CTLA-4 (so they prevent the negative signal that CTLA-4 could cause), or it could cause the activation of dendritic cells by inhibition of T regs (through the binding of the antibody with the CTLA-4 on the Treg surface, we leave the B7 on dendritic cells without a receptor to bind to, and this generates a phenomenon called reverse signalling in APC cells that causes their activation and the inhibition of tolerization, a mechanism through which these cells stop presenting the antigens of the tumor to T cells, especially if they are infiltrated, and so they stop activating them; in this way, APCs start triggering the activation of T cells again). The tolerization can also be inhibited directly by the binding of the antobody with the CTLA-4 on the surface of the tumoral cell (so that it doesn’t inhibit infiltrated dendritic cells anymore). Another mechanism through which tremalimumab is able to activate T cells is by preventing the activation of the Tregs, so that effector T cells can proliferate and activate (it does it by binding to the CTLA-4 present on the surface of Tregs). Moreover, the binding of the CTLA-4 directly on the surface of the tumor could cause the activation of apoptosis in these cells or the triggering of ADCC. So in general, these strategies of immunotherapy have the purpose to induce the reactivation of the suppressed immune system against the tumor to overcome the immune escape operated by cancer. Toxic effects of checkpoint inhibitors involve the general side effects related to a monoclonal antibody therapy (like diffuse inflammation of the lung parenchyma, so pneumonitis, gastrointestinal toxicities, hepatotoxicity, pain in the site of injection, problems at the thyroid, nephritis, …), but they are very rare. We could also have another strategy to prevent the binding between the CTLA-4 checkpoint inhibitor and its ligand, and we don’t use an antibody but a recombinant receptor, called CTLA- 4 Ig: we use it to inhibit T cells, so it does the opposite of anti-CTLA-4 mAbs. It is used for the treatment of rheumatoid arthritis, that’s why we need to inhibit T cells. It inhibit the activation of T cells by binding to CD80/CD86 (B7), so that CD28, a T cell co-stimulatory receptor, won’t be able to bind to them (CD28 and CTLA-4 share the same ligand on APCs). In this way, this recombinant receptor is able to reduce the proliferation of synovial recirculating T cells and to reduce the production of inflammatory mediators. We have two molecules that use this strategy: Abatarcept, made of the external portion of the CTLA-4 receptor and the Fc portion of an IgG1, and Belatacept (it is the same as abatarcept but it presents two amino acid substitutions). The second one is used for the immunosuppression of renal transplantation. - ANTIBODIES NOT USED AGAINST CANCER: Denosumab: it is a mAb used against osteoporosis. It is a fully human antibody of the IgG2 isotype, it targets a protein called RAK ligand. Since RANK Ligand is part of the TNF receptor family, high specificity of this mAb is demonstrated by the fact that denosumab does not bind to other TNF receptors, like TNF-alpha, TNF-beta, TRAIL or CD40 ligand. Many different factors can affect osteoclast activity, but RANK Ligand is one of the most important because it is required to mediate or permit the effects of these factors on bone resorption. Several factors (for example the parathyroid hormone [PTH], TNF,  interleukin [IL]-1) stimulate the expression of RANK Ligand by cells of the osteoblast lineage and other cells (like activated T cells), resulting in increased bone loss. Evidence from gene deletion and other studies indicates that RANK Ligand is an essential mediator of osteoclast activity, as it is a key factor regulating osteoclastogenesis and bone resorption, especially in a condition of low levels of macrophage colony- stimulating factor (M-CSF). The RANK Ligand polypeptide is a type II transmembrane protein found on the surface of expressing cells as well as in a proteolytically released (cleaved) soluble form. RANK Ligand is expressed (both in a transmembrane and soluble form) from the osteoblast lineage cells. Additionally, RANK Ligand is expressed in various cell types, including, but not limited to: lymphoid cells; basal epithelial, luminal epithelial, and stromal cells from normal prostate and non-malignant breast tissue, including mammary epithelial cells; tumor cells including breast cancer cells, prostate cancer cells, and subsequent bone metastases; dendritic cells, activated T cells and B cells, osteoclasts, mesenchymal cells, proliferative chondrocytes, early osteocytes, and periosteal cells; and synovial tissue, cardiomyocytes, vascular smooth muscle cells, endothelial cells, and the GI tract. RANK Ligand subsequently binds to its receptor, RANK, on immature and mature osteoclasts, which leads to maturation of prefusion osteoclasts to multinucleated osteoclasts, and finally to activated osteoclasts. RANK is a member of the TNF receptor family and is expressed on osteoclasts and osteoclast progenitors, but it has also been observed on cartilage cells (chondrocytes), mammary gland epithelial cells, and trophoblast cells. In premenopausal state, estrogen regulates the expression of RANK Ligand and, in particular, as estrogens levels decline during the menopausal period, RANK ligan expression increases; the elevated RANK Ligand levels lead to an increase osteoclast formation, function and survival, inducing an increase in bone loss, bone weakness and ultimately leading to osteoporosis and fractures. Anti-osteoclast effects are normally exerted by OPG, the natural endogenous inhibitor of RANK Ligand. This is why denosumab is considered an PG mimetic: the inhibition of RANK ligand leads to an inhibition of osteoclast formation, function and survival, which leads to the inhibition of bone resorption and to the block of the disease. This mAb is especially used on women in post-menopausal period that do not respond to other anti-osteoporosis treatments. Deosumab is also used in cases of bone metastasis of some tumors, because we could have bone resorption also in this case. Omalizumab: humanized antibody (5% murine) able to bind circulating IgE antibodies, which are regulators of the phenomenon of allergy, so this antibody is mainly used for the treatment of asthma. Allergies are usually treatment with antihistaminic drugs, but asthma is a really complex disease (it presents multifactorial causes), and so sometimes asthma patients do not respond to normal treatments. Omalizumab is able to form small, biologically inert complexes with IgE by binding to the Fc portion of the immunoglobulins. In this way they prevent the degranulation of mast cells and basophils that IgE normally mediate through the binding with the FcepsilonRI receptors; the release of pro-inflammatory cytokines from basophils and mast cells is thus reduced, as well as the levels of eosinophils in blood, tissue and sputum of the patients. It is used for patients with moderate to severe allergic asthma whose asthma is poorly controlled with inhaled corticosteroids and inhaled long acting β2 agonist bronchodilators. Abciximab: it is used to prevent platelet aggregation. Platelets normally get activated through the binding of GPIIb-IIIa receptor with von Willebrand factor, and then they get aggregated thanks to the binding of the same receptor with fibrinogen. This induces the formation of the clot: when this doesn’t happen physiologically, we could undergo some complications, especially when it happens in veins, so antiplatelet drugs are useful to avoid it. Abciximab is a chimeric mAb which targets the GPIIb-IIIa receptor with high affinity and low dissociation rate. It has a short plasma half-life of 10-30  minutes, so it needs continuous infusions; adverse reactions involve hemorrhages, thrombocytopenia, constipation, arrhythmias. It is used in unstable angina as an adjuvant for coronary thrombolysis therapy or in angioplasty surgery to prevent ischemic complications. Bamlanivimab, Etesevimab, Casirivimab and Imdevimab: they are anti-Sars-cov2 antibodies. Bamlanivimab is a neutralizing monoclonal antibody that targets the receptor-binding domain (RBD) of the S protein of SARS-CoV-2, which is the protein that binds the ACE2 receptor of the cells of the airways. Etesevimab is another neutralizing monoclonal antibody that binds to a different but overlapping epitope in the RBD of the SARS-CoV-2 S protein. Casirivimab and imdevimab are recombinant human monoclonal antibodies that bind to nonoverlapping epitopes of the S protein RBD of SARS-CoV-2. They are used for the treatment of mild to moderate COVID-19 in non-hospitalized patients (they don’t work in severe disease cases because in that case the cytokine storm caused by the infection has already started) with laboratory confirmed SARS-CoV-2 infection who are at high risk for progressing to severe disease and hospitalization. They are able to prevent the viral binding or the viral fusion with the host cell. Since they can bind different epitopes, they are frequently used in combination. - ANTIBODY VARIATIONS: we could have some antibody variations that we could use for therapeutic purposes: they are derive from the antibody molecule but they have a different structure. One example is diabodies: they are made of two variable regions directed against two different antigens, bound together by a peptide linker. Usually, one variable portion is directed against the target antigen and the other against an antigen of an effector cell (cross- link with effector cells). They can be expressed at high yield in bacteria. Then we could have small antibody fragments (scFv), which are smaller molecules that are able to penetrate the tissues more rapidly than normal antibodies, or we could couple antibody fragments to form dimers and tetramers and thus obtaining an increased avidity and cross-linking. Howe are scFV produced? In a scFv expression vector, the cDNAs that encode the heavy-chain variable region (VH) and the light-chain variable region (VL) of an antibody are assembled via the sequence encoding a linker peptide. Common linkers of 14–15 amino acid residues are long enough to span the distance between the N- and C-termini of the variable domains in a scFv. However, using linkers of 3–12 amino acid residues in length will result in the formation of diabody. When two polypeptide chains, VHA-VLB and VHB-VLA, linked by the short linkers are expressed in the same cells, dual-functional antigen-binding sites are formed by crossover pairing of the variable light-chains and heavy-chains. Another particular example is the one of nanobodies: they were discovered in camels; they are a particular type of antibody that lack the light chain, but they have the same antigen affinity as their four-chain counterpart. The domain which recognizes the antigen is called nanobody (VHH) and its structure makes it more resistant to heat and pH. Methods were developed to clone the VHH (Variable heavy camelid domains) repertoire of an immunized dromedary (or llama) in phage display vectors, and to select the antigen specific VHHs from these 'immune' VHH libraries. VHH are obtained by lymphocytes obtained by the peripheral blood of immunized camels. The technique to produce the library is very similar to the phage display method. Recombinant Nanobodies are small (15kDa) and strictly monomeric. They bind the target with nM affinity, are stable and easy to manipulate. Moreover, the Nanobodies often bind to epitopes that are less immunogenic for conventional antibodies, such as the active sites of enzymes. Due to their small size, they also target areas that are not accessible to standard antibodies. Another advantage is that they generally bind conformational epitopes and that they are well expressed in bacterial expression systems so that they are cheaper and easier to produce in all kinds of formats than standard monoclonal antibodies. So we can engineer antibodies to produce multiple types of constructs which have particular characteristics that makes them better than normal antibodies: we can bind them to enzymes, toxins, reporter molecules for detection of signals (diagnostic), … in general, taking advantage  of the high affinity of the variable regions of an antibody to build up very complex but very potent biotechnological drugs or diagnostic tools. There are at least 5 nanobodies under clinical trial and more than 20 under pre-clinical trials, mainly for the treatment of rheumatoid arthritis, airway viral infections and osteoporosis. We could also have immunoconjugates: they are constructs in which an antibody is bound to a chemotherapeutic drug, which in this way can be delivered only to the cells to which the antibody will binds. The consequence is that the administration of the chemotherapeutic drug will have no off-target toxicity. We have 3 methods to attach cytotoxic drug to variable regions of mAb: we couple the drug to lysine moieties in the mAb, or we generate aldehyde groups by oxidizing the carbohydrate region and induce a subsequent reaction with amino-containing drugs or drug derivatives, or we couple drugs to sulfhydryl groups by selectively reducing the interchain disulfides near the Fc region of the mAb. A promising immunoconjugate is BR96- doxorubicin conjugate a mouse/human chimeric mAb which targets antigens over-expressed on surface of human carcinoma cells of breast, colon, lung, and ovary; it presents disulfide reduction attaches mAb to the drug. A lot of other clinical trials of engineered antibodies are currently undergoing for the treatment of cancers but also chronic autoimmune diseases. One last example comes from the field of immunotherapy: TNFRSF18 is a receptor of T cells that is able to induce their activation even after immune escape operated by tumors. So we can produce an antibody that is able to stimulate this receptor by binding to it to induce the activation of T cells. BIOSIMILARS Biosimilars are the copies of biotechnological products, but they are not exactly the same as their biotech drug counterpart. They can also be called biogenerics, follow-on biologics/protein or subsequent entry biologic. Biosimilars are finely regulated: in 2005, the official biosimilar definition stated that “the active substance of a similar biological medicinal product must be similar, in molecular and biological terms, to the active substance of the reference medicinal product”. In 2015 a new definition was given: “a biosimilar is a biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product (reference product) in the EEA (European economic area). Similarity to the reference material product in terms of quality characteristics, biological activity, safety and efficacy based on a comprehensive comparability exercise needs to be established”. To demonstrate that a biosimilar as the same characteristics of safety and efficacy of the reference product, the so-called “comparability exercise” can be performed. For chemical drugs, a generic drug is the exact copy of the molecule of the original drug. For biotechnological drugs it is different because they are really complex products so when we reproduce them we will never obtain the same exact copy. For every pharmacological product we have a patent: the patent it’s an exclusive right of the pharma company that produced the drug to sell the product for 20 years, starting from the marketing of the drug (which generally occurs after some years from the patent application), preventing others to exploit the invention without the consent of the pharma company. The patent was invented as a way to provide the opportunity to the pharma company to recoup investments in the product that was produced, which generally is really costly, especially in the first steps of the development. Initially, the patent term used to start from the date of filling of the application from the pharma company, however the drug cannot be marketed without the FDA approval, therefore, since, as we said, this could take some years, by the time the drug hit the market the patent may only have a few years of protection left. Hence, a patent-term-extension mechanism was created in order to compensate for the time lost in obtaining market approval, so that the drug innovators remain incentivized to develop new drugs. The patent is usually associated to a period of data exclusivity, which refers to the period during which clinical trial results, detailing the approved drug's toxicology and efficacy, cannot be used by generic entrants for subsequent marketing approval. So, data exclusivity is a protection of drug clinical data submitted to the FDA (or EMA) for market approval. Under the protection of data exclusivity, such data cannot be relied on by other companies for a limited period of time for obtaining market approval without the holder’s authorization; however other companies are not precluded from generating their own data to obtain market approval. For example, in the US and Taiwan, a new molecular entity ("NME") is given five years of date exclusivity. However, after those five years are up, other companies can apply for market approval using the same data, and thereby avoid the costs associated with generating their own data. Therefore, such data exclusivity doesn’t ultimately prevent other companies from obtaining market approval, but it can slow them down a bit to incentivize the original holder. After the expiry of the patent, other companies can access the data of the clinical trials and so they can produce the drug without having the need to produce new data with their own clinical trials. Therefore, for biosimilars there is no need for a lot of new clinical trials, unlike an original biotech drug: biosimilars are a way of producing drugs without having to waste a lot of time and money on trials. Despite leading the field in the biosimilars market, Europe has faced challenges relating to uptake of these copies of biotech drugs. Uptake varies significantly between countries in Europe, with some, such as Italy and Spain, having relatively low use of biosimilars compared with countries where there is high acceptance of biosimilars, such as Austria, Germany, The Netherlands and Sweden. Which are the EMA guidelines for biosimilars? Since the EU approved the first biosimilar medicine in 2006, the EU has pioneered the regulation of biosimilars. Over the past 10 years, the EU has approved the highest number of biosimilars worldwide, amassing considerable experience of their use and safety. Biosimilars approved through EMA can be used as safely and effectively in all their approved indications as other biological medicines, so the guidelines have the purpose to make these drugs the most reliable as possible. For the EMA, a biosimilar is a biological medicine highly similar to another biological medicine already approved in the EU (the so-called ‘reference medicine’). Because biosimilars are made in living organisms there may be some minor differences from the reference medicine. These minor differences are not clinically meaningful, so no differences are expected in safety and efficacy compared to the reference product. Natural variability is inherent to all biological medicines (“the process is the product”, in fact we know that even the same company can produce slightly difference final products, so obviously products produced by different companies are different from one another), so strict controls are always in place to ensure that it does not affect the way the medicine works or its safety. Biosimilars are similar but they are not identical to their original counterpart, because they are produced in different cell lines and through different manufacturing processes. However, these small differences in substrate and manufacturing process may affect patient’s safety and the clinical efficacy of the product, that’s why they must be finely regulated and controlled. Which differences could we have between the original biotech product and its biosimilar? For example we could have differences in the glycosylation moiety (glycosylation happen in the same amino acid but with different carbohydrates), but this is usually allowed as it doesn’t change the efficacy nor the immunogenicity of the product. Biosimilars are approved according to the same standards of pharmaceutical quality, safety and efficacy that apply to all biological medicines approved in the EU. The aim of biosimilar development is to demonstrate biosimilarity, so high similarity in terms of structure, biological activity and efficacy, safety and immunogenicity profile. Immunogenicity, in particular, is very important to regulate: it must be the same, if not lower (if possible), than the original product, because we know that there are some safety and efficacy concerns regarding the immunogenicity of the biotech drug. So, no differences in safety or efficacy are expected between an approved biosimilar and its reference product. But biosimilar must demonstrate no significant difference from its reference product also for what concerns the pharmacokinetics and pharmacodynamics: robust analytical, toxicologic, PK/PD, and immunogenicity studies in comparison to reference product must be performed. However, a lower number of comparative effectiveness clinical trials can be conducted in patients in a disease for which the reference product is licensed, and there is no need to demonstrate efficacy in all indications. In fact, by demonstrating biosimilarity, a biosimilar can rely on the safety and efficacy experience gained with the reference medicine. This avoids unnecessary repetition of clinical trials already carried out with the reference medicine. So, the period of clinical trial testing for biosimilars is shortened: we can see that for the biosimilar pathway, the analytical studies take a longer time than clinical studies, while in the original drug pathway the clinical studies weight more in the development of the final product because we have to assess the efficacy and the safety of the drug for the first time. In biosimilars, once you established that the efficacy and the safety of the product is the same as its original counterpart, you don’t have to carry out any more studies because we already know that the original biotech drug is efficacious and safe on a specific population. So clinical trials for biosimilars are conducted in sensitive patient population with sensitive endpoints and they are designed to detect a difference, if there is one. This shortens the time of production of biosimilars: to produce an original product it takes around 12 years, while for the production of biosimilar only 7-8 years are generally required. The similarity of the biosimilar to the original product is assessed through comparability, thanks to the comparability exercise. The comparability exercise is the act to compare any issue related to the biosimilar with the reference product. In fact, from the aminoacidic sequence to the secondary and tertiary structures, to the glycosylation patterns, to the efficacy in animal models and to the final formulation… any step is compared to the ones of the production of the originator. This helps us to assess the similarity of the biosimilar to the original product, from a structural, physicochemical and functional point of view (bioassays are critical for this last point). This allows us also to perform, as we said before, comparative clinical studies: after we demonstrate, through the comparability exercise, that the biosimilar is similar to the original product, we know that the biosimilar molecule will be active against the pathology towards which the original product was produced, so the clinical studies won’t be performed on the patients of the same disease, but they will be performed just in a population of particular patients. For example, if I am producing a biosimilar of trastuzumab, we won’t be testing the molecule on any breast cancer patient but just on particularly sensitive patients. This means that to evaluate the overall survival rate of the molecule on the populations of patients we will be using the data generated by the clinical trials of the reference medicine and we won’t be producing new data in another clinical trial, so that we save money and time. However, keep in mind that the goal of comparative clinical studies is to assess whether the biosimilar is different from the reference, not to demonstrate safety and efficacy, so these characteristics, as well as immunogenicity, must be assessed in the ways we talked about before, independently from the comparability exercise. Immunogenicity, from a clinical point of view, must be evaluated by the analysis of the antibody formation and cytokine levels. Ultimately, only clinical studies and post-authorization pharmacovigilance to monitor potential immunogenicity will provide definitive evidence for product comparability to the innovator product in relation to safety and efficacy. When a biosimilar product is produced, we could expect that the biosimilar is not similar to the original drug (for example something went wrong during the production process), thus the biosimilarity cannot be demonstrated and the comparability test failed. In this case we have two possibilities: either the biosimilar is not actually similar, effective and safe and so it cannot be produced and marketer, or the biosimilar that we produced functions even better than the originator (“biobetter”). This is something that is not so rare because, since the patent lasts 20 years, in the meantime we could have made some developments in the production field and so some technical improvements could allow the production of a product that is even better than the original one. The biobetter is a different molecule than the original drug, in fact if it is authorized it is marketed as a different drug, but with the same target. This new drug could have some chemical or molecular modifications that make it work better from a pharmacodynamic point of view, or it could have a better pharmacokinetic profile (better absorbance, longer half-life, less immunogenicity, …), but it surely has a higher price. In general, it is improved in safety, efficacy or dosing regimen (because is it has an enhanced effect on the target for a longer period of time, it can be administered at lower doses and so also the side effects will be reduced). However, usually the development of a biobetter is reached thanks to small changes, while bigger changes in the original molecule induce to a product with no clinical effects and great safety concerns. If a biosimilar is highly similar to a reference medicine and has comparable safety and efficacy in one therapeutic indication (so against one pathology), safety and efficacy data may be extrapolated from other indications already approved for the reference medicine (so from other applications of the drug). Extrapolation needs to be supported by all the scientific evidence generated in comparability studies (quality, non-clinical and clinical). Extrapolation means that we can use the biosimilar for the same disease against which we had produced the original product without the need for clinical trials to prove its efficacy in these diseases. If we demonstrate that the biosimilar molecule matches the reference one through all the information generated in the biosimilar development program, we can assess that the biosimilar molecule is expected to behave the same way as the reference molecule in all indications and patient populations. Extrapolation is not a new concept, but a well-established scientific principle used routinely when biological medicines with several approved indications undergo major changes to their manufacturing process (for example to introduce a new formulation). In most of these cases, clinical trials are not repeated for all indications and changes are approved based on quality and comparability studies. Safety of biosimilars is monitored through pharmacovigilance activities, in the same way as for any other medicine. There is no specific safety requirement applicable only to biosimilars because of their different development route. Over the last 10 years, the EU monitoring system for safety concerns has not identified any relevant difference in the nature, severity or frequency of adverse effects between biosimilars and their reference medicines. Biosimilar competition can offer advantages to EU healthcare systems, as it is expected to improve patients’ access to safe and effective biological medicines with proven quality. However, EMA does not regulate interchangeability, switching and substitution of a reference medicine by its biosimilar. These fall within the remit of EU Member States. Interchangeability refers to the possibility of exchanging one medicine for another medicine that is expected to have the same clinical effect. This could mean replacing a reference product with a biosimilar (or vice versa) or replacing one biosimilar with another (by the physician). Since the biosimilar is a less expensive drug, it would be preferred to substitute the reference product with it (because this is the aim of biosimilar drugs, to save money). The replacement can happen by: switching (when the prescriber, so the physician, decides to exchange one medicine for another medicine with the same therapeutic intent) or substitution (practice of dispensing one medicine instead of another equivalent and interchangeable medicine at pharmacy level without consulting the prescriber, so it's a decision made by the pharmacist hence it is “automatic”). However, for biosimilars cannot be interchanged through substitution. For generics (small molecules), pharmaceutical equivalence equals to therapeutic equivalence (because it is the same exact molecule as the original small molecule, so sometimes, depending on local or institutional rules, pharmacists may be even required to substitute the original with the generic; physicians should be able to do that too, expecting the same benefit and no increased risk in patients). For biologics, the pharmacist cannot decide to interchange the biosimilars with the original products, because the drug is not the same as the original one and because usually biologics are not sold in pharmacies. So, this means that only the physician can replace an original drug with its biosimilar, so for biologics only switching is possible. The switching could happen between two reference drugs (so between two different molecules of the same class), it could happen between a reference and its biosimilar (normal switching), or from a biosimilar to its reference (reverse switching), from a biosimilar of a drug to another biosimilar of the same drug (cross-switching), from a reference drug to the biosimilar of another drug of the same class (intraclass switch), or vice-versa, from the biosimilar of drug to the biosimilar of another drug of the same class (intraclass cross-switch), or there could be multiple switching (back and forth from the reference to the biosimilar). Physicians decide whether to perform one of these switches solely for a single patient’s case, because the prior decision on whether to allow interchangeable use and substitution of the reference biological medicine and the biosimilar is taken at national level (so it is something that is not dependent on EMA). In Italy AIFA has stated that the physician choses the treatment with either a biotech drug (originator) or a biosimilar and the prescription of biosimilars is a first choice for the treatment of naïve patients (then, if it doesn’t work, we can perform a switching). This is because patients that need to undergo a treatment with a biotech drug are generally patients that need to be treated for their whole life, so we prefer to start with biosimilars and then change to other medicines with some differences, but this doesn’t cause any change in the therapy regimen. In Italy we have the example of epoetins: the switching to biosimilar or between biosimilars does not show any additional adverse effects or reduced efficacy. Which are the clinical impacts of biosimilars? They have lower costs and they increase access to medications of patients (thanks to the prescriber behaviour change), and this has helped saving up to 40% of money invested. Moreover, they induced a change in the paradigm of considering study designs and relevant endpoints (extrapolation concept), they cause dispensers and prescribers to consider substituting interchangeable products and they require providers to be aware of the pharmacovigilance issues, such as naming and immunogenicity items. Immunogenicity of a biosimilar must systemically be investigated, however the predictive value of non- clinical studies for the evaluation of immunogenicity in human is low, so this means that the comparison of the antibody response of the biosimilar to the reference product in an animal model may be a part of the comparability exercise, but still clinical studies will be required. Immunogenicity may have to be assessed individually for each indication or patient population, so an optimal antibody-assay strategy is needed (assays must be validated throughout product development, we must use screening assays that are highly sensitive, specific, precise, reproducible and robust, and an assay for the detection of neutralizing antibodies must be available). Immunogenicity has to be addressed in the Risk Management Plant (RMP). Generally, if the biosimilar has slight modifications from the originator, they are not more immunogenic that the latter. The switching between a biotech drug and its biosimilar, so a molecule with some slight changes, generally does not induce harmful immunogenicity, moreover, experience shows that a harmful immune response is unlikely also after a change in the manufacturing process of a biological medicine, since comparability studies prove that the batch from the new process is of the same quality and free of impurities or aggregates that can trigger immunogenicity. We can compare the generic drugs (small molecule drugs) to biosimilar drugs: