1. Anti-IgE effect of the mAb omalizumab
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Figure 1. The upper part of the figure shows the initial phase of the allergic cascade in the absence of omalizumab.
After their stimulation, B cells evolve into plasma cells which release immunoglobulins, in this case IgE. IgE binds to its receptors located on the surface of effector cells (mast cells and basophils). These effector cells will then be prepared to recognise and bind to allergens, and start the crosslinking effect and the subsequent degranulation and mediator release.
┘ When omalizumab is present, it binds to free IgE, blocking its ability to bind to the receptors located on the surface of the effector cells, thereby aborting the process. More recently, additional effects of omalizumab have been reported (Domingo C. Omalizumab for severe asthma: efficacy beyond the atopic patient? Drugs 2014; 74:521-33).
Anti-IgE effect of the mAb omalizumab
Effector cells
Mast cell
Basophil
Absence of omalizumab
B-Lymphocyte
Plasma cell
Free IgE X
Presence
of omalizumab
Blockade effect
Non active
Immune complexes
IgE-Omalizumab
Domingo Ch. Drugs 2017 1

2. Different mechanisms via which mAbs exert their anti-IL effect.
(A) IL blockade
(B) IL receptor blockade
mAb Anti-IL IL
mAb Anti-ILR
Figure 2. (A) Anti-IL monoclonal antibodies bind to the interleukin, preventing it from binding to its receptor. Mepolizumab and reslizumab bind to IL-5.
┘ (B) Anti-IL receptor monoclonal antibodies bind to the cell receptor. Benralizumab in a monoclonal antibody against IL-5 receptor (benralizumab also has a direct cytotoxic effect on eosinophils).
mAb: monoclonal antibody 
Anti-ILR: anti interleukin receptor
Domingo Ch. Drugs 2017

3. Sensitization – adaptive immunity
Allergens
IL-4
B lymphocyte
Plasma cell
Th0 lymphocyte
Th2 lymphocyte
Mast cell, basophil
Figure 3.- Sensitization phase.
This phase starts with the contact between the allergen and the dendritic cell or antigen presenting cell (APC). Then, the antigen is presented to the naive T lymphocyte which will differentiate into a Th2 lymphocyte, which then transmits the information to the B lymphocytes, which undergo an immunoglobulin switch and start to express IgE instead of IgM on the cell surface. These are then transformed into plasma cells, responsible for IgE release.
During this phase, the patient doesn’t have any clinical symptoms.
IL-13
Modified from: Domingo C. Omalizumab for severe asthma: efficacy beyond the atopic patient? Drugs. 2014; 74:
Domingo Ch. Drugs 2017

4. Acute phase of the allergic reaction
Allergens
Mast cell, basophil
Th0 lymphocyte
Th2 lymphocyte
IL-4
IL-13
Figure 4. Acute phase of the allergic reaction.
This phase follows the sensitization phase. In addition to promoting the allergic cascade through the APC and/or the direct stimulation of B lymphocytes, the allergens will be bound to the specific IgE the plasma cells have been releasing. These IgE will be bound to their high affinity receptors located on the surface of the effector cells.
The new events occurring are indicated in red.
B lymphocyte
Histamine, tryptase prostaglandins leukotrienes, cytokines
Plasma cell
Allergic symptoms
Modified from: Domingo C. Omalizumab for severe asthma: efficacy beyond the atopic patient? Drugs. 2014; 74:
Domingo Ch. Drugs 2017

5. Acute phase of the allergic reaction: the crosslinking effect
Allergen
Blood analysis:
Total IgE
Specific IgE
Skin Prick test
Cell membrane
Histamine
Figure 5.
This figure illustrates the effect of the crosslinking as well as the changes occurring in the internal part of the IgE receptor, necessary for degranulation.
The first image shows the cell membrane of mast cells and basophils and the receptors for IgE.
┘ After sensitization has occurred, free IgE molecules bind to their receptors
┘ Once an allergen arrives, it is captured by two IgE molecules.
┘ Then, a crosslinking effect occurs, causing changes in the intracellular part of the receptor that induce degranulation of histamine vesicles.
┘ This is the reaction observed in the skin prick test.
┘ Finally, we can measure the allergic reaction by the concentration of total IgE as well as of specific IgE.
IgE receptors
(FCɛRI)
Domingo Ch. Drugs 2017

6. Late phase of the allergic reaction
Allergens
Cytotoxic proteins, enzymes, leukotrienes
Allergic symptoms
Remodelling
Eosinophil
IL-5
IL-4
B lymphocyte
Plasma cell
Histamine, tryptase prostaglandins leukotrienes, cytokines
Th0 lymphocyte
Th2 lymphocyte
Mast cell, basophil
IL-13
Figure 6. Late phase of the allergic reaction.
The late phase of the allergic cascade is characterized by the secretion of IL-5 which recruits eosinophils. This is similar to the situation in the chronic phase (see next slide).
The new events occurring are indicated in red.
Domingo Ch. Drugs 2017
Modified from: Domingo C. Omalizumab for severe asthma: efficacy beyond the atopic patient? Drugs. 2014; 74:

7. Chronic phase of the allergic reaction
Allergens
Cytotoxic proteins, enzymes, leukotrienes
Allergic symptoms
Remodeling
Eosinophil
IL-5
IL-4
B lymphocyte
Plasma cell
Histamine, tryptase prostaglandins leukotrienes, cytokines
Th0 lymphocyte
Th2 lymphocyte
Mast cell, basophil
IL-13
Figure 7.
The chronic phase of the allergic cascade.
It mimicks the late phase of the allergic cascade since there is an eosinophil recruitment. It is basically characterized by the internal feedback that helps to perpetuade the loop. At this step, free IgE are able to bind to their receptors located on the APC membrane; this event will contribute to activate the allergic cascade. The new events occurring are indicated in red.
Domingo Ch. Drugs 2017
Modified from: Domingo C. Omalizumab for severe asthma: efficacy beyond the atopic patient? Drugs. 2014; 74:

8. Bronchial epithelium disruption
Innate immunity in asthma (I)
Allergens, Virus, Pollution
Bronchial epithelium disruption
Bronchial epithelium
TSLP, IL-25, IL-33
ILC2
IgE
B Lymphocyte
IL-4
IL-13
IL-13
Mucus production
Figure 8. Innate immunity was originally considered to comprise a barrier (the bronchial epithelium) and certain cells, basically macrophages. The present interpretation of the pathophysiology is quite different. The disruption of the epithelium entails the destruction of its cells, which then release mediators. Although not an active function (that is, mediators are not strictly speaking secreted), the process confers on the epithelium a kind of “secretory” function. Allergens , viruses and pollution can contribute to this bronchial epithelium disruption, or may sometimes be its main cause. These epithelial cells thus release some mediators (TSLP, IL-25, IL-33) which will stimulate type 2 innate lymphoid cells (ILC2). These ILC-2 will release the Th2 cytokines that stimulate IgE production and eosinophil recruitment.
IL-5
Eosinophil
Domingo Ch. Drugs 2017

9. Bronchial epithelium apoptosis
Innate immunity in asthma (II)
ILC3
Bacteria
IL-33
TSLP
IFN-γ
Bronchial epithelium apoptosis
NKT
IL-17
Neutrophil
infiltration
IL-5
IL-4
IL-13
B Lymphocyte
IgE
Eosinophil
Figure 9. Following the process described above, TSLP and IL-33 stimulate Natural Killer T cells (NKT). These NKT cells release Th2 IL which favors IgE production and eosinophil recruitment. They also release IFN-γ which, in turn, contributes to bronchial epithelium apoptosis, thus creating a feedback loop.
The innate lymphoid cells type 3 (ILC3) can be stimulated by bacteria; they release IL-17 which contributes to neutrophil infiltration.
Smooth muscle cell
Domingo Ch. Drugs 2017

10. What releases IL-5? Figure 10.
The cells which release IL-5 are mainly Th2 lymphocytes, eosinophils and mast cells. Mast cells release the highest amounts of IL-5.
By reducing the activity of Th2 lymphocytes and mast cells, omalizumab notably decreases IL-5 concentration. This is the mechanism that partially explains why omalizumab has an eosinopenic effect on the bronchial mucosa.
Domingo Ch. Drugs 2017
Domingo C. Omalizumab for severe asthma: efficacy beyond the atopic patient?. Drugs (2014) 74:

11. Allergens, Virus, Pollution
Representation of the interrelations between innate and adaptive immunity in asthma
Allergens, Virus, Pollution
Allergens
Bacteria
TSLP
IL-33
IL-25
Eosinophil
MUCUS
PRODUCTION+ AHR
Cytotoxic
proteins,
enzymes,
leukotrienes
ILC3
IL-17
NEUTROPHIL
Histamine, tryptase
PGD2
LTD4
Cytokines
IL-4
IL-5
ILC2
NKT
IFN-γ
Th0
Lymphocyte
Th2
APC
Serum free IgE
Mast cell, basophil
IL-4
IL-5
IL-13
Figure 11.
This figure shows the interrelations between innate and adaptive immunity.
The allergic cascade is represented on the left hand side and the innate immunity on the right.
As the figure shows, both arms finally produce the same interleukins (IL-4, IL-5 and IL-13).
┘ In this first screen we see the allergens being captured by the antigen-presenting cell (APC).
┘ The APC then present the allergen to the Th0 lymphocyte which in turn differentiates into a Th2 cell.
┘This Th2 lymphocyte releases IL-4, IL-5 and IL-13.
┘The IL-4 promotes the contact between Th0 and Th2 lymphocytes.
┘The IL-4 also promotes the contact between Th2 and B lymphocytes.
┘B lymphocytes differentiate into plasma cells.
┘Plasma cells release IgE that bind to their affinity receptor on effector cells.
┘These effector cells then release mediators such as histamine, LKT4, PGD2.
┘Allergens go directly to their high affinity receptors on effector cells,
┘ Some other allergens can directly stimulate B lymphocytes.
┘IgEs can directly bind to their high affinity receptors located on the membrane surface of the APC.
┘IL-5 recruits eosinophils that release toxic substances.
┘IL-13 facilitates mucus production.
┘On the right hand side (innate immunity), viruses, allergens and pollution can disrupt the bronchial epithelium.
┘ The bronchial epithelium then release mediators – alarmins (IL-33, TSLP, IL-25) – that stimulate type 2 innate lymphoid cells (ILC2s) and NKT cells.
┘ These ILCs finally release IL-5, IL-13 and under certain circumstances IL-4, that is, the same ILs as adaptive immunity.
┘These mediators released by the disrupted bronchial epithelium can also directly stimulate the APC, thus promoting the allergic cascade.
┘ Finally, bacteria can stimulate ILC3 which will release IL-17 and in turn recruits neutrophils.
AHR: airway hyperreactivity.
B Lymphocyte
Plasma cell
Domingo Ch. Drugs 2017

12. Basic description of eosinophil biology relevant to the lung
APC - iEos
Leukotrienes
Eosinophil
CHEMOKINES
(EOTAXIN)
CCR3
Specific
Basic
Proteins
ECP
EPO
EDN
iEos
Antigen
APC
BRONCHIAL
MUCOSA
Th0 lymphocyte
rEos
IL-5
Lung Parenchyma
Th2 lymphocyte
IL-13
IL-4
GM-CSF
EOTAXIN
IL3
IL5
BONE
MARROW
Figure 12.
This figure describes eosinophil biology.
The first image shows the first part of the allergic cascade and the sites influencing eosinophil biology.
┘ Eosinophils are mainly synthesized in the bone marrow. For the differentiation into eosinophils, the eosinophil progenitor (EosP) requires the presence of some mediators among which IL-5 is one of the most important. Initially considered to have an active function, the role of IL-5 is now considered to be more permissive than instructive. Thus, its presence is indispensable for EosP to differentiate into eosinophils. If IL-5 is absent or blocked, the number of EosP in blood will increase and subsequently, the number of EosP available to transfer to the bronchial mucosa will be higher than when bone marrow eosinophilopoiesis is not interrupted. This has therapeutic implications because if IL-5 is not totally blocked, some free IL-5 can induce eosinophilopoiesis in bronchial mucosa, and then the clinical effect of the treatment would be lower than expected.
┘ From the bone marrow, eosinophils are released into the blood stream where they travel all around the body and will be retained in the target organ, in this case the bronchial mucosa. They are captured by vascular adhesion molecules (VCAM1) whose expression on the membrane surface of the endothelial cells is facilitated by IL-4, one of the T2 cytokines.
┘ Once in the bronchial mucosa, eosinophils need to be activated in order to start releasing mediators which have toxic effects for the bronchial mucosa.
┘ Eosinophils have also been found in the lumen of the airways, their origin obviously being the bronchial mucosa. Here they have several functions, one of them being to act as antigen-presenting cells. They are also able to follow the inverse route, re-entering the bronchial mucosa from the lumen and travelling to the regional lymph glands. Since all these eosinophils come from the blood stream after being produced (or induced) at the bone marrow level, they are called induced eosinophils (iEos).
┘ Eosinophils are present in organs such as the gut or mammary glands, but were not initially believed to be present in the lung. It has recently been observed that there is a population of eosinophils residing in the lung parenchyma, which have been called resident eosinophils (rEos). In the rat, rEos have a different shape from blood eosinophils and so it was initially hypothesized that they might represent a different cohort of cells. In any case, in humans, these differences are not evident. Their function is still unknown but they seem to regulate the activity of the antigen-presenting cells.
B lymphocyte
Plasma cell
VCAM-1
LUNG VESSEL
Domingo Ch. Drugs 2017

13. X Allergens Blockades Domingo Ch. Drugs 2017 13 Seasonal Perennial
Figure 13. This figure shows that perennial as well as seasonal allergens can cause chronic asthma. Patients with chronic severe asthma due to seasonal allergens can also benefit from anti-IgE treatment.
Allergens
Seasonal
Perennial
Blockades X
Effector cells
Mast cell
Basophil
APC
Antigen-presenting
cell
Non-active
immune complexes
IgE-Omalizumab
Lymphocyte switch
Th0-Th2
Free IgE
Eosinophil
B-Lymphocyte
Plasma cell
Domingo Ch. Drugs 2017 13

14. Effect of IgE blockade
Figure 14.- This figure shows the effects of the IgE blockade.
┘ The anti-IgE effect of omalizumab was initially considered to be due only to binding of free IgE, preventing IgE from binding to its receptors located on the effector cells (mast cells and basophils). This is true, but it is not the whole story.
┘ The result of this first action is a decrease in the concentration of free IgE.
┘As the red arrow show, the decrease in free IgE reduces the number of high and low affinity receptors located on the APC.
┘ This reduces the number of high affinity receptors on Th0 lymphocytes and on Th2 lymphocytes (in blue).
┘ The immediate consequence is a decrease in the release of IL-4 and IL-5.
┘All of these downstream effects reduce the production of IgE.
Modified from: Domingo C. Omalizumab for severe asthma: efficacy beyond the atopic patient? Drugs. 2014; 74:
Domingo Ch. Drugs 2017

15. Severe Asthma Treatment
Uncontrolled GINA IV or GINA V- treat by phenotypes
Allergic Phenotype
(Clinical symptoms + IgE * +
Positive SPT and/or specific IgE)
Non-Allergic Phenotype (Absence of clinical symptoms +Negative SPT+ Negative specific IgE
Without eosinophilia
With eosinophilia
Eosinophilic Phenotype
Non-Eosinophilic Phenotype
Continue
Omalizumab
Controlled
Uncontrolled
OCS and/or Alternatives
Total IgE *
Total IgE 30-75*
anti-IL-5
Uncontrolled
Alternatives
Continue
same option
Controlled
OCS
Continue anti-IL-5
Controlled
Uncontrolled
anti-IL-5
Omalizumab
Omalizumab
anti IL-5
Uncontrolled
Figure 15. Decision tree for uncontrolled severe asthma treatment.
The guidelines advise treatment according to phenotypes. This decision tree is based upon  the different phenotypes patients may have. It should be noted that the allergic phenotype is not defined only by a positive skin prick test, nor is the eosinophilic phenotype defined by an elevated blood eosinophil concentration (the number of peripheral blood eosinophils is frequently high in allergic patients too). The phenotype is a clinical description that includes clinical symptoms plus laboratory test results.
The first general criterion used in this algorithm is the differentiation between allergic and non-allergic phenotypes. In allergic patients, omalizumab will be the first option, regardless of the blood eosinophil concentration. Anti-IL-5 may be an alternative in those patients in which omalizumab has failed and the blood eosinophil concentration is elevated. Since omalizumab has shown to be definitively effective when blood IgE concentration is above 76 IU/mL, in allergic patients with a high number of eosinophils and relatively low blood IgE concentration (IgE between 30 and 75 IU/mL), either omalizumab or an anti-IL-5 monoclonal antibody can be prescribed. If the calculated dose of omalizumab exceeds the dose advised in the dosing tables (due to weight or blood IgE concentration > 1500 IU/mL), a trial should be performed with the highest authorized dose.
In the non-allergic phenotype arm, specifically in the eosinophilic phenotype, when the anti-IL-5 agent has failed, omalizumab can be tried if blood IgE concentration is ≥ 30 IU/mL (compassionate use).
The alternative treatment includes weight loss as well as therapies which are not systematically included in the guidelines but may be tested by experienced research groups. In some cases (neutrophilic asthma), oral corticosteroids are not expected to work although a trial course may be recommended.
Omalizumab
IgE ≥30
IgE < 30
Uncontrolled
OCS and/or
Alternatives
Controlled
Continue
Omalizumab
Domingo Ch. Drugs 2017
Alternatives: weight loss as well as therapies not systematically recommended by the guidelines (azithromycin, methotrexate, thermoplasty).
In some countries, only a high blood IgE concentration is required to prescribe omalizumab, without skin prick test or specific IgE value.* IgE units: IU/mL